UNIVERSITÉ DU QUÉBEC À MONTRÉAL

MÉCANISMES MOLÉCULAIRES ET CELLULAIRES DE L'APOPTOSE INDUITE PAR L'ACROLÉINE:

IMPLICATION SUR LA SANTÉ HUMAINE

THÈSE PRÉSENTÉE À LA FACULTÉ DES SCIENCES COMME EXIGENCE PARTIELLE

EN VUE DE L'OBTENTION DU GRADE DE PHILOSOPHUE DOCTOR (Ph.D.) EN BIOCHIMIE

PAR

ANDRÉ TANEL, M.SC.

AVRIL 2007

UNIVERSITÉ DU QUÉBEC À MONTRÉAL Service des bibliothèques

Avertissement

La diffusion de cette thèse se fait dans le respect des droits de son auteur, qui a signé le formulaire Autorisation de reproduire et de diffuser un travail de recherche de cycles supérieurs (SDU-522 - Rév.01-2006). Cette autorisation stipule que << conformément à l'article 11 du Règlement no 8 des études de cycles supérieurs, [l 'auteur] concède à l'Université du Québec à Montréal une licence non exclusive d'utilisation et de publication de la totalité ou d'une partie importante de [son] travail de recherche pour des fins pédagogiques et non commerciales. Plus précisément, [l 'auteur] autorise l'Université du Québec à Montréal à reproduire, diffuser, prêter, distribuer ou vendre des copies de [son] travail de recherche à des fins non commerciales sur quelque support que ce soit, y compris l' Internet. Cette licence et cette autorisation n'entraînent pas une renonciation de [la] part [de l'auteur] à [ses] droits moraux ni à [ses] droits de propriété intellectuelle . Sauf entente contraire , [l 'auteur] conserve la liberté de diffuser et de commercialiser ou non ce travail dont [il] possède un exemplaire. »

REMERCIEMENTS

J 'a imera is tout d ' abord exprimer ma grat itude à ma directrice de recherche, la

Dr. Diana Averill-Bates, qui , grâce à son soutien et à sa superv is ion, m ' a permis de

réa li ser les travaux présentés dans cette thèse.

Je ti ens tout patticulièrement à remerc ier ma femme Pauline pour sa

co mpréhension et son encouragement tout au long des ix années de recherche et

surtout pendant la rédaction. Fina lement, j e déd ie cette thèse à ma mère et mon père,

a ins i qu ' à mes deux sœurs Andréa et Eliane, et à mes deux frères Eli e et Toni , en

gui se de remerciement pour leur présence continue ll e, leur suppo rt mora l et leur

confi a nce en mo i.

Je voudra is également remerc ier mes co ll ègues étudiants, Tatiana Souslova et

Kamin i Hitesh, qui m'ont grandement a idé dans les deux premi ères années de mon

docto rat par des conversations enrichi ssantes et pa r les beaux moments passés

ensembl e. De plus, j e veux remercier Mi che l Mari on pour so n support au ntveau

technique ain si que M. Bertrand Fournier (SCA D, UQA M) pour son ass istance dans

les analyses stati stiques de mes articles . Un grand remerc iement po ur la bourse

d ' excellence F rancine Beaudoin-Denizeau, Fondation UQA M, qui m 'a été accordée

pour fé li citer mon doss ier académique.

Le fin ancement nécessaire à 1 'é laboration des trava ux présentés dans cette

thèse a été fo urni par les Instituts de Recherche en Santé du Canada (IRSC) et le

Conse il de Recherche en Sciences Nature ls et en Génie (C RSNG). De plus, j e veux

remerc ier la Fondati on UQAM, les Fond s à l'access ibilité et à la réuss ite des études

(FA RE), le programme d'aide financ ière à la recherche et à la créatio n (PAF ARC), le

département de chimie de I' UQAM, le centre in teruni vers ita ire de recherche en

tox ico log ie (C IRTOX) et le centre de recherche en tox ico logie de 1 ' env ironnement

(TOXEN) pour les bourses d 'exce llence qu ' il s m·ont accordées.

TABLE DES MATIÈRES

LISTE DES FIGU RES ..................... . .. ... . . ... ......... . ... .. ......... ........ .. .. . VIl

LI STE DES ABBRÉYIATION S . . . ... .... . . . ... .. .. .. ............. ....... . ... . ......... . X l

RÉSUMÉ .. .... .. .. ...... .. ... ..... .. .... ... ........ .... ........... ... . ...................... . XV I

CHAPJTRE 1 .......................................................... . ... ........ . ....... . .

INTRODUCTION .... . .... . . .. ............. . ... .. .. . . . . ..... ... . .. . . . .. . . ........ .. ..... .. .

1. 1. L'ACROLÉINE . .. .. ... ... ... . . . ....... .. . . . ... . . ... . ..... . .. . .... . . . .... . .... ... .... .

1. 1.1. Généralités ......... . . . .. ........ ............ ... ....... . ..... .. ........ . . . ....... .

1. 1.2 . Propriétés physiques .. .. . ... . .. . . .. ........ ...... . .. .. .... . ..... ..... .. . .......... . 3 1. 1.3. Propriétés biochimiques . .. .. ... . . . .... ........ ..... ............... . . ....... .. .. 3

1.1 .3. 1. La réaction d'addition de Michae l. ......... . . ..... ................. . 3

1.1.3 .2. Interactions au niveau cellula ire: méca ni smes de toxicité .... .. 4

1.1.3 .2. 1. Modifications des ac ides nucléiques par 1 ' acroléine .. . 4

1.1 .3.2.2. Modifications des ac ides aminés par 1 'acrolé ine .. . .. .. 5 1.1 .3.2 .3. Interactions avec les thi ols et les facteur de

transcription................... ............... .... .. ......... ............ 7

1.1 .3.2.4 . Phys iopathologies assoc iées à l'acrolé in e. . ............ 9

1.1.3.2 .5. Métaboli sme de l'acroléine....... . ........ .. ....... . ..... 12

1. 1.4. Protection ce llulaire des effets néfastes de l'acroléine .... ..... . ......... .. 14

1.1.4. 1. Le glutathion (GSH)......... . ... ... ... . .. .... . . . .. . .. .. . ... . .. . .. ..... 15

1.1.4.1.1. Biosynthèse du GS H.. .................. .... .. .. .. .. .. .. ... 17

1.1.4 .2. Modulation du glutathion intrace llula ire par des composés

synthétiques. ... . . ........... ..... .... .. ... ...... ...... .. ............. . .. ....... .. 18

1.1.4 .2. 1. Le N-acéty i-L-cysté ine (NAC)...................... ... .. 18

1.1.4.2.2. Le 2-oxo-4-thiazol idine carboxy late (OTC).. . . .. .. .... 19

1.1.4.2 .3. Le L-buth ionine sul fox imine (BSO)... . .... . .. . .. ........ 20

IV

1.2. MÉCAN ISMES DE MORT CELLULA IRE....... ..... . . ... . .. .... .. ... . ........ 21

1.2 . 1. Nécrose . ... .. ........... ... . ......... . ... .. . .. .... . .. ................... .. ..... .... 2 1

1 .2.2. A po ptose............. ............. . ....... . ...... ... . .. .. . . . . ..... .... ... .. .. ..... 22

1 .2.2. 1. In trod uction. . ..... .. ..... ........ . ... . ... .. .. ... ...... ....... ....... . .. 22

1.2.2.2. Voies de signali sation de l'apoptose . . .. . . . . . ............ .. . . . . . . . . . 24

1.2.2.2. 1. La vo ie des récepteurs.......... . ...... .. ......... .. ....... 24

1.2 .2.2.2. La vo ie mitochondriale. . .. . .... .. . . . . . . . . . . . . . . . .. . . . . . . . . . 28

1.2 .2.2 .2. 1. Mitochondri e, potenti el membranaire

et libération du cytochrome-c.. .. . .. . .. . . . . . . . .. . .. . .. . .. 28

1.2.2.2 .2.2 . La famille de Bc l-2.... .... .. .... .. .... . .. .... 32

1.2.2.2.3. La voie du rét iculum endoplasmique .... ....... .. .... ... 34

1.2.2.3. Les caspases........ .. . .. .. .. ..... ......... .. ................ . ... . .. .... 36

1.2.2 .3.1. Nomenclature. ............ ... ... . .................... .... ... 36

1 .2.2.3.2 . Structure des caspases.. ..... .. .. ....... .... .. ...... .. .... ........ 36

1.2.2.3.3. Act ivation des caspases . . .. ... . .. . .. .... .. . . .. ..... .. .. . .. 38

1.2.2.3.4. Les substrats des cas pa es................................... 38

1.2.2.3.5. Régulat ion des caspases....... .. . .. . . .. .. . ... . ... .. ......... 39

1.3. LES YOlES DE SIGNALJSA TION DES MAPK, AS K 1, AKT ET P53.. .. . 42

1.3 . l Les voies de signali sation d ' apoptose médi ées par MAPK et ASK 1.... 42

1.3.2 La voie de survie AKT.. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..... 46

1.3.3 Le facteur de transcription p53 et son rôle dans l' apoptose .. . . ... ..... . .. . 46

1.4. PRÉSENTATION DU PROJET.. ..... ... . .. . .. .... ........... ... . ..... .... ........ 49

l .4. 1. ln trod uction............. . ... .... .. ..... . .... .. ........ . ............. ......... .... 49

1.4.2. Modèle ce llula ire.............. .... ............................... .. .... .. .. . ... 50

1 .4.3. Objectifs du projet........ . ... .... . .. ..... ............. . ...... . ...... ............ 5 1

1.4.4. Approche expérim entale....... ... .... .. . ..... ... .... ........... ................ 52

CHAPITRE II : RÉSULTATS EXPÉRIM ENTAUX.................................... 56

2. 1. Préface.............. . .... .... .. . .. .... ... ... .............. . ........ .... .. .. . .......... .. . 56

v

2 .2. A r tic 1 e l. .. .. .. .. . .. .. .. .. . . .. .. . . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . . . . .. .. .. .. .. .. 58

THE ALDEHYDE ACROLETN IN DUCES APOPTOSIS VIA

ACTIVATION OF THE M ITOCHONDRIAL PATI-IWA Y............... 58

RÉSU MÉ... ... . .. . .... . .. ........ . ......... . ... . ....... ... .... . ...... . .... 60

ABSTRACT.. .. . ..... ... . . . . .. . .. . . . . . . . .. . . . . . ....... . .. . .. .. . . .. . .. . ... 6 1

1 NT R 0 DU CTI 0 N .. .. .. . .. . . .. . .. .. . . .. . .. . .. . . . . .. . .. . .. . . . . .. .. .. .. . .. 6 2

MA TERIALS AND METHODS ....................... ......... .. .. .. 64

RESULTS. .... .. .. . . .. . .. . .. . .. . . . . . . . .. . . .. . . . .. . ..................... ... 70

DISCUSSION.. ............ . .... . ...... . .. . . ... ... ... . . .... . . . .... .. ... .. 74

FIGURE LEGENDS ..... .... . ........ .. ............. . ...... . ........... ... 9 1

REFERENCES... . ....................................... . ... .. . . ........ 96

2.3 . Art icle 11 .... . . . .. .. .... .. ..... ... .. ... ...... .... . . ... . .. .. ...... . .. . .. ........ .... . ... . 103

ACTIVATION OF THE DEATH RECEPTOR PATHWAY

OF A PO PTOS IS BY THE ALDEHYDE ACROLE I .......... .. .. .. .... .

RÉSUMÉ .... ... . ... ... . .. . . . .... . ....... . .. .. ...... . . . ... . . ........ ..... . .

ABSTRACT .................................. . ... . . .. . .... ....... .. .... . .

INTRODUCTION ... . . ..... . .. . ... ... .. ... ... .... .............. ....... ..

MA TERJALS AND METHODS ................................ .. . .. .

RESULTS . . .... . ...... . . . ....... .................. .... .. .... . ... . . .. . .. . ..

DISCUSSION . . .............................. .. ............ ..... .... ... . .

FIGURE LEGENDS ..... .. ...... ..................... . ..... . ... .. ... ... .

REFERENCES .... .. . ........ . ... . ... . ... ..... .. . .. . ..... . . .... ...... ... .

2 .4. Artic le Tl J. ............................................................ ... ....... ...... .

P38 AND ERK MITOGEN-ACTlVATED PROTEIN KINASES

MEDIATE ACROLEIN-INDUCED APOPTOS IS IN CH!NESE

HAMSTER OVARY CELLS .. .. . . .. .. . .... .... .... . . . ..... .. . . ............... .

RÉSUMÉ ......... . .. .. ... .. ...... . .... . .. ... . ........... . . .. .......... .. ..

ABSTRACT .... .. ............... . ............................... . ....... .

103

105

106

107

11 0

11 5

119

134

139

149

149

15 1

152

INTROD UCTION ........ ............ . ........ . .......... .. .. . ......... .

MATERlALS AND METHODS .............. oo .......... oo .... oooooo

RESUL TS ............ .. ......... 00 .... 00 ..... ...... .. 00 ........ oo •• 00.00 • •

DISCUSS ION ... . .................. . .... ... ... .... ........... ........... .

FIGU RE LEGENDS .. 00. 00 ... .. . . 00. 00 .. .. 00 ... . 00 . 00 .............. 00 000

REFERENCES .. ...... ..... ...... ...... .. .. ......... ... ... .. . ....... .. . ..

2.5. Article IV ............ . ........ .. ... . . . .............. . . ... .......... . .. . . .... ... . ...... .

INH IBITION OF ACROLElN-fNDUCE D APOPTOS I BY THE

ANTIOX IDANT N-ACETYLCY TEINE .. 00 .... 00 .... 00.00 .... 00 .... 00. 00 ..

RÉ UMÉ ...... .. .. ..... ... ... ... . ..... ... .. ............... ...... ........ . .

ABSTRACT ........................ . .............. .. .. . ............. . .. . .

INTRODUCTION ............................................. . ...... . . .

MATERlALS AND METHODS .... .. . .. .................. .......... .

RESULTS ..... .. . .. . .......... . ...... . ...... . ... . .... .. . .............. . .. .

DI SCUSS ION .............. .......... .... ... ......... ................... .

FIGURE LEGENDS ..... . ............. . . . . ... ............... . .. . ...... oo

REFERENCES ..... ........... .. . . ..... . ..... .. . .... . ........ ... ........ .

CHAPI TRE lU : CONCLUSION .. 00 .......... 00 .... 00 .......... 00 00 .. 00 .... 00 ........ .

3. 1 Induction de l'apoptose par l' acro léine selon la littérature .. 00 00 00 00 0000 00 00 00 ..

3.2. Les mécani smes d'action de l' apopto e induite par l' acrolé ine .. 00 0000 00 00 00 ..

3.2. 1. Induction de la vo ie mitochondriale de l' apoptose par l'acro léine ......

3.2.2. Induction de la vo ie des récepteur de mort par l' acro léine .. 00 .... 00 000

3.2.3. Im plicati on de la voie de signali sati on des MAPK dan l' apoptose

V I

153

157

162

166

183

187

199

199

200

20 1

202

205

210

215

220

226

244

244

247

247

250

induite par l'acroléine ..... 00 00.00 •• 00.00 00 •• 00 . 00 .... 00.00 .... 00 ... 00.00. 00 •• 00.00 .... 252

3.2.4. Induction de la vo ie de survie AKT pa r l'acroléine .. 00 0000 00 00000000 00 .. .. .. 254

3.2.5. Pho phorylation de la p53 par l' acroléine .. 00 .. 00 00 0000 .. 00 00 .00 .... 00 ..... 256

3.2.6 . L'antioxydant N-acéty lcystéine inhi be l' apoptose induite par

l'acrolé ine .. 00.00 . ...... 00.00. 00.00 .... 00. 00 ........... . . 00 . ....... 00 •••••• • 00 •••• 00. .... 256

V II

3.2.7 . Pertinence de l'acroléine dan s le traitement des cancers.......... .. ..... 260

A EXE........ ........ ..... ....... .... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... .. .. ... 263

ANTI-TUMORAL EFFECT OF NATIVE AND IMMOBILIZED

BOVINE SERUM AM INE OX IDASE IN A MOUSE MELANOMA

MODEL........ ..... ................... ..... ...... .. ....... .............. ... ...... ..... .................... 263

RÉFÉRENCES........................... .... ... . .. . ... ............ .... . . ......... .. ....... 274

-----------------------------------------------------------------·----------------------------------,

LISTE DES FIGURES

INTRODUCTION

1. 1. La réaction d'addition de Michae l entre un énolate et l 'acro léine. . ........ . .. 4

1.2. Des cycles formés par la réaction entre la désoxyguanos ine de 1' ADN

et l ' acro léine ..... .. ..... ... .. ....... . .... .... . .. . ......... . . .. . . .. . . . ... . ............. . . . .. .

1.3. Les structures de acroléine-lys ine et acroléine-hi stidine ...................... . .

1.4. Une illustration d' une glutathi olation d' une protéinepar l ' acroléine .... ... .. ... .

1.5. La réaction d' un thiol avec l 'acroléine ..................... .. .... . ..... . .. ........ .

1.6. Voie du métabolisme de l ' acroléine proposée chez la poule .. ......... ..... .. .

1.7. Mécanismes de détox ificati on cellulaire de l ' acroléine impliquant

5

6

7

7

13

le glutathion.. .. . .. . . . . .. . .. . .. . ... .. . .. . . . . .. . . . . . . . . . . .. . . . . . . . . . . ... . . ... . . . .. . . . . . . . . ... . 16

1.8. Acti vation des caspases par les récepteurs de mort . . . . . . . . . . . . . . . . . . . . . . . . . . ... . 26

1.9. Les deux principales voies conduisant à l'apoptose; la vo ie des récepteurs

de mort et la vo ie mitochondriale.. ....... ........... ................................... ..... ...... ........ 27

1.1 O. La régulati on de l 'apoptose au niveau de la mitochondri e. ..... ............... 29

1. 11 . Induction de l 'apoptose.. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... .. .. . . . . .. 30

1.12. Représentation schématique des membres de la famille Bc l-2. .. . . .. . .. . .. . . 33

1.13. Signalisation apoptotique de la vo ie du rét iculum endoplasm ique en

réponse à un stress. . ... .. . .. .. .. .. . . .. ........ . ...... . . . . . ............. .. .. . ............. .. 35

1. 14. Structure et activation des caspases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . ....... ... 3 7

1.15. Inhibition de l 'apoptose au niveau des caspases. . . . . . . . . . . . . . . .. . .. . .. . . . . .. . . 4 1

1.1 6. Signalisation des di fférentes MA PKs, ERK, JNK et p38 .. ... .. .. ...... ....... 43

1.17. Vo ies de signali sation d' apoptose impliquant p53 et ASK 1.. . . . .. . .. . .. . .. . . 45

1.18. Signalisation de p53 suite aux dommages ...... .................. ...... ...................... 48

ARTICLE J

Figure 1. 1 nduction of cytotoxicity by acrolein ....................... . ..... ....... .. ..... 79

ix

Figure 2. M orpholog ica l analys is of apopto i and necros i in ce li s

fo llowing exposure to acrolein .. . ..... .. . .. . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . ... 80

Figure 3. Acrolein induces depolari zati on of the mitochondri almembrane

and liberation ofcytochrome-c.. ... .. . .. . .. . .. . ... ... .. . .. . .. . .. . .. . .. . ... . . . .. . ... . . .... 8 1

Figure 4. Act ivation of initiator caspase-9 and cleavage of procaspase-9

by acrolein .. .. .... .. . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . .. . . . . . . . .. . . . . . . . . . . .. . .. 83

Figure 5. Acrolein cleaves procaspase-3 but inhibits enzymati c activi ty

of caspase-3. . .................... ... .. . ................... .. ............................. ... 85

Figure 6. Acrolein activates caspase-7 and cleaves procaspase-7 ........ .. .... ... ..... 87

Figure 7. Inhibition ofapoptos is induced by acrolein by a spec ifie inhibitor

of caspase-9. .... .... ...... .............. ... .............. . .. .. ..... ... .. ... . .. . .......... .. . 89

Figure 8. Acrolein causes cleavage of ICA O.......... . ............ .. ................. . 90

ARTICLE II

Figure 1. Acrolein induces externalization of pho phatidy lserine .................... ... .

Figure 2. Acrolein induces translocation ofF DD ....... . ............ ...... .... .. ... .

Figure 3. Acti vation of initiator caspase-8 by acrolein ... . .. ... .. ............ .. ..... .

Figure 4. FasL express ion is increased by acrolein whereas FasR express ion

i not affected .. .. ................ .... . .. ........... .... . .. . ......... ... . .. ... . . . .. .... .. .. .

Figure 5. Activation of caspase-8 and caspase-7 by acrolein is decreased

124

125

126

127

by an inhibitor of Fas receptor .. . ... .... .. . .......... . ... . . . . . . . . . . . . . . . . ... . . . . . . . . . . ... . 128

Figure 6. A n inhibitor of Fas receptor decreases apoptos i induced by

acrolein .. ........ ... ... ... .. . ... ... ... ....... .. . .. . ..... .. . ... ... .... .. . .. . .. .. ...... . . . .. ... 129

Figure 7. Acrolein in duces cleavage of bid to t-bid... . .. ... . .. ............. ....... ... 130

Figure 8. Acti va tion of caspase-9 by acrolein i decreased by inhibi tors of

caspase-8 and Fa R......... . ....... .... .. .......... . .. . .... ............... .. . . .... ... .. . .. 131

Figure 9. C leavage of PARP by acrol ein is inhibited by an inhibitor of

caspase-8. . .. . ................... . .. .. . . ....... . ... . ...... . ... ........................ .. . .. .. 132

x

Figure 1 O. Inhibition of caspase-8 decreases acrolein- induced chromat in

condensation........................ .... . .. ..... . . . ..... . . ........ .... . . .. . . .. . ... . . . . . ... .. 133

ARTICLE III

Figure 1. Acrolein induces act ivat ion ofp38 and ERK MAPI<.s, the JNK

substrate c-jun and ASK 1. ......................... . . ... ... ..... .. .................. ... ... 172

Figure 2. Inhibition of p38 and ERK. MAPKs decreases acrolein-induced

chromatin condensation ..... ..................... .... ..... .. ... ........ ....... ........................ ... .. .... . 174 Figure 3. Acro lein-induced activat ion of caspase-9 and caspase-7 and

cleavage of ICAD are decreased by inhibitor of p38 and ERK MAPKs .. . ...... . 175

Figure 4. Acrole in induces activation of AKT ..... . .... . ... . . . . .. ... ... .............. . 177

Figure 5. Inh ibition of PJ3K/A KT pathway by LY294002 or Wortmannin

enhances necrosis induced by acro lein ............................................. ... .. ... .. .. . 178

Figure 6. Effects of AKT inhibitors on acrolein-induced act ivati on of

caspases-9, -8 and -7 and cleavage of 1CA D.. .. . .. . ... . .. .. . . . . . . . ......... ..... . .. . .... 180

Figure 7. Acrolein induces phosphorylation of p53 at er 15 and ser46 as

we il as act ivation of AS K 1.. .. ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . .... 182

ARTICLE IV

Figure 1. NAC protects ce lis aga inst acrolein cytotox icity........ . ..... ...... .... .. . 232

Figure 2. Acrolein causes severe depletion of intrace llular glu tathione levels:

effect ofNAC .... . .. . .. ............ . .. ... .. ......... . .... .... . .... . ... . . .... .. ... ................ 233

Figure 3. Morpholog ical analys is of apoptos is and necros is in ce li s

fo llowing exposure to acrolein: protective effect ofNAC. ....... .. . . . . . . . ... ... . . . . 234

Figure 4. Acro lein-induced trans location of Bad to the mitochondria

is inhibited by NAC........ ... ...... .......................................... . ... . .... .... 235

Figure 5. Acro le in promotes trans location of Bax to mitochondria..... .... .... ... .... 236

X l

Figure 6. NAC inhi bits acro lein-i nduced trans locati on of Bc l-2 to the cytoso l. . . 237

Figure 7. NA C in hib its acro lein- induced depo larizati on of the mitochondr ia l

membrane. ..... . . .. .... . .... . . . ... . .. . ............... . .. . ... ............... . ...... . ... ...... 23 8

Figure 8. Acro lein- induced liberation of cytochrome-c to the cytoso l is

not affected by NAC.... . .. .... . ... . .... . ....... . ........ . . . . . .. . . .. ........ .. . .. . . . . . . . ... 239

Figure 9. Activation of ini tiator caspase-9 by acro le in is inhi b ited by NAC .. .. . . 240

Figure 1 O. NAC inhi bits activat ion of effector caspase-7 and ini t iator

caspase-8 by acro le in ...... ... . .. . . ... .. . ... .. ..... . ................. .. . . ... . . . . .... . ...... 24 1

Figure 1 1. NAC inhib its acro le in- induced c leavage of PARP. ........... ......... .. 242

Figure 12 . Proposed schema for NAC-induced inhibition of acro lein-induced

apoptosis .. . .. . . . ......... . . . . . . .... ........................ . . . .... . . . .... .. ... ... . .......... 243

CONCLUSION

Figure 3 .1 . Schéma rep résentant les vo ies de s igna lisation dans 1' induct ion de

l' apoptose par l' acro lé ine: inhi bit ion par le NAC.. .. . . .. . . . .. . . . . . . . .. . . . . . . . .. . . . . . 248

AFC

A IF

ALMA

A RE

AlF

A KT

AMC

AM Pc

ANT

A P-l

A PAF-1

ASKI

ATM

ATP

ATR

BH

BIR

BRCA I

L -B SO

BSA

BSAO

CA D

CA RO

CHAPS

Chk l

LISTE DES ABRÉVIATIONS

Amino tritluorocoumarin

Facteur inducteur d' apoptose

Ac ide mercapturique

Élément de réponse aux antioxydants

Facteur inducteur d' apoptose

Protéine kinase 8

A mino méthy lcoumarin

A dénos ine monophosphate cyc lique

Translocateur d 'adénine

Protéine acti vatri ce-1

Facteur acti vateur de protéase apoptotique

Kinase régulatri ce du signal apoptotique

Protéine mutée d' ataxia-te langiectasia

A dénos ine triphosphate

Protéine similaire à Rad3

Domaine homologue à Bcl-2

Répétition de IAP du baculov irus

A ntigène 1 du cancer de sein

L-buth ion ine-(S,R)-sul phox i mi ne

A lbumine du sérum bov in

A mine oxydase de sérum bovi n

A DNase caspase-dépendante

Domaine recruteur de ca pase

Ac ide 3-[(3 -cholamidopropy l)di methy lammonio]-2-hydroxy-1-

propanesul fon ique

Kinase de vérificati on 1

Chk2

Cl-IO

CK

CD

Cr mA

Cyt-c

DO

DED

DI ABLO/Smac

DI SC

DNA-PK

EGF

EGFR

ERK

EO R

FA DO

Fas

Fa sR

FasL

FBS

FCCP

FDP-Iys ine

bFGF

FLICE

FLIP

F 1-1

GA DD45

Kinase de vérification 2

Ovaire de hamster chinois

Créatinine kinase

Cyclophiline D

Cytokine modifi ant de la réponse

Cytochrome c

Domaine de mort

Domaine effecteur de mort

x Ill

Protéine liante de I' IAP avec un déri vé de la mitochondrie

acti vateur de caspase

Le complexe inducteur de s ignal de mort

Kinase DNA -dépendante

Facteur de croissance épiderm ale

Récepteur du facteur de cro is ance épiderm ale

Kinase régulée par des stimuli extrace llula ires

Espèce réactive de 1 'oxygène

Protéine associée au Fa avec un domaine de mort

Proté ine assoc iée au fi broblaste

Récepteur Fas

Ligand Fas

Sérum foeta l bov in

P-trifluoromethoxy-phenyl-hydrazone

NE -(3 -formy 1-3,4-deshydropipérid ino) lys ine

Facteur basique de crois ance de fibroblaste

Caspase-8, une proté ine assoc iée au li gand Fas

Protéine inhibi tri ce de FLI E

Hormone stimulante des fo lli cul es

Protéine induite par le dommages à l' ADN et arrêtant la

croissance

GA POI-l

y-GCS

GPx

GS H

GSSG

GST

HDL

HK

4-1-INE

HOCL

1-IPMA

HSPs

IAP

ICAD

IGF

IK

IKK

IL-l

.INK

LDL

LPO

MAPKs

MAPKK

MAPKKK

MDM2

MEK

a.-MEM

MOPS

Glycéraldehyde-3-pho phate déshyd rogénase

y-glutamyl-cystéine-synthéta e

Glutathion-péroxydase

Glutathion réduit

Glutathion disulfure

G 1 utath ion-S-transférase

Lipoprotéine de haute densité

1-lexokinase

4-hydroxynonénal

Acide hypochloreux

Acide 3-hydroxypropylmercapturique

Protéines de choc thermique

Inhibiteur de protéine apoptot ique

Inhibiteur de CA O

Facteur de croissance ressembl ant à l' in suline

Inhibiteur du facteur NF-KB

Kinase du K~ inductib le

1 nter leukine-l

Kinase du terminus-N de c-jun

Lipoprotéine de faible densité

Péroxydation lipidique

Protéines kina e act ivées par les mitogènes

MAPK kinase

MAPK kinase kinase

Murine doubl e minute

MAPKK de ERK

Milieu essenti el minimum alpha

Acide 3-(N-morphol ino )- propane sul fo n ique

XIV

AC

A DH

F-KB

0

N rf2

o2·-oH·

ONOO­

u-OH-PdG

y-OH-PdG

OTC

PA RP

PB S

PdG

PIKK

PI 3-K

PKB

PMSF

PS

PTP

PVDF

RA IDD

RE

N -acétylcystéine

N icotinamide adénine dinucléotide

Facteur nucléa ire-KB

Oxyde nitrique

Facteur de transcripti on nucléa ire déri vé de érythroide

Su peroxyde

Radical hydroxy

Péroxyn itrite

Isomères 6a et 6P de 3H-6-hydroxy-3-(P-D-

xv

2'désoxyri bofuranosy l)- 5, 6, 7, 8-tétrahydropyrido[3 ,2-a] purine-

9-one)

Isomères Su et sp de 3H-8-hycl roxy-3-(p-o -

2'désoxyri bofuranosy 1)-5 ,6, 7, 8-tétrahycl ropyrim iclo[3 ,2-

a]purine-9-one ou 1 ,N(2)-gamma-hydroxypropano

déoxyguanos ine

2-oxo-4-thiazo lidine carboxy late

Poly -A DP-ribose-po lymérase

Tampon phosphate sa i in

1 ,N2 - ( 1 ,3-propano )-2'-clésoxyguanos ine

Kinase similaire à la phosphoinos itide-3-k inase

Phosphatidy linos ito l-3-k ina e

Protéine kin ase B

Fluorure de phénylméthy lsul fony l

Phosphatid y lséri ne

Pore de transition de perméab i 1 ité

Difluorure de polyv iny lidene

Protéine assoc iée au RI P par un DD

Réticulum endoplasm ique

RIP

sos SOS-PAGE

SEM

SMAC

TNF-a

TN FR

TRAOO

VOAC

VLDL

Wt

Récepteur interagissant de protéine

Sodium dodesy lsul fa te

XV I

Électrophorèse sur ge l de polyacrylam ide en présence du SOS

Erreur standard de la moyenne

Activateur mitochondri al des caspases

Facteur de nécrose tumorale-a

Récepteur du facteur de nécrose tumorale

Protéine associée au TNF-R 1 avec un domaine de mort

Canal ionique voltage-dépendant

Lipoprotéin e à très fa ible densité

Wortmannine

RÉSUMÉ

L'acroléine est un aldéhyde a.,p-insaturé extrêmement réactif et ell e est un produit de la péroxydation lipidique. Ell e est un po lluant env ironnemental qui a été impliqué com me un facteur de risque de développer plusieurs malad ies resp irato ires (bronchite, asthme) et neurodégénératives (A lzheimer) ainsi que l'athérosclérose. L' acroléine qui se trouve à des concentrations élevée au niveau de l' hippocampe des patients atte ints de l' Alzhe imer est proposée comme marqueur biochimique pour le pronostic de la malad ie. L'acroléine est uti 1 isée comme herbicide et elle est générée par la fumée de cigarette, la cuisson d ' hui le, le métaboli sme de certa ins agents ant icancéreux, lors de la péroxydation des 1 ipides et après expos iti on aux rayons UV. L' acro léine est libérée lors des feux de forêt, ce qui représente un grand risque pour les pompiers et les seco uri stes. JI a été démontré que 1 'acro lé ine est un fort irritant des muqueuses et du système respiratoire. L'acro lé ine est produite lors de la désamination oxydative par la spermine oxydase . Les produits d ' oxydation des polyamines sont impliqués dans l' inhibition de la proli fé rat ion cellulaire, l'apoptose, et l' inhibition de la synthèse de l'ADN et des protéi nes. Ai nsi, à cause de l' impact impo11ant de l'acrolé ine sur la santé humaine, nous avons entrepris cette étude afin de déterminer, et ce pour la première fois , les mécani smes moléculaires impliqués dans la mort ce llulaire induite par cet aldéhyde.

Le premier obj ectif visait à défi nir l' implication de la vo ie mitochondri ale dans l' apoptose induite par l'acroléine. Cette étud e est réalisée chez les cellules ovariennes de hamster chinois (CHO). L'acroléi ne induit l' apoptose en entraînant la baisse du potentiel membranaire m itochondria l, la 1 i bération du cytochrome-C, l'activation de la caspase-9 initiatrice et la caspase-7 exécutrice. D'a ill eurs, l' acro léine a inhibé l'activité enzymatique de la caspase-3 exéc utrice tout en c li vant la fo rme proenzyme de la protéine. L'activation des caspases-7 et -9 a été confirmée par le c livage de leurs proenzymes. L' apoptose induite par l'ac roléine a été inhibée par un inhibiteur de la caspase-9 mais pas avec un inhibiteur de la caspase-3. L' induction de l' apoptose par l'acroléine a été confirmée morpholog iquement par la co ndensati on nucl éaire de la chromatine et par le cli vage de l' inhibiteur de la DNase act ivée par les caspases (ICAD). Le cli vage de I' ICAD libère l'endonucléase CA O qui emmène la fragmentation de 1 'ADN . Ces résu ltats démontrent que 1 'acroléine induit l' apoptose par la voie mitochondriale.

Le deuxième objectif visait à étab lir l' implicat ion de la vo ie des récepteurs de mort dans l' induction de l' apoptose par l' ac rolé ine. L expos ition des cellu les ovariennes de hamster chinois (CHO) à l' acro léi ne entraînait la translocation de l'adapteur de domaine de mort de Fas (FADO) à la membrane plasmique et l'activation de la caspase-8 ini tiatr ice. Kp7-6, un antagoni ste du récepteur Fas, bloquait les évènements apoptotiques en aval de la caspase-8 , comme l'activation de la caspase-7 exécutrice et la condensation de la chromat ine nucléa ire. L'acro léine induit une transduction cro isée entre la vo ie de signali at ion des récepteurs de mort et

XV III

ce lle de la mitochondrie en clivant la protéine Bid en sa forme tronquée t-bid qui se transloque à la membrane mitochondriale pour stimuler cette voie. L ' inhibition spécifique du récepteur Fas ou de la caspase-8 inhibait parti ellement l 'activation de la caspase-9 par l ' acroléine. Ces résultats démontrent que l ' acroléine active la voie du récepteur Fas en amont de la voie mi tochondriale. La caspase-9 demeurait active malgré l ' inhibition du récepteur Fas et de la caspase-8 suggérant que l 'acroléine peut induire la vo ie mitochondriale indépendamment de la vo ie des récepteurs de mort.

Le troisième objectif était d' examiner l ' implicat ion des proté ines kinases activées par des mitogènes (MAPK) dans l ' apopto e induite par l ' acroléine. Quelques études ont démontré que l ' acroléine active les MAPKs et d 'autres ont rapporté que l ' acroléine induit l 'apoptose. Cependant, aucune étude n a examiné le lien entre l ' activation des MAPK et l 'apoptose induite par l 'acroléine. Nous avons démontré pour la première fo is que l ' apoptose induite par l ' acroléine est dépendante des MAPK. Une heure d' exposition à l ' acroléine induisa it une forte phosphory lation de la kinase régulatrice extracellulaire (ERK), de la p38 et du substrat de la kinase N­termina le de c-jun (JN K), c-jun, chez les ce llule Cl-10. L' inhibition de la condensati on de la chromatine induite par l ' acroléine à l 'a ide de l ' inhibiteur de ERK, leU 126, et l ' inhibiteur de la p38, le SB203580, démontre clairement l ' implication de ces deux voies dans l ' apoptose induite par !" acro léine. En plu s, le UO I26 et le SB203580 inhibaient l ' act ivat ion des caspases-7 et -9 et le cli vage de I ' ICAD induites par l 'acroléine. D'autre part, les voies de JNK et de la protéine kinase 8 (AKT) semblent être impliquées dans la survie contre l ' agression par l 'acroléine, puisque des inhibiteurs pharmaco log iques de ces deux voies, le SP600 125 , le L Y294002 et le Wortmannin changeaient la mort par apoptose en nécrose. Enfin, l ' acroléine a entraîné la phosphory lation de p53, une proté ine responsable de la transcription de plusieurs gènes impliqués dans l ' apoptose dont ceux de Bax et le ligand Fas.

Le quatrième objectif visait à déterminer l ' implication du N -acéty lcystéine (NAC), un précurseur du glutathion, dans la protection contre la toxicité engendrée par l ' acroléine. L ' expos ition des cellules Cl-IO à une concentrat ion non-cytotoxique d' acroléine (4 fmol/cel l), baissa it le glutathion intracellulaire de 45 % de son taux initial. Le NAC augmentait le niveau du glutathion intracellulaire et otfrait une protection contre la cytotox icité et l 'apoptose induite par l 'acroléine. Le NAC affecta it l ' apoptose induite par l ' acroléine en inhi bant la vo ie mitochondriale. Le NAC inhibait la translocation de bad à la mitochondrie et la baisse du bcl-2 mitochondriale induites par l 'acro léine. D 'autre part, le NAC inhibait la baisse du potentiel membranaire, le clivage de la procaspase-9, 1 'activat ion des caspases-9, -7 et -8 ainsi que le clivage de PARP. L ' inhibition parNAC de l ' apoptose induite par l ' acroléine était confirmée morph ologiquement par la baisse de la condensation de la chromatine nucléaire. Ces résultats suggèrent que NAC peut être utili sé comme un antidote pour traiter les gens exposés à l 'acroléine.

En conclusion, ces résultats ont permis une meilleure compréhension de la tox icité de l ' acroléine, un des produits majeurs de l 'oxydation des po lyamines

X IX

impliqués dans la régulation de la proliférati on cellulaire et la croissance des tumeurs. En plus, cette étude a permis de proposer une exp li cat ion possible de l ' act ivité pharmacologique et/ou de la toxicité du cyc lophosphamide, un agent anticancéreux, qui est métabo li sé en acroléine et en un mélange de phosphoramides. Cec i ouvre la vo ie au déve loppement de l ' enzymothérapie par l 'am ine oxydase du sérum de bovin (BSAO) pour lutter contre les cancers. Le milieu cancéreux contient des concentrations élevées en polyamines qui sont des molécules nécessa ires à la division et la proliférati on ce llulaire. Ces polyamines seront oxydées par la BSAO et libéreront les prod uits d'oxydation dont l ' acroléine et le péroxyde d'hydrogène qui sont impliqués dans l ' inhibition de la proli fé rat ion ce llulaire, l ' apoptose, et l ' inhibition de la synthèse de l ' A DN et des protéines. Ce faisant, la taille de la tumeur régresse et la santé du pat ient s'améliora. D 'autre part, les résultats de ce projet permettent d'envisager la poss ibilité d'utiliser les compo és qui augmentent le ni veau de glutathion (GS!-1) dont le AC dans des cas de toxic ité à l 'acroléine, molécule qui prend de l 'ampleur dans la recherche sur la maladie d' Alzheimer.

Mots clés: acroléine, apoptose, mitochondrie, voie des récepteurs, MAPK, NAC.

1.1. L'ACROLÉINE

1.1.1. Généralités

CHAPITRE 1

INTRODUCTION

Le 26 avril 1914, M. Thomas Edison écrit une lettre à M. Henry Ford et je

c ite : "The injurious agent in cigareLtes cames principally fi'o m the burning paper

wrapper. The substance thereby formed, is ca/Led "Acrolein". ft has a violent action

on the nerve centers, producing degeneration of the cells of the brain, which is quite

rapid among boys. Unlike most narcotics this degenera/ion is permanent and

uncontro!Lable. 1 employ no persan who smokes cigare/Les". Cette cé lèbre lettre de

l' inventeur du télégraphe montre bien la noc ivité de cette substance et le dan ger du

tabagisme. De nos jours, 1 'acroléine soul ève de pl us en pl us d' intérêt dans la

recherche surtout au niveau des malad ies respirato ires et neurodégénérat ives.

L'acro léi ne (2-propenal) est la plus é lectroph ile des aldéhydes a ,f3-insaturées.

Elle est extrêmement volatile, piquante, lacrymogène et infl ammable. L' humain est

exposé à l' acroléine de multiples façon s et elle est un polluant omniprésent dans

l' environnement (Takeuchi et al. , 200 1; Li et al. , 1999). Cet aldéhyde est dix foi s

plus toxique que le formaldéhyde qui est largement étudi é en tox icolog ie de

l' environn ement (Sun et al., 2006) .

L'acroléine est utilisée comme herbi cide aquat ique depuis plus de 40 ans

(Bowmer et al. , 1976), car elle est cytotoxiq ue pour les ti ssus des plantes et se

décompose rapidement dans l'eau (Kissel et al., 1978). Son utili sation s'étend à la

synthèse des polymères de l'acrylate, de l'ac ide acrylique, de l' immunosuppresseur

FR90 1483 (Maeng et Funk, 2001) et de la cytotox ine lepadiformine (Greshock et

Funk, 200 1 ).

,---~~~~~~~~~~~~-------------- -- ------- ----~~~~~~-

2

L' acrolé ine se trouve dans tous les types de fumée, notamment la fumée de

cigarette (25 à 468 ~Lg par unité) (Esterbauer et al. , 199 1; Carm ines et Gawor ki ,

2005; Fuji oka et Shibamoto, 2006), l'émiss ion des moteurs et les feux de forêts ce qui

représente un ri sque pour les pompiers et les seco uri stes (Caux et al., 2002).

L' acrolé ine est auss i libérée dans la vapeur de la cui sson d ' huile dont on a rapporté

des cas sévères de toxicité (Beauchamp et aL. , 1985). En plus, ell e peut être produite

par l' oxydation des lipides et former de l'acrylamide, un puissant cancérigène, suite à

la cui sson de la patate par de l' huile végétale (Ehling et al. , 2005) . Durant la

combustion de matér iaux organiques, par exemple le diese l et le plast ique, l' ac rolé ine

e t libérée en grandes concentrat ions (G hilarducc i et a l. , 1995 ; Uchida et al. , 1998).

"ln vivo" , l'acroléine est un des métabo lites des agents anti cancéreux

cyc lophosp hamide et ifosfamide et e lle semble responsab le de l' effet thérapeutique

( chwerd t et al. , 2006; Kehrer et Biswa l, 2000) et de l' hémorrag ie cystique causée

par une expos ition chronique à ces agents (Batista et al. , 2006). De plus, e lle est

formée à partir de l'oxydation de la thréonine par la myé loperox idase du neutrophile

aux sites d ' inflammation (S hao el al. , 2005; Vas ilyev et al., 2005; Anderson et al. ,

1997). L'acrolé ine est produite par l'oxydation des polyamines (Hoet et Nemery,

2000; Agostinelli et al., 1996), par le métaboli sme de l' a ll y lamine qui est une toxine

card iovasculaire (He el aL., 1998) et par l'oxydati on de lipides insaturés par 1 ' ozone

(Med ina-Navarro et al, 1999). Ell e est auss i fo rmée par des réacti ons photochimiques

dans l'a ir pollué. Se lon une étude, le tabag isme est une source s ignifi cati ve

d ' acroléine à l' in téri eur des maisons mal aérées de l' Il e du Prince-Edouard du Canada

(G ilbert et al. , 2005). On estime à 5% la concentrat ion de l' ac roléine dans l'a ir pollué

par rapport au total des aldéhydes, notons que le fo rmaldéhyde représente le taux le

plus élevé.

L'acrolé ine est un irritant sensori el puissa nt. Une co urte exposition à 0.67

ppm endommage les muqueuses olfact ives et respirato ires (Cassee et al. , 1996).

D'autre part, une courte expos ition à 6 ppm peut emmener une baisse de 50% dans le

ta ux de respi ration chez le rat et une inhibi tion accrue des mouvements des c il s

3

(Bergers et al., 1996; Cassee et al., 1996) . Une expo iti on prolongée à 10 ppm

d' acroléine peut entraîner la mort d ' une personne et des cas d' irritation sévère des

poum ons et de la trachée ont été rapportés avec une expos iti on a llant de 0. 17 ppm à

0.43 ppm (Agency fo r Tox ic Substances, 1989). Des analyse récentes montra ient des

concentrati ons au-delà de 3 ppm da ns 1 0% des fe ux à la vi Il e de Boston aux États­

Uni s (Tre itman et al. , 1 980). Notons qu 'on retro uve un e concentrat ion de 0.04 à 0.08

ppm d'acrolé ine dans l' air ambiant (Costa et Amd ur, 1996) . On est ime la

concentrati on d 'acroléine inhalée en fum ant ou en res pirant dans un environnement

de fumeurs à 80 ~-tM dans le fluid e du tractus respirato ire (E iseri ch et al., 1995). Sa

production annue lle aux États-Uni s est estimée à 60 millions de li vres sans compter la

partie utili sée pour la synthèse de l'ac ide acrylique (Huang et al., 2002).

1.1.2. Propriétés physiques

L'acroléine est un liquide de couleur blanche ou jaune. Ell e a une odeur trè

désagréable qui peut être sentie à aussi peu qu e 0.2 ppm . La fo rmul e chimique est

C3H40 et la masse molaire est de 56.06 g/mol. La pre sion de vapeur de l' ac rolé ine

est de 220 mm Hg à 20°C et son coeffic ient de parti tion Log Kow est -0.01 (Agency

for Tox ic Substances, 1989) . Pour l' acroléine, 1 ppm représente une concentration de

2.29 mg/m3.

1.1.3. Propriétés biochimiques

1.1.3.1. La réaction d'addition de Michael

L'acro lé ine est capable de réagir selon la réaction d' additi on de Michae l

(Kaminskas et al., 2005; Maeda et Kraus, 1997) qui est une façon class ique de fo rmer

des 1 iaisons carbone-carbone (F igure 1 . 1 ). JI s ' ag it s implement de la conjuga ison d ' un

énolate nucléoph ile à un composé carbonyle a , ~- i n saturé te l que l' ac roléine.

L' acro léi ne peut réagir avec les ac ides nucléiques, les acides aminés et les thiols par

la réaction d'addi tion de Michae l, et de ce fa it créer de li a i ons protéine-acroléine-

4

proté ine (B urcham et Pyke, 2006), ADN-acro léin e-ADN (Sanchez el al. , 2005 ;

Kozekov et al., 2003) ou protéine-acrolé in e-ADN (M inko et al. , 2005) .

0 0'1 R)L'-x;t-R énolate

/ riL_ acroléine ·-rrh

0 t Figure 1.1 : La réacti on d' addit ion de Mi chae l entre un énolate et l' acrolé ine (Maeda et Kraus, 1 997) .

1.1.3.2. Interactions au niveau cellulaire : mécanismes de toxicité

1.1.3.2.1. Modifications des acides nucléiques par l'acroléine

L'acro lé ine réagit avec les bases azotées de l' ADN (Pawlowicz el al. , 2006;

Kitamura et al. , 2006; Talœ uchi et al., 2001 ; Kehrer 2000). L'acro léine réagit avec le

désoxyguanos ine (dG) de 1 ' ADN pour forme r des exocycles stéréo isométriques dans

les tissus humai ns (Ch ung et al., 1999; De Los Santos el al., 200 1) entraînant une

substitution de la guanine en thymine ou en adénine lors de la réplicati on de 1 'ADN .

Il a été démontré que la fo rme 1 (voir Figure 1.2) e t dominante après une réaction

entre l' acroléine et le 2' -désoxyguanosine de l' ADN tandi s que la forme 3 est la

stru cture la plus stable (Khull ar et al. , 1999 ; Kanuri et al. , 2002) . Ces exocyc les

prévi ennent la fo rmati on des ponts hydrogènes Watson-Cri ck et ainsi inhi bent la

synthèse de l'ADN jusqu 'à 70% et emmènent des erreurs durant la réplication chez

Escherichi a co li (Yang et al. , 200 1 ). Ces nouve ll es 1 iaiso ns peuvent contribuer à la

mu tagenèse spontanée et jouent un rôle dans le vie illi ssement et le cancer (Yang et

al., 2002; Chung el al. , 1999). L' addition de l'acrolé ine à la guanine se fa it par la

réaction d' add ition de Michael (Nechev et al., 2002 ; Pan and Chung, 2002). Une

récente étude démontrait par simulation d' énergie libre (L\G) que laN -alky lation de la

5

guanine éta it plus favorisée que la 0 6-alky lat ion (Ba lu el al. , 2002). L ' acroléine

stimule la ca rcinogénèse de l 'appare il urinaire chez le rat (Cohen el al. , 1992). Elle

est une inhibitrice potentielle des enzymes de réparation de l ' ADN, par exemp le la

poly-ADP-ribose-polymérase (PARP) (Grafstrom et al., 1994 ; Dypbukt et al., 1993).

Cela exp lique sa génotox icité et son activ ité mutagénique chez la drosophile (S ierra

el al. , 199 1) et les bactéries (Parent et al., 1996; Van Beerendonk et al., 1998) .

0 O H 0 8

H O fL~~ ~ N ~

HO~(::('z, )o 0 N N ..._ H

O H

1!

0 ÎOH

r --!--NH ~OH Hop ___ll· -~ ) 0 N N H

O H

2

O H:

1a

r--1 ~ HOl--o---J.JL . -~ - _ _) ~ H

OH 3.

Figure 1.2: Des cycles formés par la réaction entre la désoxyguanosine de l 'ADN et l ' acroléine (Yang el al., 2001, 2002 ; Khull ar et al. , 1999) . Il a été démontré que la forme 1 est dominante tandis que la forme 3 est la structure la plus table (Khullar et al. , 1999; Kanuri et al., 2002).

1.1.3.2.2. Modifications des acides aminés par l'acroléine

L 'acro léi ne réagit avec le groupe sul fhyd ry l de la cysté ine (Starkenmann et

al., 2005), l ' imidazo le de l ' hi st idine et l ' amine de la lysine des protéines (Kitamura el

al. , 2006; Takeuchi et al., 2001; Kehrer, 2000). D'autre part, l 'acroléine mod ifie les

résidus histidine de l 'anhydrase carbonique (Tu et al., 1989) pour former deux

nouveaux composés, le Afl-acétyl-~-formy l éthy lhi stidine (F igure 1.38) et le Afl­

acéty i-N r-formylethylhistidine (F igure 1.3C) (Uchida et al. , 1998). Bien qu ' il éta it

6

proposé que la réacti on de l' acroléin e avec les groupements amme fo rme les

propanals ~-substi tu és (R---N H---CH2---CH2---CHO) et la base de Schiffs (R---N I--l-­

-C l--1 2---CI-h ----CH-----N---R), une structure acroléine- lys ine ou W-(3 -fo rm yl-3,4-

dehydropiperidino) lys ine (FDP-Iys ine) a été identifi ée comme le produit majeur de la

réacti on (Esterbauer et al. , 199 1) (F igure 1.3 A ). Une récente étude a démontré le

potenti el é lectrophilique de la FDP-Iys ine (Furuhata et al., 2002). A insi la structure

N-a -acety i-FDP-Iys ine, générée par la réaction de la N-a-acétyl-lys ine avec

l' ac rolé ine, a été cova lement liée à la glycérald ehyde-3-phosp hate déshydrogénase

(GA PDH). Notons que la GA PDH est responsab le de la prod uction du nicotinamide

adenine dinucléotide (NADH). La structure FDP-Iys in e réag issa it auss i avec le

glutathi on pour fo rmer un nouveau conjugué proté ique (F igure 1.4) (Furuhata et al.,

2002).

A

HOOC~~O Ac-NH

B HOOCY?=\

Ac- NH N~~O

c Ac-NH'Ç O~N~N

Figure 1.3: L'acroléine réagit avec le groupe suiA1yd ryl de la cysté ine (Starkenm ann et al., 2005), l' imidazo le de l' hi stidine et l'amine de la lys ine des protéines (Kitamura et al., 2006; Takeuchi et al., 2001 ; Kehrer, 2000). Cette figure illustre les structures de l' acroléine-lys ine (A) et l'acro léine-hist idine (B et C). A, i\f'-acetyi-W-(3-form yl-3,4-dehydropiperidino) lys ine ou j\P -acéty i- F OP-lys in e. B, iV'-acety l-~­

fo rmylethy lhi stidine. C, 1\f-acéty i-Nr-fo rmylethylhi st idi ne. (Uchida et al. , 1998).

N H2

1 A crolein

""""' L y s ......,.....

UCHO N 1

-...- L y s ........,...

F D P-Iysine

7

SG (JCHO N

1 GSH ..

,.,..,.,. Lys ....,.,..

Figu•·e 1.4: Une illustration d' une glutathio lat ion d' une protéine par l 'acroléine (Furuhata et al., 2002) . L'acroléine réag it avec le groupe amine de la lys ine des protéines pour former la FDP-Iys ine, le produit majeur de réaction (Ki tamura et al. , 2006; Takeuchi et al., 2001 ; K ehrer, 2000). La tru cture FDP-Iy ine réagit ensui te avec le glutathion pour former un nouveau conj ugué proté ique (Furuhata et al. . 2002).

1.1.3.2.3. Interactions avec les thiols et les facteurs de transcription L 'acroléine est la plus réactive des aldéhydes a.,f3- insaturées. Elle se lie

rapidement aux molécules nucléophiliques comme le glu tathi on qui est un thiol

essentiel pour la défense antioxydante. On peut vo ir dan la Figure 1.5 la réaction

d' un thiol (glutathion) avec l ' acroléine par la réact ion d' add it ion de Michael.

RSH + ~-k-z ~SR H H

acrol ein

Figu•·e 1.5: Réaction d' un thiol (exemple: glutathi on) avec l 'acroléine (Tacka et al.,

2002). L ' acroléine, une molécule électrophili que, e lie rapidement aux molécules

nucléophil iques comme le glutathion qui est un thiol essentiel pour la défense

antioxydante. L'acroléine réag it avec le groupe ul fhyd ry l de la cysté ine selon le

mécanisme d'addi tion de Michae l (Starkenmann et al., 2005).

8

De plus, l ' acroléine inacti ve l ' enzyme glu ta thi one réductase qui régénère la

form e réduite du glutathion (GS H), lorsque le GS H est oxydé en disul fure de

glutathi on (GSSG) (N unoshiba et Yamamoto, 1999) . Ces réacti ons vont diminuer les

concentrati ons intracellulaires des thi ols, créant un déséquilibre dans la balance

d'oxydo-réducti on (Finkelstein et al. , 2001 ; N unosh iba et Yamamoto 1999) .

La balance d' oxydo-réduction des thiol est cri t ique pour plusieurs fonctions

cellulaires (A rrigo, 1999) et des changements indui ts par l ' acroléine semblent affecter

plusieurs vo ies de signali sation. Notons qu ' il y a un grand nombre de gènes et de

facteurs de transcription sensibles aux changements oxyda-réd ucteurs dont le facteur

nuc l éa ire-K~ (NF-K~) et la protéine acti vatri ce-1 (A P 1) (A llen et T resini , 2000;

A rri go, 1999). La régulation des facteurs de transcription a susc ité un grand intérêt

ces derni ères années. Ces protéines se lient au promoteur des gènes cibles

déc lenchant la transcripti on. La régulati on se fa it par activation de ces facteurs dans

le cytoso l pour une redi stribution dans le noyau, ou en affectant leur liaison au

promoteur. Ce processus requiert souvent de la cys téine au domaine de liaison à

l ' A DN. En se liant à un acide aminé nucléophile comme la cysté ine d ' un facteur de

transcription, 1 ' acroléine peut directement interférer avec cette transcripti on, ou

indirectement en diminuant le glutathi on intracellulaire. Plusieurs gènes et facteurs de

transcription impliqués dans le cycle ce llulaire et l ' apoptose ont été identifiés. "ln

v itro", 1 acroléine inhibe quelques-uns de ces facteurs notamment le facteur nucléa ire

kappa beta (N F-KB) et la protéine acti vatrice-1 (A P- l ) (Horton et al., 1999; Li et al.,

1999). Cec i se produit par liaison à une cystéine sur la so us-uni té p50 et/ou la sous­

unité p65 dans le cas du facteur N F-KB (Kumar et al. , 1992 ; To ledano et al., 1993).

Plus récemment, on a démontré que l ' acroléine pourrait réag ir avec la kinase

inductible K~ ( !KK) et supprimer sa foncti on et de ce fait inhiber l ' activité du NF-KB .

Normalement, I' IKK enlève l ' inhibiteur kinase ( IK) du NF-KB par phosphory lati on et

ainsi act ive le facteur de transcription NF-KB (Valacchi et al., 2005)

9

L 'acroléine inacti ve la disul fure-i somérase qui participe à la maturation des

proté ines nouve llement synthétisées en perm ettant une bon ne formation des ponts

disul fures au niveau du rét iculum endop lasmique (Ca rbone el al., 2005 ; Li u et Sok,

2004). En plu s, l ' acroléine inhibe la thioredox ine et la thioredox ine-réd uctase qui

jouent un rôle dans la proli férat ion cellulaire et la régulation de plusieurs facteurs de

transcripti on qui sont sensibles aux condit ions d' oxydoréduction (Yang et al., 2004).

Une exposition des cellules A549 provenant d' un carcinome de poumon à une

concentrat ion non-léta le d'acroléine ( 150 f mol/cell ule pour une heure) diminue le

glutathi on intrace llulaire de 80%. De plus, l 'acro léine augmente la transcripti on de

l ' enzyme de synthèse de GSH, la y-glutamy lcystéine synthétase (y-GCS). En ce

fa isant, la y-GCS aide à rétablir le ni veau de glu tath ion réd ui t. Notons que cette

enzyme appartient à la famille des éléments de réponse aux ant ioxydants (ARE).

Cette activation de la transcription de la y-GCS est médiée par 1 ' act ivat ion du facteur

de transcripti on nucléaire déri vé des érythroides (N rf2) qui se lie aux gènes de la

phase Il et induit leur transcripti on (Tirumalai et al., 2002). Uchida ( 1999) a rapporté

que 1 ' acroléine est capable d' indu ire 1 'express ion de la gl utathion-S-transférase

(GST) qui est une enzyme impliquée dans la détox ificat ion de nombreux

xénob iotiques.

1.1.3.2.4. Physiopathologies associées à l'acroléine

L'acroléine augmente l 'express ion des gènes de mucines M UC5AC et

MUC58 qui emmènent une hypersécréti on de mucus (801·chers et al. , 1998, 1999).

Cette hypersécréti on est responsable de plusieurs maladies respirato ires notamment la

maladie chronique pulmonaire obstructi ve (Hogg 200 1 ), l ' asthme, la fibrose cystique

(W itschi et al. , 1997; Samet et al., 1994), la bronchi te chronique et l 'emphysème

(Saetta, 1999).

En parallèle, l 'acroléine peut altérer la fonct ion des neutrophiles humains en

inhibant leur apoptose et en accentuant la libération de l ' interleukine-8

10

(chimioattracteurs des neutrophiles). Cette altérati on favori se le processus

d' inflammati on des vo ies respiratoires (Akgul et al., 200 1; Finke lstein et al. , 200 1)

puisque l 'apoptose est l 'étape clé dans la réso lution de l ' inflammati on (Has lett 1999;

Fadok et al., 1998). En plus, l 'acroléine inhibe la NA DPl-1-oxydase des neutrophil es

qui est essentielle dans la défense cel lulaire contre les pathogènes (Nguyen et al. ,

200 1 ). Plusieurs études ont démontré des effets multiples de l 'acroléine sur la

fonction des macrophages alvéo laires, notamment une inhibition de la phagocytose

(Low et al., 1977). De plus, l 'acroléine inhibe la production de superoxyde dans les

macrophages alvéo laires de rat et dans les neutrophil es humains ( guyen et al., 200 1;

Witz el al. , 1987). L 'acroléine induit un taux élevé de mort ce llulaire, une producti on

altérée de cytokines ainsi qu ' une inhibition de la synthèse des macromolécules et de

l ' act iv ité ATPase dans les macrophages (L i et al. , 1997, 1999), ce qui est

probablement la cause de l ' immunosuppress ion pulmonaire (L i et Holain, 1998). De

nouve lles observations démontrent qu ' à un tau x de respirati on norm al de 2 à 4 Llm in,

un enfant peut théoriquement recevo ir une dose immunosuppress ive d'acroléine ( 10-

30 ~Lg) pour une heure d'expos ition, une dose qui co rrespond à une expos ition typique

à la fumée secondaire de cigarette dans un restaurant. Cette dose supprimerait la

réponse des lymphocytes T au niveau des poumons des fumeurs (Lambert et al.,

2005) .

Des liaisons acro léine-protéines ont été trouvées chez des personnes souffrant

de néphropathie diabétique (S uzuki and M iyata, 1999), de la maladie d'A lzheimer

(Se idler et al., 2006; Willi ams et al., 2006; Kawaguchi- iida et al., 2006; Se idler et

Squire, 2005 ; Arlt et al., 2002 ; Pocernich et al., 200 1; Ca lingasan et al ., 1999) et de

l ' i chém ie cérébrale (Ad ibhatl a et al., 2003), qui sont des malad ies assoc iées avec

une élévati on de la peroxydation lipidique. On a suggéré l ' utili sat ion de l 'acro léi ne

comme marqueur biochimique en clinique pour le prognostic de la maladie

d ' A lzheimer (Ca lingasan et al. , 1999), des lés ions diabétiques g lomérul aires (S uzuki

et al., 1999), de l ' athérosclérose (Biswal et al., 2002 ; Uchida et al. , 1998), du stress

oxyda tif chez les nouveaux-nés (Tsukahara et al., 2004 ), du diabète du type 1 (Hata et

Il

al. , 2006), de l' in suffisanèe rénale chronique ( lgarashi el al., 2006), et pour le

d iagnostic des accidents cérébraux (Tomitori et al. , 2005). Le sui vi peut se fa ire en

testant la concentrat ion du conjugué acrol éin e-l ys in e dans l' urine du pat ient (Yan el

al. , 2006). Au ni veau de l' hippocampe des patients attei nts de la maladie

d' Alzheimer, l'acroléine atteint 2 nM (Love ll el al., 2000, 200 1 ). Récemment, on a

observé que l'acroléin e éta it augmentée au ni vea u de la moe ll e épinière sui vant un

traumati sme (L iu-Snyder et al., 2006 ; Luo et Shi , 2004). De récents résultats

suggèrent que l'acrolé ine joue un rô le critique dans l'athérogénèse chez les humains

et ceci, en altérant le déplacement et le nettoyage du cholestérol à partir de la paroi

artéri ell e et en rendant moins stab le la plaque d'athérome déjà frag il e (Vindi s et al. ,

2006). Le prob lème du nettoyage du cholestérol est dü au fa it que l' acroléine interfère

avec le transport de la lipoprotéine de haute densité (I-IDL) en mod ifi ant la protéine

apoA-1 à la surface du HDL (S hao et al. , 2005). En plu s, l' acro léine est impliquée

dans le développement de l' angiopathie dont souffrent les fumeurs, et le sang de ces

gens ainsi que ce lui des diabét iques contient des concentrat ions é levées en acroléi ne

(M isonou et al. , 2006).

La peau est auss i une des c ibles de l' acroléi ne. pui sque la molécul e est fo rmée

de faço n end ogène suite aux irradiations des lipides pa r les rayons ul trav iolets du

oleil (Niyati-Shirkhodaee et Shibamoto, 1992). Dans la peau, l'acro léine peut causer

l' hypersensibilité non-immunologique (Verrier et al. , 1999 ; Coverly et al. , 1 998),

l' infl ammation, le développement de cancer, le vie illi ssement, la cytotox icité et la

génotox icité (G rafstrom et al. , 1994; U.S . Department of 1-l ea lth and 1-luman

Sciences, 1993) . L' acrolé ine est capab le d'act iver le récepteur du facteur de

cro issance épiderm ale (EGFR) chez les kérati nocytes humains, emmenant l' act ivat ion

des protéines ki nases act ivées par les mitogènes (MA PKs) et ainsi induire la mort

cellulaire par a po ptose (Takeuch i et al. , 200 1 ). Mentionnons ici que 1 'act ivat ion des

MAPKs peut prendre différentes vo ies de signali sation et ainsi induire l' apoptose

dans le cas des kératinocytes humains (Takeuch i el al., 200 1) et 1' inhibition de

l' apoptose chez les neutrophiles humains (Finke lste in et al. , 200 1 ).

12

Enfin, l ' acrol éine semble changer le phénotype bénin des tumeurs de co lon à

ce lui de malin et ceci probablement en inhibant la synthèse du p53 (Zarkov ic et al.,

2006).

En général, on remarque que l ' acroléine induit la mort ce llulaire par nécrose

(Rudra et Krokan, 1999) et cause une inhibi tion de la proliférati on ce llulaire

(Agostinelli et al., 1994, 1996; Biswal et al. , 2002). Toutefois, l ' acroléine peut

induire l ' apoptose ou l ' inhiber dans le même type cellulaire et cec i dépendamment de

la concentration et le type cellulaire utili sé. Par exemple, une fa ible concentration en

acroléine (< 10 ~-tM ) induit l 'apoptose chez les neutrophil es tandis qu ' une plus grand e

concentration (> 10 ~-tM) l ' inhibe (F ink.elsetin et al. , 2005).

1.1.3.2.5. Métabolisme de l'acroléine

Jusqu 'à récemment, il n' y ava it pas eu d'étude déta ill ée sur le métabolisme de

1 ' acroléine chez aucune espèce. On rapporta it que 1 ' acroléine était métabol isée en

glyc idaldéhyde, glyceraldéhyde et l ' ac ide acry lique dans les microsomes de fo ie de

rat (Pate! et al., 1980). Sharp et al. proposa it la vo ie métabolique ci-dessous (Figure

1.6) pour l ' acroléine chez la poule. Le métabolisme et la distribution du 2,3-C 14 de

l ' acroléine étaient étudiés chez 10 poules qui ont reçu oralement des doses de 1.09

mg/kg de masse corporelle par j our pour 5 j ours. La radioactivité était mesurée dans

les œufs, les excréments et l ' air expiré.

Il 0 1 Il

II 2C= C- -Il

13

, ,?~ ,,-~:: ,- [ ,J~c,,J, J [ "' ~L~-o"] [ /O" 11 J

I-I 2C - CH - C-H

1 ,3-PRO PANEDIOL 3-1-I YDROX YPROP IONALDE I-I YDE ACR YLI A CID G L YC IDA LDEHYDE

1l GLY CE RIC ACID

1l @L YCERA L DEIIYDE]

1l @LYCEROL -3 -P H OS P I-I AT'D~ GLY EROL

1l [ ENOL-PYRU V AT E]

1l ~ AM INOA ' IDS

TR IGLYCE RID ES

[I' YRUVAT'D ~ 1- A TATE

~::~tO~> eATHA 'D CREATINE

1l I IOLEST ERO L

AM INO AC IDS == @XALOACETATE] --"'---------~ ITR ATE]

TCA CYCLE ~co2 AM INO ACIDS

! ~URINES] - ALLANTOIN

Figure 1.6: Voie du métabo lisme de l' acro lé ine proposée chez la poule, en é tudiant la radi oactiv ité du 2,3- 14C de l'acro lé ine . Les composés entre pa renth èses sont des in te rmédia ires qui ne sont pas iso lés (S harp et al ., 2001 ). Le méta bo lisme et la di stributi on du 2,3-C 14 de l' acro lé ine éta ient étud ié c hez 10 poules qui ont reçu o ra lement des doses de 1.09 mg/kg de mas e corpore lle par j our pour 5 j ours. La radi oacti vité éta it mesurée dans les œ ufs, les excréments et l' a ir expiré .

14

1.1.4. Protection cellulaire contre les effets néfastes de l'acroléine

La glutathi on-S-transférase (GST) li e l' ac roléine au glutathi on (GS I-I ) qui est

impliqué dans la défense cellulaire et ainsi éviter à la ce llule les effets indés irables de

1 'acro léine (Meacher et Menzel, 1999; Berhane el al., 1994; Eder et al., 1982).

D'autre part, l'acroléine induit la GST et la GS H-péroxydase in vivo suite à une

agress ion vasculaire chez le rat pour contrer la ba i se accru e de la GS l-1 -péroxydase et

de la GST au début de l'agress ion (Yousefipour el al., 2005) . L' aldéhyde­

déshydrogénase-! est une enzyme importante dans la défense ce llulaire contre les

a ldéhydes tox iques dont l'acroléine chez l' humain (Lindahl et Peterson, 199 1;

Burcham et Fontaine, 2001 ). Cependant, l'acrolé ine entraîne une inhibition de

l' enzyme a ldéhyde déshydrogénase-! chez l' humain (Ren el al., 1998; Bunting et

Townsend , 1998), ce qui bloquera une des vo ies d' éliminati on de l'acroléin e.

L'enzyme 0 6-alkylguanine-ADN-transférase enl ève la modifi cati on apportée

à la pos ition 0 6 de la guanine après une alkylati on par l'acrolé ine (Ca i et al., 2000;

Balu el al., 2002) . De plus, récemment, on a découvert que la ce llule possède un

mécani me de réparation pour contourner les domm ages infligés à la guanine. En fa it,

la proté ine Rev 1 aj oute un rés idu C en face du G al ky lé par l'acrolé in e et la

polymérase Ç continue la répli cation et ain si la mutati on est réparée (Wolfle el al.,

2005 ; Washington el al., 2004).

L' hydral az ine, qui est un antihypertensi( est un piégeur efficace de l' acroléine

(Burcham et Pyke, 2006; Liu-Snyder el al. , 2006; Kaminskas el al., 2004; Burcham et

al., 2000). En plus, la carnos ine, un puissant anti oxydant, est auss i un piégeur de

l' ac roléine (A idini el al., 2005). Une nouve ll e étude vient de démontrer que la

v ita mine Cou l'ascorbate peut protéger des lipoproté ines te lles que les lipoproté ines

de très fa ible densité (VLDL) et les lipoprotéines à fa ibl e densité (LDL) contre

l' oxydati on par l' ac rolé ine. Les VLDL oxydées seront phagocytées par les

macrophages, une étape c lef dans l' initi ati on de l' ath érogénèse (A rai el al., 2005).

15

1.1.4.1. Le glutathion (GSH)

L'acroléine a été identifi ée comme un initi ateur et un produi t de la

peroxydation lipidique (Bi swa l et al. , 2002; Uc hid a, 1999) . De plus, ell e induit la

producti on de l' oxyde nitrique (NO), du superoxyde (02·-), elu peroxynitrite (ONOU)

et du peroxyde d' hydrogène (H20 2) (M isonou et al. , 2006). Comme ce processus

d'oxydation des 1 ipides ex iste naturell ement chez toute les ce llules et qu ' i 1 est

augmenté dans plusieurs pathophysiolog ies, un potenti e l de toxicité ex iste après une

exposition continue à un agent oxydati f te l que 1 ' acro léine.

Le glutathion (GSH), le plus important antioxydant intrace llula ire, peut

détox ifi er l' acroléine, une réaction catalysée par la T (F igure 1.7) . Trois produits

de détoxification sont fo rmés de la réacti on entre r acrolé ine et le GS H, so ient les

deux produits majeurs, l' ac ide 3-hydroxypropylmerca pturique (HPMA) et l' ac ide 2-

carboxyethylmercapturique (CEMA) , et l' ac ide merca pturique (A LMA) (Athersuch

et ai., 2006) . De plus, le cycle du glutathi on est le mécani sme central pour éliminer le

peroxyde d' hydrogène (H20 2) qui est auss i produi t dans la ce llule en présence

d' acrolé ine (Luo et al. , 2005).

D'autres substrats métabolisés par le glutathi on incluent les larges molécules

d' hydroperoxydede 1 ipides, formés par 1 'attaq ue de rad icaux 1 ibres sur les 1 ipides

membranaires, ain si que des produits de la réaction cataly ée par la lipooxygénase

(Heffner et Repine, 1989). L' enzyme clé du cycle d'oxydoréducti on responsable de la

réducti on du 1-120 2 est la glutathione péroxydase (G Px) (Hashicla et al ., 2002) (F igure

1.7). Cette réaction requiert spéc ifiquement le glutathion réd ui t (GS H) qui sert de

donneur d'é lectrons. Le glutathion di sul fure (GSSG) fo rm é au cours de la réacti on est

réduit de nouveau en GS H par la glutathion réd uctase (G R) (Halli we ll et Gutte rid ge,

1999), qui utilise le NADPH généré par le cyc le de pentose phosphates (C PP) qui

sert de donneur d 'électron (Rahman et al. , 1999). Des ce llules non tressées

maintiennent un rapport intracellula ire GS H:GSSG élevé pour assurer une

di sponibilité élevé de GSH (Deneke et Fanburg, 1989).

16

Détoxification

L-BSO

'-yccs

Glu + Cys ______. Glu-Cys+ Gly

NAC / ~ CAT

OTC NADP+. :;s~GSH ( H202

G&PD~cNADPH ~GSSG' 2H,O

Yz Oz+BzO

HS"'.. HO CHzO 0

1 H 1 Il Hz A C N CH C C

-1' "'- / y \ 1 "'- / "cH 0 C N C 1

OH Hz H Hz

0 NH2

Glutathion

GSH : glutathion L-BSO : L-buthionine sulfoximine NAC: N-acétylcystéine OTC : 2-oxo-4-thiazolidine carboxylate GPx: glutathion peroxydase GS: glutathion synthétase OST: glutathion -transférase GR: glutathion réductase y-GCS : y-glutamylcystéine synthétase CA T : catalase G6PD: glucose 6-phosphate déshydrogénase

Figure 1.7: Mécanismes de détoxication cellulaire de l'acroléine impliquant le glutathion (adapté de Anderson, 1997; Rahman et al., 1999; Logan et al., 2005). Le GSH détoxifie l'acroléine à l'aide de la GST et le peroxyded'hydrogène (HzOz) à l'aide de la GPx. Le glutathion disulfure (GSSG) formé au cours de la réaction est réduit de nouveau en GSH par la glutathion réductase (GR) (Halliwell et Gutteridge, 1999), qui utilise le NADPH généré par le cycle des pentose phosphates (CPP) qui sert de donneur d'électron (Rahman et al., 1999).

17

Le GSl-1 est essentiel pour la défense antioxyclante, il réagit directement avec

les radicaux intermédiaires des réactions non-enzymatiques. L ' élimination du

superoxyde 0 2·- par GSH entraîne la formation des rad icaux thio l (GS·) et de 1-120 2

par l ' intermédiaire de plusieurs étapes, qu ' on appelle une réaction de propagati on

radicalaire.

En plus de son rôle comme cofacteur du GPx et de la GST, le GS H est

impliqué dans d' autres processus métaboliques, tel le maintien de l ' acide a c01·bique

sous sa fo rme réduite, la communication entre les ce llul es et la prévention de

1 ' oxydati on des groupements thi ols des protéines et des 1 iaisons croisées entre les

protéines (Halliwell et Guttericlge, 1999) . Le G 1-1 parti cipe à la synthèse des

précurseurs de la synthèse protéique et de 1 ·A ON et au transport d'ac ides am inés

(Oiry et al. , 1999). Le GSI-1 peut chélater les ions de cui vre et ainsi diminuer leur

disponibilité pour générer des radicaux libres . Par ailleurs, le GS I-I j oue un rôle cl ans

le repliement protéique et dans la dégradation des protéines avec 1 iaison disulfure.

Le GSH protège les cellules épithéliales pulmonaires de l ' oxydati on et de

l ' inflammation (Rahman et al., 1999, 2000). Une récente étude a démontré que le

GS H peut auss i atténuer le développement de l 'athérosclérose (Rosenblat et al.,

2002) . Enfin, on le retrouve dans le cytoso l à des concentrat ions de l 'ordre du

millimolaire chez les plantes, les animaux el les bactéri e aérobiques (A nderson,

1997).

1.1.4.1.1. Biosynthèse du GSH

Le GSH est synthéti sé en deux étapes. Premièrement, 1 'enzyme y­

glutamy lcystéine synthétase (E 1) catalyse la fo rmati on du premier lien pept idique,

ensuite le produit est converti en GSH par la glutathi on synthétase (E2) (Équation 1)

(Ancier on, 1997 ; Rahman et al, 1999):

18

Équation 1:

El L-glutamate + L-cystéine + ATP ~ L-y-glutamyl-L-cystéine + ADP + P,

E2 L-y-glutamyl-L-cystéine +glycine + ATP --+- GSH + ADP + P1

Les cellules peuvent produire la cystéi ne nécessaire à partir de la méthionine ou

bien elles peuvent la prendre dans les flu ides avo isinants. Souvent, les ce llules

utili sent la forme disulfure (cystine) et la rédui sent en cysté ine à l ' intérieur de la

ce llule. Pour sa part, la y-g lutamylcystéine synthétase est rétro-inhibée par le GSH et

ne semble pas être saturée à des niveaux ce llulaires norm aux de cysté ine (Ha ll iwell et

Gutter idge, 1999). U ne élévation en cysté ine peut promouvo ir la synthèse du GS H

dans certaines circonstances.

1.1.4.2. Modulation du glutathion intracellulaire par des composés

synthétiques

L 'acroléine et les EOR, lorsque générées en excès et avec un mécanisme de

défense insuffisant, peuvent engendrer des dommages à l 'ADN, aux lipides

membranaires, aux protéines et aux carbohydrates (G illi ssen et Nowa! , 1998). Chez

l ' humain, plus particulièrement dans les poumons, plusieurs maladies ont a oc iées

aux métabolites oxygénés qui ont un rô le prédominant dans la pathogénicité. Par

conséquent, une approche thérapeutique consisterait en une augmentat ion de produits

antioxydants dans l 'organisme, tel le N -acéty l-L-cysté ine (NAC), le 2-oxo-4-

thiazo lidine carboxy late (OTC) et le chélateur desferroxami ne (G illi son et Nowak,

1998).

1.1.4.2.1. Le N-acétyl-L-cystéine (NAC)

Le NAC qui est un dérivé thiol , augmente le ni veau de GSH intracellulai re

chez les cellules animales. Le NAC est désacéty lé intrace llulairement afin de céder

19

une cystéi ne (Cotgreave, 1997). Cette dernière peut ensuite être rap idement utili sée

pour la synthèse du GS H (Issels et al. , 1988). À cause de son groupement SH, le

NAC élimine éga lement le peroxyded ' hydrogène (H20 2), les radicaux libres

hyd roxy les (OH") et l'acide hypochl oreux (HOC!). De plus, le NAC inhibe la

propriété immunosuppress ive de l'acroléine (La mbert et al., 2005) mai s réduit la

production ce llulaire des méd iateurs pro- inflammatoire tel s que le facte ur de nécrose

tumorale (TNF-a) et l' interleukine-( (IL-l ) (G illi ssen et Nowak, 1998). Le NAC

inhibe l' activation des facteurs de transcripti on "redox-sensiti ve", comme le AP-l

(Lo et al ., 1995) et le NFKB (Schreck et al ., 199 1 ), et bloque l' act ivat ion des MAPK.s

(Chen el al., 1995 ; Sekharam et al., 1 998) . Par ai lieurs, le N AC est uti 1 isé

c l iniquement pour traiter les surdoses d 'acétam inophène, un médicament détoxi fi é

par le GS H (Perry et Shannon, 1998). Le NAC est auss i un agent chimioprotecteur

réduisant les effets secondaires de nombreux composé cytotoxiq ues. Il st imule le

système immunita ire, prév ient le cancer, détox ifi e les ce llules des métaux lourds,

traite la bronchite et la toux des fumeurs , prév ient le maladi es cardi aques et ralentit

la progress ion du SIDA (De la Fuente et al, 2000; Droge, 1999; Faintush et al, 1999) .

Le NAC diminue le potentiel mutagénique de l'AD oxydé (Ma lins et al., 2002), et

peut augmenter l'expression de l' ARNm du p53 (Li u et al. , 1998). Enfin , le NAC

peut inhiber complètement l' apoptose induite par la fumée de cigarette au ni veau des

ce llules épithéli ales des alvéo les (Hoshino el al., 200 1 ).

1.1.4.2.2. Le 2-oxo-4-thiazolidine carboxylate (OTC)

L'OTC est converti intracellulairement en S-carboxy-L-cystéine à l' a ide de

l' enzyme 5-oxo-L-pro linase et ensuite décarboxylé spontanément pour libérer la L­

cysté ine (Dizdar el al. , 2000) nécessai re pour la ynthèse du glutathi on (Rosenblat et

al, 2002). L'OTC augmente le taux de surv ie des patients atte ints de SIDA (O iry et

al. , 1999), protège les lymphocytes du sang (après expo ition au peroxyde

d' hyd rogène (Cors i et al., 1998) et protège les poumons de l' hyperoxie chez les rats

20

(Levy et al., 1998). Néanmoins, les effets bénéfiq ues de ces molécul es (NAC et OTC)

sont res tre ints "in vivo" par les grandes concentrat ion nécessaires pour avo ir des

effets biologiques, par leur métaboli sme extrace llula ire rapide et par la faible

concentrati on plasmique (Oi ry et al., 1999).

1.1.4.2.3. Le L-buthionine sulfoximine (BSO)

Le L-BSO est un inhibiteur spécifique de la y-glutamylcystéine synthétase (GCS)

(G ri ffi th et Meister, 1979). JI est utili sé afi n de démontrer l' implication d ' un stress

oxydatif lors de l'étude de composés toxiques (Sak ura i et al. , 1998). Le BSO

augmente la susceptibilité à la mort cellulaire induit par plusieurs stress, tels que le

stress oxydat if (Przybytkowski et A veri 11 -Bates, 1996) , le choc thermique (Lord­

Fontaine et Averiii-Bates, 1999 ; Mitchell et Ru s o, 1983) et des agents anticancéreux

(A nderson et al., 1997). Ce dernier inhibe la synthè e du GS H plutôt que la réserve

intracellulaire en GSH. Le 10 à 20% de G H restant dans la cellule suite à un

traitement avec le L-BSO se retrouve à l' intéri eur des mitochondries (A nderson,

1997). Ces dernières, ne pouvant pas synthéti er le GS H, l' importent à pa1iir du

cytosol. De ce fait, les cellules ayant subit un traitement avec le L-BSO s' avèrent être

plu sensibles face à une attaque de radicaux libres (A nderso n et al, 1999). Enfin, la

diminution du GSH réd uit l' act ivité enzymat ique de la gluthat ion péroxidase (F iohe

L, 1982 ; Lord-Fontaine et Averill , 1999; No mura et al., 1999).

La ce llule se dote d' un mécanisme de défen e très bien régulé impliquant des

protéases afin de s' autodétruire en cas de dérèg lement en faveur des dommages. Un

dérèg lement peut être causé par des substances toxiques tels qu e l' acrolé ine ou

l' acétaminophène et 1 ou la diminution des défenses ce llulaires. D' ailleurs, il est

connu que les dommages ce llulaires qui nui sent à la fo nction norm ale de la cellule

sont à l' ori gine de plusieurs cancers. Ainsi, si le dommages infligés à la ce llule par

les ERO sont plus importants que la défense ce llul aire, la ce llul e s·autodétruit par

apoptose ou meurt par nécrose.

2 1

1.2. MÉCANISMES DE MORT CELLULAIRE

1.2.1. Nécrose

La mort cel! u laire nécrotique est un processus pass if résu !tant d'une séquence

d'événements cellulaires et ti ssulaires, qui urvient à la suite de perturbati ons

profondes de l'env ironnement cellulaire. Elle se produit dans des conditions

pathologiques te lles que l'hypox ie sévère, la pri vat ion de glucose, le traumati sme, la

variati on de l'osmolarité, de la température de l'env ironnement, et du pH , ainsi qu'à

l'expos ition à des tox ines et des agents infecti eux. La nécrose conduit à une perte

cellulaire toujours secondaire à des conditions pathologiques (Choi et al ., 1999) et

elle se caractéri se de façon constante par une réaction inflammato ire.

Les anomali es morphologiques observées dans la nécrose résultent des

perturbati ons des fonctions cellulaires. A u ni veau de la ce llule, deux phénomènes

principaux conduisent irréversiblement à la mort de ce llules nécrotiques affectées :

la perte de l'intégrité membranaire et l'altérat ion des fo ncti ons mitochondriales. Un

des premiers événements est la rupture de la membrane pl asmique et donc la perte de

sa capac ité à réguler les gradients osmotiques et ioniques. Ce la engendre une entrée

mass ive d'eau accompagnée d'électrolytes, provoquant ainsi un gonflement de la

ce llule et une augmentation de la perméabilité aux molécules extrace llulaires. A insi

les morpho logies ce llulaires sont rapidement modi fiées et vont s'accompagner d'une

augmentati on du vo lume cellulaire, d'un gonflement des organites et du noyau.

Dans le cytoplasme, les organites commencent à dégénérer et de profondes

perturbations de l'acti v ité mitochondriale sont observées. La mitochondrie et le

réti culum endoplasmique se dilatent, des vacuoles intracytopl asmiques se forment et

les po lysomes se détachent du réticulum endoplasmique. L es organites

intracellulaires sont dispersés dans l'espace extrace llulaire. La structure nucléaire est

éga lement perturbée, le noyau est gonflé et la chromatine digérée par des protéases et

des endonucléases notamment des sérines protéases et ainsi l'A DN nucléa ire va être

22

dégradé de manière' aléatoire ' (Bicknell et Cohen, 1995 ; Dong el al., 1997) générant

des f ragments dépourvus d'extrémité 3' sortante.

La masse de ce llules nécrotiques peut se limi ter à une nécrose localisée, ou

évo luer vers une nécrose di ffuse qui résulte de l'acti on d'enzymes lysosomiales

déri vées de la ce llule elle-même (autolyse) ou de ce ll es vo isines (hétérolyse). La mort

ce llulaire par nécrose déclenche souvent une forte réaction inflammato ire loca le. Les

cellules nécrotiques lysées libèrent des substances dont les leucotri ènes qui sont

générés par la peroxydation des lipides membranaires, et des composants de la

mitochondrie activant le complément (Giclas et al. , 1979) qui ne sont pas encore

identifiés (Kagiyama et al., 1989). Ces substances indui sent une réacti on

inflammato ire typique de la nécrose (Kroemer et al, 1998) et les débris ce llulaires

sont alors phagocytés par les cellules immuni ta ires de l' inflammati on.

1.2.2. Apoptose

1.2.2.1. Introduction

La mort cellulaire programmée fait partie intégrante de la physiologie normale

d'un organisme. Ainsi au cours des nombreuses mitoses et di fférenciations cellu laires

qui permettront de créer un organisme à partir d'un œuf, il es t nécessa ire d'éliminer

continuellement les ce llules superflues ou potentiellement dangereuses. Ce

phénomène d'élimination sélective des ce llules est méd ié par un processus appelé

APOPTOSE. Le nom apoptose fait référence à la chute programmée des feuilles à

l'automne; apo pour éloignement et ptose pour chute. La not ion d'apoptose a été

introd uite en 1972 par K err et co ll. pour dés igner une form e de mort cellulaire

totalement différente de la nécrose, tant d'un point de vue morphologique que

biochimique. La nécrose était la seule forme de mort ce llul aire connue à cette époque.

L 'apoptose est une réponse hautement conservée des eucaryotes unice llulaires

jusqu'aux mammifères (M aarouf et al. , 1997 ; A mei en el al., 1995). L 'apoptose

surv ient naturellement au cours de l'embryogenèse, du renouve llement ti ssulaire et

23

lors du vieilli ssement. Cependant, elle peut éga lement se produire dans diverses

conditions pathologiques. De plus, un dysfoncti onnement de mécanismes régulateurs

de l'apoptose va donner lieu à de graves patholog ies. A insi, un défaut d'apoptose va

entraîner des syndromes proliférati fs assoc iés à un proce su de tumori sation, tout

part i cu 1 ièrement au niveau des ti ssus à renouve llement rapide comme le système

immunitaire ( lymphome, leucémie). A l'inverse, une acti vat ion anormale va donner

1 ieu à un phénomène de dégénérescence, comme ce la e t observé au cours de

nombreuses maladies neurodégénérati ves te lle que la maladie d'A lzheimer, la

maladie de Parkin son, ou la sc lérose latérale amyotrophique (Thompson, 1995).

L 'apoptose est considérée comme une mort cellulaire "ordonnée ", procédant

par di fférentes phases (Duva l! et Wy llie, 1986; Kerr et al., 1972). L 'un des points

morpho log iques caractéristiqu es de l'apoptose est l' importante condensation à la fo is

du noyau et du cytoplasme ce qui induit une diminution signifi cative du vo lume

ce llulaire. Ensuite, l 'ADN est clivée en f ragments réguliers d'environ 180 paires de

base ( Wy llie et al. , 1984) . La membrane plasmique va bourgeonner et conduire à la

formation de corps apoptotiques renferm ant une part ie du cytoplasme de la ce llule.

Afin de fac iliter la reconnaissance des corps apoptot iques par les phagocytes, la

ce llule va signaler son état apoptotique à son env ironnement notamment grâce au

changement de locali sation des molécules de phosphatidy lsérine qui passent d'une

ori entati on cy toplasmique vers une orientati on ex trace ll ula ire. L 'un des points

majeurs de l'apoptose est que l'intégri té de la membrane plasmique n'est j amais

altérée au cours du processus, ce qui permet d'éviter tout déversement du contenu

ce llulaire et ainsi prévenir tout dommage infligé aux ti ssu avo isinant. Toutefo is, l'on

peut noter que l'inflammation n'est pas nécessairement totalement absente durant

l'apoptose, en raison du relargage de l' interl eukine-! et -1 8 dans l'environnement,

mais il s'ag it d'une inflammation régulée.

24

1.2.2.2. Voies de signalisation de l'apoptose

De nombreux signaux très di ffé rent , phys io logiques ou patholog iques,

in tracellula ires ou extrace llul aires, peuvent déc lencher l'apoptose. Les dommages

cellulaires, te ls que les agents cytotox iques, les rad iations ioni santes et les chocs

thermiques, induisent des signaux apoptotiques. L'apoptose peut être induite, par

exemple, par la carence en facteurs de croissance et par certa ins récepteurs nucléaires

(récepteurs aux glucocorticoïdes) acti vés par leur li ga nd . T rois vo ies princ ipales

peuvent être décrites dans l'inducti on de l' apopto e: l' acti vation des récepteurs

membranaires de "mort", comm e le récepteur a socié au fibroblaste (FasR) ou le

récepteur du facteur de nécrose tumorale (TNF-R), l' act ivat ion de la vo ie

mitochondriale et cell e de la vo ie du réti culum endoplasmique. Les s ignaux vont être

in tégrés par la cellule et de nombreuses vo ies de s ignali sat ion vo nt être ainsi acti vées

abouti ssant à l'activat ion des caspases

1.2.2.2.1. La voie des récepteurs

De nombreux stimuli tels que la radiati on et des agents chimiothérapeutiques

sont capables d'indu ire l'apoptose. Toutefo i i 1 ex iste un e fa m i li e de récepteurs

spéc iali sés dans l'induction de la mort cellulaire programmée: les récepteurs de mort.

Une fo is stimulés par leurs ligands, ces récepteurs induisent l'act ivati on des caspases

(Lo ngth orne et Williams, 1997). Cette vo ie d'acti vation est impliquée dans

l'é limination des ce llules potentie llement dangereuses pour l'organi sme et notamment

les lymphocytes autoréacti fs. Les récepteurs de mort appartiennent à la famille du

récepteur du facteur de nécrose tum orale (TNF-R) (Nagata, 1997). Les TNF-R

peuvent promouvo ir, selon le contexte ce llul ai re, soit la survie, so it la mort. Par

exemple, les récepteurs CD27 (Camerini el al., 199 1) et CD30 (Durkop et al., 1992)

sont impliqués dans la survie cellulaire. Parmi les membres de la fa mille impliquée

dans la mort ce llulaire, il convient de c iter Fas (Oehm et al. , 1992) . L' in te raction du

récepteur Fas à son ligand (FasR/FasL) , au même titre que cell e du

25

granzymeB/perforine joue un rôle prépondérant dans la cytotox icité des lymphocytes

T (L Tc) du système immunitaire. Ces deux méca ni smes coopèrent pour permettre

l'élim ination de la ce llule cible par un processus d'apoptose (Cohen, 199 1; Go lste in et

al. , 199 1 ). Au cours de la réponse immunitaire, le récepteur Fas va éga lement être

impliqué dans l'éliminati on des cellules infectées pa r des virus ou des paras ites, ou

des ce llules tumorales . Bien que l'express ion et le rôle de la protéine Fas dans le

système nerveux ne so ient pas c lairement déft ni s, ell e a été impliquée au cours de

patholog ies neurodégénérati ves te ll es que l'i schémi e ou la malad ie d'Alzheimer.

Ce paragraphe décrira surtout le méca ni sme d'activat ion du récepteur Fa . Une

fo is stimulé par son ligand spéc ifi que (FasL), le récepteur Fas se triméri se et recrute

une proté ine adaptatrice, la protéine assoc iée au Fas avec un domaine de mort

(FA DO) (Chinnaiyan et al. , 1995) (F igure 1.8). Le FA DO présente la parti cul ar ité de

posséder, en plus de son domaine de mort ce llulaire (DD), un domaine effecteur de

mort cellulaire (DED). Le DED est nécessa ire et suffisa nt pour induire l'apoptose.

Dans certa ines conditions, le TNF-R 1 rec ru te éga lement F AD D. Ce coupl age est

cependant indi rect et se fa it par l'interm édiaire d'une protéi ne comportant un DD, la

protéine assoc iée au TNF-R 1 avec un domaine de mort (TRA DD) (Hsu et al., 1995)

(F igure 1.8). En définiti ve le FA DO représente le po int de convergence des vo ies de

signalisati on induites par Fas ou TNF-R 1. Par la suite, le FA DO peut recruter la

procaspase-8 (Boldin et al., 1996; Fernandes-A lnemri el al. , 1996) ou la procaspase-

10 (Vincenz et Dixit, 1997) par l'interm édi aire de ces DEDs et ainsi ini tier la cascade

apoptotique (F igure 1.9) (Hirata el al., 1998). La procaspase-8 sera c li vée en caspase-

8, qui acti vera ensuite les caspases effectrices -3, -6 et -7. Ce caspases effectri ces

c li veront leurs substrats correspondants dont l' inh ib iteur de I'ADNase caspase­

dépendant (I CA D) et entraîneront la mort par apoptose (Los et al. , 1999) . Cette

signalisa ti on est vraie auss i pour la procaspase-1 0 (Los el al., 1999) .

Le TRADD peut auss i interagir avec le DD de "receptor in teract ing protein "

(R1P) (Hsu et al. , 1996a) (F igure 1.8), une serine/thréonine kinase qui peut induire,

26

selon les circonstances, soit l'activation de NF-KB soit l'apoptose (Hsu et al., 1996a).

Le RIP interagit avec la protéine associée au RIP par un DD (RAIDD) (Duan et Dixit,

1997) qui lui-même lie directement la procaspase-2 mais pas avec les caspases 1, 3, 4,

6, 7 ou 9 (Duan et Dixit, 1997) (Figure 1.8). La caspase-2 activera ensuite la caspase-

3 effectrice et conduira à l' apoptose (Tyagi et al. , 2006).

Fas TNF-R1 TNF-R1

P•ooa•p••• 8ADtJÜ 8 ou10 g

~~rl TRADD

~FADO Pro casp:ase

8 et10

0 Don1aine de mor-t: (DO)

0 Domaine effect.eur· de mon tDED)

• Domaine de recnJternent de• ca• pases (CARO)

Figure 1.8: Activation des caspases par les récepteurs de mort. Les récepteurs de mort appartiennent à la famille du récepteur du facteur de nécrose tumorale (TNF-R) et ils comprennent plusieurs récepteurs dont le FasR et le TNF-Rl (Rathmell et al. , 1999).

-c Gl 0 :a ;::1 ii u loo. ;, Gl '0 > c 'GI

Il:: ......

.-.. c Ill

.2 :c ... ~ a '3 'GI g) )( 'GI w ~

Stimuli pasunt p~r les récept• urs de mort

Fas-L

~

,~·~o FUPs Q JO -DD- -+

p,o C~5 p-.i4t 1

~ //~ [~] Caspases

initiatrices D ~

Cupacu effectrices

'

tB Id

Protéayse de substr~ts

1

Stimuli indiptnd1nt du r éu~teurs de mort

Bel ·!

/

27

\ f ytochromt c

A

-...::::==::' Ap at ·1 • J ;

pro. 0

1.-.J...--L ........ .j cnpan 9 " 0 M

~

Figure 1.9 : Les deux principales voies conduisant à l'apoptose : la voie des récepteurs de mort et la voie mitochondriale. L'activation des caspases initiatrices -8 et -9 entraîne l'activation des caspases effectrices -3 , -6 et -7 qui cliveront leurs substrats correspondants pour emmener la mort par apoptose. Notons que la caspase-8 peut activer la voie mitochondriale par l' intermédiaire de la protéine proapoptotique Bid. Adapté de Los et al. , 1999

28

1.2.2.2.2. La voie mitochondriale

1.2.2.2.2.1 Mitochondrie, potentiel membt·anait·e et libération du cytochrome-c

La mitochondrie j oue un rô le c lé dans la régula ti on de l'a po ptose (Brenner et

al , 1998; Kroemer, 1997). Cette vo ie est acti vée par de multipl es stimuli notamment

le c hoc the rmique, la radiation, l' hypoxie, la pri vat ion en sérum et en facteurs de

cro issance, et par des agents chimiothérapeutiques co mm e l'étopos ide. Ces fac teurs

causent un déba lancement entre les fac te ur anti-apoptot ique et les facte urs pro­

apoptiques en fave ur de ces derniers (W il son, 1999). En effet, la phase effectri ce de

l'apoptose comporte la diminution du po tent ie l membrana ire (il 'Pm) qui pe rm et

l'ouverture des pores de transition de perméab ilité (PT P) spécia li sés (Vander 1-I e iden

et aL. , 1997) e t la libération de molécul es a poptogènes de l' espace intermembrana ire

vers le cytopl asme (F igure 1.9, 1. 1 0, 1. 11 ) (Kohler et al., 2002 ; Kluck et aL., 1997;

Yang et al. , 1997). Ces mo lécules sont le cytochrome-c (Cyt-c), le facteur inducteur

d ' apoptose (A IF) (F igure 1. 10), l' acti va teur mi tochond ri a l des caspases ( mac)

(F igure 1. 11 ) et les proté ines de choc th ermiqu e, HSP 10 e t HSP60. Dans un

compl exe appe lé "apoptosome", le cy t-c se li e au fac teur act ivateur de protéase

d ' apoptose (A paf) afin d ' acti ver la procaspase-9. Deux mo lécul es de procaspase-9

c li vées se 1 ient ensemble pour fo rme r la caspase-9 acti ve (F igure 1.9). La caspase-9

ac ti vera ensuite les caspases effectrices -3, -6 e t -7. Ces caspases effectri ces c li veront

le urs substrats correspondants dont 1' 1 CAO et entraîneront la mort par a poptose (Los

et al. , 1999). Cette phase de libérati on des fac teur pro-apoptotiques de la

mitochondrie est sous le contrô le des me mbre de la fa mill e Bc l-2 (F igure 1. 1 0).

A in s i, Bc l-2 et Be l-XL sont capables de bloquer la sortie du cyt-c dans le cytoplasme

(Kluck et al., 1997; Vander He iden et al. , 1997), a lors que les proté ines pro­

apoptotiques te lles que Bax et Bak peuvent l'induire (Jurgensme ier et al. , 1998).

Apoptotic Stimulus

Pore Formation

29

Figure 1.10: La régulation de l' apoptose au niveau de la mitochondrie. Cette organite joue un rôle important durant l' apoptose. Les membres anti-apoptotiques de la famille Bcl2, tels que Bcl-2 et Bel-XL, inhibent la mort cellulaire au niveau mitochondrial en maintenant la fonction physiologique de l'organite normale et en prévenant la libération du cytochrome-c et du facteur inducteur d'apoptose (AIF). Mais, les protéines pro-apoptotiques telles que Bax stimulent l' apoptose. Un des canaux libérant le cyt-c et l' AIF est formé de plusieurs protéines dont le canal ionique voltage-dépendant (VDAC), le translocateur d'adénine (ANT), la cyclophiline D (CD), la créatinine kinase (CK) et l' hexokinase (HK) (Bortner et Cidlowski, 2002).

• perforine

Gr.lnzyme-8

' • '

AIF ( Facteur inducteur d'apoptose)

Apoptose

APAFl (Facteur activateur de protéase d'apoptose) SMAC (Activateur mitochondriale des caspases) ICAD (Inhibiteur de I'ADNase caspase-dependant) PARP (poly ADP-ribose polymérase) IAP (Inhibiteur de protéine apoptotique)

30

Choc thennique, radiation, déprivation en sérum, agents chimiothérapeutiques

Figure 1.11: Induction de l' apoptose par la voie des récepteurs de mort et la voie mitochondriale. L' activation des caspases initiatrices -8 et -9 entraîne l' activation des caspases effectrices -3, -6 et -7 qui cliveront leurs substrats correspondants pour emmener la mort par apoptose. Adapté de Los et al., 1999; Salvesen et Duckett, 2002; Stennicke et al. , 2002; Pharmingen; Sigma.

3 1

Il est admis que la libération du cytochrome c ainsi que la perte du ~'Pm induite

par Bax/Bak peuvent être régulées par le PT P. Le pore PT P est un canal oligo­

protéique constitué au niveau de la membrane ex terne de la mitochondrie par la

porine VDAC, sur la membrane interne par I'ANT et d'une protéine matri cielle la

cyc loph i 1 ine D (CD) (F igure 1. 1 0) (Kroemer el al., 1998). De pl us, i 1 a été montré

que Bax pouva it interagir avec VDAC (Nari ta et al., 1998 ; himizu el al., 1999) et

ANT (M arzo et al. , 1998; Shimizu el al. , 1999). Il semble que Bax/Bak puissent

induire so it un changement de conformati on du canal V DAC afin de former un pore

perm ettant le passage des di fférentes molécules apoptogènes, so it interagir

directement avec VDAC et participer ainsi à agrandir le pore.

Il semble que la mitochondrie j oue le rôle d' in tégrateur de · di fférents ignaux

et qu'une fo is le seuil atteint, la totalité du cyt-c e t li bérée en une seule étape.

Toutefo is toutes les mitochondries d'une même cellule ne vont pas être parfa itement

synchronisées. Tout récemment, l'inva lidati on du gène codant pour le cytochrome ca

confirmé l'importance cruciale de cette protéine dans l'apoptose (L i el al., 2000). Les

cellules Cyt c-l- provenant d' un embryon de souris ne peuvent pas induire l'activation

de la caspase-3 en réponse à différents stimuli pro-apoptotiques. Enfin, les ce llules

Cyt c-l- sont rés istantes à l'apoptose indui te par le U.V. ou l'étopos ide et très peu

sensibles aux effets pro-apoptotiques de la pri vati on en facteur de croissance ou de la

staurosporine (Li et al., 2000). De plus, A paf-1 reste sous form e monomérique dans

des conditions où l'apoptosome devrait se former. Cec i implique qu 'aucune autre

protéine cellulaire ne pui sse remplacer le Cyt c pour l'o ligoméri sati on d'Apaf-1 et

pour l'acti vation de la caspase-3 induite par un tress ce llulaire ou par un agent ciblant

la 111 itochondrie.

Le facteur A IF est auss i une des molécules apoptogènes libérées de la

mi tochondrie. L' A IF transloque au noyau, condense la chromatine et fragmente

l ' A DN, indépendamment des cascades impliquant les caspases. (S u in el al., 1999).

32

Durant l'apoptose, la proté ine SMAC qui est un inhibi teur des lAPs est libérée

de la mitochondrie, afin de potenti a liser l' apopto e. Enfin , la relâche de Smac est

inhibée à l' aide du Bci-2/Bcl-XL (MacFarl ane eL al .. 2002).

L'activation des caspase induite pa r le cytochrome c cytoso lique assoc ié à

Apaf-1 , ou l'apoptose induite par les récepteurs de mort, ne sont pas des mécani smes

tota lement indépendants (F igure 1.9). En effet, un nouvea u membre pro-apoptotique

de la famill e Bcl2, la protéine Bid (Wang et al., 1996), permet de fai re le lien entre

les récepteurs de mort et la libération du cytochrome c de la mitochondri e. Le Bid est

directement c li vé par la caspase 8 et le fragment C-tenninal prod uit transloque à la

mitochondri e et permet la libération du cytochrome c (F igure 1. 9) (L i el al., 1998 ;

Luo et al., 1998) .

1.2.2.2.2.2 La famille de Bcl-2

Les protéines de la fami ll e Bcl-2 jouent un rôle majeur dans la régul at ion des

caspases et plus généralement de l'apoptose . Si l'on se réfère à leurs fo ncti ons

bio logiques, on peut c lasser les membres de la fa mille Bc l-2 en deux sous-famill es,

les membres anti-apoptotiques te ls que Bc l-2, mais auss i Be l-XL (Bo ise et Thompson,

1997), Bc l-w (G ibson et al., 1996), BAR (Zhang el al., 2000) et les membres pro­

apoptotiques comme Bax (Oitva i el al. , 1993), Bak (Chinenden et al. , 1995;), Bel XS

(Bo ise et Thompson, 1997), Bad, Bid (Yang et al., 1996) et Bim (O'Connor et al.,

1998). Cene fa mille se caractérise par la présence de domaines homologues à Bcl-2

1 à 4 (B H 1 à 4) (Kelekar et Thompson, 1998) . La di strib ution de ces doma ines

permet de regrouper cette fam ille en trois classes, la classe contenant BI-l l à BH4, la

c lasse contenant BH 1 à BH 3 et la classe contena nt seulement B!-13 (F igure 1.12).

Il semblerait que la balance entre la vie et la mort cellula ire soit influencée par

le type et la proporti on de dimères anti - ou pro-apoptotique fo rmés (O itva i et

Ko rsmeyer, 1994; Sedlak el al., 1995). Ainsi par exemple, lorsque Bax est

préférentiellement exprimé, des homodimères Bax-Bax se fo rmeront et condu iront à

33

la mort ce llulaire. En revanche, si c'est Bcl-2 qui est majoritairement exprimée, alors

il y aura survie ce llulaire. Cependant un arti cle récent semble indiquer que, pour de

raisons structurales, des homodimères Bcl-2 ne pourraient pas se fo rmer et que Bc l-2

jouerait son rôle anti-apoptotique sous forme monomériqu e (Conus et al., 2000) .

Les membres ne comportant qu'un domaine B l-1 3, comme Bid, ne peuvent pas

former des homodimères et ne possèdent pas d'act iv ité intrinsèque. En fa it, il s

exerceraient leur rô le pro-apoptot ique en formant des dimères avec des membres anti­

apoptotiques réduisant ainsi la capacité de ceux-c i à exercer leurs effets protecteurs et

dans le même temps favoriseraient la const itution de dimères pro-apoptotiq ues

(Ke lekar et al. , 1997; Sattler et al., 1997).

Bcl-w Mc:l-1 Al Boo

Bax Bak B a d

dl

Mtd ( Bok ) Div a

B ik Bld Bif'Y'I Hrl~ ( OPS ) e.I J< B n i p 3

E:· nip..:o L

Régulation

Cirnè r i:>ation -------

a2 a3 a4

For n atio n du : ana l d 5 èl6

BH3 1 BH'I 1

BH3

EH2

Ancre rn em br ana ire

a7

Figut·e 1.12: Représentation schémati que des membres de la fam ille Bc l-2. Les domaines BH 1 à Bl-14 sont des motifs très conservés . a l -a7 représentent les héli ces alpha identifiées dans Bel-XL. aS et a6 forment une structure hydrophobe entourée par 5 hélices amphipatiques . Le domaine entre a 1 et a2 contient une boucle de régulation. Il est à noter que tous les membres de la famille Bcl-2 ne possèdent pas le domaine d'ancrage membranaire. Adapté de Tsujimoto eL a l. , 2000.

34

1.2.2.2.3. La voie du réticulum endoplasmique

La vo ie du réticulum endoplasmique est la tro isième vo1e d ' inducti on

d'apoptose nouve llement identifiée. Cette vo ie e t in duite suite à l'accumulati on

excess ive de protéines nouvellement synthéti sées et qui sont mal repliées

(Kadowaki et al., 2004) . Cette voie acti ve principalement la caspase-1 2 initiatrice

qui acti vera à son tour la caspase-9 (Kadowaki et al., 2004). La caspase-7 et la

ca lpaine qui est activée par le calcium sont auss i impliquées dans l'activation de la

caspase-1 2 et l'exécution de l'apoptose par cette vo ie (Szegezdi et al. , 2003)

(F igure 1 .13).

35

c bannel

t

Ill) th ~•divation

Figure 1.13: Signalisation apoptotique de la voie du réticulum endoplasmique (RE) en réponse à un stress. Trois voies majeurs d ' induction de l'apoptose par la voie du RE ont été rapportées: (1) la voie caspase-12-dépendante; (2) la voie de la kinase régulatrice du signal apoptotique et de la kinase c-jun (ASK/JNK), qui induisent la libération du cyt-c de la mitochondrie et l' activation de la caspase-9; (3) la voie Bap31/caspase-8 qui induit aussi la libération du cyt-c de la mitochondrie et l' activation de la caspase-9 (Momoi, 2004).

1.2.2.3. Les caspases

1.2.2.3.1. Nomenclature

36

Une nouvelle nomenclature proposée par A 1 nemri et al. ( 1 996) regroupe

désormais les protéases apoptogènes sous le nom de A PASE. Le C repré ente la

cystéine du site actif (QAÇxG) et ASPA SE définit la spécificité stricte de c livage des

substrats de cette famille de protéases après un ac ide aspartique (F igure 1. 14a). À ce

jour 14 caspases ont été identifiées, mais i 1 ne fait aucun doute que cette 1 iste n'est pas

exhaustive. Notons qu 'on a classé les caspases sou trois groupes: il y a les caspases

initi atrices (2, 8, 9, I 0, 12), les caspases effectrices (3 , 6, 7) et les caspases impliqués

dans l' inflammation (1 , 4, 5, 1 I , 13) (F igure 1.14b) . Enfin on a la caspase- 14 qui est

impliquée dans la différenciation des kératinocyte (Ko hler el al., 2002).

1.2.2.3.2. Structure des caspases

Toutes les caspases ont une structure très conservée comprenant un

prodomaine N-terminal de ta ill e variable, un domaine qui deviendra après c livage la

grande sous-unité ( 17-21 k.Da) qui porte le s ite act if et un domaine qui deviendra

après clivage la petite sous-unité ( 10-1 4 k.Da) (F igure 1.1 4a). Certains membres de la

fami lle des caspases possèdent un domaine de li aiso n entre la grande et la petite sous­

unité. Les prodomaines sont vari ables, à la fo is dans leur ta ille et dans leur séquence.

A insi les caspases -3, -6 et -7 ont un pet it prodomaine alors que les caspases -1 -2, -

4, -5, -8, -9, -10, -Il , -1 2 et -1 3 possèdent un grand prodomaine. Les caspases à petits

prodomaines sont souvent regroupées sous le nom de caspases effectrices. Ces

caspases sont act ivées par des caspases dites initiatri ces . Les prodomaines semblent

jouer un rô le dans les interactions proté ines-proté ine . Ainsi les prodomaines des

caspases -8 et -1 0 contiennent des DEDs qui sont des structures permettant la 1 iaiso n

de la caspase aux molécul es adaptatrices FADO (Bo ldin er al. , 1995 ; Chinnaiya n et

al. , 1995) ou TRADD (Hsu et al., 1995). Certai nes autres caspases (caspases -1 , -2, -

4 et -9) possèdent un domaine de recrutement des caspases (ou "caspase recru itment

37

domain": CARD) (Hofmann et al. , 1997). Ces domaines CARDs jouent un rôle dans

l'interaction entre caspases ainsi qu'avec une grande variété de molécules adaptatrices

ou régulatrices.

QACxG Où.~~ .. ~

lijlel

procas pase

enzyme partiellement clivée

caspase mature et active

pro-domal e : grande sous~unité (p20) ; domaine de liaison : • pettte sous-unité (p10)

32-56kDa

A8p-X QACXG Aap·X

N-==~~~~========~[j~--~~~ Prodomaln (2-25 kDa)

l'nitiator caa ases

Capa-.·2 C.apaae-8 Caspa-.-8 Caapua-10 mC.spase-12 (?)

lùne aetlvatora

Cupa .. -11 c .. pa .. -4 c .. .,. .. -s mCapa .. ·11 c..~ .. ·13

Large aubunlt (17~1 kDa)

Effector caa

c .. .,. .. -3 c • ..,. ..... c .. .,. .. -7

(10'-13 kDa) l Small: aubuntt

ActiVe caapase

Figure 1.14: Structure et activation des caspases. Les caspases contiennent quatre domaines principaux qui sont clivés en deux étapes afin de donner une caspase active sous forme de dimères. a) Adapté de Rathmell et al. , 1999 et b) Adapté de Kohl er et al. , 2002.

38

1.2.2.3.3. Activation des caspases

La conversion de la caspase à l 'état de zymogène en une enzyme mature

nécess ite au moins deux clivages au ni veau de lien A p-X. Ce cli vages success ifs

ont lieu de manière séquentielle : tout d'abord coupure entre la grande et la petite

sous-unité (donc il y a libération de la petite sous-uni té du reste de la molécule) sui v ie

par la libérati on du prodomaine (F igure 1.1 4a). Le caspa es vont pouvo ir s'auto­

act iver et/ou être acti vées par d'autres caspases. Ce derni er concept introduit la noti on

de cascade d'acti vat ion. A insi une fois les caspases initiatri ces acti vées, elles vont

pouvo ir cli ver d'autres caspases encore à l'état de zymogène, notamment les ca pases

effectrices -3, -6 et -7. Ce type d'activat ion en cascade perm et probablement la

régulati on et l'amplification du signal apoptoti que.

1.2.2.3.4. Les substrats des caspases

Le rôle des caspases est principalement exécuti f. Elles vont acti ver des

molécules qui partic iperont à la destruction ce llulaire. Le caspases sont des enzymes

extrêmement sé lecti ves. Les substrats cli vés par les caspases comprennent des

protéines du cytosquelette (a-fodrine, actine, ge lsoline) (V ill a et al., 1998), nucléa ires

( lamines), ainsi que des protéines impliquées dans la réparati on de l'ADN comme la

poly A DP-ribose polymérase (PA RP) (Sa lvesen et D ix it, 1997; Germ ain et at. , 1999)

et la suppress ion tumorale comme la rét inoblastome (Rb) (Chang et Y ang, 2000). De

plus, des protéines impliquées dans la transducti on du signal, dans l'express ion de

gènes, dans la régulati on du cyc le ce llulaire (p27K ip 1 ), dans les maladies génétiques

ou des protéines de régulation de l'apopto e ( ICAD) ont auss i des substrats des

caspases. Notons que le complexe ICAD/CAD, une fo is cli vé, libère la protéase CAD

qui entre dans le noyau pour condenser la chromatine et rragmenter l ' A DN (Chang et

Yang, 2000).

39

1.2.2.3.5. Régulation des caspases

Etant donné les effets dévastateur que pourra it avo ir une acti vati on

inappropriée des caspases, il n'est pas surprenant que cette étape soit étro itement

modul ée. En fa it, non seulement l' acti vation, mais auss i l'acti vité et la production des

caspases, sont régulées à plusieurs niveau x. La cas1 ase-3, par exemple, est fortement

exprimée dans de nombreuses cellules lymphoïdes et myé loïdes matures a lors qu 'ell e

n'est que fa ibl ement présente dans l'épithélium mammaire et dans les neurones

normaux (Krajewska et al., 1997) .

La p35 , qui est une des protéines de bacul ov irus, possède un large spectre

d ' inhibition, en inhibant à la fois les caspases initiatri ces et effectrices (Figure 1.15)

(Bortn er et Cidlowski , 2002).

La cytokine modifi ant la réponse (Crm A) est issue d'un gène précoce du virus

cowpox. Sa surexpress ion permet d'inhiber l'a popto e induite par la pri vation en

facteurs de croissance et par le CD95 (o u récepteur Fas) ou le TN F (Figure 1. 15)

(Tewari et Dixit, 1995; Gagliardini el al., 1994). La Crm A inhibe l'activité

protéo lytique des caspases -2 et -8 (Bortner et Cidlowsk i, 2002). Ces deux proté ines

virales (Crm A et p35) sont des inhibiteurs compétiti fs. Une fo is clivées par une

caspase, elles vont se lier à ces enzymes et ain i empêcher la dégradati on de

nouveaux substrats (Komiyama et al. , 1994; Bump el al., 1995).

La stimulati on des récepteurs de mort va conduire au recrutement de

molécules adaptatri ces puis au cl iv age de la fo rme zymogène de caspases initiatri ces

( caspases -8 et -1 0) et à l'activation des ca pa ses effectri ces. Cette étape de

recrutement peut être régulée par une protéine inhibitri ce de la caspase-8 (FUP)

(Ra per et al., 1998; lrmler et a l. , 1997) .

Les baculov irus possèdent une autre proté in e capable d'inhiber l'apoptose:

l' inhibiteur de proté ine apoptotique ( IAP). Au moins 5 homologues ont été identifi és

chez les mammifères . Il s'agit de NA IP, de XIAP et de la survivine (Li ston et al. ,

1996), de c lAP-I , et de clAP-2 (Rothe el al., 1995). L'effet protecteur des lAPs est dCt

40

à leur capacité d' inhiber l'acti vation et donc l'acti vité de certa ines caspases. Ainsi,

XIAP, c lAP-I et ciAP-2 inhibent les caspases 3, 7 et 9 mais pas les caspases 1, 6 et 8

(Deveraux et al., 1997-1 999; Roy et al. , 1997 ; Sun et al., 2000 ; Takahashi et al. ,

1998). La NA IP, pour sa part, est incapable d'inhiber les caspases 1, 3, 6, 7 ou 8 (Roy

et al. , 1997). Les lAPs, à l'exception de la survivine, sont caractéri sées par 3

domain es de répétiti on de JAP de la bacul ov iru (B IR) da ns leur part ie N-terminale et

un domain e d'interaction protéine-proté ine contenant un atome de zinc dans leur

parti e C-terminale. La survivine ne contient que les domain es B!Rs et la plupart des

carc in omes humains la surexpriment ce qui a comme conséquence la survie et la

croissance des ce llules tumorales (Choi et al., 2003). De plus, il semble que clAP-I et

c iAP-2 puissent se lier au facteur assoc ié au TNF-R 1 et 2 (TRA F-1 et 2) grâce à leur

motif BIR (Rothe et al., 1 995 ; Roy et al. , 1997). Cec i implique que les elAPs, ainsi

que les vlAPs (Vucic et al., 1998), puissent exercer des effets inhibiteurs sur

l'acti vati on des caspases en aval des récepteurs de mort (Wang C.Y. et al. , 1998). Par

a ill eurs clAP-I , ciAP-2 et XJAP ont été décrites comme des inducteurs de l'activation

deN F-KB (Chu et al., 1997). Cec i pourra it notamment contribuer à l'effet protecteur

de N F-KB sur l'apoptose induite par le TNF-u (You et al. , 1997; Stehli k et al., 1998 ;

Wang C. Y. et al., 1 998).

Fas Ligand

crmA/p35

Active Caspasea-2,8

UV, Stress, Growth Factor Deprivation

.. • • • • • •• cytC

+

41

Figure 1.15: Inhibition de l'apoptose au niveau des caspases. Les IAP inhibent la caspase-9 (initiatrice) et les caspases -3 et -7 (effectrices). De plus, les ciAP-1 et ciAP-2 se lient au facteur associé au TNF-R 1 et 2 (TRAF-1 et 2) grâce à leur motif BIR (Rothe et al. , 1995; Roy et al. , 1997). Ceci implique que les elAPs puissent exercer des effets inhibiteurs sur l'activation des caspases en aval des récepteurs de mort (Wang C. Y. et al. , 1998). CrmA est un inhjbiteur des caspases initiatrices (caspase- 2 et -8), avec toutefois une capacité limitée d' inhiber les caspases effectrices. Un inhibiteur spécifique de la caspase-8 a été identifié et nommé "FLICE­inhibitory proteins" (FLIP) qui inhibe l'apoptose en se complexant avec le complexe inducteur de signal de mort (DI SC) qui regroupe le récepteur de mort, le F ADD et la procaspase-8 (aussi nommé FLICE). Enfin, le p35 isolé du baculovirus possède un large spectre d' inhibition, en inhibant à la fois les caspases initiatrices et effectrices (Bortner et Cidlowski, 2002).

1.3. LES VOIES DE SIGNALISATION DES MAPK, ASKI, AKT ETP53

1.3.1. Les voies de signalisation d'apoptose médiées par MAPK et ASKI

42

Il ex iste deux vo ies di stinctes de s ignalisati on de l' apoptose à partir du

domaine intrace llulaire du récepteur Fas : la première vo ie est ce lle qu 'on a

développée à la section de la vo ie des récepteurs (sect ion 2-2-2.1) et qui implique la

cascade des protéines adaptatrices qui interag issent via des domaines d ' homologie, et

qui finalement communiquent le message aux caspases (F igures 1.8 et 1.9) ; la

deuxième implique, quant à elle, une cascade de protéines kinases . La cascade des

protéines kinases activées par les mitogènes (MA PK) est multifonctionnelle et a été

conservée durant l' évo lution (Takeda el al., 2003) . Il y a tro is différentes cascades qui

convergent vers la kinase régulée par des stimuli extracellula ires (ERK), la kinase du

terminus-N de c-jun (JNK) ou la p38 MAP kinase (F igure 1. 16). Ces MAPK sont

régulées par différents stimuli (Pearson et al. , 200 1; Kyriakis et A vruch, 200 1 ).

Souvent, la vo ie ERK est connue comme étant anti-apoptotique et de survie et

ell e est act ivée par des facteurs de croissance (Lee er al., 2006; Park et al. , 2003)

tandi s que les vo ies de JNK et de la p38 MAP kinases sont connues comme étant pro­

apoptotiques (Sarker et al. , 2003 ; Assefa et al. , 2000). Les JNK et p38 sont activées

par le facteur de nécrose tumorale (TNF-a), le peroxyded ' hyd rogène, les rayons UV,

les rayons X, le choc thermique et la pri vat ion de sérum et de fac teurs de croissance

qui entraînent l' activation de la protéine kinase régul atri ce du signal apoptotiq ue

(ASK I) (F igure 1.16) (Takeda el al., 2003 ; Rain gea ud et al. , 1995; Xia et al. , 1995 ;

Kyri aki s et Avruch, 1996; Verheij et al., 1996). Les JNK et p38 MAPK induisent la

translocation de Bax vers la mitochondrie et la libérat ion du cytochrome-c (Tsuruta et

al,. 2004; Park el al., 2003) activant de ce fa it la voie mitochondria le, tandis que ERK

inhibe l' activité de la caspase-8 (Park et al., 2003) , inhibant ainsi l'apoptose. En plus,

la p38 peut phosphoryler le facteur pro-apoptotique p53 aux serines -1 5, -37 et -46, et

--------------------- --

43

a insi induire l' apoptose (Bulavin el al., 1999; Kim el al., 2002; Sanchez-Prieto et al.,

2000).

Growth Factor etc.

Plasma membrane

MAPKKK

MAPKK

Raf

l ml ~~

Growth

Oxidative stress TNF, Fas ligand

etc.

Survival Differentiation

Apoptosis

Figure 1.16: Signa li sation des différentes MAPKs, ERK, JNK et p38 . Suite à une stimulation par un facteu r de croissance, la MAPK kinase kinase (MAPKKk) Raf active la MAPKK, MEK 1 et 2, qui acti ve ERK pour induire la survie ce llulaire. Cependant, une stimulation par un li gand Fas, un ligand TNF, un stress oxydatif ou un stress du RE activera la MAPKKK ASK 1 qui activera à son tour des MAPKK qui activeront la JNK et la p38. Cette cascade emmène l"a poptose de la ce llule (Takeda et al., 2003) .

44

Notons que la phosphorylation à la sérine 46 es t spéc ifique à la p38 MAPK (B ulavin

et al., 1999) . Le facteur de croissance ép id ennale (EGF) pourrait protéger les cellules

de l'apoptose en act ivant la vo ie des ERK 1/2 et en s'o pposant à la vo ie de la p38

MAPK (Kanasak i et al. , 2000).

Des effets contradi cto ires ont été rapportés pour ces di ffé rentes MAPK. La

p38 MAPK peut intervenir dans les mécanismes de surv ie ce llulaire. En effet,

l' hormone stimulante des fo lli cu les (FSH) induit la survie et l'arrondi ssement des

ce llules de granulosa de rat (Maizels et al., 1998). Dans ce cas, la FSH induit la

phosphorylation de la p38 MAPK, via la protéine kinase A (PKA) . La

phosphorylat ion de la p38 MAPK induit la phosphory lati on de la protéine HSP-27

qui induit la réorgani sation des filaments d'actin e et leur stabilité (Huot et al. , 1 997).

Une récente étude a auss i démontré que la proté in e - RK peut être impliquée dans la

mort ce llul aire des kératinocytes humains malins expo ées aux chélateurs de fer (Lee

et al., 2006) . La voie JNK peut auss i être impliquée dans la survie cellul aire des

macrophages (Himes et al. , 2006) contra irement à on caractère apoptogène discuté

plus haut.

Une importante MAP kinase kinase kinase (MAPKKK) du stress apoptotique,

la kinase régulatr ice du s ignal apoptot ique (ASK 1) a été identifi ée. Elle active les

deux voies de signali sation JNK et p38 (lchij o el al. , 1997) (F igures 1.1 6, 1.17). La

surexpress ion de ASK l dans des cellules épithéliales incubées avec très peu de sérum

induisa it la mort cellulaire par apoptose. Tandi s que la surexpression du mutant

ASKl , ASK I-K709R, rédu isait s ignificativement l' apoptose induite par le récepteur

TNF-a. Cec i suggère que ASKI joue un rôle majeur dans l' induction de l'apoptose

par les cytokines ou par différents stress (lchijo et al., 1997; Tobi ume et al. , 1997).

45

Figure 1.17: Voies de signalisation d' apoptose impliquant p53 et ASKI. (1. Curtin et Cotter, 2003; 2. Takeda et al. , 2003: 3. Bras et al., 2005; 4. Tsuruta et al. , 2004; 5. Bulavin et al. , 1999). Les deux voies de signalisation des récepteurs de mort passant par FasR et TNFR convergent à l' activation de ASK 1. En plus, les espèces réactives de l' oxygène (EOR) peuvent entraîner l'activation de ASKl. Ensuite, ASKI active les deux voies JNK et p38 qui sont impliquées dans la transcription des gènes des protéines apoptotiques dont Bax, Bim, le ligand Fas et le récepteur Fas. D'autre part, p53 qui est induite suite aux dommages infligés à l'ADN par des composés toxiques tel que l' acroléine joue un rôle important dans la transcription du Bax, Bim, ligand Fas et du récepteur Fas.

46

1.3.2. La voie de survie AKT

L' AKT est une protéine pro-oncogène et qui est activée par la

phosphoinos itide-3 kinase (P13K) et cec i par phosphory lation. L'activati on de la

protéine AKT est impliquée dans la croissance et la survie ce llula ire et l' inhibition de

l' apoptose (Asse! in et al. , 2001 ; Down ward , 1998). L 'A KT inhibe l'apoptose en

phosphory lant plusieurs protéines c ibl es dont la protéine pro-apoptotique Bad. Ce

fa isant, la protéine Bad sera reconnue par la protéine 14-3-3 qui induit sa dégradati on

et ainsi enl ève l' inhibition de Bad sur la proté ine anti-apopto tique Bel-XL (Sarker et

al., 2003; Franke et al. , 1997; Bellacosa et al., 1998). Ain s i, l' inhibition de AKT

induit la mort par apoptose chez les cellules cancéreuses.

1.3.3. Le facteur de transcription p53 et son rôle dans l'apoptose

p53 est le facteur de transcription le plus étudié. Il est étroitement re lié à

l' arrêt du cycle ce llulaire et l' apoptose en réponse aux stress cellulaires comme les

dommages de l'A DN (Evan et Littlewood, 1998). Il a été décrit la premi ère fois en

1979 et il est le premier gène suppresseur de tumeur à être identifié. La princ ipa le

fo ncti on du p5 3 est l'élimination des ce llules en proli fé rat ion anormale pour prévenir

le déve loppement néoplas ique. Une dérégul at ion de la fo ncti o n du p53 est retrouvée

dans la grande majorité des tumeurs humaines.

Des dommages à l' ADN , une forte augmentat ion d'A M Pc ou des radi ations

UV induisent la phosphorylation de la proté ine p53 et de ce fa it son acti vation

(A msterdam et al., 1996; 1997) (F igure 1.17). Au ni veau de l' ADN, il est bien établi

que la protéine p53, suppresseur de tumeurs, joue un rôle essentie l dans la réponse

suite à des cassures doubl e-brins, en modul ant l' express ion de certains gènes. Le

fac teur de transcription p53 induit la transcription de plus ieurs gènes qui sont

impliqués dans sa régul ation négative te ll e que la murine do uble minute (MDM 2),

dans le contrôle de la réplication et de la réparat ion de l' ADN te ll e que la proté ine

induite par les dommages à l'A DN et arrêtant la croissance (GA DD45), transactive

47

des proté in es impliquées dans l'arrêt du cyc le ce llul aire (p2 1 ), qui inhibent les kinases

cyc lines-dépendantes et provoque alors l'arrêt en G 1 (Tokin o et Nakamura, 2000).

D'autres gènes sont c iblés par le p53 dont ceux impliqués dans l' inhibition de

l' angiogénèse (la thrombospondine) et dans l' induction de l'apoptose (Bax) (Tokin o

et Nakamura, 2000). La p53 active la transcripti on de Bax (F igure 1.17), qui induit la

sortie de cyt-c des mitochondries. Le cyt-c ainsi libéré va acti ver les cascades de

caspases et induire l'apoptose (Levine, 1997; Li et al., 2000). La p5 3 peut auss i

activer la transcription du récepteur Fas et inhi ber ce lle des protéines anti­

apoptotiques telles que Bcl-2 et les lAPs (Curtin et Cotter, 2003).

Il y a trois principaux régul ateurs de la p53 , la proté ine mutée d ' atax ia­

te lang iectas ia (ATM), la protéine s imilaire à Rad3 (ATR) et la kinase DNA­

dépendante (DN A-PK) (Zhu et Zhang, 2003). Le ATM, ATR et DN A-PK qui

appartiennent à des cascades di stinctes phosphorylent la p53 (F igure 1. 18) (Baatout et

al., 2002; Abraham, 2001 ) pour inhiber sa dégradati on par la Mdm2 et ainsi

prolonger son effet anti -prolifératif. Les ATM , A TR et DNA-PK appartiennent à la

famill e des phosphoinositides 3-kinase-related kinases (PIKK). Ell es jouent un rô le

c lé dans la régulati on de la prolifération ce llulaire et la surve ill ance génomique. Les

acti vités kinases de ATM et ATR sont induite en répon eau bri s à l' ADN et elles

phosphorylent plusieurs effecteurs du point de contrôle du cyc le ce llula ire. L' A TM

phophoryle les kinases de vérificati on 1 et 2 (Chkl et 2), l' anti gène 1 du cancer du

se in (BRCA 1 ), p53 à la sérine 15, Mdm2. L' ATR phosphoryle la kin ase de

vérificati on 1 (Chkl ) (Das et Dashnamoorthy, 2004) . La DNA- PK apparti ent auss i à

la fa mill e des PIKK et elle est activée en répon e au bri s à l' ADN. La DNA-PK

phosphoryle p53 aux résidus sérine 37 et sérine 15 (Das et Dashnamoorthy, 2004;

A chanta et al., 2001 ). Enfin, la Mdm2 qui est une proté in e possédant une activité E3

ubiquitine ligase dégrade la p53 afin d 'assurer un e autorégul ati on négative du cyc le

de la p53 (Maya et al., 2001 ). En plus, la protéine p53 peut être phosphory lée par la

p38 MA PK (Curtin et Cotter, 2003; Bulav in et al. , 1999; Kim et al., 2002; Sanchez-

48

Prieto et al., 2000) et JNK (Curtin et Cotter, 2003), ce qui fa it le li en entre les voies

des MAPK, de la p53 et de l' apoptose (F igure 1.1 7).

DNA dsbs

~ ATM

ATR

Accumulation i Transactivating function

~ G1 phase arrest Apoptosis

Figure 1.18: Signa li sation de p53 suite aux dommages infligés à l' ADN . Les bri s de double br in de l'ADN (dsbs) seront reconnus par ATR et ATM qui phosphoryleront Chk 1 et 2 a insi que la p53 à des sites bien défini s. La p53 pho phory lée ne sera plus reconnue et dégradée par son inhibi teur la MDM2. En plus, la fo rme phosphorylée est la forme active de la protéine qui va transcrire les protéines pro-apoptotiques Bax, Bim, le récepteur FasR et son ligand Fas (Baatout et al. , 2002; Abraham, 200 1 ).

49

1.4. PRÉSENTATION DU PROJET

1.4.1. Introduction

L'apoptose est un processus phys iolog ique de mort ce llulaire au cours duquel

des mécani smes complexes sont activés pour aboutir à la destruction de la cellul e. Ce

« suic ide cellulaire » est un événement clé en bio logie car il permet l'équ ilibre entre

prolifération et dégénérescence cellulaire dans les organi smes pluricellulaires, c'est-à­

dire le maintien de leur homéostas ie. De plu s, l'apoptose est un mécani sme «

s ilencieux » pour l'organisme et il permet donc la régulation des populations

cellulaires tout en respectant l'intégrité de l'organi sme. Cette mort cellulaire régulée,

induite et régie par une large variété de stimuli exogènes et endogènes, conduit à

l' activation de la fa mille des protéases à cystéine, les caspases. Elles jouent un rôle

fondamental dans le processus apoptotiq ue.

Il y a un intérêt cro issant à dé fin ir les mécani smes d ·a po ptose assoc iés à la

tox icité de faibles doses de différents composés chim iques (Orrenius et Zhi votovsky,

2006). Cec i inclut les polluants envi ronnementaux te ls que la toxine aq uatique

tributyltin (Reader et al. , 1999; Jurkiewicz et al., 2004) et les pesticides

organochlorés (Okoumassoun et al. , 2003) qui ont des impacts impo1tants sur la santé

humaine et d 'autres espèces.

La 1 iste des pathologies 1 iées à un dysfonct ionnement de 1 ' apoptose est en

croissance. La compréhension des mécani smes qui régul ent ce type de mort ce llula ire

peut a ider à identi fie r des sites d 'action potentiels pour envisager di ffé rentes

stratégies thérapeutiques, so ient activatrices, so ient inhibitrices du processus

apoptotique. Le traitement de la leucémie aiguë promyé locytai re par le trioxyde

d' arsenic représente un exemple d' utili sation thérapeut ique d' agents inducteu rs de

l' apoptose (F iorijn E, 1950). D'autre part, on a le traitement des lymphomes par le

cyc lophosphamide qui entraîne l' apoptose à des dose déterminées, et on pense que

50

1 ' ef fet thérapeutique de ce médicament est dCJ à son métabolite 1 ' acroléine (Schwerdt

et al. , 2006).

Dans notre recherche, on s' intéresse à l ' acroléine car elle est produite par la

réaction enzymatique de l ' amine oxydase avec les polyamines cellulaires (spermin e,

permidine) nécessa ires à la division et à la proli fé ration ce llulaire. Cette oxydation

enzymatique génère auss i le peroxyde d' hydrogène qui est connu pour son potentiel

apoptotique. Ainsi, ces propriétés de l ' amine oxydase de générer deux produits

tox iques, l ' acroléine et le l-b02 lu i confèrent des caractéri stiques pertinentes dans le

traitement de cancer. C'est pourquoi des études pré-cliniques sur des modèles

ce l! ulaires s' avèrent essentielles pour que 1 'enzymothérapie pu isse être val idée et

peut-être utili sée de façon efficace en clinique. En plu s, on s' intéresse à étudier

l ' acroléine car elle est un polluant ominéprésent de notre environnement et un

marqueur important dans le pronostic de la maladie d ' A lzheimer.

En général, on remarque que 1 ' acroléine induit la mort ce l! ulaire par nécrose

(Rudra et Krokan, 1999) et cause une inhibition de la proli férati on ce llulaire

(Agostinelli et al. , 1994, 1996; Biswal et al., 2002). Toutefois, l ' acroléine peut

induire l 'apoptose ou l ' inhiber dans le même type ce llulaire et ceci dépendamment de

la concentration et du type cellulaire utili sé. Par exemple, une fa ible concentration en

acroléine (< 10 ~-tM) induit l 'apoptose chez les neutrophil es tandi s qu ' une plus grande

concentration (> 1 0 ~-tM ) 1' inhibe (Finkelsetin et al ., 2005). Cependant, les

mécanismes d' inducti on de l ' apoptose par l 'acroléine ne sont pas encore compri s.

1.4.2. Modèle cellulaire

Pour pouvoir utiliser un nouveau médicament en c linique, chercher une

protecti on contre les effets toxiques d' un polluant environnemental , les mécani smes

d' action ce llulaires et moléculaires doivent tout d' abord être compri s. Les études in

vitro utili sant des lignées ce llulaires présentent comme avantage de générer

rapidement des résultats fiables et utiles tout en perm ettant un meilleur contrôle des

51

conditions expérimentales, contrairement aux expéri ences sur les an1maux qu1

présentent souvent des problèmes li és à t·hétérogénicité ce llulaire. La culture

ce llulaire limite l ' utili sation d' animaux de laboratoire qui es t considérée de moins en

moins justifiée par les organismes qui veillent à la protection des animaux, et elle est

relati vement peu coüteuse en comparaison. La plupart des ce llules norm ales ont une

durée de v ie limitée (20 à 100 générations). Les ce llules de rongeur et celle de la

plupart des tumeurs mises en culture continue, peuvent subir une transformati on

spontanée ou induite, qui leur confère un ca ractère immortel. Cette transformation

provoque l 'aneuploidie et une augmentati on de la tum origénicité. Ces lignées sont

bien caractéri sées et présentent une population de cellules homogènes. Ces ce llules

sont cultivées dans un environnement qui peut être fac ilement contrôlé (température,

pH , oxygène, etc) . Dans le cadre de cette étude, les di f férents types

d 'expérimentations ont été effectués sur des ce liu les d' ovaire de hamster chinois

(CHO) (Ling et T hompson, 1973). Le phénotype des ce llules C HO a été très bien

ca ractéri sé et ces dernières comptent parmi les types ce llulaires les plus utili sé dans

la recherche in vitro. Cependant, l ' utilisation de culture ce llulaire comporte des

limitati on . Elle ne peut refl éter la complex ité biologique des ti ssus ou organismes

entiers. Les interactions cellulaires ex istant dans les ti ssus se perdent lors de la

propagation sur un substrat bidimensionnel. Cependant, la culture cellulaire est utile

pour étudier et comprendre les vo ies de signali sation ce llulaire, les bases moléculaires

de pathologies ou les mécanismes de tox icité. Enfin, il y a de plus en plus d' intérêt à

déve lopper les modèles de culture de cellules en trois dimensions pour la poursuite

des études lorsque les mécanismes recherchés ont été caractéri sés par les cu !tu res

cellulaires conventionnelles.

1.4.3. Objectifs du projet

La première hypothèse est que l ' acro léine induit l ' apoptose et ceci par la vo ie

mitochondriale et 1 ou par la vo ie des récepteurs. La deux ième hypothè e est que

52

l' acroléine induit des dommages à l'ADN, ce qui entraînera l'act ivat ion de la p53. La

p53 induira ensuite la transcription de Bax a insi que ce ll e du récepteur Fas, act ivant

de ce fa it la voie mitochondriale et la vo ie des récepteurs, respectivement. La

troix ième hypothèse est que la vo ie des récepteur peut act iver la vo ie mitochondriale

par l' intermédia ire de la caspase-8, ain si que le vo ies de p38 MAPK et JNK.

L'apoptose induite par l'acro lé ine peut être inhibée par les vo ies AKT et ERK suite à

1 ' ac ti va ti on des récepteurs tyrosines kinases. La quatri ème hypothèse est que

l' acroléine engendre un stress oxydati f, en diminuant le GS H, un puissant

antioxydant intracellulaire, ainsi on examinera l' effet protecteur des modulateurs de

g lutathion sur l' apoptose induite par l' acroléine.

L'objecti f général de cette étude est de comprendre les mécani smes

molécul aires impliqués dans l' induction de l' apoptose par l'acrolé ine. Les objecti fs

spécifiques de l'étude sont de déterminer ( 1) l' implication de la voie mitochondri a le,

(2) la vo ie des récepteurs de mort et (3) la signa li sat ion des MAPK et de la p53 dans

l' apoptose induite par l'acroléi ne, et (4) le rôle du glutathion dans la protection de

l' apoptose.

1.4.4. Approche expérimentale

Objectif 1 : Ce projet déterminera si l' acroléi ne entraîne l' apoptose et ou la

nécrose. L' apoptose est révélé par le colorant Hoe cht No. 33258 en microscop ie à

fluorescence. Le co lorant 1-l oescht No. 33258 s' interca le dans l' ADN des ce llules et

fluoresce proportionnellement à la condensation de la chromatine qui est une

caractéri tique importante de l' apoptose. La nécrose e t révé lée par l' iodure de

propidium qui entre dans les ce llules dont les membranes sont endommagées et

co lore le noyau. De plus, la propriété de l' acroléine à act iver la voie mitochondria le

de l'apoptose, en induisant une baisse du potenti e l membranaire, en libérant le

cytochrome-c vers le cytoso l et en translocant les protéines pro-apoptotiques Bax et

Bad du cytosol vers la mitochondrie sera investiguée. La baisse de la protéine anti-

53

apoptotique Bcl-2 au ni veau de la mitochondrie sera analysée. La détection de ces

proté ines se fera par la méthode de l ' immunobuvardage de type We tern en utili sant

les anticorps spécifiques correspondants après le f ractionnement sous-cellul aire. La

pureté des différents compartiments (cytoso l, mitochondrie, membrana ire) sera

vér ifi ée par des marqueurs spécifiques de chaque f raction ce llulaire. La ca lnexine est

spéc ifique aux microsomes, la GST-n 1 au cyto ol et la cytochrome oxydase est

spéc ifique aux mitochondries. Les bandes de protéines seront ensuite révélées par

développement sur un film . La quantificati on de ces bandes protéiques par un

densitomètre au laser permettra de ca lculer leurs concentrations relatives par rapport

aux cellules non exposées à l ' acroléine. La baisse du potentiel membranaire

mitochondriale sera analysée en cytométrie de flux par la sonde rhodamine-123. On

détermin era si l 'acroléine active les caspases initiatri ces (ex: caspase -8 et -9) et les

caspases effectr ices (ex: caspases -3, -6 et -7) par fluorimétr ie. Cela consiste à

étudier la cinétique des réact ions enzymatiques en utili ant les substrats fluorescents

et spécifiques à chaque caspase. Si la caspase est act ivée, le substrat est c li vé, et on

aura augmentation de la fluorescence du fragment cli vé qui se ra détectée par un

lecteur de m icroplaque à fluorescence à des longueures d' onde précises d ' exc itation

et d 'émiss ion. Ensuite le clivage de chaque caspase sera étudié par immunobuvardage

de type Western pour détecter la forme pro-caspase et le fragment clivé. En utilisant

des inhibiteurs pharmacologiques des di fférentes caspase , on déterminera leurs

implications dans l ' exécution de l ' apoptose. Le cli vage de I' ICA D et de la PARP,

deux importants substrats des caspases, sera examiné en immunobuvardage de type

Western dans un lysat cellulaire tota l.

Objectif 2: En infligeant des dommages à l ' ADN par expos ition à l ' acroléine,

la p53 peut induire l ' act ivation du récepteur Fas conduisant de ce fait à l 'activation de

la vo ie des récepteurs. Ainsi, on examinera l 'augmentation de l 'express ion du

récepteur Fas et de son li gand FasL au ni veau de la membrane plasmique, la

trans locati on de FADO du cytoso l vers la membrane plasmique par

54

immunobuvardage de type Western dans les fraction s sous-cellulaires. On détectera le

c l iv age de la pro-caspase-8 et 1 'appar iti on de son fragment par i mmunob uvardage de

type Western , et 1 'act ivation de la caspase-8 par Il uori métri e. Enfin , 1' uti 1 isat ion

d' inhibiteurs pharm aco logiques spécifiqu es de la caspase-8 et du récepteur Fas

déterminera l' importance de la vo ie des récepteurs de mort et de la vo ie

mitochondriale dans l' induction de l' apoptose induite par l' ac roléi ne. Cec i, en

déterminant leurs effets sur l'activation des différentes caspases, le cli vage de la

PARP et la condensation de la chromat ine pa r l'acrolé in e. Le cli vage de Bid dans le

cytoso l en sa fo rme tronqué t-B id qui se transloq ue à la membrane mi tochondri a le

sera auss i détecté en immunobuvardage de type Western dan les fractions sous­

ce llulaires. L' externalisation des phosphatidylsérines, des phospholipides de la

membrane plasmique, pour présenter l' état apoptot ique de la cellule au milieu

environnant pour qu ' elle so it phagocytée par un macrophage, sera détectée à l' aide du

cytomètre à !lux par l'annexine V (Nardini et al. , 2002). L' an nexine V co upl ée à un

1luorochrome reconnaît la molécule de phosphatidylsérine qui se présente à la face

externe de la membrane plasmique et marque ainsi la membrane par fluorescence.

Objectif 3 : Pour démontrer l' implica ti on de la vo ie de signali sation des

MAPKs dans l' induction de l'apoptose par l' ac roléin e, on analysera par

immunobuvardage de type Werstern l' expression des formes phosphorylées (actives)

ou non-phosphorylées de ERK 1 et 2, c-jun , et p38 MA P kinase. En plus, on détectera

l' act ivation par phosphorylat ion de ASK 1 (Takeda et al., 2003) qui est une

MAPKKK (MAPK ki nase kinase) qu i active .J K et p38 MAPK. S' il y a

phosphorylation des différentes MAPK, on utili era des inhibi teu rs de chacune des

tro is cascades des MAPKs afi n de confirmer l' impl ication de différentes kinases dans

l' apoptose induite par l' acro léine. Les inhibiteurs envisagés seront le PD 98059 et le

UO 126 qui sont des inhibiteurs de MEK (L in et al., 2004), le SB203580 qui est un

inhibiteur de p38 MAPK (Li u et Hofmann , 2004) et le SP600 125 qui est un inhibiteur

de JNK (Naruishi et al. , 2003). Les effets des inhi biteurs seront analysés sur

1 ' activat ion des différentes caspases, le cl iv age de l' 1 CA D et la condensat ion de la

55

chromatine par l 'acro léine. Enfin, on analysera l ' express ion de AKT (proté ine kinase

B) qui est impliquée dans l ' inhibition de J'apoptose par phosphorylation de Bad

(Sarker et al. , 2003). S' il y a phosphory lation, on utili sera des inhibiteurs

pharmacologiques afi n de confirmer son implication dans l ' apoptose induite par

l 'acroléine. Les inhibiteurs env isagés seront le L Y294002 et le wortmannin qui sont

des inhibiteurs de la vo ie PI3K-AKT (H u et al. , 2000; Takeda et al., 2004). Enfin, on

examinera l 'express ion de la protéine suppresseur de tumeur la p53 qui est induite par

des dommages à l ' A DN causés par des compo és toxiques tel que l ' ac roléine et cec i

par immunobuvardage de type Western . L 'act ivat ion par phosphory lation de la p53

sur les sérines -1 5 et -46 sera auss i détectée par immunobuvardage de type Western .

Objectif 4: Compte tenu que l ' acroléine diminue de façon marquée l 'express ion

d' un antioxydant important, le g lutathion, la capacité des thiol s N-acétylcystéine

(NAC) et L-2-oxothiazolidine-4-carboxylate (OTC) à détoxifier la ce llule de

l ' acroléine et ainsi inhiber l ' apoptose sera investiguée. Le AC et I 'OTC sont utili sés

pour leur capacité à augmenter le glutath ion et les th iols intracellulaires. La

concentrat ion du glutathion est mesurée par spectrophotométrie à une longueur

d' onde précise. D 'autre part, l ' habil eté de la diminution du glutathion par le L­

buthionine sul fox imine (BSO) (inhibiteur de synthèse du glutathion) à augmenter

1' induction de 1 ' a po ptose et la cytotoxicité, sera déterminée. La cytotox icité est un

test qui mesure la capacité des ce llules à proliférer dans des pétr is après une

incubation avec un toxique. A près l ' expos ition, les cellules seront diluées et déposées

dans des pétris. Enfin, on comptera les co lonie fo rm ées après une semaine de

culture.

En conclusion, on s' attend que ce projet apporte une meilleure compréhension

de l 'apoptose induite par l 'acroléine afi n d'exp lorer le potentiel d 'utili ser la BSAO

dans le traitement des cancers. En plus, les mécani smes explorés dans ce projet

apporteront une meilleure compréhension de la tox icité de l ' acro léi ne qui es t

impliquée dans la malad ie d' A lzheimer et la tox icité du cyclophosphamide, et ainsi

trouver des moyens de protection.

2.1. Préface

CHAPITRE II

RÉSULTATS EXPÉRIMENTAUX

Ce chapitre inclut quatre articles scientifiques décrivant les résultats

expérimentaux de ce projet, que j 'ai réali sé au co urs des quatre dernières années dans

le laboratoire du Dr. Diana A. Averill.

Le pt·emier manuscrit s'intitule: "The aldeh yde acro lein induces apoptos is v1a

act ivation of the mitochondrial pathway, Biochim Biopflys Acta 1743 (3): 255 -67,

2005 ; par André Tanel et Diana Averi ii-Bates" . Ce manuscrit a été rédigé par moi­

même et a été rév isé par le Dr. Diana Averiii-Bate . Pour ce qui est des expériences

de cet article, je les ai effectuées en entier. Cette étude porte sur le mécani sme de

mort cellulaire par la voie mitochondriale induit par l' acroléine chez les cellules

d'ova ire de hamster chinois (C HO).

Le deuxième article s'intitule: "Activation of the dea th receptor pathway of

apoptos is by the aldehyde acrolein, Free Radical Bio/ogy and Medicine 42 (6): 798-

810, 2007; par André Tane l et Diana Averi ii -Bates ". Ce manuscrit a été rédigé par

moi-même et corri gé par Dr Diana Averi ii-Bates. Pour ce qui est des expéri ences de

cet a1ticle, je les ai effectuées en entier. Cette étude porte sur le mécanisme de mort

ce llulaire par la voie des récepteurs de mort indui te par l'acro léine chez les ce llules

CHO.

Le tt·oisième article s' intitule: "p38 and ERK milogen-act ivated prote in kinases

mediate acrolein-induced apoptos is in Chinese hamster ovary cell s, Cell Signal 19

(5) : 968-977, 2007; par André Tan el et Diana A veri 11-Bates " . Ce manuscrit a été

réd igé par moi-même et a été rév isé par Dr Diana Averi ll- Bates. Pour ce qui est des

57

expériences de cet articl e, je les ai effectuées en entier. Cette étude porte sur

l' implicati on de la vo ie des MAPKs dans l' inducti on de l' apoptose par l'acroléine

chez les ce llules CHO .

Le quatrième article s'intitule: "Inhibi tion of acrolei n-induced apoptos is by the

antiox idant N-acetylcysteine, J Pharmaco/ Exp Th er 32 1 ( 1) : 73 -83 , 2007; par

André Tanel et Diana Averi ll-Bates". Ce manuscrit a été rédigé par moi-même et a

été révisé par Dr Diana Averiii-Bates. Pour ce qui est des expéri ences de cet art icle, je

les ai effectuées en entier. Cette étude porte sur le mécani sme de mort ce llulaire par la

vo ie mitochondri ale induit par l'acroléin e et la protection apportée par un

prétraitement avec l'antioxydant N-acétylcystéine (NAC) chez le ce llules CHO.

D'autres contributions: "Anti-tumoral effect of nat ive and immobilized bov ine

serum am ine oxidase in a mouse melanoma madel. Biochem Pharmacol 69 ( 12):

1693 -704, 2005. Par Averiii-Bates DA, Cheri f A, Agostinelli E, Tanel A et Forti er G.

Ce manuscrit a été rédi gé par Dr Diana A ve ri 11 -Bates et le expériences ont été

effectuées par An issa Cherif et moi-même. Dr Diana Averiii-Bates et Dr Guy Fortier

sont les directeurs de cette étude. Dr Enzo Agosti ne lli a fourni l'enzyme BSAO. En

ce qui concerne ma partie, j ' ai effectué la mi eau point du tes t de l' annexine V et j 'a i

effectué les expériences de la fi gure 7 et du tab leau 2.

2.2. ARTICLE 1

THE ALDEHYDE ACROLEIN INDUCES APOPTOSIS

VIA ACTIVATION OF THE MITOCHONDRIAL

PATHWAY

André Tanel and Diana A. Averill-Bates 1'2

Département de chimie et de biochimie, TOXEN, Université du Québec à Montréal CP 8888, Succursale Centre Ville, Montréal, Québec, H3C 3P8, Canada.

1To whom correspondence should be addressed: Tel: (514) 987-3000 (ext4811) Fax: (514) 987-4054 Email: averill.diana@uqam.ca

2Formerly Dr Diana A. Bates

Acknowledgments: Financial support was obtained from the Natural Sciences and

Engineering Research Council of Canada (DAB). The authors thank Stéphanie Lord­

Fontaine and Michel Marion for technical assistance and Bertrand Fournier (SCAD)

for statistical analysis.

Abbreviations: AFC: amino trifluorocoumarin; AMC: amino methylcoumarin; BSA:

bovine serum albumin; BSAO: bovine serum amine oxidase; CAD: caspase activated

DNase; CHAPS: 3-[(3 -cholamidopropyl )dimethylanunonio ]-2-hydroxy-1-

propanesulfonic acid; CHO: Chinese hamster ovary; Fas: fibroblast-associated; FBS:

feta l bovine serum; FCCP: P-trifluorometboxy-phenyJ-hydrazone; ICAD: inhibitor of

59

caspase acti vated DNase; MEM: mm1mum essenti al medium ; MOPS : 3-(N­

morpholino)-propane sul fo nic ac id ; PARP: po lyADP-ribose polymerase; PBS :

phosphate-buffered sa line; PM SF: phenylmethylsufonyl tluorid e; PTP: permeability

transition pore; PVD F: polyvinylidene difluorid e; SOS-PAGE: sodium dodecy l

sulphate-po lyacry lamide ge l electrophores is; SEM: standard error of mean.

Keyword s: Acrolein , apoptos is, caspase, mitochondri a, ce ll , cytotox icity.

60

RÉSUMÉ

L 'acroléine est un aldéhyde a.,p-in saturé extrêmement réacti f et elle est un produit de

la peroxydati on lipidique. Elle est un polluant environnemental qui est impliqué dans

plu ieurs maladies respiratoires. L 'acroléine est produite lors de la désamination

oxydative par la spermine oxydase. Les produi ts d' oxydati on des polyamines sont

impliqués dans l ' inhibition de la proli fé ration cellulaire, l ' apoptose, et l ' inhibition de

la synthèse de l ' A DN et des protéines. La présente étude expl ore le mécanisme de

mort ce llulaire induit par l 'acroléine. Acroléine induit l ' apoptose en entraînant la

baisse du potentiel membranaire mitochondri al, la li bérat ion du cytochrome-c,

l ' acti vati on de la caspase-9 initiatrice et la caspase-7 exécutrice. Par ailleurs,

l ' acro léine a inhibé l 'activité enzymatique de la ca pase-3 exécutrice sans empêcher

cependant le clivage de la proenzyme. L ' acti vati on des caspases -7 et -9 a été

confirmée par le clivage de leurs proenzymes. L ' apoptose indui te par l ' acroléine a été

inhibée par un inhibiteur de la caspase-9 mais pas par un inhibiteur de la caspase-3.

L ' induction de l ' apoptose par l ' acroléine a été confi rm ée morphologiquement par la

condensation nucléa ire de la chromatine et par le cli vage de l ' inhibiteur de la DNase

acti vée par les caspases (JCAD). Le cli vage de IICA D libère l ' endonucléase CA O

qui emmène la f ragmentation de l ' ADN. Ces résul tats démontrent que l 'acroléine

induit l ' apoptose par la vo ie mitochondriale.

6 1

ABSTRACT

Acrole in is a highly react ive a,p-unsaturated aldehyde, which is a product of li pid

peroxydat ion. lt is an environmental pollutant that ha been irnpli cated in multiple

respiratory diseases . Acro lein is produced by the enzymati c oxidati ve dearnination of

spermine by amine oxidase . Oxidation products of polyamines have been invo lved in

inhibi tion of ce ll proli feration , apoptosis , and inhibition of DNA and prote in

synthes is. The present study investigates the mechani sm of ce ll death induced by

acrole in . Acrolein induced apoptosis through a decrease in mitochondrial membrane

potential, liberation of cytochrome-c, acti vatio n of initi ator caspase-9 and acti vat ion

of the effector caspase-7. However, acrole in inhi bited enzymatic act ivity of the

effector caspase-3, although cleavage of pro-caspase-3 occurred. The act ivati on of

caspases-9 and -7 was confirmed by cleavage of their pro-enzy me form by acro le in.

Apoptos is was inhibited by an inhibitor of caspase-9, but not by an inhibitor of

ca pase-3. Induction of apoptosis by acrolein was confirmed morpholog ically by

condensation of nuclear chromatin and by cleavage of inhibitor of caspase activated

DNase (ICAD), which leads to liberat ion of CA D that causes DNA fragmentat ion.

These results demonstrate that acrolein causes apopto is th rough the mitochondrial

pathway.

62

INTRODUCTION

Acrolein is a highly reactive, u,p-unsaturated aldehyde and humans are

exposed to thi s compound in multiple situat ions [ 1]. Acrolein is one of the tox ic

produ cts of endogenous lipid peroxidation (LPO) [2]. Aero le in is a major component

of cigarette smoke and a constituent of automoti ve exhaust [3]. lndustrially, acrolein

is used as a herbicide as weil as a starting material for acrylate polymers and in the

produ cti on of acrylic acid [4]. Acrolein reacts with cyste ine, hi stidine and lys ine

res idues of proteins [5 , 6] . Acrole in protein adducts have been demonstrated 1n

di abetic nephropathy [7], Alzheimer's di sease [8, 9] and atherosc leros is [ 1 0].

Apoptosis is a phys iolog ica l cell death process whi ch plays an importa nt ro le

du ring development and maintenance of ti ssue homeostas is [Il , 12]. A balance

between ce ll death and ce ll pro liferati on is required to mainta in a cellular homeostatic

state. Devi ation from thi s cellular balance di srupts the normal state and can lead to

hum an di sease [1 3]. A family of cytoso l ic cy te in e proteases, the caspases, stored in

most cell s as zymogens, play an essenti al role in th e executi on of apoptos is. The

caspases are di vided into api cal ( -2, -8 , -9 and -1 0) and executioner subsets ( -3, -6 and

-7) [ 14]. The zymogen of caspase-9 is acti vated via a post-mitochondri al route [ 15].

The mitochondrial pathway is triggered by growth factor deprivation, ionizing

radi ati on and some anticancer dru gs such as cyc lophosphamide and etopos ide [1 6].

Cytochrome-c is released from mitochondri a into th e cytoso l, where it interacts with

dA TP and apoptos is protease activa ting fac tor (A pa fi) and pro-caspase-9 in the

apoptosome complex in the cytoso l. This leads to conversion of pro-caspase-9 to

act ive caspase-9 [17]. Anti-apoptotic (Bcl-2) and pro-apoptoti c (Bax) member of the

Bcl-2 fa mil y are thought to control apopto i by modulating cytochrome-c release

from mitochondri a [18] . Once acti vated, caspase-9 activates effector caspases such as

caspases-3, 6 and 7 [14]. Effector caspases then cleave specifie cytoplasmic,

63

cytoske leta l and nuclear protein substrates such as polyADP-ribose polymerase

(PARP), lamins and inhibitor of caspase act ivated DNa e (ICAD). The ce l! then

exhibits the characteri st ic morphologica l featu re of apo ptos is such as chromat in

condensation, ce l! blebbing and fo rm at ion of apoptot ic bod ies [ 19].

Acrole in has been shown to induce apoptosis in ce l! types such as human

a lveo lar macrophages [20], hu man keratinocytes [21 ] and hu man branchial epithe lia l

ce ll s 1-lB E 1 [22]. ln contrast, acrolein inhibited neutrophi l apoptos is [23] and induced

oncos is/necros is rather than apoptos is in murine proB lymphocytes [24]. Thus,

acro lein-induced apoptosis appears to be ce il type dependent. Most of these stud ies

confinned induction of apoptos is by means of endpoints occurring later in the

apoptotic cascade such as DNA fragmentat ion, without determining the mechanisms

occurring upstream of these events.

Acrolein is a metabolic product of cyc lophosphamide [25] and could be

invo lved in its ant icancer action. Lt is a Iso a prod uct of the ox idation of polyamines by

amine oxidases along wi th hydrogen peroxyde[26]. Ou r previous studies suggest that

amine ox idase could prove to be useful in ca ncer treatm ent [27]. Targeting

polyamines has emerged as a promisin g therapeuti c strategy since they play an

important role in the development and main tenance of neo plastic growth [28].

Moreover, rap idly grow ing tissues such as tumors have elevated levels of polyamines

[29]. To take advantage of this di ffe renti ai effect between normal and tumor cell s,

toxic products such as H20 2 and aldehydes could be generated in s itu by amine

ox idases fo r the selective killing of tumor ce ll s [30]. However, the molecul ar

mechanisms in vo lved in acrolein- induced apoptos is in cancer ce ll s are not

understood. Thi s study invest igates the ability of acrolein to act ivate apoptos is via the

mitochondrial pathway in proliferating Chinese ham ter ova ry (CI-10) ce ll s which are

known to induce tum ors.

64

MATERIALS AND METHODS

Cel/ culture

Cl-IO ce lls (AuxBl) [3 1] were grown 111 monolayer in m1111mum essential

medium-Alpha (a-MEM) (G ibco Canada, Burlington, ON , Canada) plus 10% fetal

bovine serum (FBS) (G ibco Canada) and 1% penici llin (50 units/mL)- streptomycin

(50 ~Lg/mL) (Flow Laboratories, Mississauga, ON, Canada), in ti ssue culture flasks

(Starstedt, St Laurent, QC, Canada), in a humidifi ed atmosphere of 5% C02 in a

water jacketed incubator at 37°C [32]. The ce ll were grown to near confluence and

were then incubated for 24 h with fresh culture med ium. Confluent ce ll s were then

harvested using citrated phosphate-buffered saline (0. 14 M NaCl, 0.01 M sod ium

phosphate, 0.015 M sod ium citrate) , was hed by centrifu gation ( 1 OOOg, 3 min) and

resuspended in a-MEM fo r experimental studie .

Clonogenic cytotoxicity assay

Cytotoxicity was eva luated as the abi li ty of ce ll s to proliferate to form

macroscopic co loni es after a toxic insult with acro lein, using a c lonogen ic cell

urvi va l assay. CHO ce lls (1 05/mL) were incubated with acrole in (Aldrich Chemi ca l

Co, Mi lwa ukee, Wf), in a final vo lume of 1.0 mL in a-MEM containing 10 % FBS

for 1 h at 37°C. The ce lls were then washed three times by centrifugation ( 1 OOOg, 2

min) to stop the incubation [32]. The cell s we re resuspended and diluted to the

appropri ate concentration with a-MEM plus FBS and plated in ti ssue culture dishes

(60 x 15 mm), which were incubated at 3 7°C in an atmosphere of 5% C02 fo r 8 da ys.

The dishes were then washed with PBS , fixed with 95% eth ano l and sta ined with

methylene blue before counting macroscop ic co lonies (>50 ce ll s). Cytotoxic ity was

expressed as the mean number of co loni es obta ined relat ive to the mean number of

co lonies obta ined in the control. Two hundred ce ll s were seeded in the control plates,

but where there was a loss of ce l! survival , ce ll s were plated at severa! different

65

densities to ensure that countab le co lonies wo uld be obta in ed, and the results were

corrected accordingly. We have previously demonstrated that, in thi s system, there is

linearity between the number of cell s plated and co loni es fo rm ed over th e range of 10

to 104 [32].

Morplzological analysis ofapoptosis

To visualize nuclear morphology and chromat in condensation by flu orescence

mi croscopy [33], cell s were seeded and cultured to near confluence in tissue culture

d ishes containing SmL of a-M EM and 10% FBS. Cell s were incubated with acrole in

fo r 4 h or 24 h. Dishes were washed tw ice with PBS and Hoechst (33258) (0.06

mg/mL) was added fo r 15 min at 37°C to sta in apoptotic ce ll s. The di shes were

wa hed with PB S and propidium iodide (50 ~Lg/m L) was added to sta in the necroti c

cell s. Observations were made by flu orescence mi croscopy (Carl Ze iss Ltd, Montrea l,

QC) and photographs were taken by di gital camera (camera 3CCD, Sony DXC-950P,

Empix lmaging !ne, Miss issauga, ON). Images were analysed by Northern Ec lipse

software. Ce ll s were classified using the fo ll owing cri teria: a) li ve cell s (normal

nuclei, pale blue chromatin with organized structure) ; b) membrane- intact apoptotic

ce ll s (bright blue condensed or fragmented chromatin) ; c) necrotic ce lls (red , enl arged

nuclei with smooth norm al structure [33]. The fract ions of apoptotic and necrotic

ce ll s were determined relative to total ce ll s (obta ined using bright fie ld illumination).

A minimum of200 cell s were co unted per dish.

Caspase inlûbitors

Treatment of ce ll s with inhibitors of ca pase was performed on confluent

ce lls in monolayer. The specifie inhibitor used were: Caspase-3 lnhibitor V, Z­

DQMD-FMK; Caspase-9 lnhibitor 1, Z-LEHD- FMK and the general Caspase

lnhibitor 1, Z-VAD-FMK (Ca lbiochem, La Ja ll a, A. The ce ll s were exposed to 10 or

20 ~M concentrations of inhibitors and to va ri ous concentrations of acrolein fo r 4 h.

66

Ce ll s were analysed for apoptos is by flu orescence m1 croscopy using Hoescht

sta ining.

Determination of caspase activity by.fluorescence spectroscopy

Freshly harvested CHO cells (0.5 x 1 06) were resuspended 111 a.-M EM and

incubated with acrolei n in a final volume of 1.0 mL at 37°C. After the appropriate

ti me, the ce il s were washed three times with co ld PB S by centri fugat ion ( 1 OOOg, 3

min) to stop the incubation. The ce ll s were re uspended in 50 ~Li of PBS and 25 ~Li

were depos ited into 96-well plates and lysed by freez in g at -20°C for 20 minutes.

Fifty ~Li of reaction buffer (20 mM piperaz ine-N,N'-bi s(2-ethanesulfonic ac id)

(PIPES), 100 mM NaCI, 10 mM dithiothreito l, 1 mM EDTA, 0.1% 3-[(3-

cholam idopropyl)dimethylammonio ]-2-hydroxy-1-propanesul fo n ic ac id (C HAPS),

10% sucrose, pl-I 7.2) was added and stabilized at 37°C [34]. The ki netic reaction was

started after add ition of 25 f.i.l of the appropriate ca pase substrate at 37°C using a

Spectra Max spectrofluorimeter (Spectra Max Gem ini , Molecular Deviees,

Sunnyva le, CA).

Caspase-3 activity was measured by cleavage of the flu orogeni c substrate N­

acetyi-Asp-G lu-Vai-Asp-am ino-4-methylcoum arin (Ca lbiochem, La Joli a, CA) to

produce amino methylcoumarin (AMC) with Àmax exc itation at 380 nm and Àmax

emiss ion at 460 nm. Caspase-7 act ivity was measured by cleavage of the flu orogenic

substrate 1 MCA-VDQVDGWK(DNP)-N!-1 2 with Àmax exc itation at 325 111n and

Àmax emiss ion at 395 nm. Caspase-9 act ivity was measured by cleavage of the

substrate Ac-LEHD-AFC to produce AFC with Àmax exc itation at 4 15 nm and Àmax

emiss ion at 490 nm .

Flow cytometry analysis of mitochondrial membrane potential

To measure .0.\jfm, the fluorescent probe rhodamine 123 was u ed. Freshly

harvested CHO ce il s ( 1 06) were resuspend ed in a.-M EM and incubated with acrolein,

67

or the pos itive control p-trifluorom ethoxy- phenyl-hydrazo ne (FCC P), in a fin al

volume of 1.0 mL at 37°C. After one hour of incubati on, th e cell s were washed three

times with co ld PBS by centri fugation ( 1 OOOg, 3 min) to stop the incubati on. The

ce ll s were resuspended with PB S and then incubated with th e tluorescent probe (800

ng/mL) for 5 minutes. Samples were washed and stored in the dark at 4°C until the

time of analys is (usually within 5 min). Propidium iodid e (Pl ) was added to ce l!

suspensions stained with rhodamine l 23 to identi fy dead ce l! s. Ce lis were analyzed

with a FACScan fl ow cytometer equipped with an argon laser emitting at 488 nm

(Becton Dickinson, Oxford , UK) . Data was acquired and analysed using Lys is Il

sofu;ya re (Becton Dickinson). Mean fluorescence intensity of 20,000 cells was

ca lcul ated for each sample and co rrected for autoflu orescence obta ined from samples

of unl abeled cell s. The analyser threshold was adju ted on the fo rward scatter (FSC)

channel to exc lude no ise and subce llular debri s. Photomultipli er settings were

adj usted to detect rhodamine 123 flu ore cence on the FL 1 detector and Pl

flu orescence on the FL2 detector. ln each case, the photomultiplier voltage was set so

that the signal peak from non-stained ce lls (mostl y due to autoflu orescence), feil

within the first decade of the logarithmic amplifier. Light scatter parameters were

used to establish size gates which detect apoptoti c ce ll s, thus exc luding dead ce lls

[35]. Populati ons of dead cell s hi ghly flu oresce in FL-2 because they incorporate Pl ,

thus 2 populati ons of ce ll s appear on the computer screen. For each sample, onl y the

li ve ce ll s are se lected and the ir mean flu orescence in FL-1 is analysed. Apoptoti c

ce li s, whi ch undergo a decrease in m itochondri a l membrane potentia l, incorporate

less of the rhodamine 123 dye, therefore emitting less flu orescence on the FL-1

detector.

Subcellular ji-actionation and immunodetection of cytochrome-c, caspases and

ICA D

68

Following treatment with acrolein , ce li s were washed in buffer A ( 100 mM

ucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4) and resu pended in buffer B [buffer A

plus 5% Percoll , 0.01 % digitonin and a cocktail of protease inhibitors: 10 f!M

aprotinin, 10 f!M pepstatin A, 10 f!M leupeptin, 25 ~tM ca l pain inhibitor 1 and 1 mM

phenylmethylsulfonyl fluoride (PMSF)]. After 30 min incubation on ice, lysates were

homogenised using a hand porter (Kontes gia s CO, Dual! 22, Fisher, QC, Canada).

Un broken cells and nuclei were pelleted by centi fugation at 2500 g for 10 min. The

upernatant was centrifuged further at 100 000 g fo r 1 h. The resultant supernatant

was des ignated as the cytosolic fraction , which was used for detection of cytochrome­

c. For immunodetection of caspases and ICAD, who le ce l! lysates were used.

SDS-polyacrylam ide ge l e lectrophores is (S OS-PAGE) of cellular proteins was

carried out according to Laemmli [36]. Proteins (30 ~tg) were quantified according to

Bradford [37] and then so lubili sed in Laemmli sample buffer. The sampl es were

boi led for 5 min at 1 00°C and loaded onto a 15% acrylamide ge l. Electrophores is was

carri ed out at a constant vo ltage of 125 V. Ce li ular protei ns were transferred

electrophoretically to a polyv inylidene difluoride (PV DF) membrane using a

MilliB iot Graphite Electroblotter 1 apparatus (Milli-pore, Bedford, MA). The transfer

buffer contained 96 mM glyc ine, 10 mM Tris and 1 0% methanol. The transfer was

carri ed out for l.S h at constant amperage of 80 mA/gel. Hydrophobie or nonspec ific

sites were blocked overnight at 4°C with 5% powdered skim milk in Tri s-buffered

saline (50 mM Tris and 150 mM NaC I) containing 0.1 % Tween 20 (TBS-T).

Membranes were washed four times for 15 min in TBS-T. The blots were probed

with the primary antibody: anti-cytochrome-c (B D Biosc iences Canada, Mississauga,

ON), anti-caspase-3, anti-caspase-9, anti-lCA D, anti-tubulin (Santa Cruz

Biotechnology, Santa Cruz, CA) and anti-caspase-7 (Ce ll Signaling Technology,

Beverly, MA) in TBS-T, 1% bovine serum album in (BSA) for 1 h at room

temperature. Membranes were washed fo ur times for 15 min and incubated for 1 hat

room temperature with peroxidase-conjugated secondary antibody ( 1:1 000) in TBS-T

69

containing 5% milk powder. Secondary antibod ies consisted of horserad ish

perox idase (HRP)-conjugated goat anti-mouse, anti-rabbit and anti-goat lgG

(B iosource, Camarill o, CA). PVDF membranes were washed four times for 15 111111

and cytochrome-c, caspases and ICAD were detected us ing the ECL plus

chemiluminescence kit (Perkin Elmer, Boston, MA). For verification of equ ivalence

in protein loading, the blot was probed with the anti-tubulin antibody and by

co lorat ion of the gels using Coomass ie blue. Protein express ion was quanti fied using

a scanning laser densitometer, re lat ive to ~-tubulin (Mo lec ul ar Dynamics, Sunnyva le,

CA) .

Statistical analysis

Statist ical di ffe rences between control and treated groups fo r ce ll survi val and

ICA D cleavage were detennined by a two-tai led unpaired Student' L test. Statist ical

compari sons fo r the glutathione assay were made with one-way ANOVA whi ch

measures the linear contrast of means. An adj ustment was made to limit the

famil ywise error rate (FWE) to 5% by ca lcul at in g an adj usted p-va lue whi ch is a

simulated based p -value obta ined from the multi va ri ate t distributi on (number of

simulations = 1000 000) [38). Two-way ANOV A vv ith a p-adjusted value obtained

usin g the Bonferroni-Holm method (a stepwise method) was u ed to compare ce ll s

treated with acro le in versus control, using the 1-l oescht test. Data fo r caspase act ivity

and caspase cleavage were analyzed for signifi ca nt diffe rences using a one-way

ANOV A with a Bonfe rroni-Holm post-test correction for multiple comparisons. For

ex periments invo lvin g caspase inhibitors, adj usted p-va lues were obtained using one­

way ANOV A fo ll owed by the Dunnett test.

Values are expressed as means ± SEM. Difl"erences were considered

stati stically significant atp < 0.05 .

70

RESULTS

We established the concentrations of acro le in whi ch were ab le to induce

cytotoxicity in CHO ce li s (F ig. 1 ). Cytotoxic ity wa induced at concentrations of 50

fm o l/ce ll of acro le in and hi gher. A concentrati on of about 1 80 to 190 fm o l/ce ll ( 18-

19 11M) was s ufficient to decrease the fraction of surviving ce ll s to 10% . Above 200

fmol /ce ll , there was a marked decline in ce l! surviva l to 5 logarithm s of ce l! killing at

350 fm o l/cell . lt shou ld be noted that the acro le in co ncentrat ion was expressed as

fmo l/ce ll because the ce l! density was different fo r certa in tests [39].

Subseq uently, we investigated the type of ce l! death (i.e. apoptos is or

necrosis) induced by acro le in in proliferating C l-IO ce ll s. Morpho logical analys is

demonstrates that acro lei n induces both a poptosis and necros is in ce ll s (Fig. 2).

Apoptosis is characte rized by early and prominent condensati on of nuc lear ch romat in

[33]. Apoptosis induced by acro le in was reveal ed by condensation of ch romat in with

the flu orescent probe Hoescht (b lue-green) whereas necros is was revealed by the

flu orescent probe Pl (red). Acrolein (50 fm o l/ce ll , 4h) (Fig. 2C) induced apoptos is

and necrosis in 21% and 4% of ce ll s, respecti ve ly (F ig. 2E, 2F). A higher

concentration of acro lein ( 100 fmol /ce ll , 4h) (F ig. 2 0 ) sw itched the mode of ce l!

death from apoptosis (9%) to necrosis (20%) (F ig. 2E, 2F). lt should be pointed out

that induction of apoptos is occurred at lower concentrati ons of acro le in (~ 30

f mo l/ce ll , 4h) (F ig. 28 , 2E), relative to inducti on of necrosis (~ 50 fmol/cell , 4h)

(F ig. 2C, 2F) . Very few dead cell s were seen in untreated contro ls (F ig. 2A). When

ce ll s were exposed to acrolein for 24 h, higher level s of toxicity occurred by

apoptos is and necrosis (Fig. 2E, 2F) . Maximum leve ls of apoptos is were induced by

30 and 50 fmol/cell of acro le in , whereas 100 fmo l/ce ll of ac ro lei n caused essentia lly

onl y necros is.

The subsequent step was to determ ine whether ac ro le in could induce

apoptosis v ia activation of the mitochondri a l pathway. The ab ility of acro le in to a lter

71

mitochondrial membrane potenti a l and to induce liberati on of cytochrome-c from the

mi tochondria to the cytoso l was determined. Expos ure of ce lls to acrolein ( 10 to 30

.fillol!ce ll ) for 1 h led to a decrease in rhodamine 123 flu orescence in the FL 1 channel,

relati ve to untreated control ce ll s (Fig. 3A, 38 ). Thi s represented a decrease in

mi tochondrial membrane potenti al by 25% in the presence of 30 j inol/ce ll of acrolein,

relati ve to contra is (Fig. 3B). The magni tude of the decrease was similar to that

induced by FCCP (5 ~-tM), whi ch was used a a pos itive control fo r membrane

depolari sati on (F ig 38 ). The mitochond rial perm eability transition pore (PTP)

complex is considered to play an important role in the release of pro-apoptoti c

proteins such as cytochrome c. Opening of the PTP can be inhibited by compounds

such as cyc losporine A (40]. lndeed, cyclosporine A part iall y inhibited apoptos is

induced by acrolein (Fig. 3C), further confirming the role of membrane depolarisati on

in acrolein-induced apoptos is. Acrolein (4 to 30 f mol/ce ll ) caused the liberati on of

cytochrome-c after 1 h (F ig. 3D). Exposure to acrolein ( 1 to 50 jin ol/cell ) for 1 h

caused activation of caspase-9, which is the initiato r caspase of the mitochondrial

path way (Fig. 4A). However, after 2 h, th ere was no acti va ti on of caspase-9, but

instead, inhibition of the enzyme. ln fact, caspase-9 acti vity was lower than the

control leve! fo r concentrations between 20 and ISOjinol/ce ll , a lthough thi s was only

significant at the highest dose. We fo und that acrolein cleaved procaspase-9 as a

function of increas ing concentrati on from 2 to 50 j mol/ce ll afte r a 1 h in cubati on

(F ig. 48, 4C). This was apparent as a decrease in th e intensity of bands (F ig. 48 ) for

the pro-enzyme fo nn of caspase-9 (F ig. 4C). There was a corresponding increase in

the quantity of cleavage fragment of 35 k.Da fo r caspase-9 (F ig. 48, 4D), with

increas ing acrole in concentration.

We subsequentl y determined the ability of acrole in to acti vate effector

caspases. However, the enzymatic acti vity of the major downstream effector caspase-

3 was inhibi ted by low concentrations of acrolein (5 to 20 jmol/ce ll ) (F ig. SA).

Caspase-3 acti vity was rapidly inhibited after expo ure to acrole in for onl y 5 min

72

(F ig. SA). lndeed, exposure of ce lls to 20 f mo l/ce ll of acro le in fo r 1 h compl ete ly

inhibited caspase-3 activity (F ig. SA). A lthough caspase-3 act ivity was inhibited,

procaspase-3 was cleaved (F ig. 58 , SC) to it two fragments pli and pl 7 (F ig. 58,

5 0 ). C leavage of pro-caspase-3 is necessary fo r the fo rm ati on of the acti ve d imeric

fo rm of ca pa se-3 [ 4 1].

S ince effector caspase-3 was not act ivated , we determined whether other

downstream caspases could be activated, in arder to exp la in the acro le in-induced

chromatin co ndensation (F ig. 2C). We eva luated the effect of acro le in on act ivity of

caspase-7, w hich is a downstream caspase th at ca n c leave PA RP. T hu s, 10 fm ollce ll

of acro le in , w hi ch inhibited caspase-3 after 5 min of expos ure (F ig. SA), activated

caspase-7 after 2 h (F ig. 6A). S imil ar to caspase-3, c leavage of pro-caspase-7 is

necessary fo r the fo rmat ion of the acti ve dimeric fo rm of caspase-7 [4 1]. lndeed,

c leavage of procaspase-7 (F ig . 68, 6C) to its acti ve fragment p20 (F ig. 6B, 6 D) was

induced after 1 h, by 10 to 50 ji11o l/ce ll of acro lein. H igher co ncentrat ions of acro le in

(~ 100 jmo l/ce ll ) were needed to act ivate caspase-7 afte r 1 h, whereas lower

concentrations ( 10 and 50 fm o l/ce ll ) ac ti vated the enzyme after 2 h of acro le in

exposure (F ig. 6A). H igher doses of acro le in (> 100 ji11o l/ce ll ) did not acti vate

caspase-7 after 2 h (F ig. 6A).

T he subsequent step was to confi nn the imp li cat ion of caspase-9 in acro le in

induced apoptos is. Therefore, the ability of a gene ra l inhib itor of caspases and of

spec ifi e inhibitors of caspases-3 and -9 to inhi bit apoptosis induced by acro le in was

eva lu ated. The leve! of apoptos is was partia lly inhi bited by the genera l inhibi to r of

caspases (f ig. 7A). T he inhibi to r of caspase-9 dec reased ce l! death by apoptos is by

about 70% (F ig. 7 A). However, the inhibi to r of ca pase-3 had no effect on apoptosis

induced by acro lein (F ig. 7B). Hydrogen peroxydewas used a a pos itive contro l to

show that the inh ib ito r of caspase-3 wa ab le to inhi bit per x ide- induced apoptosis

(F ig. 7C).

73

Acro le in (~50 fmol /ce ll ) indu ced c leavage of the caspase substrate ICAO, the

inhibito r of caspase activated ONase (CAO), afte r 2 h (F ig. 8), thereby liberat ing

CAO w hich can cause ONA fragmentation in the nuc leus.

74

DISCUSSION

The new finding of the present study is th at acrole in is capable of inducing the

mitochondrial pathway of apoptosis invo lving liberat ion of cytochrome c and the

act ivation of certain caspases in proliferating ce il s. We provide new informat ion on

the molecul ar mechani sms by which acrolein induces apo ptos is. Induction of

apoptos is by acrolein was confirmed morpholog ica ily by the co ndensation of nuclear

chromatin after 4h , a later event in the apoptotic cascade, as we il as by severa! earlier,

upstream events associated with apoptos is at th e mitochondrial and post­

mi tochondri al levels. At the mitochondrial leve!, acrole in caused a decrease in

membrane potential , foilowed by the liberati on of cytochrome-c into the cytoso l, after

1 h. At the post-mitochondrial level, cytochrome c is an essenti al component of the

cytoso li c apoptosome complex, along with Apafl and pro-ca pa e-9. Acrolein indeed

induced the activation of initi ator caspase-9 after 1 h and the effecto r caspase-7 after 1

to 2h. Caspase act ivation was fo ilowed by cleavage of ICA D after 2h. Thi s leads to

the li beration of the protease CAO, wh ich can cause later events such as chromatin

condensation and fragmentation of DNA in the nucleu .

Act ivat ion of these caspases was confirmed by the cleavage of their pro­

enzyme fo rms and the generat ion of appropriate cleavage fragments, as we il as by the

increase in their enzymatic activities. The role of caspase-9 was a Iso confirmed using

a spec ifi e caspase-9 inhibitor. At present, there is no spec ifi e inhibitor of caspase-7

a v ai labie commercially.

The implicat ion of the PTP and cytochrome-c release in acro lein induced

apoptos is was assessed using cyclosporine A. Although the mechani sms of

cytochrome c release are not completely understood, it i considered that cytochrome

c can be re leased from mitochondria via the PTP as we il as by formation of channels

on the membrane involving Bax [42]. The inhi bition of apoptos is by cyclospo rine A

supports a role for PTP opening and release of cytochrome c in acrolein-induced

75

apoptos is. The parti al inhibition by cyclosporine A uggests that acrolein could also

induce apoptos is by alternative mechani sms to the mitochondri al pathway and/or th at

cytochrome c can be released by severa! di ffe rent mechani sms.

Our findin gs show that caspase-3 is not essenti al for the execution phase of

acrolein-induced apoptosis in proliferating ce ll s. Thi s was demonstrated by the lack

of activation of its enzymatic activity and the lack of inhibition of nuclear chromatin

condensati on by a specifie inhibitor of caspase-3. lt was reported that MCF-7 ce ll s,

whi ch lack caspase-3, can still undergo apoptosis [4 1]. lndeed, acrolein-induced

apoptos is in CHO cell s appears to be medi ated through acti vat ion of the effector

caspase-7. ln addition to caspase-3, caspase-9 is abl e to d irectly cleave pro-ca pase-7,

leading to subsequent activation of caspase-7 [43]. ln our study, cleavage of ICA D is

likely to be mediated by caspase-7 [44]. lt is often di ffi cult to distingui sh between the

involvement of caspase-3 and -7 in apoptos is since they share similar protein (PARP,

ICAD) [19] and peptide (Ac-DEVD-AMC) ubstrates [45].

A surprising findin g in our study was that pro-caspase-3 was indeed cleaved

in cells, yet the enzymatic activity of caspase-3 was markedly inhibited by acrole in .

The cleavage of pro-caspase-3 is likely to result from the proteo lyti c acti on of th e

active caspase-9 [46]. Even though pro-caspa e-3 undergoe cleavage to generate

caspase-3 in cell s, enzymatic activ ity appears to be immedi ately inhibited. This could

ari se through direct alkylation of its active site cysteine res idue by acrolein . The

inhibition of caspase-3 by acrole in is in agreement with other studi es. Inhibi tion of

caspase-3 acti vity occurred in neutrophil s after 2 to 8 h of treatment with 10 ~-tM

acrolein [23]. Activities of caspases-3, -8 and -9 were inhibi ted 12 h fo llowing a 30

min exposure to acrolein (5 to 40 ~-tM) in murine proB lymphocytes [24]. For each of

the caspases-3, -7 and -9, cleavage of the pro-enzyme form into cleavage fragments

occurred in CHO cell s. However, ali of the caspase enzymes contain a nucleophilic

acti ve-site cysteine residue and potenti ally they could ali be inhibited by acrolein [23 ,

47]. Acrole in appears to be causing some act iva ti on ofcaspases-7 and -9, while at th e

76

same time inhibiting their activity. lt appears th at caspases-7 and -9 are not res istant

to inactivation by acrolein, but rather, they may be Jess susceptible to inactivati on

than caspase-3. Although acrolein appears to eventually inhibit enzymati c act ivity of

each of these 3 caspases, caspases-9 and -7 were suffi cientl y acti vated to trigger

caspase-dependent downstream events such the ICA D cleavage and chromatin

condensation. The fact that acrole in completely inhibits caspase-3, whereas caspases-

7 and -9 undergo some leve! of acti vati on in Cl-I O ce lls could also be expla ined by

more restri cted access of acrolein to the acti ve site cysteine res idues of caspases-7

and -9. It was reported that the major structural di ffe rences among th e caspases occLu·

at the active sites, whereas the rest of the structures are very similar. These structural

di fferences at the act ive sites could affect access to the acti ve site as we il as

interactions with other molecules and could explain the di ffe rences in the substrate

spec ificities among the different cas pa ses [ 41].

It has recently been reported that proteo lyti c cleavage is neither required nor

suffic ient fo r acti vati on of monomeric initi ator ca pases such as caspase-9 [48].

Dimerizati on, rather than cleavage, appears to be th e most important step for caspase-

9 activation and form ati on of an active site. 1-1 0\vever, cleavage of the prodomain

stabili ses the active form of caspase-9. There are severa! similarities between

activation of caspases-7 and -9. lnterestingly, th e caspase-9 dimer only contains one

acti ve site. The catalytic apparatus in th e other domain is di sabled due to steric

clashes. This inacti ve domain is almost identica l to that of the zymogen fo rm of

caspase-7, indicating that some structural similariti es ex ist between caspases-7 and-

9. A iso, identical confo rmational changes leadin g to fo rm ati on of the active sites was

reported fo r caspases-7 and -9, even though they are acti vated by diffe rent

mechani sms: proteo lysis versus d imerisa ti on, respecti v ely [ 48]. 1-I owever,

interdomain c leavage by initiator caspases is needed fo r activa ti on of executi oners

such as caspases-3 and - 7 [48].

77

In general, different studies have reported va riable tï ndings in that acrolein

can either cause or inhibit apoptos is, or cause predom inantly necros is rather than

apoptos is. For example, acrolein (25 ~tM) caused apoptos is in i olated human alveo lar

macrophages, detected by morpholog ica l changes and D A fragmentation after 24 h

[20]. Exposure to 50 )..LM acrolein for 24 h induced atypi ca l apoptosis in primary

cu ltures of human keratinocytes [21]. Acro lein st imulated apoptos is in the human

lung epithelial ce ll li ne HBEI , as indicated by externalisati on of phosphatidy lserine

and DNA fragmentation 24 h after a 30 min expo ure to 10 to 25 ~tM acrolein [22]. ln

human neutrophi ls, however, acrolein (25 ~tM) inhibitecl the con titutive pathway of

apoptos is [23 ]. ln proS lymphoid ce ll s, the ce ll death pathway was predominantly

oncos is/necros is, with only low leve ls of apoptosis occurring at 10\-ver doses ( < 10

)..LM) [24]. These vari ab le results could be due to di fferences in biochemical factors

determining cell death pathways in different cell types, which include non­

pro li ferating primary cell cultures and ce ll s involvecl in immune responses, as we il as

pro l iferating cancer ce li li nes. Furthermore, cl ifferent methodo logies were used in

these studies, such as longer exposures to acrolein for 24 h or 48 h versus shorter

exposures to acrolein for 30 to 60 min, w ith or w ith out a 24 h-recovery period. ln

some cases, inclucling the present work, cells were expo ed to acrolein in medium

conta ining serum [20], whereas other studies exposed ce lls in serum-free medium

[2 1, 22, 23 , 24]. Acro lein can bind to serum proteins, which would likely decrease the

concentrati on ava ilable to interact w ith cell . Th is may slow or modi fy the onset of

tox icity leacling to apoptos is, rather than necros is. Necrosis appeared to be the

predominant fonn of ce ll death when ce ll s were expo ed to acrolein in serum-free

med ium [24]. ln our study acro lein did induce necrosis, but at higher doses than th ose

wh ich caused apoptos is.

One of our interests 1n exploring mechanisms of acrolein toxicity 111

pro li ferati ng ce ll s li es in the context of anticancer treatment. We reported that

acrolein and H20 2 both contr ibuted to cytotox icity induced by spermine and bov ine

78

serum amine ox idase (BSAO, EC 1.4.3.6) in HO ce ll s [49]. Since tumor tissues

contain elevated levels of polyamines, ta rgeting po lya mines could be a promi si ng

therapeutic strategy [28]. Tax ie products such as H20 2 and acro lein could be

generated in situ by amine ox idases for the se lect ive ki Il ing of tu mor ce li [ 49].

Fu rthermore, the enzyme could also act by deplet ing polyamines which are necessary

fo r tumor growth. BSAO has been successfull y immob ilized in biocompatible

po lyethylene glyco l hyd roge ls, which could be useful for in vivo stability and

de li very ofthe enzyme to tumors [50].

ln conclusion, this study clearly demonstrates that acrolein can cau e ce ll

death by both apoptos is and necros is. A small elevati on in acrole in concentration

witched the mechan ism of ce ll death from apoptosis to necros is. Acrole in induced

apoptos is through the mitochondrial pathway, in vo lvi ng cytochrome c release from

mitochondria and activation of caspases. However, thi s does not rul e out the

poss ibili ty that acrolein could induce apoptos is by alternat ive or complementary

mechani sms. Apoptos is cou ld also occur via death receptor pathways without the

in vo lvement of cytochrome c. Furthermore, a cros -ta lk pathway ex ists between the

death receptor and mitochondri al pathways, whi ch also leads to cytochrome c release.

Future studies will investigate th e role of death receptor and cross-talk pathways in

acrolein-induced apoptos is. The present findin gs may be important in exp laining the

pharmaco logical action and/or taxie side effects of cyc lophosphamide, which has

acrole in as one of its metabo lites, as we il as the tox icity of environmental exposures

to law doses ofacrolein in proli ferat ing cells.

,---------------------------- -- -------------------,

79

F igure 1: Induction of cytotoxici ty by acrolein

100

-~ 0 10 -ns > 1 ••• > llo..

:::l (/)

- 0,1 Cl)

(.)

0,01

0,001

0 50 100 150 200 250 300 350 Acrolein ( fmol/cell)

-------------------------------------------------------------------------------------------

50

40

!30 '5 20

~ 0

10

0

Figure 2: Morphological analysis of apoptosis and necrosis in cells

following exposure to acrolein

E 100 F

~ ~ h h

Ul 75

).. '5 ~ 0

25

0 0 30 50 100 0 30 50 100

Acrolein ( f mol/cell)

80

F igu re 3 : Acrolein ind uces d epola r ization of t he m itochondria l m em bra ne

a nd libera tion of cytochrom e-c

A

0 Co ntro l , No Aero lem

0 Ac rolem, JUjmoVcell

Rhodamine fluorescence

B

120 (1)=-(J 0 c:.P 100 <Il !: (J 0 ** Ill (J

80 *** *** <Il-0 0 .2~ 60 --<Il >. >:t:::

:;:; Ill 40 Cil !: - <Il <Il...., 0:: !: 20

0 0 10 15 30 FCCP

Acrolein (fmol/cell )

81

t

0

)>

(") ~

4:>.

0 (t)

:::::s

.-..

,--'

-3

0 0 :::

: ("

)

(t)

N

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0

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och

rom

e-c

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atio

n

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n ~ 0 - CD ::::1 -......., 3 0 ::::

n CD - -

0 U1 0

0

~

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f Ap

op

tosi

s

....l

. ..

..l.

N

U

1 0

U1

0

D

D

+

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(1

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'<

(1

(1

0 0

)>

)>

>------+--~ *

N

w

U1

0

(")

00

N

Figure 4 : Activation of initia tor caspase-9 and cleavage of procaspase-9

by acrolein

A 3

~ :

"" :::,., .....

0,5 :::: : :: ':::;J

. . . ~

0 1 4 10

Acrolein

1 m >r

: :

20 30 (fmol/cell)

..

+

~ ~

50 100 150

83

B 0 2 4 10 20 50 Acrolein

caspase-9 r------------------, (fmol/cell) p50 -

p35 -

c 1 20

1 0 0 = 3

~ i.ili W~l 1 ~HH

"iii 80 s::::

11111

;::·: ·= ==

Q)

.i:·. l1lll .... lllll

s:::: 60 inn ==n: :::: :

Q)

lltlt

::;:: lll ~j > ~nn

';ti ;::·:

:llm "' 40 !:!.!

~ il::: :::::

nin '.'j'

:nl~l ~iH~ ·:::·:

20 ~= i:! :;:=·=

=nn n====

11111 1nn

0 n1111

0 2 4

lili:

1@ :::::

~1 ~1 i

ii Ill !Hl! !:1:!

1 0

***

~~ Il il ;::

Ill !W!

!!Il! ;::::·

i!ljli ==n:: li:=: >== :;:;:· ;:·· ::=:=·

!!!!!!

:;:::

j}

D 3.5

3.0

~ 2 . 5 "iii c ! 2.0

s::::

Q) > 1.5 ';ti

"' ~ 1.0

0.5

1n:i O+J~~~~~~--~~~~

20 50 0 2 4 10 20 50

Acrolein (fmol/cell)

84

Figure 5: Acrolein cleaves procaspase-3 but inhibits enzymatic activity of

caspase-3

A

>. .... > .... (..) n:s

M 1

Q) tn n:s a. tn n:s u

1.2

1.0

0.8

0.6

0.4

0.2

0 0 5 10

Acrolein ( fmol/cell)

0 5 min 0 15 mi S 3Q mi ISI 6Q mi

20

85

8

caspase-3 0 2 4 50 Acrolein

.---------------------, (fmol/cell) 10 20

(3-tubulin -~.___~ __ ~.--. __________ _

c 10

~ ·-80 Ill c ! .560

~ '.Cl I'CI40

~ 20

0 0 2

D

4 10 20 50

20

cv >10

'.Cl l'Cl

~ 5

0 ~~ 0

~ ··· ~ ··· ~ ***

2 4 10

Acrolein ( f mol/cell)

.. ~

20 50

86

-----------------------------------------------------,

A

Figure 6: Acrolein activates caspase-7 and cleaves procaspase-7

>­..... > :;;

2.5

2

(.) 1.5 n:s t-

l Q)

Il> 1 n:s Q. Il> n:s u 0.5

0

-

0

"**

+ ~ h

-l<"kk * -+-

~ ~~ .• - r+

-r--~

10 50 lOO 150

Acrolein ( f mol/cell)

87

88

8 2 4 10 20 50 Acrolein

caspase-7 (fmol/cell)

p35-~===========~ p2o - LI ----------------------~

~-tubulin - LI __ .....:=.....:=.....:= ____________ _!___~

c D 120 40

100 ~ 30 11:ip201

~ c-: -

~

,_

~ ~25 ~ -~ ... 'iii 80

- 'iii 1:

~ 1 1 1: ;; a.J a.J 'E: 20 ..... 1: 60 - - --

a.J a.J -

œ· 1 1 > - --~ > 15 -- -- :tl :;; ra 1 1 ra 40

~ 10 Qj c::: -

1 1 1 - -20 -

- 5 U.l . ~ . - J 0 -- - - - 0 '10.

n 2 4 10 20 50 0 2 4 10 20 50

Acrolein (fmol/cell)

89

Figure 7: Inh ibition of apoptosis induced by acro lein by a specifie inhibitor of

caspase-9

25

Il) A Il) 20

25

Il) 8 0 ..... c.. 0 15 c..

Il) 20 0 ..... c.. 15 0

<( 10 -0

c.. <( 10 -~ 5 0

0

::::!"! 5

0

0 0 0 20 50 0 50

Acrolein (fmol/cell) 15

c Il)

Il)

0 10 ..... c.. 0 c.. <( 5

~ 0

0 DMSO 0 25 50 ale ne H20 2 (f..I.M)

90

Figure 8: Acrolein causes cleavage ofiCAD

120

100 Q) C)

** ca 80 > ca Q)

u 60 Cl <( 40 (.)

*** 20

0 0 25 50 100 200 Acrolein

45 kDa - 1 1( fmol/cell)

9 1

FIGURE LEGENDS

Fig. 1. Induction of cytotoxicity by anolein. Cytotox icity was assessed by a

c lonogenic ce ll surviva l assay . CHO cell s ( 1 05/mL) were exposed to acrolein (50 to

350.fi11 0I/cell ) fo r 1 h at 37°C in 1 mL ofa.-MEM containing 10% FBS. Acrole in was

then removed and cell s were incubated in culture dishes fo r 8 days to all ow the

format ion of macroscop ic colonies (see Methods). The control va lue represents 105

ce ll s and thi s was normalized to represent 100% ce l! surviva l. There is a signifi cant

difference between cells treated with acrolein , relat ive to untreated contra is, p <

0.00 1 (***). Data represent means and SEM from four independent experiments

performed with multiple estimations per point. When not hown, error bars li e within

the symbols.

Fig. 2. Morphologica l analysis of apoptosis and nenosis 111 cells following

exposure to acrolein. Cells (0 .3x 1 06) were seeded and cul tu red for two da ys to near

confluence in ti ssue culture di shes containing a.-M EM and 10% FBS at 37°C. Ce li s

were incubated with different concentrations of acrolein : A) 0, B) 30, C) 50 and D)

100 fmol /cell , for 4 h. Cells were stained with 1-loechst and Pl and vi suali sed by

flu orescence mi croscopy (magnifi cation 320x). The fracti ons of (E) apoptot ic and (F)

necrotic cell s fo ll owing treatment with acrolein for 4 h and 24 h are given relat ive to

total ce ll s. A minimum of 600 ce l!s were coun ted per di sh. Data represent means and

EM from four or five independent experiments perfo rmed with multiple est imat ions

per point. p < 0.05 (*), p < 0.0 l (**) or p < 0.00 1 (***) indicates a stat istical ly

signifi cant di ffe rence between treatm ent with acrole in and the control.

92

Fig. 3. Acrolein induces depolarization of the mitochondt·ial membrane and

liberation of cytochrome-c. (A) CHO cell s ( 1 06/mL) were incubated with acrolein

(30 j i11o l/cell ) (grey) fo r 1 h at 37°C in a-M EM and 10% FBS, relative to untreated

control ce ll s (white), and then analysed by llow cytometry fo r rhodamine 123

(800 ng/mL) lluorescence in channel FLI. (8) Data represent means and SEM of

rel ative lluorescence intensity of rh odamine 123 from even independent

experiments. p < 0.01 (**) or p < 0.001 (* **) indicate a stati stically significant

di ffe rence between treatment with acrolein or FCCP and the control. The abso lute

data va lue for the untreated control ce ll s from 7 experiments was 39 ± 3 (mean ±

SEM) re lat ive fluorescence units. The control va lue was des ignated as 100% and

other data values in FCCP- and acro lein-treated cell s were normali sed to this poin t.

(C) Ce ll s were incubated with acrolein, with or without 5 IJ.M cyc losporine A, for 4 h,

and then stained with Hoechst and Pl. A minimum of 600 ce ll s were counted per

di sh. Data represent means and SEM from four independ ent experiments performed

with multiple estimations per po int. p < 0.05 (*) indicates a stat istically significant

difference between treatment with acrolei n with or without cyc losporine A. (D) Ce ll s

( 106 /mL) were incubated with acro lein (4 to 30 j i11 ol/ce ll ) fo r 1 h in a-MEM

containing 10% FBS. lmmunodetection of cytochrome-c wa carried out by SOS­

PAGE, using 13- tubulin as a load ing control (not shown). A representative ge l is

shown from four independent experiments. Ex pres ion of cytoch rome-c was relat ive

to the untreated contro l, des ignated as 1. Data repre ent mea ns and SEM fo r ge ls

from fo ur independent experiments. p < 0.00 1 (***) in dicates a stat istica lly signifi cant

difference between acrolein treatment and control.

Fig. 4. Activation of initia tor caspase-9 and cleavage of procaspase-9 by acrolein.

(A) CHO cell s (O .Sx 106 /mL) were incubated with acrolein ( 1 to 150 jinol/ce ll ) for 1

or 2 h in a -M EM with 10% FBS. Caspase-9 acti vity was measured in ce l! lysates

using the flu orescent substrate Ac-LEHD-AFC. Ca pa e-9 act ivity was expressed

93

relative to the untreated control, des ignated as 1. Data repre ent means and SEM from

eight independent experiments performed with multiple estimations per point. p <

0.05 (*) or p < 0.01 (**) indicates a stati stically significant difference between

acrolein treatment and the control. (B) Ce lls (1 06 /mL) were incubated with acrolei n

(2 to 50 .fillol /ce ll ) for 1 h in a-MEM containing 10% FBS. lmmunodetection of

procaspase-9 and its cleavage fragment (35 kDa) was carri ed out by SOS-PAGE,

using !3- tubulin as a loading control. A repre entat ive ge l i shown from four

independent exper iments. Densitometric ana lys i of express ion of (C) procaspase-9

and (D) the cleavage frag ment are relative to the untreated control. p < 0.001 (* **)

indicates a statistically significant difference between treatment with acrole in and the

control.

Fig. 5. Acrolein cleaves procaspase-3 but in hi bits enzymatic activity of caspase-3.

(A) CHO cell s (0 .5x 106 /mL) were incubated with acro lein (5 to 20 .fillollce ll ) for 5,

15, 30 and 60 min in a-MEM containing 10% FBS . Caspase-3 activity was measured

in ce ll lysates using the fluorescent substrate Ac-DEVD-AMC . Caspase-3 act ivity

was expressed relative to the untreated control, des ignated as 1. Data represent means

and SEM from seven independent experi ment performed with multiple est imations

per point. p < 0.01 (**) or p < 0.001 (***) indicates a stati stically significant

difference between acrole in treatment and the control. (8) Ce lls ( 106 /mL) were

incubated with acro lein (2 to 50 .fi11ol/ce ll ) for 1 h in a-MEM contai ning 10% FBS.

lmmunodetection of procaspase-3 and its cleavage fragme nts ( Il and 17 kDa) was

carried out by SOS-PAGE, using !3---tubulin as a loadin g control. A rep resentative ge l

is hown from four independent experim ents. Densitometri c ana lys is of express ion of

(C) procaspase-3 and (0) the cleavage fragments are re lative to the untreated control.

p < 0.05 (*) , p < 0.005 (**) or p < 0.001 (***) indicates a tati stically significant

difference between treatment with acro le in and the control.

94

Fig. 6. Acrolein activates caspase-7 and cleaves procaspase-7. (A) Cl-IO ce ll s

(O.Sx 106 /mL) were incubated with acrolein ( 1 0 to ISO .fil1ol/ce ll ) fo r 1 and 2 h in a­

MEM conta ining 10% FBS. Caspase-7 act ivi ty was measured in ce lll ysates usin g the

flu orescent substrate MCA-YDQYDGWK-(DNP)-N l-1 2. Caspase-7 acti vity was

ex pressed relat ive to the untreated control, designated a 1. Data represent means and

SEM from seven independent experiments performed with multiple estimations per

po in t. p < 0.05 (*) or p < 0.00 1 (***) indicates a stati stica ll y significant di ffe rence

between acro lein treatment and the control. (B) Ce ll s ( 106 /mL) were incubated with

acro lein (2 to SO.fino l/cell) for 1 h in a-MEM containing 10% FBS. lmmunodetection

of procaspase-7 and its cleavage fragment (20 kDa) was carried out by SOS-PAGE,

using (3-tubulin as a loading contro l. A rep resentat ive ge l is shown fro m fo ur

independent experiments. Densitometri c analys is of express ion of (C) procaspase-7

and (D) the cleavage fragment are relati ve to the untreated control. p < 0.05 (*), p <

0.005 (**) or p < 0.001 (***) indicates a stati st ica ll y signi ficant differe nce between

treatment with acrolein and the contro l.

Fig. 7. Inhibition of apoptosis induced by acrolein by a specifie inhibitor of

caspase-9. Treatment with (A) l 0 ~LM of caspase-9 inhibi tor 1, Z-LEHD-FMK, 10

~M of general caspase inhibitor 1, Z-YAD-FMK, (B) 10 or 20 ~LM of caspase-3

in hi bitor Y, Z-DQMD-FMK and (C) 10 ~LM of caspase-3 inhibitor Y, Z-DQMD­

FMK (Ca lbiochem, La Jolla, CA) was performed on confluent cells in monolayer.

Ce ll s were in cubated with (A, B) acrolein (20 and 50/illol/ce ll ) or (C) 1-1 20 2 (25 and

50 ~M) fo r 4 h with inhibi tors present. The fract ion of apoptoti c ce lls (Hoechst) is

given relat ive to tota l ce lls. A minimum of 600 ce lls was counted per dish. Data

represent means and SEM from five independent experiments perfo rmed with

multiple estimations per point. (A, C) p < 0.00 1 (***) indicates a statistica lly

signifi cant difference between cell s exposed to acrolein with and without caspase

inh ibitors.

95

Fig. 8. Acrolein causes cleavage of ICAD. HO ce ll s ( 106 /mL) were incubated

with acrole in (25 to 200 .finol/ce ll ) fo r 1 h in a-M EM containing 10% FBS .

lmmunodetection of ICAD was carried out using SOS-PAGE. Ex press ion of ICA D

wa relati ve to the untreated control, des ignated as 1 00%. Data represent means and

EM from four independent experiments perfo nned with multiple estimati ons per

point. p < 0.01 (**) or p < 0.00 l (***) indicates a statistica lly significant di fference

between acrole in treatment and the contro l.

96

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2.3. ARTICLE II

ACTIVATION OF THE DEATH RECEPTOR PATHWAY

OF APOPTOSIS BY THE ALDEHYDE ACROLEIN

André Tanel and Diana A. Averill-Bates 1'2

Département des sciences biologiques, TOXEN, Université du Québec à

Montréal, CP 8888, Succursale Centre Ville, Montréal, Québec, H3C

3P8, Canada.

1To whom correspondence should be addressed:

Tel: (514) 987-3000 (ext 4811)

Fax: (514) 987-4647

Email: averill.diana@uqam.ca 2Formerly Dr Diana A. Bates

Running title: Acrolein, death receptor and apoptosis

Acknowledgments: Financial support was obtained from the Canadian Institutes for

Health Research (DAB). André Tanel gratefully acknowledges financial support from

the Bourse Francine Beaudoin-Denizeau, Fondation UQAM. The authors thank

Bertrand Fournier (SCAD UQAM) for assistance with statistical analyses .

Dedication: This paper is dedicated to the memory of Professor Francine Beaudoin­

Denizeau, and to her endless energy and enthusiasm in the pursuit of scientific

excellence in biochemistry and environmental toxico logy (Ma.rch 25 , 2004).

104

Abbrevi ati ons: AFC: amino trifluoroco um arin ; AMC: amino methylco umarin ; BSA:

bov ine serum album in ; CHAPS: 3-[(3 -cholam ido propyl)d i methylammon io ]-2-

hydroxy-1-propanesulfonic acid; CHO: Chinese hamster ovary; DI SC: death­

inducin g signalling complex; FADD: Fas assoc iatin g prote in with death domain ; Fas:

ft brob last-assoc iated; FasL: Fas li gand ; FasR: Fas receptor, FBS: fetal bov ine serum ;

4-HNE: 4-hydroxynonenal; JCAD: inhibitor of caspase act ivated DNase; MEM:

minimum essentialmedium; MOPS : 3-(N -morph olino)-propane sul fo nic ac id ; PARP:

polyA DP-ribose polymerase; PB S: phosphate-buffe red sa line; PM SF:

phenylmethylsufonyl fluoride ; PS : pho phatidylserine; PVD F: polyviny lidene

difluoride; SOS-PAGE: sodium dodecy l sulphate-polyacrylamide ge l electrophores is;

SEM: tandard error of mean. TNF-R: tumor necros is fac tor receptor.

Kevwords: Acrolein, death receptor, apoptos is, caspase

105

RÉSUMÉ

Les aldéhydes hyperacti fs comme l ' acroléine sont des composés po lluants

maj eurs de l 'env ironnement et y constituent un grand ri sque. L ' acroléine est un

produit de perox idation lipidique impliquée dans plusieurs pathophys iolog ies incluant

les maladies neurodégénératives et respiratoire . Bien que 1 ' acroléine induit

l ' apoptose dans plusieurs types cellulaires, les mécanismes biochimiques impliqués

demeurent inconnus. Cette étude explore la vo ie des récepteurs de mort dans

1' inducti on de 1 'apoptose par 1 'acro léine. L ·exposit ion des cellules ovariennes de

hamster chinois à l ' acroléine a entraîné la translocat ion de l ' adapteur de domaine de

mort de Fas (FA DO) à la membrane pl asmique et l ' acti vation de la caspase-8

initiatrice. Kp7-6, un antagoniste du récepteur Fas, a bloqué les évènements

apoptotiques en aval de la caspase-8 , comme l ' acti vat ion de la caspase-7 exécutri ce et

la condensati on de la chromatine nucléa ire. Acroléine acti ve la vo ie de crosstalk entre

la vo ie de signali sation des récepteurs de mort et ce lle de la mitochondri e en clivant la

protéine Bid en sa forme tronquée t-bid qui ' est transloqué à la membrane

mitochondriale pour stimuler cette vo ie. L ' inhibition spéc ifique du récepteur Fas ou

de la caspase-8 a inhibé partiellement l 'acti vat ion de la caspase-9 par l' acroléine. Ces

résultats démontrent que l ' acroléine act ive la vo ie du récepteur Fas en amont de la

vo ie mitochondriale. L a caspase-9 est demeu rée act ive malgré l ' inhibition du

récepteur Fas et de la caspase-8 suggérant que !" acro léine peut induire la voie

mitochondriale indépendamment de la vo ie des récepteurs. Ces résultats nous

emmènent à mieux comprendre le mécanisme de tox icité de l ' acroléine, un po lluant

omniprésent de notre environnement.

106

ABSTRACT

Reactive a lpha,beta-unsaturated a ldehydes such as ac ro le in are majo r

components of common environmental pollutants. As a toxic by-product of lipid

peroxidation, acro le in has been implicated as a poss ible med iator of ox id ative

damage to ce lls and tissues in a wide va riety of di sease states, including

atherosc leros is, neurodegenerative and pulmonary di seases. A ltho ugh acro le in can

induce apoptotic cell death in various ce ll types, the biochemical mechanisms are not

understood. T hi s study investigates the implicat ion of the death recepto r pathway in

acro le in-induced apoptosis . Exposure of C hinese hamster ovary ce ll s to ac ro le in

caused translocation of adaptor protein Fas-assoc iated w ith death doma in to the

cytop lasmic membrane and caspase-8 act ivat ion. Kp7-6, an antagoni st of Fas receptor

activation, blocked apoptotic events down stream of ca pase-8, such as caspase-7

act ivat ion and nuc lear chromat in conde nsat ion. Acrolein act ivated the crossta lk

path way between the death receptor and mitochondri a l pathways . Bid was c leaved to

truncated-bid , w hich was trans located to mitochondri a. Act ivation of the

mitochondri a l pathway by acro le in was confïrm ed by caspase-9 act ivat ion. Inhibiti on

of act ivat ion of e ither the Fas receptor or caspase-8 partially decreased ac ro le in­

induced caspase-9 activation. T hese fïndings indicate that ac ro lei n activates the Fas

receptor pathway, which occurs upstream from the mitochondrial pathway. Caspase-9

act ivation st iJl occurred despite inhibition of the Fas receptor pathway, suggest ing

th at ac ro lein co u id trigger the m itochondria l pathway independently of the receptor

pathway. T hese fïndings improve our understa nding of mechani sms of toxicity of the

react ive a ldehyde ac ro le in, which has w idespread implication s in multiple disease

states which appear to be med iated by ox idative stress and lipid peroxidation.

Keywords: Acro le in, dea th receptor, apoptos is, ca pase, m itochondri a

107

INTRODUCTION

Acrolein is a highly reactive, a ,p-unsaturated a ldehyde and humans are

exposed to thi s compound in multipl e contex ts [ 1]. lt is a ubiqui to us environ mental

pollu tant whi ch is present in food. Acrolei n is fo und in the va pours of overheated

cooking oi 1 and severe hu man toxic ex posures have been reported [2]. ft is used

industrially as a starting materi a l for acrylate polymers and in the production of

acrylic ac id , and as a herbicide [3]. Acrole in is also a metabo li c productofthe widely

used anticancer drug cyclophosphamide and has been implicated in its tox ic side

effects [ 4].

As we il as be ing a ubiquitous environm ental pollutant, acrolein is one of the

tox ic aldehyde by-products of endogenous 1 ipid perox id ati on, together with 4-

hydroxy-2-nonenal [5; 6]. Lipid peroxidati on is a de leteri ous chain reaction occurring

mainly in bio logica l membranes, resulting from oxidati ve stress. Acrole in a lso reacts

with glutathione [7; 8]. ln fact, acrole in causes more rapid and severe depletion of

thi s important cellular antioxidant when compared to the we ll-known ox idant H20 2

[9]. Furtherm ore, acrolein and its glutathione adduct, glutathi onylpropionaldehyde,

were shown to cause fo rmati on ofoxygen radica ls [1 0], which may be responsible fo r

inducti on of lipid perox idati on by acro lei n. Together, these studi es show that acrole in

causes an oxidati ve redox imbalance.

Ox idati ve stress and lipid perox id ati on have been implicated in a va ri ety of

human disease states. Since acro lein is an exogenous product of lipid peroxidati on

and can itself induce lipid peroxidation, thi s reacti ve a ldehyde may have a poss ible

role as a mediator of oxidative damage to cells and tissues. lndeed, acrolein has been

implicated in the development of multipl e disease states in vo lving oxidati ve stress,

such as atheroscleros is [6; Il] , vari ous lung diseases including chroni c obstructive

pulmonary di sease (COPD) [ 12], co lon carc inogenes is [ 13], diabetic nephropathy

[1 4], head trauma during ag ing [15] and Alzheimer's di sease [1 6]. Acro lein has been

108

used as a biomarker to evaluate ox idative stress-induced damage to retina [ 17] and to

erythrocytes [18] , as weil as in Alzheimer ' s disease [1 9; 20].

Acrole in is a component of smoke and is generated during fo rest and house

fi re and is a constituent of automobile exhaust. lt is a major component of cigarette

smoke and appears to contribute to its toxicity (2 1; 3]. Acrole in may contribute to

c igarette smoke-induced inflammatory processes in the lung by increas ing neutrophil

recruitment and reducing neutrophil c learance by apoptos is (2 1]. lt in creases th e

ex pression of mucin gene transcripts (MUC5AC and MUC5B) , whi ch leads to

excess ive mucus secreti on [22; 23], a hall mark in the pathogenes is of severa! a irway

di seases including COPD [24], asthma and cystic fibros is [25].

Apoptos is is a normal phys iolog ical process that plays an essential role in

development and the maintenance of homeostas is in multi ce ll u lar organ isms (26; 27].

A ba lance between cell death and cell proli fe rat ion is req uired to maintain a ce llular

homeostati c state . Dev iati on from thi s ce llular ba lance di srupts the normal state and

can lead to human disease [28 ; 29]. Apoptosis has many important repercussions in

human health. Ce ll accumulation via insuffïc ient apoptos is can contribute to

conditi ons such as cancer, inflammation and autoimmune di sease. ln contrast,

exce sive apoptos is can play an important ro le in neurodegenerati on, AlOS, eye

di sorders, osteoporosis and heart fai lure [30; 3 1]. Apoptot ic cell death has been

implicated in the mechani sms of toxicity of different chemica ls and environmental

pollutants [32], such as the aquatic toxin tributyltin (33 ; 34] and the orga nochlorine

pesticide heptachlor [35], wh ich have important implications in human health as weil

as that of other species.

Two of the major pathways of apoptos is are the death receptor and

mitochondri al pathways. There are severa! death receptors located at the cytop lasmic

membrane of cells, which are involved in the in duction of apo pto is, such as Fas

(CD95, AP0-1 ) and tumor necros is fac tor recepto r (TNF- R) (36]. The Fas receptor

mediates apoptoti c signal ling after binding of its natural li gand , Fas (FasL) [37]. FasL

109

binding induces Fas receptor trimerization. T his is followed by the recruitment of a

cytop lasmi c adapto r protein, Fas-associated dea th doma in (F ADD) [38] and pro­

caspase 8, to the cytopl asmi c s ide of the membra ne, to fo rm a death-inducing

s igna lling complex (D ISC). The initi ator caspase-8 th en becomes auto-act ivated [39],

thereby initiating the cystei ne protease cascade by direct act ivat ion of effector

caspases such as caspases-7 and -3. Caspase-8 ca n a l o act ivate the mitochondrial

pathway of apoptos is through c leavage of the proapoptot ic prote in bid to gene rate its

truncated fo rm, t-bid . T-bid then trans locates to the mitochondria l membrane and

inte racts w ith the proapoptotic protein Bax to induce mitochond ri a l membrane

permeabi li zation and activation of caspase-9 and the mitochondrial pathway [40].

Downstream events in the apoptotic cascade inc lu de the cas pa e-med iated c leavage

of protein substrates such as polyADP-ribose po lymera e (PARP) and inhibito r of

caspase act ivated DNase (!CAO). T he ce l! th en exhibits the characteristic

morpholog ical featu res of apoptos is such as chromatin conde nsation, cytoske leta l

c hanges, nuclear me mbrane breakage, ce l! blebbing and fo rm at io n of apoptotic bodies

[3 1; 4 1]. A poptotic bodies are then ph agocytozed by macrop hages or ne ighboring

ce ll s, thus avo iding inflammato ry damage to adjacent tissues.

G iven the w idespread exposure of humans and other spec ies to ac ro le in , as

weil as its implication in multiple cli sease states in vo lv in g x idative stress, it is

important to understand its mechani sms of tox icity. Acro le in ha been shown to

induce apoptos is in severa ! different ce l! types [42 ; 43; 44], however, the molecul ar

mechani sms and biochemical pathways invo lved are not we il understood. Recently,

acro le in was shown to act ivate the mitochondria l pathway of apoptos is in C hinese

hamster ovary (CHO) ce ll s [45] . With the a im of advanc ing our knowledge of thi s

ubiquitous toxic compound, the present study in vesti gates whethe r ac ro lein can ca use

apoptos is through act ivat ion ofthe death receptor s igna llin g pathway .

---------

110

MATERIALS AND METHODS

Cel/ culture

CHO ce lls (A uxBI) [46] were grown 1n monolayer in minimum essential

medium-Alpha (a.-MEM) plus 10% feta l bov ine serum (F BS) (G ibco Canada) and

1% penicillin (50 units/ml)-streptomyc in (50 ~g/ml ) (F low Laboratories,

Miss issauga, ON, Canada), in ti ssue culture fl asks (Sarstedt, St La urent, QC,

Canada), in a humidified atmosphere of 5% C02 in a water jacketed incubator at

37°C [47]. The ce lls were grown to near confluence and then incubated for 24 h with

fresh culture medium . Confluent cells were then harvested using citrated phosphate­

buffered saline (0. 14 M NaCI, 0.01 M sodium phosphate, 0.015 M sodium citrate),

washed by centrifugation ( 1 OOOg, 3 min) and resuspended in a.-MEM plus 10% FBS

for experimental studies.

Determination of apoptotic cell dea th by Annexin V-FITC staining

Externalized phosphatidylserine (PS) on the outer surface of the cytoplasmic

membrane becomes labelled by flu oresce in-l abell ed Annex in Y (BD Biosc iences

Canada, Miss issauga, ON, Canada), whi ch has a hi gh affi nity for PS-containing

phospholipid bilayers [48]. CHO ce ll ( 1 x 106/ml) were incubated for 2 h with

acrolein or hyd rogen peroxide, and then \.vashed twice with PBS and resuspended in

1 ml of bind ing buffer ( lü mM Hepes/NaO H, pH 7.5, 140 mM NaC I, and 2.5 mM

CaCb). Five hundred J..il of cell suspension were then incubated with 5 ~t l of Annex in

Y -FITC and 10 ~tl of propidium iodide (Pl ) for 10 min at room temperature in the

dark. The population of annexin V-posit ive cell s was eva luated by flow cytometry.

Data were co ll ected using a FACS scan equipped with an argo n laser emitting at

488 nm and analyzed using Lysis Il software (Becton Dickinson, Oxford , UK).

Morphologica/ analysis of apoptosis byfluorescence microscopy

Ill

Ce ll s were incubated with acro lein for 4 h in tissue cu lture dishes containing

5ml of a-MEM and 10% FBS. Dishes were washed twice with PBS and Hoechst

(33258) (0.06 mg/ml) was added for 15 min at 37°C to sta in apoptotic ce lls. The

di she were washed with PBS and Pl (50 ~Lg/ml ) was added to stain necrotic cells.

Observations were made by fluorescence microscopy (Ca rl Zeiss Ltd , Montreal , QC,

Canada) and photographs were taken by di gital camera (camera 3CCD, Sony DXC-

950 P, Empix lmag ing lnc, Mississauga, ON , Canada) . Images were analysed by

orthern Eclipse software. Cells were classified using the fo llowing criteri a: a) live

cell s (normal nuclei, pale blue chromat in with orga ni zed structure); b) mem brane­

intact apoptotic cell s (bright blue condensed or fragmented chromat in); c) necrotic

ce ll s (red, enl arged nuclei with smooth normal structure [49]. The fractions of

apoptot ic and necrotic cells were detennined relat ive to tota l ce ll s (obta ined us1ng

bright field illuminat ion). A minimum of 600 ce l! vvas counted per di sh.

Treatment ofcells with inhibitors ofca!>.ïJase-8 and Fas receptor

Confluent ce lls in monolayer were pretreated with the fo ll owing irreversible

inhibitors: Caspase-8 Inhibitor Il, Z-I ETD-FMK and Fas/FasL antagoni st Kp7-6

(Ca lbiochem, La Jo li a, CA, USA). The ce ll s were ex posed to a 10 ~LM concentration

of the inhibitor Z-IETD-FMK or to 1 mM of Kp7-6 and then to var ious

concentrations of acro lein fo r 1 or 4 h.

Determination of caspase activity by fluorescence .spectroscopy

Freshly harvested CHO cells (0.5 x 1 06) were incubated with acrolein in 1.0

ml of a-MEM plus 10% FBS at 37°C. A fter the appropri a te ti me, ce li s were washed

three times with co ld PBS by centri fugat ion ( 1 OOOg, 3 min), resuspended in 50 ~LI of

PBS and 25 ~LI and were deposited into 96-we ll plates and lysed by freezing at -20°C

fo r 20 min. Fifty ~LI of reaction buffe r (20 mM piperaz ine-N,N'-b is(2-ethanesul fon ic

ac id) (PIPES), 100 mM NaC I, 10 mM dithi othreito l, 1 mM EDTA, 0.1% 3-[(3 -

11 2

cholamidopropy l)dimethylammonio] -2-hydroxy-1 -p ropanes ulfo ni c acid (C l-l APS) ,

10% sucrose, pH 7.2) was added and stabilized at 37°C [50]. The kinetic reaction was

started after addition of 25 ~tl of the appropri ate caspase substrate (Ca lbiochem) at

37°C using a spectrotluorimeter (Spectra Max Gem ini , Molecular Deviees,

unnyva le, CA, USA) [45].

Caspase-8 act ivity was measured by cleavage of the l·luorogenic substrate Z­

IETD- amino-4-tritluoromethylcoumarin (caspase-8 substrate 11 ) to produce 7-amino-

4-tritluoromethylcoumarin (AFC) with Àmax exc itat ion at 4 15 11111 and Àmax

emiss ion at 490 nm . Caspase-9 activity wa measured by cleavage of the substrate

Ac-LEHD-AFCto produce AFC. Caspase-7 act ivity wa measured by cleavage ofthe

tluorogenic substrate 1 MCA- VDQVDGWK(DNP)-N I-h with Àmax excitation at 325

nm and Àmax emiss ion at 395 nm.

Subcel/ular fractionation and ùnmunodetection r~l Fas receptor (FasR), Fas ligand

(FasL), FADD, caspase-8, bic! and PARP

Fo ll owing treatment with acrolein, ce ll s were washed in buffer A (100 mM

sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4) and res uspended in buffer B [buffer A

plus 5% Percoll , 0.01% digitonin and a cocktail of protease inhibitors: 10 ~LM

aprotinin, 10 ~LM pepstatin A, 10 ~LM leupept in , 25 ~LM ca l pain inhibitor 1 and 1 mM

phenylmethylsulfonyl tluoride (PMSF)] [51]. After 30 min incubat ion on ice, lysates

were homogenised using a hand potter (Kontes g lass CO, Duall 22, Fisher, QC,

Canada). Unbroken ce ll s and nuclei were pe ll eted by centifugati on at 2500 g fo r 10

min . The supernatant was centrifuged at 15 000 g fo r 20 min and the resulting pellet

was designated as the mitochondrial fracti on, whi ch was used for the detection of t­

bid [34]. A flll1her centrifugation of the supernatant fract ion at 100 000 g for 1 h

resulted in a pellet designated as the microsomal fract ion for the detection of FasR

and FADO, whereas the supernatant was des ignated a the cyto olic fraction , wh ich

11 3

was used fo r the detection of bid . Whole ce ll lysates were used fo r immunodetection

of FasL, caspase-8 and PARP.

Separati on of cellular prote ins (30 ~Lg) [52] was ca rri ed out by SDS­

polyacrylamide gel electrophores is (S OS-PAG E) [53], u inga 10% (PARP) or a 15%

(FA DO, cytochrome-c oxidase, GST-nl , caspase-8 , Fas R, FasL, ca lnex in , bid)

acrylamide ge l [45]. Ce llular proteins were tran fe rred electrophoretically to a

po lyv inylidene difluoride (PVDF) membrane using a MilliB iot Graphite

Electroblotter 1 apparatus (M illipore, Bedfo rd , MA) [45]. Membranes were probed

with the primary antibodi es: anti- FasR, anti-FADD (Stressgen, San Diego, CA,

USA), anti-caspase-8, anti-bid, anti-PARP (Sa nta Cruz Biotec hnology, Santa Cruz,

CA, USA) and anti-FasL (Ca ltag Laboratori es, Burlingame, CA, USA). Secondary

antibodi es consisted of horseradi sh peroxidase (1-1 RP)-conjugated goat anti-mouse,

anti-rabbit and anti-goat lgG ( 1:1000) (Biosource, Camarilla, CA, USA) . Proteins

were detected using the ECL plus chemiluminescence kit (Perkin Eimer, Boston, MA,

USA) and film s (Fuji medical X-ray film , Düsserld orf, Germany) were scann ed with

a Laser Scanning Densitometer (A lpha lnnotech Co rp ., San Leandro, CA, USA).

Purity of mitochondrial , cytoso lic and microsomal fract ions was verified using

antibodies to cytochrome-c oxidase (Sa nta Cruz Biotechnology), GST1r 1

(Ca lbiochem) and ca lnexin (BD Biosciences Canada), respective ly. Caspase-8 , PARP

and FasL express ion in whole ce l! lysates was qua ntified us ing IPGEL softwa re,

re lati ve to f3-tubulin loading contra is.

Statistical analysis

Stati stical differences between control and treated groups were detennined

using a one-way ANOVA whi ch measures the linear contrast of means. An

adju tment was made to limit the familywise error rate (FWE) to 5% by ca lcul at ing

an adjusted p -va lue which is a simulated ba ed p-va lue obta ined fro m the mul tivariate

1 di stributi on (number of simulations = 1 000 000) [54]. Then, the Bonfe rroni-Holm

11 4

meth od (a ste pw ise meth od) was used to contro l th e FWE. For compari son between

a treatment with ac ro lein in presence or absence of an inhibitor, stati stica l differences

were determined by a two-tailed unpa ired Student's t test.

Values are expressed as means ±SEM. Differences were considered

stati sti ca lly s ignificant atp < 0.05.

11 5

RESULTS

Induction of apop tosis by acrolein

The induction of apoptos is by acrolein was eva lu ated by Annex in V labell ing

of externalised PS on ce l! membranes (F ig. 1 ). There was a 2.5 to 3-fo ld increase in

Annex in V positive cell s foll owing exposure to 20 and 30 fm ol/cell of acrolein (F ig.

1 8 , 1 C), compared to untreated controls (F ig. 1 A). Hycl rogen perox ide also increased

Annex in V pos itive ce lls and was usee! as a pos itive control (F ig 1 D, 1 E). lt shoulcl be

noted th at the acro lein concentration was ex pressed as f mol/cell because the ce l!

density was di ffe rent fo r certain tests [55].

Acrolein induces translocation of FADD, cleavage ofprocaspase-8 and activation of

init iator ca. pase-8

The ability of acrole in to activate the death receptor path way of apoptos is was

investigated in CHO cell s. The adaptor protein FA DD, whi ch all ows the Fas receptor

and procaspase-8 to form the membrane DI SC complex, was recruited to th e

cytoplasmic membrane after 30 to 60 min (F ig. 2). The translocati on of FADD to the

membrane (Fig. 2A, 2B) resultee! in a corresponding reduction in levels of FADD in

the cytoso l (F ig. 2A, 2C), as weil as acti vat ion of caspase-8, an initi ator caspase (f ig.

3A). The cleavage of the inactive pro-enzyme fo rm is a major step in generation of

the acti ve fo rms of caspases. Exposure of cell s to acrolein resulted in cleavage of

procaspase-8 as a function of increas ing concentrat ion from 20 to 30 j mol/ce ll after

30 or 60 min (Fi g. 38 , 3C, 3D). Thi s wa apparent as a clecrease in the intensity of the

ba nd (F ig. 3 B, 3C) fo r the pro-enzyme fo rm of caspase-8 (55 kDa) and a

corresponding increase in the quantity of the 20 kDa cleavage fragment (F ig. 38 ,

3D).

Blockade of Fas receptor activation partially inhibits acrolein-induced apoptosis

11 6

The next step was to determine whether acti vat ion of caspase-8 by ac ro le in

wa dependent on Fas receptor s igna lling. First of a il , we co nfirmed that CHO cell s

indeed express the Fas receptor on the ir ce ll membranes (Fig. 4A). Furthermore,

expos ure of ce li s to acro le in at apoptosis-induc ing co ncentrat ions ( 10 to 50 jmol/cell)

did not affect the leve! of Fas receptor express ion (Fig. 4A , 4B) . There was,

however, an increase in Fas li gand (FasL) express ion w hen ce ll s were exposed to

ac ro le in (F ig. 4C, 40) .

The subsequent step was to determin e the ability of an inhibitor of Fa

receptor activation to bl ock acro le in-induced caspase-8 activati on and other apoptot ic

events downstream of caspase-8. Kp7-6 is an irrevers ible inhibitor of Fas receptor

activat ion. lt can bind to the Fas receptor and Fa L, therefore preventing the Fas L

from fonning a stab le complex w ith the Fas receptor [56]. Once caspase-8 is

activated, it directly processes downstream caspases such as caspases-3, -6 and -7 . It

was previously reported that acro lei n can activate caspase-7, but causes inhibition of

caspase-3 in these ce ll s [45 ]. Oownstream eve nts fro m caspase-8 th at were

investigated in the apoptot ic cascade inc luded the effector caspase-7 and chromat in

condensation in the nuc leus. Kp7-6 effect ive ly inhibited activation of initiator

caspase-8 by acrolein (F ig. SA) and partia lly inhibited activation of effector caspase-7

(F ig. 58).

Morphological analys is demonstrates that ac ro le in induces both apoptosis and

necros is in ce ll s (Fig. 6B). Apoptos is was revea led by condensation of chromat in

w ith Hoescht 33258 (b lue fluorescence) whereas necros is was revea led us in g PI (red

fluorescence) . Kp7-6 a lso inhibited acro lei n-induced nuclear ch romatin condensation

by about 60% (F ig. 60, 6E). The Fas receptor inhibitor did not affect contro l ce ll s

(F ig. SA, SB , 6C, 6E, 88).

A croie in activa tes the crosstalk pathway

11 7

Once procaspase-8 is autoacti vated at the DISC to become a mature acti ve

enzy me, two di stinct downstream path ways have been identifi ed for activati on of

apoptos is. ln one route, caspase-8 directl y proce se downstream effector caspases

uch as caspases-3, -6 and -7. Through an altern ati ve route, caspase-8 can acti vate a

crosstalk pathway between the death receptor and mitochondri al pathways, by the

cleavage of bid, a pro-apoptotic member of the 8 cl-2 fa mily. Exposure of ce lls to

acrole in for 30 or 60 min caused cleavage of bid (F ig. 7) to form t-bid, which

tran locates to the mitochondri a. This was apparent as a decrease in leve ls of bid in

the cytoso l with in creasing acrole in concentrat ion (F ig. 7 A, 78) and an increase in the

leve! of the cleavage fragment t-bid in the mitochondri al ti·acti on (F ig. 7A, 7C) . Once

loca li sed to the mitochondrial membrane, t-bid can acti vate the mitochondrial death

pathway. Thi s occurs by the release of cytochrome-c from mitochondri a and

acti vati on of another initiator caspase, caspase-9.

Caspase-8 is a maj or upstream ca~pase in acrolein-induced apoptosis

The subsequent step was to determine the role of ca pase-8 in activati on of the

mitochondri al pathway in acrolein-induced apoptosis. To achieve thi s, the ability of

an irreversible inhibitor of caspase-8, Z-IETD-FMK, was used to assess the effect of

caspase-8 activation on acrolein-induced caspase-9 act ivat ion, a downstream event in

the mitochondrial pathway. Acrolein induced the act ivati on of caspase-9, which was

partially decreased by the caspase-8 inhibitor (F ig. 8A). The acti vati on of caspase-9

by acrolein was also partially dimini shed by inhibition of Fas receptor activation by

Kp7-6, confirming the role of the Fas receptor upstream o t' th e mitochondrial path way

(F ig. 88). The role of initiator caspase-8 was in vesti gated in the acti vation of oth er

downstream apoptoti c events, such as cleavage of the caspase substrate PARP and

chromatin condensation in the nucleus. Expo ure of cell s to acrolein led to cleavage

of the DNA repair enzyme PARP ( 1 16 kDa), to fo rm a 85 kDa fragment, whi ch was

also diminished by the caspase-8 inhibitor (F ig. 9) . Z-I ETD-FMK al so decreased

acrolein-induced chromatin cond ensati on, a late stage event in apoptoti c ce ll death ,

11 8

by abo ut 60% (Fig. 1 OD, 1 OE). The caspase-8 inhibitor did not affect control cells

(Fig . 8A, 9, 1 OC, 1 OE) .

11 9

DISCUSSION

This study shows fo r the first time that acrolein can induce apoptos is 1n

proliferating ce ll s through act ivation of a death receptor pathway. Induct ion of

apoptos is by acrolein was confirmed by annex in V label ling of externalised PS and

morphologica lly by condensation of nuclear chromat in. Exposure to acrolein led to

recruitment of FADD and procaspase-8 to the cytop lasmi c membrane, resulting in

caspase-8 process in g. Once activated , the initi ato r caspase-8 processed the

downstream effector caspase-7. We confirmed that the Fas receptor was invo lved in

acrolein-induced apoptos is, by increased expres ion o t' FasL by acrolein and also with

Kp7-6 , an inhibitor of Fas receptor acti vat ion. Kp7-6 inhibited downstream apoptoti c

events in the death receptor pathway, such as act ivat ion of caspases-8 and - 7, and

chromatin condensati on in the nucleus.

lt was prev iously reported that acrolein can act ivate the mitochondria l

pathway of apoptos is in Cl-IO ce ll s [45]. Th is was apparent as a decrease in

m itochondri al membrane potential , 1 i beration of cytoch rome-c from 111 itochondria

into the cytoso l and the subsequent activat ion of initi ator caspase-9. lt is possible that

acrole in could activate the mitochondrial pathway direct ly, and/or by death receptor

signalling through the crosstalk pathway involving caspase-8 med iated cleavage of

the proapoptotic protein Bid . Our findings support both of these scenarios. The

crosstalk pathway was indeed act ivated by acro le in and the post-mitochondria l

activat ion of caspase-9 was part ia ll y decrea ed by an inhibi to r of caspase-8 . The fact

that there was on ly partia l inhibition of caspase-9 activation suggests that the

mi tochondria l pathway could a lso be st imulated by acrole in independent ly of the

death recepto r pathway. Thi s is a lso supported by the partia l inhibition of acrolein­

induced chromatin condensation by Z-LEHD-FMK, an inhibitor of caspase-9 [45].

The caspase-8 inhibitor, Z-I ETD-FMK, also caused partial inhibition of chromat in

120

condensat io n, suggesting that acro le in can act iva te both the death receptor and

mi tochondrial pathways directly .

Acro le in was shown to medi ate apoptos is via the effector caspase-7, rather

than through caspase-3 [45]. Severa! stud ies have r po rted th at acro le in inhibits

activity of the majo r effector caspase-3 [45 ; 57 ; 58] , probably through inhibition of its

active s ite cyste ine res idue. T he effecto r caspase-7 ca n be act ivated by bo th of the

initi ato r caspases, caspase-8 and caspase-9 [59]. Both of the effector caspases-3 and-

7 share s imil ar prote in substrates and a re able to c leave the caspase substrate PARP

[4 1; 60].

The concentrations ( 10-50 fmo l/ce ll or 10-50 ~LM ) used in this study appear to

be re levant to in vivo levels of acro le in . T he phy io log ica l leve! of acro le in in the

serum of a norma l human was estimated to reach 50 ~LM [6 1]. Hi gher leve ls of

acro le in were reported fo r certa in di sease states. Acro le in is estim ated to reach 80 ~LM

in the respiratory tract lining fluid s as a resul t of c iga rette smoke [43 ]. ln our study,

exposure to l 0-50 ~M acro le in induced apoptot ic ce l! death after re lative ly sho rt

expos ure times of 1 to 4 h. Expos ure to the e concentrati ons, o r even lower

co ncentrat ions of acrole in for much longer time , such a days (as li ke ly occurs in

vivo) , would therefore be expected to cause w idespread ce ll death. Furthermore, it

wou ld be expected that acrolein concentrat ions in vivo wou ld inc rease w ith tim e after

injury, s in ce lipid peroxidation is a cha in react ion process, and in ad diti on, dead cells

wo uld liberate acro le in from the cytoso l into the extrace llul ar env ironment.

T he modes of ce l! death induced by different stresses a nd toxic compounds

are dependent on dose and depe nd on the susceptibility of indi v idua l ce ll types [32).

Acro le in-induced apoptos is is c learl y ce ll type depende nt. ln ne utrophil s, low doses

of ac ro le in were shawn to inhibit caspase-8 act ivity [57 ; 58]. Di ffe rent studi es have

re ported variab le findi ngs in that acro le in ca n e ither cause o r in hibi t apoptos is, o r

ca use predominantly necros is/oncos is rather than apoptos is . Fo r example, acro le in

(25 ~LM) caused apoptos is in iso lated human a lveo lar macrophages, detected by

12 1

morpholog ical changes and DNA fragmentation a1ter 24 h [42]. Acrolein act ivated

the mitochondri al pathway of apoptos i in Cl-I O cell s [45 ]. Acro lein stimulated

apoptos is in the human lung epitheli al ce ll line HBE 1, as indicated by externali sation

of pho phatidylserine and DNA fragmentati on 24 h a lter a 30 min exposure to 10 to

25 ~LM acrolein [43 ]. Exposure to 50 ~tM acrolei n fo r 24 h induced aty pica l apoptos is

in primary cultures of human keratinocytes [44]. ln human neutrophil s, however,

acrole in (25 ~LM) inhibited the consti tutive pathway of apoptos is [2 1]. ln proB

lymphoid cell s and human neutrophil s, the ce l! death pathway was predominantly

oncos is/necros is, with only low levels of apopto is occurring at lower doses ( < 10

~M) [57; 58]. These va ri able resul ts could be cl ue to differences in bi ochemi ca l

fac tors determin ing ce l! dea th pathway in cl iffe renr ce l! types, wh ich incl ude non­

proli fe rat ing primary ce l! cul tures and cell s in vo lved in immune responses, as weil as

proli fe rating cancer ce l! !ines.

ATP appears to play an acti ve role in detennin ing the type of cel! death

occurring under various metaboli c condi tions [62]. Dep leti on of ce llular ATP levels

by an ATP synthase inhibitor, o li gomycin , med iated the switch from apoptos is to

necros is in leukemi c ce lls [63 ]. Cell s treated with known apoptos is inducers,

staurosporin (STP) and anti-CD95, died exc lusively by necros is when pretreated with

o li gomyc in . Apoptoti c characteri stics were not observee! when cell s were depleted of

ATP below a criti cal leve!, but were restored upon glucose addi tion. Severa! studies

have reported the inhibitory effect of acro le in on TP prod ucti on. Exposure to 10 uM

acrolein fo r 24 h in rat pneumocyte Il L2 cell s [64], and fo r 4 h in myocytes [65], led

to decreased ATP levels. lntracellular ATP concentrations were also decreased in

murine FL5. 12 proB lymphocytes, reaching 0% o l: control 24 h afte r a 30 min

exposure to 40 ~LM acrolein [57] and in mi tochondr ia of neuronal PC 12 ce ll s 24 h

afte r exposure to 10 ~M acrolein [66]. The type of ce l! death in proB lymphocytes

and PC 12 ce li s und er these conditions was main ly necros is. To our knowledge, ATP

leve ls have not been determined under conditions where acro lei n induces apoptos is.

122

The antioxidant glutathi one appears to have an important role in the

detox ification of acrolei n [67]. This react ion can be spontaneo us or catalyzed by the

detox ifi cation enzyme glutathione S-transferase ( · T) [68] via the reacti on of

Michael addition [69]. Moreover, acrole in can al o be metabo lized by th e rat liver

a ldo-keto reductase [70] and by aldehyde dehydrogenase [7 1].

Acro le in is generated as a product of the enzymatic act ion of amine ox idases

on natura lly occurring polyamines such as sperrnine and spermidine [7; 72].

Polya mines play an essential role in cellular proliferati on and differentiation [73).

The reason fo r the generation of a hi ghly tox ic product as acrolein from such a basic

and important phys iological process is certainl y in triguing and is not presently

known.

ln conclusion, this is the first detailed study to demonstrate that the

mechani sm of acrolein-induced apoptos is is medi ated by the Fas receptor pathway as

we il as the m itochondrial pathway in pro! iferat ing ce li . The se fi nd ings are relevant

to the tox icity of acrolein in many contexts, in c ludin g the pharmacological act ion

and/or tox ic side effects of the anti cancer agent cyc lophosphamide, the regul at ion of

ce llular proliferation and tumor growth by po lyamines, Alzheimer' s di sease, as weil

as the toxic ity of environmental exposures to low do e of acrole in .

123

ABBREVIA TI ONS

AFC: amino trifluorocoumarin ; AMC: amino methylco umar in ; B A: bov ine serum

albumin ; CHAPS: 3-[(3 -cholamidopropyl)d imethylam moni o]-2-hydroxy- 1-

propanesul fo ni c ac id ; CHO : Chinese hamster ova ry; DISC: death-i nducin g signal ling

complex; F ADD: Fas associating prote in with dea th domai n; Fas: fibroblast­

assoc iated; FasL: Fas li gand ; FasR: Fas receptor, FBS: feta l bov ine serum ; ICAD:

inhibitor of caspase activated DNase; MEM: minimum essential medium ; MOPS : 3-

(N-morpholino)-propane sulfonic ac id ; PARP: polyA DP-ri bose polymerase; PB S:

phosphate-buffered sa line; PMSF: phenylmeth ylsufonyl fluoride ; PS :

phosphatidylserine; PVDF: polyvinylidene difluoride; SOS-PAGE: sodium dodecyl

sulphate-po lyacrylamide ge l electrophores is; SEM: standard error of mean. TNF-R:

tum or necros is facto r receptor.

124

Figure 1: Acrolein induces externalization of phosphatidylserine

A B -r------·-·------·--·,

E

35 ***

30 *** + 25 *** > s::: ·x 20 Q) s::: s:::

15 < ~

10

5 1

0 Control 20 30 50

Acrolein Hz Oz (!mol/ce li) (J..!M)

B 2.5

0 0 2 Z.o ïii 4: t:~

2 Cll1.5 t: t: ·- "' Q) .... >.c .. E 1 "' Q) OiE cr:

0.5

D

0

Calnexin ---+

Figure 2: Acrolein in duces translocation of FADD

A 0 20 30 Acrolein (fmol/cell)

! --- Membrane FADO

~================~ 1---Cytoso lic FADO

~------------------------~

*

20

*

**

30

c

.... 0

100

>.0 80 ~0 1/)4: ~~ ë .!::! 60 ·;;;a >Il)

·- 0 -:; >. 40 Qi u cr:

20

Acrolein (fmolfcell) 0 20 30

Cyt. Micro. Cyt. Micro. Cyt. Mito.

' GST-n1- Cytoc.h rome-c---+ ox1dase

125

Il) 100 Il)

c..

'0 80 >. ..... ·~ 60 QJ ..... t::

QJ 40 > :;:: ra Qi 20 a::

A

1.5 >. ..... ·:;:: :;:: u ra

1 00 1

QJ f/) ra c.. f/) ra

0.5 u

0

Figure 3: Activation of initiator caspase-8 by acrolein

~ h

0

20

10 30

Acrolein (fmol/cell)

D

~3 ~

30

.... 0

.?;­(jj 2 t:: QJ ..... t::

QJ

> :;:: ra Qi a::

Acrolein (fmol/cell) 0

*** **

50

**

20 30

126

127

Figure 4: FasL expression is increased by auolein whereas FasR ex pression is

not affected

A 0 10 30 50 A crolein (fmollcell) r------------------,1 +-- Membrane FasR

(48 kDa)

B '------------------'~ - Ca lnex in (44 kDa)

0::: 120 (/) cu

LI.. 100 ..... 0

~ 80 ëii c: ~ 60 .... c: ~ 40 > :;::: cu 20 a:;

0::: 0

0 10 30 50 Acrolein (fmol/cell)

c 0 10 30 Acrolein (fmollcell )

1

- Fas l (35 kDa)

1- p-tubu lin

D

3 ...J (/) *** cu 2.5 LI.. ..... 0 *** >. 2 .... ëii c: 1.5 ~ .... c: ~ > :;::: cu 0.5 a:;

0::: 0

0 10 30 Acrolein (fmol/cell )

-------------------------------------------------------------------------------------------------------

128

Figm·e S. Activation of caspase-8 and caspase-7 by acrolein is decreased by a n

inhibitor of Fas receptor

A 2 ***

D · lnh 1 !SI + lnh FasR

~ 1.5

·;;: :;:::; (..) co

CIO 1

C1) (/) co a.

** * -t

r+ f ~H ~~~ r-f-

-~ ~ 1

(/) co (.) 0.5

0 ~ ~ ~ 0 5 15 30 50 100

Acrolein (f mol/cell)

B

*** 1.5 ***

~~

~ ·;;: :;:::; (..) co ..... 6 (/) co a. 0.5 (/) co (.)

0 0 5 15 30 50

Acrolein (fmol/cell)

129

Figure 6: An inhibitor of Fas receptor decreases apoptosis induced by acrolein

Control

-lnh

+ lnh FasR

0 30

Acrolein {fmol/cell)

Figure 7: Acrolein induces cleavage of bid to t-bid

A Acrolein (fmol/cell) 0 20 30 .---------------------,

Cytosolic bid - 1

(22 kDa) ~-=========~ Mitochondrial t-b id

(15 kda) 1 -"'C

..c

B

100

0 75 >.

:'.: rn c: .!!! 50 c: Cl> > ~ 25 Qi ~

0 20

c

30

"'C3

..c

..!. -0

~2 rn c: Cl> -c:

Acrolein (fmol/cell)

130

Figure 8: Activation of caspase-9 by acrolein is decreased by inhibitors of

caspase-8 and FasR

A

1.5

0 5

B

1.5 ***

~ ·;;: tl 1 C'CS

0'1 d> ~ a.

8 0.5

0

0 5

***

15 30 50 Acrolein (fmol/cell)

***

15 30 50

Acrolein (fmol/cell)

o- inh CS

0 + lnh CS

***

100

100

131

132

Figure 9: Cleavage ofPARP by acrolein is inhibited by a n inhibitot· of caspase-8

B

100 a.. e::: <( a.. 75 -0 >. ~ rn 50 r:::: Q) ..... r:::: Q)

20 > ~ cu a; e:::

0

A Acrolein Inh of CS

PARP 116 kDa --+

Cleaved PARP 85 kDa --+

+

p-tubulin --+ 1 .......... - - .........,

0

c Ln 00

1 a.

2

0 1.5 z. ïjj

~ 1 ..... r:::: Q)

> :.;::::; 0.5 cu Q)

e:::

***

o~~~--~--~--~~--

30 0 30 Acrolein (fmol/cell)

Figure 10: Inhibition of caspase-8 decreases acrolein-induced chromatin

condensation

Control Acrolein (30 fmol/cell)

-lnh

+ lnh ca

E 20

. !!! ••• 1/) 0 .... a. 0 a. 10 <t ~ 0

~q,

0 Control DMSO + lnh C-8

+ + Acrolein 30 fmol/cell

133

~----------- ------ - - -----------------------

134

FIGURE LEGENDS

Fig. 1 Acr·olein induces externalization of phosphatidylserine. Apoptosis was

analysee! by fl ow cytometry using Annexin V-FlTC in CHO ce ll s fo llowing treatment

with acrole in . Cells ( 1 061Inl) were either (A) untreated control , or they were treated

with (8) 20 fmoll~ and (C) 30 fmo ll~ acrolein or (D) 50 ~tM hydrogen peroxide fo r 2

h. Ce lls were subsequently stained with Ann ex in V-F lTC (x-ax is) and PI (y-axis) .

Twenty thousand cells were then analyzed usin g a F ACS scan to determine the

percentage of Annexin y +_labelled ce lls. One representative ex periment is shown

from six independent experiments. (E) P<O.OO 1 (***) indicates a statistically

signifi cant di fference between treatment with acrole in or H20 2 and the control.

Fig. 2. Acrolein induces translocation of FADD. CHO ce ll s ( 106 /ml ) were

in cubated for 1 h with different concentrations of acrole in (20 and 30 fm ol/ce ll ) in u­

MEM containing 10% FBS. Immunodetection of FADD (28 kDa) in membrane and

cytoso lic fract ions was carried out by Western blotting, using ca lnex in as a loading

contro l for the membrane fraction and GSTn-1 for the cytoso l ic fraction . (A) A

representative blot is shown from four independent ex periments. Densitometric

analyses of the expression of (B) membrane FADD and (C) cytoso lic FADD are

re lat ive to the untreated control. P<0.05 (*) or P<O.O 1 (**) indicates a stati stically

significant di ffe rence between treatment with acrolein and the control. (D) Purity of

mitochondrial, cytoso lic and mi crosomal frac ti on wa verifiee! using antibodies to

cytochrome-c oxidase, GSTir 1 and ca lnex in, respective ly.

Fig. 3. Activation of initiator caspase-8 by acrolein. (A) CHO ce ll s (0.5x 1 06/ml)

were incubated with acrolein ( 10 to 50 f mol/cell ) fo r 1/2 h or 1 h in u-MEM with

10% FBS. Caspase-8 act ivity was measured in ce l! ly ates using the flu orescent

substrate Z-I ETD-AFC. Caspase activity was exp ressed relative to the untreated

135

contro l, des ignated as 1. Data represent means and EM from eight independ ent

experiments perfo rmed with multiple estimati ons per poin t. (B) Ce ll s ( 1 06/ml) were

incubated with acrolein (20 and 30 fm ol/ce ll ) fo r 1 h in cr -M EM containing 10%

FBS . The immunodetection of procaspase-8 (55 kDa) and its cleavage fragment (20

kDa) was carri ed out by Western blotting, using f)-tubulin as a loading control. A

representati ve blot is shown from fo ur independent experiments. Densitometric

analyses of the express ion of (C) procaspase-8 and (D) the cleavage fragment are

relative to the untreated control. P<0.05 (*) or P<O.O 1 (**) indicates a stati sti cally

signifi cant di fference between treatment with acrolein and th e control.

Fig. 4. FasL expression is increased by acrolein whereas FasR expression is not

affected. CI-1 0 ce ll s ( 106 /ml) were incubated fo r 1 h with di ffe rent concentrations of

acrole in ( 10 to 50 fmol/cell ) in cx-M EM containing 10% FBS. lmmunodetecti on of

membrane FasR (48 kDa) and FasL (35 kDa) was carri ed out by Western blotting,

using ca lnexin and f)-tubulin , respective ly, as loadin g controls. Representat ive blots

are shown for (A) Fas R and (C) FasL from fo ur independent experiments.

Densitometri c analyses of the expression of (B) FasR and (D) FasL are relati ve to the

untreated control. (E) P<O.OO 1 (***) indicates a tati stica lly s ignificant di fference

between treatment with acrolein and the control.

Fig. S. Activation of caspase-8 and caspase-7 by acrolein is decreased by an

inhibitor of Fas receptor. Pretreatment of ce ll s with 1 mM of Kp7-6 (Fas/FasL

antagoni st) was perfo rmed for 1 h before additi on of acrole in . (A) CHO ce ll s

(0.5x 1 06/ml) were then incubated with acrole in (5 to 100 fm ol/ce ll ) for 1 h (caspase-

8 assay) or (B) with acrolein (1 to 50 fm ol/ce ll ) fo r 2 h (caspase-7 assay) in cx-MEM

with 1 0% FBS. Acti viti es of caspases-8 and -7 were measured in ce l! lysate using

the flu orescent substrates Z-IETD-AFC and MCA -VDQV DGWK(DNP)-NH2,

respecti vely. Caspase act ivity was expressed relat ive to the untreated control,

136

designated as 1. Data represent means and SEM from eight independent experiments

perfo rmed with multipl e estimati ons per poin t. P<0.05 C' ), P<O.O 1 (**) or P<O.OO 1

(*** ) indicates a stati stica lly significant di ffe rence between treatment with acrole in

and the control. P<0.05 (~) , P<O.O 1 (~~) or P<O.OO 1 (~~~) indicates a stati stica lly

signifi cant di fference between cells exposed to acrolein , with and without Fas

receptor inhibi to r.

Fig. 6. An inhibitor of Fas receptor decreases apoptosis induced by auolein.

Contluent ce ll s in monolayer were pre-incubated for 1 h with (C, D) or without (A,

B) 1 mM of Kp7-6 and then incubated with (A, C) 0 or (B, D) JO jmol/ce ll of

acrolein fo r 4 h, with inhibitor present (C, D). Ce ll s were sta ined with Hoechst 33258

and PI and visuali sed by flu orescence microscopy (magnificat ion 320x). The fraction

of (E) apoptotic ce lls (blue Hoechst 33258 flu orescence) is g iven relati ve to tota l

ce ll s. Data represent means and SEM from fi ve independ ent experiments perfo rmed

with multiple estimations per point. P<O.OO 1 (***) in dicates a stati sti ca ll y significant

diffe rence between treatment with acrolein and the control. P<O.OO 1 CH~) indicates a

stati stica lly significant di fference between ce ll s exposed to acro lein , with and without

Fas receptor inhibitor.

Fig. 7. Acrolein indu ces cleavage of bid to t-bicl. CHO ce li s ( 106 /ml) were

incubated for 1 h with different concentrati ons of acrolein (20 and 30 fm ol/cell ) in a­

MEM containing 10% FBS. lmmunodetecti on of-' cytoso lic bid (22 kDa) and

mitochondri al t-b id ( 15 kDa) was carried out by We tern blotti ng, using GSTn-1 as a

loading control fo r the cytoso lic fraction and cytochrome-c ox idase fo r the

mi tochondrial fraction. (A) A representati ve blot is shawn from fo ur independent

ex perim ents. Densitometric analyses of the express ion of (B) cytoso lic bid and (C)

mitochondrial t-bid are re lative to the untreated control. P<0.05 (*), P<O.O 1 (**) or

137

P<O.OO 1 (* * *) indicates a stat ist ically sign iti cant diffe rence between treatment with

acrolein and the control.

Fig. 8. Activation of caspase-9 by acrolein is deueased by inhibitors of caspase-8

and FasR. Pretreatment of ce ll s with (A) 10 ~LM of Caspase-8 lnhibitor 11 , Z-!ETD­

FMK, or (8) 1 mM of Kp7-6 was carried out for 1 h before addition of acrolein . (A,

B) CHO ce ll s (0.5x 1 06/inl) were then incubated with acrole in (5 to 100 jmol/cell) for

1 h in a-MEM with 10% FBS. Caspase-9 act ivity was meas ured in ce l! lysates, either

with or without inhibitor pretreatment, using the nuorescent substrate Ac-LEHD­

AFC. Caspase-9 activity was expressed re lat ive to the untreated control, designated as

1. Data represent means and SEM from eight independent experiments performed

with mul tip le est imat ions per point. P<O.OOI (***) indicates a stati stically significant

difference between treatment with acrolein and the control. P<0.05 (~) , P<O.O 1 (~~)

or P<O.OO 1 ( ~~~) indicates a stati stically sign ificant cl ifference between ce li s exposed

to acrolein, with and without caspase-8 inhibitor or l· as receptor inhibitor.

Fig. 9. Cleavage of PARP by acrolein is decreased by an inbibitor of caspase-8.

(A) Cell s ( 1 06/ml) were incubated with acro le in for 2 h and immunodetection of

PARP ( 11 6 kDa) and its cleavage fragment (85 kDa) wa carried out by Western

blott ing, using ~-tubulin as a load ing control. A representat ive blot is shown from

fo ur independent experiments. Densitometri c analyses of the ex press ion of (B) PARP

and (C) the cleavage fragment are relat ive to the untreated control. Data represent

means and SEM from five independent experim ents performed with multiple

estimations per point. ? <0.05 (*)or P<O.OO 1 (***) indicates a stati stically signifi cant

difference between treatment with acrol ein and the contro l. P<O.OO 1 CN~) indicates a

statistically signiticant difference between ce lls expo ed to acro lein, with and without

caspase-8 inhibitor.

138

Fig. 10. Inhibition of caspase-8 decreases acrolein-induced chromatin

condensation. Confluent ce lls in monolayer were pre-incubated for 1 h with (C, D)

or without (A, B) 10 flM of Caspase-8 lnhibitor Il , Z-I ETD-FMK, before add ition of

acrole in . Ce ll s were then incubated with (A, C) 0 or (8 , 0) 30 fmo l/ce ll of acrolei n

fo r 4 h, e ither with or without the caspase-8 inhibi tor present (C, 0 ). (E) The fraction

of apoptotic ce lls (Hoechst) is given relative to tota l ce ll s. Necrotic ce ll s were

visuali sed using PI (red fluorescence) . Data represent means and SEM from five

independent experiments performed with multiple estimat ions per point. P<O.OO 1

(***) indicates a stati stically significant di ffe rence between treatment with acrolein

and the control. P<O.OO 1 CH~) indicates a stati sti ca ll y significant di ffe rence between

cells exposed to acrolein, with and without caspase-8 inhibitor.

139

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S . .J . Orderi ng the cytochrome c- initi ated caspase cascade: hiera rchical activat ion of

caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cel!. Biol.

144:2 18-292; 1999.

[60] Cohen, G .M. Caspases: the executioners of apoptos is. Biochem. J. 326: 1-1 6;

1997 .

[6 1] Satoh, K. ; Yamada, S. ; Ko ike, Y. ; lga ras hi , Y. ; Toyokuni , S.; Kumano, T .;

T akahata, T. ; Hayakari , M .; T such ida, S .; Uchida, K. A !-hour enzyme-linked

147

immunosorbent assay for quantitat ion of acro lein- and hyd roxynonenal-modified

protei ns by epitope-bound casein matr ix method. Anal. Biochem. 270: 323 -328 ; 1999 .

[62] Chan, K. ; Truong, 0 .; Shangari , N. ; O'Brien, P.J. Drug-induced mitochondri al

toxicity. Exp ert Opin. Drug Metab. Toxicol. 1: 655-669; 2005.

[63] Leist, M.; Single, B.; Castoldi , A.F. ; Kuhnle, S.; N icotera, P. lntracellular

adenosine triphosphate (ATP) concentrat ion: a witch 1n the dec ision between

apoptos is and necrosis. J Exp. Med. 185: 148 1-1 486; 1997.

[64] Monteil , C. ; Le Prieur, E.; Buisson, S. ; Morin , .J.P .; Guerbet, M. ; Jouany, J.M.

Acrolein toxic ity: comparative in vitro study with lung s lices and pneumocytes type

ll cellline from rats. Toxicology.133:1 29-l 38 ; 1999.

[65] Toraason, M. ; Luken , M.E. ; Breitenstein, M.; Krueger, J. A. ; Biagini, R. E.

Comparat ive toxicity of ally lamine and acrolein in cul tu red myocytes and fibroblasts

fro m neonatal rat heart. Toxicology. 56: 1 07-117; 1989.

[66] Luo, J.; Robinson, J.P. ; Shi , R. Acrolein-induced ce l! death in PC 12 ce lls: role of

mitochondri a-medi ated oxidati ve stress . Neurochem. Jnt. 47:449-457; 2005 .

[67] Zitting, A. ; Heinonen, T. Decrease of red uced glutathi one in iso lated rat

hepatocytes caused by acrolein, acrylonitrile, and the thermal degradation products of

styrene co polymers. Toxicology. 17:333 -34 1; 1980.

[68] Berhane, K. ; Widersten, M. ; Engstrom, A.; Kozarich, J.W. ; Mannervik, B.

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products

148

of radi ca l reactions and lipid perox idati on by human glutathione transferases. Proc.

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[69] Eder, E.; 1-lenschler, D.; Neudecker, T. Mutageni c properties of allyli c and alpha,

beta-unsaturated compounds: considerati on of alkylat ing mechani sms. Xenobiotica.

12: 83 1-848; 1982.

[70] Gardner, R.; Kazi, S.; Ellis, E. M. Detox ication of the environmental pollutant

acrolein by a rat liver aldo-keto reductase. Toxicol. Le // . 148:65-72; 2004.

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peroxidati on by rat li ver mi crosomal aldehyde dehydrogenase. Arch. Biochem.

Biophys. 269: 11-1 7; 1989.

[72] Averiii-Bates, D.A.; Agostinelli , E. ; Przy bytkowski, E. ; Mondovi , B. Aldehyde

dehydrogenase and cytotoxicity of purified bov ine serum amine oxidase and

spermine in Chinese hamster ovary cell s. Biochem. Cel/. Biol. 72: 36-42 ; 1994.

[73) 1-l eby, 0. ; Persson, L. Molecular genetics of polyamine synthes is in eukaryotic

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2.4. ARTICLE III

P38 AND ERK MITOGEN-ACTIV ATED PROTEIN

KINASES MEDIATE ACROLEIN-INDUCED

APOPTOSIS IN CHINESE HAMSTER OV ARY CELLS

André Tanel and Diana A. Averill-Bates 1'2

Département des sciences biologiques, TOXEN, Université du Québec à

Montréal, CP 8888, Succursale Centre Ville, Montréal, Québec, H3C

3P8, Canada.

1To whom correspondence should be addressed:

Tel: (514) 987-3000 (ext 4811)

Fax: (514) 987-4647

Email: averill.diana@uqam.ca 2Formerly Dr Diana A. Bates

Acknowledgments: Financial support was obtained from CIHR (Canadian Institutes

of Health Research) (DAB). AT was the recipient of the Bomse Francine Beaudoin­

Denizeau from the Fondation UQAM for PhD studi es.

Abbréviations: AFC: amino trifluorocoumarin; BSA: bovine serum albumin;

CHAPS: 3-[(3 -cholamidopropyl)dimethylammonio ]-2-hydroxy-1-propanesulfonic

acid; CAD: caspase activated DNase; Cl-IO: Chinese hamster ovary; ERK:

150

extrace llular signal regulated kinase; FBS: fetal bov ine serum ; 4-HNE: 4-

hydroxynonenal; lCA D: inhibitor of caspase acti vated DNa e; JN K: c-Jun N-terminal

kinase; MAPK: mitogen-activated protein kinase; M EK: m itogen-activated prote in

kinase kinase; MEM: minimum essential medium ; MOPS: 3-(N-morpholino)­

propane sulfonic acid ; PARP: polyAOP-ribose polymerase; PB S: phosphate-buffered

sa line; P1 3-K: phosphoinos itide 3-kinase; PKB : protein kinase 8 ; PMSF:

phenylmethylsufonyl flu oride; PYDF: polyv inylidene ditluoride; SOS-PAGE:

sodium dodecyl sulphate-polyacrylamide ge l electrophores is; SEM: standard error of

mean; Wt: wortmannin .

151

RÉSUMÉ

L'acroléine, qui est un aldéhyde a ,l3-insaturé généré par la peroxidati on lipidique,

peut affecter les ti ssus et les ce llules et causer plusieurs patholog ies. L ' augmentation

des aldéhydes j oue un rôle important dans la pathogenèse de plusieurs maladies

notamment l ' Alzheimer, l 'athérosc lérose et le diabète. Quelques études ont démontré

que l ' acroléine active les MAPKs et d'autres ont rapporté que l ' acroléine induit

l ' apoptose. Cependant, aucune étude n'a examiné le lien entre l 'activation des

M A PKs et l 'apoptose induite par l 'acroléine. Ici, on montre pour la première fois que

l ' apoptose induite par l ' acroléine est dépendante des M A PK. Une heure d' exposition

à l ' acroléine a induit une forte phosphory lation de ERK, p38 et le substrat de JNK, c­

jun chez les ce llules CHO. L ' inhibition de la condensati on de la chromatine induite

par l ' acroléine à l ' aide de l ' inhibiteur de ERK. leU 126, et l ' inhibiteur de la p38, le

SB203580, démontre clairement l ' implicat ion de ces deux vo ies dans l 'apoptose

induite par l ' acroléine. En plus, le UO 126 et le SB2035 80 ont inhibé l ' acti vati on des

caspases-7 et -9 et le clivage de I' ICAD induites par l ' acroléine. D'autre pa1t, les

vo ies de JNK et A KT semble être impliquées dans la surv ie contre l ' agress ion par

l ' acroléine, puisque des inhibiteurs pharm aco logiques de ces deux vo ies, le

SP6001 25, le LY294002 et le Wortmannin ont changé le type de mort de l 'apoptose

en nécrose. Enfin, l ' acrol éine a entraîné la phosphory lat ion de la p53 , une protéine

responsable de la transcription de plusieurs gènes impliqués dans l ' apoptose dont

ceux de Bax et du ligand Fas. Ces résul tats fournissent de nouve lles informati ons sur

l ' apoptose induite par l ' acrol éine qui pourraient être util es pour la compréhension de

plusieurs pathologies ainsi que les effet des expos itions environnementales

impliquant l 'acroléine.

152

ABSTRACT

Acrolein , which is a highly reactive a ,f3-un saturated a ldehyde generated by lipid

perox idation, can affect cell s and ti ssues and cause va ri ous di sorders. lncreased levels

of un saturated aldehydes pl ay an important role in the pathogenes is of a number of

human di seases such as Alzheimer's disease, ath erosc leros is and diabetes . Acrolein is

a hi ghly ubiquitous toxic environmental pol lutant. Because of hu man exposure, there

is a need fo r investi gating the mechani sms in vo lved in acrolein tox icity at the ce llular

and molecular levels. Acrolein can induce ce l! death by apoptos is, although the

mechani sms are not entirely clear. The present study investigates whether mitogen­

acti vated protein kinases (MAPKs) play a ro le in acti va tion of apoptos is by acrolein .

Our findin gs show that acrolein-medi ated apoptos i is in fact MAPK-dependent in

Chinese hamster ovary ce ll s. The MAP fa mil y kin ases, including ERK and p38

kinase, and the transcription factor c-Jun were ali acti vated by phosphorylation after 1

h exposure to acrolein. Phosphorylation of ERK and p38 kinases and their blockade

by an ERK inhibitor, U01 26, or a p38 inhibitor, SB203580, respective ly, suggested

th at acti vation of apoptos is by acrolein is ERK- and p38-dependent. Thus, blockade

of ERK and p38 inhibited chromatin condensation, caspase-7 and -9 activat ion as

we il as ICA D cleavage induced by acrolein . .JNK and AKT kinases seem to be

implicated in survival pathways aga inst acro lein in sult, s ince their respective

inhibitors, SP600 125 and L Y294002/Wortmannin switched th e mode of ce l! death

from apoptos is to total necrosis. Finally, acrolein induced phosphorylati on of the pro­

apoptotic factor p53 whi ch is responsible fo r transcription of pro-apoptotic factors

such as Bax and Fas ligand . These results prov ide new info rmati on demonstrating the

implicati on of MAPKs and AKT in acro lein-induced apoptos is, and thi s info rmation

may be useful fo r understanding the pathogenes is of a number of ti ssue di seases and

environmental tox icity in response to acrolein.

Keywords: Acrolein, MA PK, ASK 1, AKT, apoptosis, caspase

153

INTRODUCTION

Acrolein, an unpleasant and troubl esome by-product of overheated organic

matter, occurs as a ubiquitous pollutant in the environment, ari sing from incomplete

combustion of plasti c materials, forest ti res, cigarette smoking and overheated

cooking oils [Beauchamp et al. , 1985]. Acrolein is also a metabolite fo rmed in the

biotransformation of ally! compounds and the widely u ed anticancer drug

cyc lophosphamide. ln addition, acrolein is used industrially as a startin g materi al for

acrylate polymers and in the producti on of acryli c ac id , and as a herbicide

[Ghilarducci and Tjeerdema, 1995] . Since acrole in was identifi ed as one of the

"unnatural" components of tobacco smoke [J ohn tone and Plimmer, 1959], a number

of reports have appeared describing the damaging effects of acrole in on the trachea l

c ili atory movement [Kensler and Batti sta, 1963] and the pulmonary wa ll [I zard and

Liberman, 1978] .

Acrolein is one of the taxie by-products of endogenous lipid peroxidation,

resulting from ox idative stress, together with the aldehyde 4-hydroxy-2-nonenal

(H E) [Adams and Klaidman, 1993; Uchi da 1999]. Ox id ati ve stress has been

implicated in chronic neurodegenerative di sorder uch as Alzheimer's disease

[Ca lingasan et al ., 1999] . Lipid perox idati on is increased in Alzheimer' s di sease

[Ramassamy et al., 1999] and it has been suggested that a ldehyde by-products such

as acrolein and 4-1-fN E could be involved in caus ing ce llular and ti ssue damage.

Acrolein protein adducts have been demon trated in Alzheimer' di sease [Ca lingasan

et al., 1999], di abetic nephropathy [Suzuki and M iyata, 1999] and atheroscleros is

[Uchida et al. , 1998]. These pathophys iologies have a li been associated with

increased lipid perox idation and alkylation of prote in res idues.

lts hi gh reactivity indeed make acrole in a dangerou sub tance for the living

ce l! . lt ha been shawn that acrolein redu ces the co lony-fo rming effi ciency of

mammalian ce ll s, fonns cyclic adducts with nucleosides in vitro and is a patent

mutagen [Esterbauer et al. 199 1]. Among ali of the a, ~-un aturated a ldehydes,

!54

acrole in is by fa r the strongest electroph ile and therefo re, shows the h ighest reacti vity

with nucleophiles, such as the sulfhydryl group of cyste ine, imidazo le group of

hi st idine and ami no group of lys ine [Esterbauer et al., 199 1 ].

Apoptos is is a naturally occurring proce s that plays an essential rote in

deve lopment and the maintenance of homeostas is in multi ce llular organi sms [Saraste

and Pulkki 2000; Hale et al. , 1996]. Apoptos is has many important repercuss ions in

human health. Ce l! accumulati on via in sufft cient apoptos is can contribute to

conditions such as cancer, infl ammation and auto immune di sease. ln contrast,

excess ive apoptos is can play an important role in neurodegeneration, AIDS, eye

di sorders, osteoporos is and heart fa ilure [Fadeel et al., 1999; Wil son, 1999].

A family of cytoso lic cysteine proteases, the ca pases, pl ay an essential rote in

the executi on of apoptos is. The caspases are di vid ed into api cal (-2, -8, -9 and -1 0)

and executioner subsets (-3, -6 and -7) [Sa lvesen and Di xit, 1997]. The zymogens or

pro-enzymes of apical caspases (-8 and -1 0) are recruited by spec ifi e adaptor

molecul es at the cytoso lique face of death receptors, whereas that fo r caspase-9 is

acti vated vi a a post-mitochondrial route [Denecker el al., 200 1]. Downstream events

in the apoptoti c cascade in clude the caspase-med iated cleavage of cytoplasmic,

cytoskeleta l and nuclear protein substrates such as polyA DP-ribose polymerase

(PARP), lamins, gelso lins, fodrins and inhibitor of caspase acti vated DNase (ICAD) .

The ce ll then exhibits the characteri stic morpholog ical fea tures of apoptosis such as

chromatin condensation, cytoskeletal changes, nuclear membrane breakage, ce il

membrane blebbing and formati on of apoptotic bod ies [Wilson, 1999; Ea rn shaw,

1995]. Apoptotic bodies are then phagocytozecl by macrophages or neighborin g cell s,

thus avo iding inflammatory damage to adj acent ce ll s and ti ssues.

While numerous candidate genes pl ay ing important roles in the signaling of

apoptos is have been identifted, the mechani sms and orcier in which they interact to

transduce signais are poorly understood. Severa! !ines of ev idence have suggested

that members of the mitogen-activated protein kinase (MA PK) fa mily are invo lved in

155

apoptot ic signaling, as we il as in control of growth and differentiation [M arshall ,

1994; Waskiewicz and Cooper, 1995; Fanger el al., 1997]. To date, three MAPK

(serine/threonine kinases) cascades that converge on extrace llular signal-regul ated

protein kinase (ERK.l /2), c-Jun N-tenninal kinases (JNKs), and p38 MAP kinase

(p38) have been weil characterized. These MAPKs are regulated by distinct stimuli

[Huot et al., 1997]. ERK is predominantly act iva tecl by growth factors or mitogens

leading to cell differenti ation, growth, and survi va l. On the other hand, JNK and p38

are preferentially act ivated in response to var ious cytotoxic stresse , including tumor

necros is factor (TNF)-a , hyd rogen peroxide (H20 2), UV li ght, X-rays, heat shock and

growth factor- or serum-withdrawa l, resulting in infl ammation and apoptos is [Huot et

al., 1997; Verheij et al. , 1996].

Apoptos is signal-regulating kinase (ASK) 1s a MAP kinase kinase kinase

(MAPKKK), which is an upstream factor that activates the SEK I-JNK and

MKK3/MKK6-p38 signaling cascades [l chij o et al. , 1997]. Overexpress ion of ASK 1

in ep ithelial ce ll s in low serum conditions induced ce ll death with apoptotic features

and ASK I-K709R, a kinase-inactive mutant of AS K 1, reduced TNF-a-induced

apoptos is, suggesting that ASKJ is a pivotai component in the mechani sm of

cytok ine- and stress- induced apoptosis [H ayakawa et a l. , 2006; lchijo et al., 1997;

Tobiume et al. , 1997].

AKT is a proto-oncogene which is acti vated by the phosphatidylinos ito l 3-

kinase (P13-K) surviva l pathway. Therefore, AKT promotes surviva l of cancer ce ll s

and tumor growth. AKT inhibits apoptos is by pho phorylat ing a number of

downstream targets. Thi s includes phosphory lat ion of the pro-apoptotic protein Bad

and its degradation by the protein 14-3-3, therefore remov ing its inhibitory effect on

th e ce ll survival factor Bel-XL at the mitochondri al membrane [Franke el al. , 1997;

Bellacosa et al., 1998]. Thus, the inhibition of AKT activation induces apopto is of

cancer ce ll s.

156

p53 is a tightly regulated transcripti on factor thar induces ce l! cycle arrest or

apoptos is in response to cellular stress, such as DNA damage [Evan and Littl ewood,

1998]. First described in 1979, and initi all y be lieved Lo be an oncogene, p5 3 was the

fïrst tumour suppressor gene to be identified. p5 3 functi ons to eliminate and inhibit

the proli fe rat ion of abnormal cell s, thereby preventing neoplastic development.

Ab rogation of the negative growth regul atory functions of p53 occurs in many,

perhaps ali , human tumours. The p53 signaling pathway is in ' standby ' mode under

normal ce llular cond itions. lt is now clea r that a number of phosphorylat ion sites on

p53 are a ltered after DNA damage, and such phosphorylat ion events have been

shown to result in alterat ions in p53 that make it more stabl e and more act ive

[Voudsen, 2000]. As a transcription factor, phosphorylated p53 induces transcription

of proapoptotic proteins such as Bax and Fas li gand and represses those of

antiapoptotic proteins such as Bcl-2 [Bras et al., 2005].

Given the widespread exposure of humans and other species to acrolein , it is

important to improve our understand ing of its mechani sms of tox icity. Acrole in has

been shown to induce apoptos is [Tanel and A veri 11 -Bates, 2005 ; Li et al., 1997;

Nard ini et al., 2002; Takeuchi et al. , 200 1 ], as we il as act ivat ion of MAPKs

[Ranganna et al ., 2002; Takeuchi et al., 200 1; Misonou et al., 2005; Pugazhenthi et

al. , 2006; Fi nkelstein et al., 200 1; Deshmu kh et al. , 2004]. 1-l owever, the implicat ion

of MAPK prote ins in the act ivation of acro lein-induced apoptosis has not been

invest igated. Since acrolein is a DNA damaging agent [Kail asam and Rogers, 2006],

it could likely cause activation of p53 . With the aim of advancing our know ledge

about this toxic compound, the present study investigates whether acrolei n-induced

apoptosis is mediated by MAPK proteins and 1 or the p53 transcripti on factor.

157

MATERIALS AND METHODS

Cel/ culture

CHO ce ll s (A uxB I) [Ling and Thompson, 1974] were grown in monolayer in

minimum essential medium-Alpha (a-M EM) (G ibco Canada, Burlington, ON) plus

10% feta l bov ine serum (FBS) (G ibco Canada) and 1% penic illin (50 units/mL)­

streptomyc in (50 ~Lg/mL) (F low Laboratories, Miss issauga, ON), in ti ssue culture

flasks (Sarstedt, St Laurent, QC), in a humidifi ed atmosphere of 5% C02 in a water

jacketed incubator at 37°C [Bates and Mackillop, 1986]. The ce lls were grown to

near confluence and then incubated for 24 h with fresh culture medium . Confluent

ce li s were th en harvested using citrated phosphate-buffered sai ine (PBS) (0. 14 M

NaCI, 0.01 M sod ium phosphate, 0.015 M sod ium citrate) , washed by centri fugation

( 1 OOOg, 3 min) and resuspended in a-MEM plus 10% FBS for experimental studi es.

In il ibitor treatment

To investigate the effect of MAPK inhibitors on acrolein-induced apoptos is,

confluent cel! cultures were preincubated for 1 h with one of the fo llowing inhibitors

prior to addit ion of acrolein: 5 ~M SB203580 (p38 inhibitor) [Deschesnes et al. ,

200 1; Me loche et al. , 2000], 10 ~M UO 126 (M EK 1/2 act ivity inh ibitor) [Favata et al. ,

1998], 50 ~M PD98059 (inhibitor of MEKI /2) [Daoud et al., 2005], 50 ~g/ 1

Ly294002 (Pl3-K inhibitor) [Me loche et al., 2000], 100 11 M Wortmannin (PI 3-K

inhibitor) [Meloche et al., 2000] , or 10 ~M SP600 125 (JN K inhibitor). Ali of the

inhibitors were purchased from Calbiochem (La .J o ll a, CA) and they were present

during acrolein incubation.

Western Blot analysis

Fo llowing treatment with acrolein, cell s were washed in buffer A ( 100 mM

sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4) and resuspended in lys is buffer B (20

----------------- --------

158

mM 3-(N-morpholino) propanesulfoni c ac id, pH 7, 10% glycerol, 80 mM b­

glycerophosphate, 5 mM EGTA, 0.5 mM EDTA, 1 mM odium vanadate, 5 mM

sodium pyrophosphate, 50 mM sodium flu orid e, 1% Triton X-1 00, 1 mM

dithiothreitol) and a cocktail of protease inhibitors: 10 ~M aprotinin , 10 ~M pepstatin

A, 10 ~M leupeptin, 25 ~LM calpain inhibitor 1 and 1 mM phenylmethylsulfonyl

flu oride (PMSF)] [Guay el al ., 1997]. After a 2 h incubati on on ice, unbroken cells

and nuclei were pelleted by centi fugation at 25 00 g fo r 10 min . The supernatant was

used for the detection of proteins .

SDS-polyacrylamide gel electrophores is (SOS-PAGE) of cellular prote ins was

carri ed out according to Laemmli [Laemmli , 1970]. Prote ins (50 ~Lg) were quantifïed

according to Bradford [Bradfo rd, 1976] and then so lubili sed in Laemmli sample

buffer. The sam pies were boiled for 5 min at 1 oooc and loaded onto a 10% (ASK 1)

or 15% acry lamide ge l (a li other prote ins) . Electrophores is was carried out at a

constant vo ltage of 125 V. Ce llul ar prote ins were transferred electrophoreticall y to a

polyv inylidene difluoride (PVDF) membrane us1ng a MilliBiot Graphite

Electroblotter 1 apparatus (M illipore, Bedford , MA) [Tanel and Averiii-Bates, 2005].

The transfer buffer conta ined 96 mM glycine, 1 0 mM Tr is and 10% methanol. The

transfe r was carried out for 1 .5 h at constant am perage of 80 mA/gel. Hyd rophobie or

nonspecific sites were blocked overnight at 4°C with 5% powdered skim milk in Tris­

buffe red sa line (50 mM Tris and 150 mM NaCI) containing 0.1 % Tween 20 (TBS-T).

Membranes were washed four times for 15 min in TBS-T. The bla ts were probed

with the primary antibodies: anti-c-jun, anti-phospho-c-j un, anti -AKT, anti-phospho­

A KT, anti-ERK 1, anti- ERK2, anti-phospho-ERK, anti-p38, anti-phospho-p38 and

anti-ASK 1 (Stressgen, San Diego, CA), anti -I CA D (Santa Cruz Biotechnology, Santa

Cruz, CA), anti-p53, anti-phospho-p5 3 (S 15) and anti-phospho-p53 (S46)

(Ca lbiochem, La Jolla, CA) in TBS-T, 1% bov ine serum album in (BSA) for 1 h at

room temperature. Membranes were washed fo ur times fo r 15 min and incubated for

1 h at room temperature with peroxidase-conjugated secondary antibody ( 1:1 000) in

159

T BS-T conta ining 5% milk powder. Secondary antibodi es co ns isted of ho rseradi sh

pe roxidase (HRP)-conjugated goat anti-mouse, anti-ra bbit and anti-goat lgG

(Biosource, Camarill o, CA) . PVDF membra nes were wa hed fo ur tim es fo r 15 min,

prote ins were detected us ing the EC L plus chemiluminescence kit (Perkin Eimer,

Boston, MA) and film s (Fuji medica l X-ray film , Düsserldorf, Gennany) were

scanned with a Laser Scanning Dens itometer (A lph a lnnotech Corp., San Leandro,

CA). Prote in express ion in who le ce ll lysates was quanti fi ed us in g 1 PGE L software,

re lati ve to !3-tubulin loading contro ls.

Morplwlogica/ analysis of apoptosis

To visua lize nuc lear morphology and chromat in condensati on by flu oresce nce

m icroscopy [Lee and Shacter, 1999], ce li s were seeded a nd cul tu red to near

confluence in ti ssue culture di shes conta inin g Sm L of a.-M EM and 10% FBS. Where

appropri ate, ce ll s were pretreated fo r 1 h w ith a spec ifi e MA PK inhibito r a nd then

incubated w ith acro le in fo r 4 h w ith inhibitor present. Dishes were washed tw ice w ith

PBS and Hoechst (33258) (blue flu orescence) (0.06 mg/mL) wa added fo r 15 min at

37°C to stain apoptoti c ce lls [Tane l and Averiii- Bates, 2005]. T he di shes w ere

washed with PBS and propidium iodide (red flu orescence) (5 0 ~Lg/mL) was added to

sta in the necroti c ce ll s. Observations were made by flu o rescence mi croscopy (Carl

Ze iss Ltd, Montrea l, QC, Canada) and photographs were taken by di g ita l camera

(camera 3CCD, Sony DXC-950P, Empix lmag ing lnc, M iss issauga, ON). Images

were analysed by Northern Ec lipse software. Ce lls were c lass ified us ing the

fo llow ing c riteri a: a) live ce ll s (norma l nuc le i, pa le blue chromatin w ith organized

tructure); b) membrane-intact apoptoti c ce ll s (b ri ght b lue condensed o r fragme nted

c hromatin); c) necrotic ce ll s (red, enl arged nuc le i w ith smooth norma l structure [Lee

and Shacter, 1999]. T he fracti ons of apoptoti c and necrotic ce ll s we re dete nnined

re lati ve to tota l ce li s ( obta ined us ing bright fi e ld i Il um ination). A minimum of 600

ce ll s was counted per di sh.

160

Determination of caspase activity by fluorescence spectroscopy

Freshly harvested CHO cel! s (0 .5 x 1 06) were res uspended in a-M EM plus

10% FBS and incubated with acrolein in a fin al vo lume of 1.0 mL at 37°C. After the

appropriate time, the ce!ls were washed three times with co ld PB S by centrifugati on

( 1 OOOg, 3 min) to stop the incubation. The ce l! s were re uspended in 50 ).li of PB S

and 25 ).1! were deposited into 96-we !! plates and lysed by freez ing at -20°C for 20

min . Fifty ).li of reacti on buffer (20 mM piperaz ine-N ,N'-bi (2-ethanesulfonic ac id)

(Pl PES) , 100 mM NaC!, 10 mM dithiothreito l, 1 mM EDTA, 0. 1% 3-[(3 -

cholamidopropyl)dimethylammonio ]-2-hydroxy-1-propanesul fo ni c ac id (CHAPS),

10% sucrose, pH 7.2) was added and stabilized at 37°C [Stennicke and Sa lvesen,

1997]. The kinetic reaction was started after additi on of 25 ~LI of the appropriate

ca pase substrate (Calbiochem) at 37°C using a spectroflu orimeter (S pectra Max

Gemini , Molecul ar Dev iees, Sunnyva le, CA) [Tanel and Averill-Bates, 2005].

Caspase-9 activity was measured by cleavage of the substrate Ac-LEHD-AFC to

produce 7-amino-4-trifluoromethylcoum arin (AFC) with Àmax exc itation at 4 15 nm

and Àmax emiss ion at 490 nm . Caspase-8 acti vity was measured by cleavage of the

fl uorogenic substrate Z-lETD-am ino-4-trifluoromethylcoum arin ( caspase-8 substrate

11 ) to produce AFC. Caspase-7 acti vity was measured by cleavage of the flu orogeni c

substrate 1 MCA-VDQVDGWK(DNP)-NH2 with Àmax exc itati on at 325 nm and

Àmax emiss ion at 395 nm.

Statistica/ analysis

Stati stical di ffe rences between contro l and treated groups were determined

using a one-way ANOVA which measures the linear contrast of means. An

adjustment was made to limit the famil ywi se error rate (FWE) to 5% by ca lcul ating

an adjusted p-va lue which is a simulated based p-va lue obtained from the multi vari ate

t di stribution (number of simulati ons = 1 000 000) [Westfa ll et a l. , 1999]. Then, the

16 1

Bonferroni-Holm method (a stepwise method) was used to contro l the FWE. For

compari son between a treatment w ith ac ro le in in presence o r absence of an inhibitor,

stati stical differences were determined by a one-way ANOV A fo ll owed by Dunnet

bilateral adjustment. For Fig. 7 a two-tailed unpaired Student's t test was used .

Values are expressed as means ± SEM. Differences were cons idered

stati stica ll y significant at p < 0.05 .

,--- - --------

162

RESULTS

Acrolein-induced apoptosis is mediated by activation ofA1APK~·

lt was reported that acro le in can act ivate MAPKs but their role in induction of

apoptosis is not known. Therefore, thi s study in vest igates the poss ible implication of

MAPK act ivation in acrolein-induced apoptosis. The changes in phosphorylation of

these kinases were examined following acrole in treatment in CHO cell s by Western

blot analys is using phospho-specific antibod ies. Treatment of ce ll s with acrolein (30,

50 fmol /cell ) for 1 h caused phosphorylat ion of both p38 (F ig. 1 A, 1 C) and ERK

MAPKs (F ig. 1 D, 1 F). The express ion of p3 8 (F ig. 1 A, 1 8) and ERK J /2 (F ig. 1 D,

1 E) in acrolei n-treated ce ll s did not change from the control. Although severa!

pub! ications have demonstrated th at activation of the J K and c-jun pathway leads to

ce l! death in brain ischemia [Whitfield et al. , 200 1; Domanska-Janik et al ., 2004], the

role of c-jun express ion and activation by extrace llular stimuli such as acro lein has

not been studied. Thus, we examined by Western blot analys is the phosphorylation of

the downstream JNK substrate c-jun by u in g anti-c-j un and phospho-c-jun

antibodies, respectively. Exposure to acrole in caused phosphorylat ion of c-j un (Fig.

1 G, 1 L), whereas expression of c-jun ( 1 G, 1 H) was unchanged.

Apoptos is signal regulating-kinase (AS K 1) is act ivated in response to various

cytotoxic stresses including TNF, Fas and reactive oxygen species such as hydrogen

peroxide. lt is an important upstream act ivator of .I NK and p38. Since, p38 and c-jun

were phosphorylated by acrolein, we asses ed wheth er acrol ein could act ivate ASK 1.

lndeed, there was a signifi cant increase in expre ion of ASK 1 upon exposure of ce ll s

to acrole in for 1 h (Fig. 1 K).

Given that severa! MAPKs were act ivated by phosphorylation fo ll owing

exposure to acrolein, we subsequently determined whether there is a link between

MAPKs and induction of apoptosis. Therefore, we examined whether inhibition of

these kinases would result in inhibition of acro lein-induced apoptosis. Exposure of

,-------------------------------------------------------------------------------------------------,

163

ce ll s to 50 fm ol/cell of acrole in fo r 4 h caused both necros1s and chromatin

condensati on (F ig. 28), a later event in the apoptoti c cascade, relative to untreated

con trois (F ig. 2A). Pretreatment of ce ll s with S8 203580, a p38 inhibitor (F ig. 2C) , or

UO 126, an inhibitor of ERK (F ig. 20 ), significantly reduced the number of acrolein­

induced apoptotic cell s from 29.26 ± 8. 12 to 5.04 ± 0.9 1 and 7.82 ± 1.67, respectively

(F ig. 2G). Moreover, SB203580 and UO 126 affo rded protection aga inst necrotic cell

death induced by acrole in (Fig. 2C, 20 , 21-I) . P098059, another ERK inhibitor, a lso

redu ced acrolein-induced apoptosis (Fig. 2E, 2G), but to a lesser extent than the ERK

inhibitor UO 126 . 1-I owever, inhibition of ERK with P098059 did not affect acrolein­

induced necrosis (F ig. 2H). When cell s were pretreated with the JNK inhibitor,

SP600 125, acrolein-induced ce ll death was swi tched l0rom apoptos is to total necros is

(F ig. 2F, 2G, 2H). Together, these findin gs suggest th at acrolein-induced activati on of

MAPKs is linked to the inducti on of apoptos is by thi s tox ic compound .

Acrolein-induced caspase activation is mediated by MAPKs

We subsequently investigated whether there could be a poss ible link between

the MA PK signaling cascade and other apoptotic events such as caspase acti vation,

during acrolein-induced apoptos is. lt was shown that caspase-3 is invo lved in MAPK­

medi ated apoptos is caused by stress-inducing agents such as cispl atin [1-Iershberger et

al. , 2002]. lt was previously reported that caspases-9 and -7 are invo lved in acro lein­

induced apoptos is, while caspase-3 was inhibited (Tanel and Averill-Bates, 2005].

Exposure of cells to 50 fm ol/cell of acrole in caused act iva ti on of initiator caspase -9

(F ig. 3A) and -8 (F ig. 38 ), and executor caspase-7 (F ig. 3C), relati ve to un treated

controls. Pretreatment of ce ll s with a p38 inhibitor, S8203580, the ERK inhibitors,

U0 126 and P098059, or a JNK inhibitor, SP600 125 inhibited acti vation of the

initiator caspase-9 (F ig. 3A), as weil as the effector caspase-7 (F ig. 3C). Furthermore,

ali of the four inhibitors inhibited cleavage of the caspase substrate lCAO by acrolein

(F ig. 30 , 3E). Caspase-8 activation by acrolein was not inhibited by any of the

164

MAPK inhibitors (Fig. 38 ). These results suggest that acro lein-induced caspase

act iva ti on is mediated in part by p38, JNK and ERK MA PKs.

Acrolein induces activation of the AKT survival pathway

Given that acrolein acti va tes the MAP Ks, whi ch are in vo lved in ce llular stress

responses, we subsequently determined wheth er acrole in could affect the survi val

pathways in cells. We examined whether acrolein co uld affect the AKT signaling

pathway. Indeed, exposure of ce ll s to acrole in (30, 50 fm ol/ce ll) resulted in a

signifi cant increase in AKT phosphorylati on (F ig. 4A, 4C), whereas express ion of

total AKT was not affected (F ig. 4A, 48) . It is li ke ly that inhibition of the AKT

surviva l pathway could increase the susceptibility of ce ll s to acrolein-induced cel!

death . The phannaco log ical inhibitors of the PI 3K/AKT signaling pathway,

Ly294002 (F ig. SC) and Wortmannin (F ig. 50), inhibited acrolein-induced apoptos is

(chromatin condensation) and switched the mode of ce l! death f rom apoptos is (F ig.

SB, SE) to near total necrosis (F ig. SC, 50 , SF). Moreover, Ly294002 and

Wortmannin inhibited caspase-9 (Fig. 6A) and -7 acti va ti on (F ig. 6C) by acro le in . In

contrast, these inhibi tors didn ' t affect caspase-8 act ivation by acrole in (F ig. 68 ).

Ly294002 and Wortmannin also inhibited cleavage of !CA O by acrole in (F ig. 60 ,

6E). These findin gs suggest that the acti vat ion of AKT by acrole in may be at !east in

part the result of a defensive response by the ce l! which activates a surviva l pathway

to protect against a cytotox ic aggress ion.

Activation ofp53 during acrolein-induced apoptosis

The ro le of p5 3 as a transcription facto r has been extensively described, as

have many transcripti onal target genes implicated in apoptos is, which include Bcl-2

proteins such as Bax and Bcl-2 [Attardi et al., 2000]. The phosphorylation status of

p53 plays an important role in the pro-apoptoti c act ivity of thi s prote in . Thus, the

abili ty of acrolein to phosphorylate p53 was in vesti gated. Immunoblots using anti-

165

phospho Ser15p53 and an anti-phospho Ser46p53 antibodies indicated that acro lein (50

jmol/cell ) caused p53 phosphorylation (F ig.7) under apoptotic conditions.

166

DISCUSSION

This study shows for the first time that acrolein-induced apoptos is In

proli fe rating ce lls is in part MAPK-dependent. Acrole in -in duced apoptosis was

described prev iously in va ri ous ce l! types [Tane l and Aver iii- Bates, 2005; Li el al.,

1997; Nardini et al. , 2002; Takeuchi el al., 200 1]. Acti vat ion of MAPKs by acrolein

was also reported in va rious ce l! types [Ranga nn a el al. , 2002; Takeuchi et al., 2001 ;

Misonou et al. , 2005; Pugazhenthi et al., 2006 ; Fin ke lste in et al., 2001 ; Deshmukh et

al., 2004]. However, previous studies did not address any poss ible lin ks between

activation of MAPK proteins in acrolein-induced apoptos is. Thi s study clearly shows

that acrolein-induced apoptosis occurred through acti vati on of the ASK llp38, and

ERK signaling pathways as upstream events fo r act ivat ion of apoptotic molecules

such as caspases -9 and -7, cleavage of ICA D and cond ensation of nuclear

condensati on.

Acrolein was shown to mediate apoptos is vi a the initiator caspase-9 and the

effector caspase-7, rather th an through caspase-3, in CHO ce li s [Tan el and A veri 11-

Bates, 2005]. Severa! studi es have reported that acrole in inhibi ts acti vity of the major

effector caspase-3 [Tanel and Averiii- Bates, 2005 ; Kern and Kehrer, 2002;

Finkelstein et al., 2005], probabl y through inhibition of its act ive site cysteine

res idue. The effecto r caspase-7 can be acti va ted by both of the initi ator caspases,

caspase-8 and caspase-9 [Siee et al., 1999]. 8oth of the effector caspases -3 and -7

share similar protein substrates and therefore caspase-7 is able to cleave caspase

substrates such as ICAD and liberate CA D, whi ch is responsible for chromatin

condensati on du ring the execution phase of apoptos is [Houde et al., 2004].

Three di stinct MAPKs, JNK, p38, and ERK have been characterized and

reported to be invo lved in apoptos is in many di ffe rent paradi gms of ce llular tox icity.

Although MAPKs play an important role in apoptos is, thi s is th e first study to show

that th ey play a role in acrolein-induced apoptosis. Acrolein was able to acti vate the

JNK substrate, c-jun, by phosphorylati on. 1-1 0\.vever, inhibition of JNK acti vity by

167

SP600 125 blocked acrolein-induced apoptosis, but in stead, the mode of ce ll death

was switched to necrosis. ln addition, SP600 125 inhibited acrole in-induced activat ion

of caspases-7 and -9 as we il as JCAD cleavage. Thi s can be expected since caspases

are not implicated in ce ll death by necros is. Our findin gs how th at JNK is act ivated,

but appears to be implicated in a ce ll surviva l ra ie under acro lein aggression. Others

studies showed that JNK is invo lved in apoptos is of lung carcin oma cells [Chuang et

al. , 2000] and murine macrophage cells [Kim and Sharm a, 2004], while it was not

assoc iated with apoptosis in HT4 neuronal ce ll s [Rockwe ll et al. , 2004] where p38

plays a major role. Moreover, JNK is also important fo r the deve lopment and urviva l

of macrophages [Himes et al. , 2006]. Thus, the fu nction of JNK in apoptos is is

controversial since it can play a role both as an activator of apoptos is or in surviva l

pa th ways.

The MAPK p38 also appears to be implicated in the activat ion of acrolein­

induced apoptos is. Acrolein induced activat ion of p38 by phosphorylation in ce lls and

SB203580, a p38 kinase inhibitor, inhibited acro lein-induced apoptos is through

inhibition of chromat in condensat ion. Furthermore, SB203580 inhibited activation of

caspases-9 and -7 by acro lein and cleavage of the caspase substrate ICA D. There was

no increase in ce ll death by necros is, as was the case fo r J K inhibition, but rather a

clecrease. These findings indicate that p38 plays a pro-apoptotic role in acrolein­

inducecl apoptos is. Although stress stimuli usuall y act ivate p38, which can lead to

apoptos is in sorne cellular models, it seems to be in·e levant in others. Act ivat ion of

p38 was required for apoptos is induced by cadmium, trop hi c facto r withdrawa l and

ischemia [Ga lan el al., 2000; Kummer et al. , 1997; Mackay and Mochly-Rosen,

1999]. On the other hand , SB203580 inhibition of p38 was unab le to prevent

apoptos is induced by UV and S-nitrosog lutathione [Franklin et al. , 1998 ; Ca ll sen and

Brune, 1999], whi ch highlights the contrad ictory co rrelat ion of p38 with apoptos is.

Sim i !ar con trad ictory results were obta ined in a recent study in HT4 neuronal ce li s,

where inhibition of p38, but not JNK, blocked apoptosis induced by cadmium

168

[Rockwell et al. , 2004] . This is consistent with our study, where inhibition of p38, but

not JNK, blocked acrolein-induced apoptos is. These di crepancies suggest the

ex istence of marked di ffe rences in regul ati on of th e stress re ponses depending on

ce l! types, albe it it is difficult to explain these diffe rence at the moment. Whether

JN K and 1 or p38 is implicated in apoptos is co ul d a lso depend on th e particular

ce llular insult in a given cel! type.

ln additi on to activation by a vari ety of growth factors, cytokines, and

mitogens, ERKs are also known to be acti vated by cytotox ic insults. This study

shows that ERK seems to be implicated in induction of apoptos is by acro lein.

Acrole in induced phosphorylation of ERK and the inhibiti on of its acti vity by UO 126

and PD98059, inhibi ted acrolein-induced apoptos is, characte rized by decreased

chromatin condensation. Moreover, UO 126 and PD98059 inhibited activation of

caspase-9 and -7 and cleavage of !CA O by acrolein . PD 98059 and UO 126 are MEK

inhibitors, and both are useful for probing the role of MEK in various biologica l

processes, particularly those involving activati on of the downstream substrate ERK.

Both inhibitors are noncompetitive with respect to the downstream MEK substrates

adenos ine triphosphate (ATP) and ERK [Favata et al. , 1998]. Although the binding

sites fo r PD 98059 and UO 126 on MEK 1 appear to over lap. their mechani sms of

action d iffe r [Dudley et al., 1995; Fava ta et al., 1998]. UO 126 is a direct inh i bi tor of

MEK activity, whereas PD 98059 inhibits MEK acti vati on. Therefore, UO 126 is a

much stronger inhibitor of the ERK pathway [Dudley et al., 1995; Favata et al.,

1998]. Researchers can exploit the di fferences and similarities of these two

compounds. For example, if an effect is inhi bited by both of these structura lly and

mechani st ically di stinct inhibitors, a much stronger argument can be made fo r MEK

play ing a ro le, than if only a single inhibitor was used. Furtherm ore, the se lect ivity of

PD 98059 fo r MEKI over MEK2 compared to the lack of preference fo r e ither by

UO 126 can be exploited to probe the relati ve roles of the two kinases. Cellular ERK

activation e ither inhibits or causes no effect on apoptos i in some cel! types [Chuang

169

et al. , 2000; Ga lan et al. , 2000], but enhances apoptos is in others [Lee et al. , 2006 ;

Ru ffe ls et al., 2004; Yang et al., 2004] . Even in th e same ce l! type, the role of ERK

act ivat ion is like ly to di ffe r, depending on ce llular in sults. ln SH-SYSY human

neuroblastoma ce ll s, for example, hydrogen peroxide induced apoptos is which was

inhibited by an ERK inhibitor [Ruffe ls et al. , 2004], whil e cadmium caused no

changes in ERK activation in the same ce l! line [Kim et al. , 2005]. Thi s suggests that

different cytotoxic insults are li kely to be linked to di ffe rent signaling molecules.

Among the MAPK kinase kinases (MAPKKKs) , ASK 1 is known to be

act ivated in response to various cytotox ic stresses, in cluding reactive oxygen spec ies

(ROS). Acrolein causes severe depletion of th e antiox idant glu tathi one [Kern and

Kehrer, 2002], which could lead to an increase in ce llular ox idat ive stress . lndeed,

acrole in caused an increase in intracellular reactive oxygen species in brain

mitochondria [Luo and Shi , 2005]. Thio l compounds uch as glutathione and N­

acety lcyste ine were shown to reduce acrolein-induced tox icity [Nunoshiba and

Yamamoto, 1999]. ASKJ can induce act ivat ion of the MAPKs, JNK and p38, as we il

as apoptos is [Tobiume et al., 2001 ]. Our findin gs clearly show that ASK I was

induced under conditions where acrolein can cau e apopto is. ASK 1 could be

responsible fo r activation of its downstream ta rgets p38 and JNK in acrolein-induced

apoptos is.

AKT (prote in kinase B or PKB) has been identifi ed as a downstream target of

growth factor receptor activation. ft is probab ly the most characterized kinase known

to promote cel! survival and to play a signifi cant ro le in glucose metabolism [Faber et

al. , 2006]. AKT was phosphorylated by acrole in and it inhibition by the inhibitors,

L Y294002 and Wortmannin , switched th e mode of ce l! death from apoptosis to

necros1s. LY294002 and Wortmannin are both inhi bitors of the lipid-mod ify ing

enzy mes known as phosphoi nos itide 3-k ina e (P I3-K), and both are useful for

probing the role of the Pl 3-K/AKT pathway in bio log ica l processes [Stei n and

Waterfield , 2000; Frum an, 1998]. Moreover, the e inhibi tors significantly decreased

170

activat ion of caspases-9 and -7, as we il as ICAD c leavage induced by acrolein .

Activation of AKT by phosphorylation prevents the pro-apoptot ic act ion of the Bcl-2

fam ily protein Bad at the mitochondri al membrane, by promoting its degradation .

Bad regulates apoptos is upstream ft·om mitochondri al-med iated caspase-9 act ivation.

These results show that AKT is implicated in the ce l! surviva l response aga inst an

acrole in insult. Akt/PKB is activated in response to many diffe rent growth facto rs,

horm ones, and external stresses such as heat shock and osmolarity [Datta et al. , 1999;

Law lor and Aless i, 200 1; Ta lapatra and Thompson, 200 1; Downward, 1998 ; Braz il

and Hemmings, 200 1).

The last nove l aspect of this study is that acrolein, which is a DNA damaging

agent [Esterbauer et al., 1991 ; Canman et al. , 1998 ; Bulav in et al., 1999], can induce

phosphorylation of p5 3. Our data documentee! for the first time thi s posttranslat ional

modification of p53 at p53 Ser15P and p53 Ser46 in acro lein-treated cell s. The p53

tumor suppressor protein is activated and phosphorylated at different serine residues

such as serine-! 5 and serine-46 in response to va ri ous DNA damaging agents such as

UV rad iat ion [Bulav in et al. , 1999). Phosphorylat ion of p53 on Ser15 reduces binding

of the mdm2 oncogene prod uct to p53 in vitro [Shi eh et al. , 1997], and binding of

mdm2 to p53 promotes rapid degradation of p53 by ta rget in g it fo r proteolytic

degradation, thereby potentia lly controll ing p53 prote in leve ls [Haupt et al. , 1997].

Effectively, acrole in induced p5 3 phosphorylat ion at erine-! 5, and as expected,

serine-46 was also phosphorylated, since act ivated p38 has been shown to target

erine-46 [Bulav in et al. , 1999]. The downstream events mediated by p53 take place

by two major pathways: cell cycle arrest and apo ptos is. As a transcripti on factor,

phosphorylated p53 induces transcripti on of proapoptotic proteins such as Bax and

Fas li gand and represses those of anti apoptotic protei ns uch as Bcl-2 [Bras et al.,

2005].

ln conclusion, the present study identifi ee! p53 , p38 and ERK as key

molecules invo lved in acrolei n-induced apoptosis and that CHO ce ll s appear to use

17 1

the AKT/PKB and the INK MAPK survival pathways to overcome ce ll death induced

by acrolein . These findings are relevant to the tox icity of acro lein in many contexts,

including the pharmacological action and/or tax ie si de effects of the anticancer agent

cyc lophospham ide, the regulation of ce li ular proliferation and tu mor growth by

po lyamines, as weil as the toxicity of environmental expo ures to low doses of

acrolein .

B

ClO (")

c. .,__ 0

~

Figure 1: Acrolein induces activation of p38 and ERK MAPKs, the JNK

substrate c-jun and ASKl

1.25

A Acrolein (fmollcell)

p38 __.. r-1 --'------'--"------,1 (43 kDa) :=========:

Phospho-p38 __.. 1 j (38 kDa)

-

Tubulin __.. j .. ~~~c~~=-----~

ClO (")

2 .5

9- 2 c. -0

***

·~ 0.75 ~ 1.5 ëii

** Q) ..... 1::

0.5 CJ.)

> :;::; ~ Qi 0.25 0::::

1:: CJ.) ..... 1::

Q)

~ 0.5 ~

o+-~0~~~3-0~~~5-0~-

Qi 0::::

0 30

D

Acrolein (fmol/cell)

Acrolein (fmollcell)

ERKl __.. :====:=======: ( 43 kDa)

ERK2 __.. 1 1 (42 kDa)

0 30

Phospho ERK __.. 1

Tubulin __.. ~csr ............. ii'. =====e9

F

***

172

H

1: 1.25 .=.. u ..... 0 >. ..... ïii 0.75 1: Q) ..... 1: 0.5 Q)

> ~ 0.25 a; rx:

0 0

G 0 30 50 Acrol ein (fmol/cell)

K

c-jun - 1....,.. ...._. - 1 (39 kD a)

Phospho c-jun - 1 .,.,. ...,_.. - 1 (39 kDa)

Tubu lin -

1

30 50

§ 4 ·~

0.. ..... 3 0 >. .....

·~ 2 Q) ..... = Q)

> :;; RI a; rx: 0

Acrolein (fmol/cell)

0 50

ASKl ------..

0

(155 kDa )

,-------- .. .. - ~~·· j

1,0 2,7

Tu bu lin ------..

***

30

Acrol ein (fmol/cell )

Fo ld Difference

*"'*

50

173

Figure 2: Inhibition of p38 and ERK MAPKs decreases acrolein-induced

chr·omatin condensation

CTL Acrolein 50 f mol/cell Acrolein + SB203580

Acrolein + U0126 Acrolein + PD98069 Acrolein + SP600126

G H

40 100 ***

.!!! 30 75 fi) o CTL

fi) o CTL 0 fi) - D+SB203580 0 EI+SB203580 Q. ... &.zo •+U0126

u 50 • +U0126 Q)

<( rl+PD98059

z ~+PD98059 - -0 C+SP600125 0 O+SP6001 25

~ ~ 0

10 0

25

o~~~~~~ o~----~~~ 0 50 0 50

Acrolein (fmollcell)

174

175

F igure 3: Acrolein-induced activa tion of ca pase-9 a nd caspase-7 a nd cleavage of

A

B

ICAD a re decreased by inhibito rs of p38 and ERK MAPKs

1.25

>. ... ·::; 1 +:: (.J

cu a> 0.75

1 Q) Ill cu 0.5 c.. Ill cu u

0.25

0

1.5

1.25 >. -:~ - 1 (.J

cu

~ 0.75 Ill cu g. 0.5 cu u

0.25

***

0 50

Acrolein (fmol/cell)

0 50

Acrolein (fmol/cell)

o CTL

0 SB203580

• U0126

Ci PD98059

B SP600125

0 CTL

0 SB203580

• U0126

!:l PD98059

Cl SP600125

c 1.5

>. 1.25 -·::;: :.;:::::; 1 i 0.75j ~ 0.5 ca u

0

0

D lnhi bit or

Acrolein + :so j mol/ce

ICAD ---<

Tu bu lin -+

E 120

c:: 100 .!2 tl)

80 tl) C1l ... c.. 60 >< C1l

0 40 <(

~ 20

0

***

li • 0 50

Acrolein (fmol/cell)

58203580 U0126 ---+ + + +

+ + +

0 50

Acrolein (fmol/cell)

0 CTL

o SB203580

• U0126

D PD98059

Q SP600 125

PD98059

+ +

o CTL

o SB203580

• U0126

0 PD98059

Cl SP600 125

+

SP600125

+ +

+

176

- --- - --------

177

F igure 4: Acrolein induces activa tion of AKT

A 0 30 50 Acrolein (fmol/cell)

AKTtotal -(59 kDa) ~===:;;;;;;;;;;;==.;;;;;;;;;;;;;;;;~

Phospho AKT -(65 kDa) ~;;;::;::===::::::=::~ Tubul in - 1.- ~

c 2 **

~ ** <(

6..1 . -0 >. -(/) 1 s::: Q) -s:::

Figure 5: Inhibition ofPI3K/AKT pathway by LY294002 or Wortmannin

enhances necrosis induced by acrolein

CTL Acrolein 50 fmol/cell

Acrolein + L Y294002 Acrolein + Wortmannin

178

%o

f A

po

pto

sis

m

.....

N

w

,J:o.

0 0

0 0

0

rn [S

I D

+

+

0 ~

r -;

-<

0

0 N

r

;:::!.

c.o

3

~

Ol

0 ::

J 0

::J

N

::J

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*

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(11

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f N

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s 'T

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..,

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0

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+

+

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~

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-1

0 0

N

r ;:::

!. c.o

3

~

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0 ::

J 0

::J

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::J

(11

0

180

Figure 6: Effects of AKT inhi bitors on acrolein-induced activa tion of caspases-9,

-8 and -7 a nd cleavage of ICAD

A

B

1,25

1

0,75

0,5

0,25

1,5

1,25 >. -:~ -0 cu co

1 Q) Il) cu a. Il) cu (.)

1

0,75

0,5

0,25

***

0 50

Acrolein (fmol/cell)

0 50

Acrolein (fmol/cell)

o CTL

~ LY294002

g Wortmannin

o CTL

a LY294002

t:1 Wortmannin

c 1,5

1,25 >. -~ 1 (.) ns ";- 0,75 Q) Il)

[ 0,5 Il) ns u 0,25

***

0 1 ' V«t=? VVe=? 0 50

Acrolein (fmol/cell)

o CTL

i!'l LY294002

g Wortmannin

D

E

lnhibitor

120

r::::: 100 .2 Ill ~ 80 .... c.. ~ 60 c ct 40 u

20

+

LY294002 Wortmannin

+ + +

+ + +

--~~~

o CTL

1:1 LY294002

8 Wortmannin

0 1 1 ~ 1 ~ 0 50

Acrolein (fmol/cell)

181

182

Figure 7: Acrolein induces phosphorylation of p53 at serlS and ser46 as weil as

activation of ASKl

0 50 Acrolein (fmol/cell)

p53 -(53 kDa)

Phospho-p53 (Ser46) -{53 kDa)

1,0 Fold Difference

Phospho-p53 (Ser15) - - •• (53 kDa)

*** Fold 1,0 5,5 Difference

Tubulin -

183

FIGURE LEGENDS

Fig. 1 Acrolein induces activation of p38 and ERK MAPKs, the JNK substrate c­

jun, and ASKl. Cells ( 106/mL) were incubated with acro le in for 1 h and

immunodetection of (A) p38 and p-p38, (D) ERK 1/2 and p-ERK, (G) c-jun and p-c­

jun and (K) ASK 1 was carried out by Western blotting, using ~-tu bu lin as a load ing

contro l. Representative blots are shown fro m five independent experiments.

Densitometric analyses of the express ion of (B) p38, (C) p-p38, (E) ERK 1/2, (F) p­

ERK, (H) c-j un , (I) p-c-jun and (K) ASK 1 are relat ive to the untreated control. Data

represent means and SEM fro m five independent experiments perfonned with

multiple estimat ions per point. P<O.O 1 (**)or P<O.OO 1 (***) indicates a statist ically

signifi cant difference between treatment with acrolein and the untreated control.

Fig. 2. Inhibition of p38 and ERK MAPKs deueases acrolein-induced chroma tin

condensation. Confluent ce lls in monolayer were pre-incubated for 1 h with (C) 5

)...t.M SB203580 (p38 MAPK inhibitor), (D) 10 !-LM UOI26 (MEK I/2 activity

inhibitor), (E) 50 !-LM PD98059 (inhibitor of MEK I/2 activation), or (F) 10 !-LM

SP600 125 (JNK inhibitor) before addition of acrole in . Ce ils were then incubated with

(A) 0 (CTL) or (B-F) 50 fmollcell of acrolein fo r 4 h. The fract ions of (G) apoptot ic

and (H) necrotic cell s are given relative to total cell s. Apoptot ic cells were visualized

by fluorescence microscopy using Hoechst dye (b lue fluo rescence) and necrotic cells

using propidium iodide (red fluorescence) (magnificat ion 320x). Data represent

means and SEM from five independent experiments performed with multiple

estimations per point. P<O.OOI (***) indicates a statist ica lly significant difference

between treatment with acro le in and the control. P<O.OO 1 (~~~) indicates a

stati stically significant di fference between ce ll s exposed to acrole in, with and without

inh i bi tor.

184

Fig. 3. Acrolein-induced activation of caspase-9 and caspase-7 and cleavage of

ICAD are decreased by inhibitors of p38 and ERK MAPKs. (A , 8 , C, D)

Pretreatment of cell s with 5 ).LM SB203580 (p38 MAPK inhibitor), 10 ).LM UO 126

(M EK 1/2 act ivity inhibitor), 50 ).LM PD98059 (inhi bitor of M EK 1/2 activat ion), or 1 0

).LM SP600 125 (JNK inhibitor) was perfo rmed fo r 1 h before add ition of acro lein.

CHO ce ll s (0.5x l 06/JnL) were then incubated with acrolein (50 fmol /cell) for (A, B)

1 h or (C) 2 h in u-MEM with 10% FBS. Activiti es of (A) caspase-9, (B) caspase-8

and (C) caspase-7 were measured in ce ll lysa tes using the flu orescent substrates Ac­

LEHD-AFC, Z-IETD-AFC and MCA-VDQVDGWK(DNP)-NH 2, respective ly.

Caspase act ivity was expressed relative to the untreated control, des ignated as 1. Data

represent means and SEM from eight independent experiments performed with

multiple estimations per point. (D) Cell s ( 106/mL) were incubated with acrolein for 2

h and immunodetection of iCAD was carried out by Western blotting, using 13-tubu lin

as a loading control. A representative blot is shown fro m tive independent

experiments. Densitometric analysis of the express ion of (E) ICAD is re lat ive to the

untreated contro l. Data represent means and SEM from five independent experiments

performed with multiple est imat ions per point. P<O.OO I (*** ) indicates a statistically

signifi cant difference between treatment with acro lei n and the untreated control.

P<0.05 (~), P<O.O 1 (~~) or P<O.OO 1 (~~~) indicates a statist ica lly signiticant

di ffe rence between cells exposed to acrolein, with and without inhibitor.

Fig. 4 Acrolein induces activation of AKT. (A) Ce ll s ( 1 06/JnL) were incubated with

acrolein for 1 h and immunodetection of AKT and p-AKT was carried out by

Western blott ing, using 13-tubulin as a loadi ng contro l. A representat ive blot is shown

from five independent experiments. Densitometric ana lyses of the expression of (B)

AKT and (C) p-AKT are relative to the untreated control. Data represent means and

SEM from five independent experiments performed with multiple estimations per

185

point. P<O.O 1 (**) indicates a statistically significant difference between treatment

with acro lein and the contro l.

Fig. S. Inhibition of PI3K/AKT pathway by LY294002 o1· wortmannin enhances

necrosis induced by acrolein. Confluent cells in monolayer were pre-incubated for 1

h with (C) 50 j.lg/1 Ly294002 (Pl3-K inhibitor) , or (D) 1 00 11 M Wortmannin (PI3-K

inhibitor) before addition of acro lein. Ce ll s were then incubated with (A) 0 (CTL) or

(B-D) 50 fmollcell of acro lein fo r 4 h. The fractions of (E) apoptotic and (F) necrotic

ce ll s are given relative to total cells. Apoptotic ce ll s were visualized using Hoechst

dye (blue fluorescence) and necrotic ce ll s usin g propidium iodide (red fluorescence) .

Data represent means and SEM from :five independent experiments performed with

multiple estimat ions per point. P<O.OO 1 (***) indicates a statistically signi:ficant

difference between treatment with acrolein and the untreated control. P<O.OO 1 ( ~~~)

indicates a statist ically signi:ficant di ffe rence between ce ll s exposed to acrolein, with

and without inhibitor.

Fig. 6. Effects of AKT inhibitors on acrolein-induced activation of caspases-9, -8

and -7 and cleavage of ICAD. (A, B, C) Pretreatment of cell s with 50 j.lg/1

Ly294002 (Pl3-K inhibitor), or 100 11M Wortmannin (PI3 -K inhibitor) was

performed for 1 h before addition of acrolein . CHO cell s (0.5x 1 06/mL) were then

incubated with acrolein (50 fmol /cell ) for (A, B) 1 h or (C) 2 h in a-MEM with 10%

FBS. Activities of (A) caspase-9, (B) caspase-8 and (C) caspase-7 were measured in

ce l! lysates and were expressed relative to the untreated contro l, des ignated as 1. Data

represent means and SEM fro m eight independent experiments performed with

multiple estimations per point. (0) Cell s ( 1 06/mL) were incubated with acrolei n for 2

h and immunodetection of lCAD was carried out by Western blotting, using !3-tubulin

as a load ing control. A representative blot is shown from five independent

experiments. Densitometric analys is of the express ion of (E) ICAD is relative to the

186

untreated control. Data represent means and SEM from five independent experiments

performed with multiple estimations per point. P<O.OO 1 (***) indicates a statistica lly

significant difference between treatment with acro le in and the control. P<O.O 1 (~~)or

P<O.OO 1 (~~~) indicates a statistically significant difference between cell exposee! to

acrolein, with and without inhibitor.

Fig. 7. Acrolein induces phosphorylation of p53 at serlS and ser46. Ce lls

( 1 06/mL) were incubated with aero le in fo r 1 h and immunodetection of p53, p-p 53

(ser iS) and p-p53 (ser46) was carried out by Western blotting, using ~-tubulin as a

load ing control. Representative blots are shown from five independent experiments.

Densitometric anal y es of the express ion of p53 , p-p53 (ser 15) and p-p53 (ser46) are

relat ive to the untreated control. Data represent means and EM from five

independent experiments perfonned with multiple estimat ions per point. P<O.OO 1

(***) indicates a statisti cally signifi cant difference between treatment with acrolein

and the control.

187

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caspase-3. FASEB J 18:1782-1784, 2004.

2.5. ARTICLE IV

INHIBITION OF ACROLEIN-INDUCED APOPTOSIS

BY THE ANTIOXIDANT

N-ACETYLCYSTEINE

André Tanel and Diana A. Averi ll-Bates 1

Département des sciences biologiques, TOXEN, Université du Québec à

Montréal, CP 8888, Succursale Centre Ville, Montréal, Québec, H3C

3P8, Canada.

Corresponding author: Dr Diana A. Averill-Bates, Département des sciences bio logiques, Université du Québec à Montréal, CP 8888, Suceur ale Centre Ville Montréal , Québec, H3C 3P8, Canada Tel : 514-987-3000 (4811), Fax: 514-987-4645 Email: averill .diana@uqam.ca

Abbreviations: AFC: amino trifluorocoumarin; AIF: apoptosis inducing factor; Apaf-1 : apoptotic protease activating factor; CAD: caspase activated DNase; CHAPS: 3-[(3-cholamidopropyl)dimethylammonio ]-2-hydroxy-1-propanesulfonic acid; CHO: Chinese hamster ovary; FBS : fetal bovine serum; FCCP: p-trifluoromethoxy-phenyl­hydrazone; GSH: reduced glutathione; ICAD: inhibitor of caspase activated DNase; MEM: minimum essential medium; MMP: mitochondrial membrane potential; MOPS: 3-(N-morpholino)-propane sulfonic acid ; AC: -acetylcysteine; PARP: polyADP-ribose polymerase; PBS: phosphate-buffered saline; PI: propiditm1 iodide; PMSF: phenylmethylsufonyl fluoride; SDS-PAGE: sodium dodecyl sulphate­polyacrylamide gel electrophoresis; PTP: permeability transition pore; y-GCS : y­glutamyl cysteine synthetase; Z-IETD-AFC : -CBZ-L-isoleucyi-L-glutamyl-L­tlu·eonyl-L-aspartic acid an1ide; Ac-LEHD-AFC: Ac-LEHD-AFC:Acetyi-Leu-Glu­His-Asp-7 -amino-4-trifluoromethylcoumarin .

Financial support was obtained from CIHR (Canadian Institutes of Health Research) (DAB). AT was the recipient of the Bourse Francine Beaudoin-Denizeau for PhD studies from the Fondation UQAM.

200

RÉSUMÉ

L 'acroléine est un aldéhyde a, ~-in saturé auquel les hu ma ins sont exposés

dans de multiples situations. C' est un po lluant environnemental qui cause plusieurs

maladies respiratoires et neurodégénérati ves dont l ' A lzheimer. La présente étude

examine l ' hypothèse que le N-acéty lcysté ine (NAC), un précurseur du glutathion,

peut protéger contre la tox icité ce llulaire engendrée par l ' acroléine. L ' expos ition des

ce llules CHO à une concentration non-cytotox ique d'acroléine (4 fmol/ce ll), a

diminué le glutathion intracellulaire de 45 % de on taux initial. Le NAC a augmenté

le ni veau de glutathi on intracellulaire et offert une protecti on contre la cytotoxicité et

l ' apoptose induite par l 'acroléine en inhibant la vo ie mitochondriale. Le NAC a

inhibé la translocation de Bad à la mitochondrie et la baisse du Bcl-2 mitochondriale

induites par l 'acroléine et cec i est démontré par immunobu vardage de type Western .

Par contre, le N AC n'a pas d 'effet sur la libérati on du cytochrome-c et la

translocati on de Bax induites par l ' acroléine. Cependant, le NAC a inhibé la baisse du

potentiel membranaire révé lée par cytométrie de fl ux, le cli vage de la procaspase-9,

l ' activati on des caspases-9, -7 et -8 ainsi que le cli vage de PA RP. L ' inhibition par

N AC de l ' apoptose induite par l ' acroléine a été conf irmée morpholog iquement par la

baisse de la condensation de la chromatine nucléa ire. Ces résultats suggèrent que

NAC pourrait être env isagé comme un antidote pour traiter les gens exposés à

l ' acroléine.

20 1

ABSTRACT

Acro lein is a hi ghly electrophilic a ,0-unsatu rated aldehyde to which humans

are exposed in many situations. ft is an environmental po llutant th at is responsible for

multiple respiratory di seases and has been implicated in neurodegenerative di seases

such as Alzheimer' s di sease. The hypothes is of the study is that the antioxidant N­

acety lcyste ine (NAC), a precursor of glutathi one, cou Id protect cell s against acrolein­

induced apoptos is. Exposure of CHO ce ll s to a non-cytotox ic dose of acrolein (4

fmo l/cell) dep leted intrace llular glutathione to 45% of initial levels. NAC, whi ch

increased intracellular glutathione leve ls by 30%, afforded protecti on aga inst

acrolein-induced cytotoxicity (loss of ce l! pro li fe rat ion) and apoptos is. NAC

protected against apoptos is by dimini shing acrolein-induced act ivat ion of the

mitochondrial death pathway. NAC inhibited acrolein-induced Bad trans locati on

from the cytoso l to the mitochondri a, as weil as Bcl-2 translocation from

mitochondria to the cytoso l, evaluated by Western blotting. HO\.Yever, NAC had no

effect on acrolein-induced Bax translocation to mitochondri a and cytochrome-c

liberation into the cytoso l. Meanwh il e, NAC inhibited depolarisation of

mi tochondrial membrane potential, eva luated by rhodamine fluorescence using flow

cytometry. NAC also inhibited procaspase-9 process ing, act ivat ion of enzymat ic

act ivity of caspases -9, -7 and -8, as weil as PARP cleavage induced by acrolein.

fnhibiti on of acrolein-induced apoptos is using NAC was confirmed morpholog ically

by diminished condensation of nuclear chromatin , eva luated by flu orescence

mi croscopy. These findin gs suggest that NAC could be potentia lly useful as a

protect ive agent for people exposed to acrolein .

202

INTRODUCTION

Acrolein is a highly reactive, a, ~-unsaturated aldehyde pollutant to which

humans are exposed in multiple situations (Kehrer and Biswa l, 2000) . lt is a major

component of smoke from cigarettes, forest and house tires, and is a constituent of

automoti ve exhaust. lt has been implicated in the deve lopment of atheroscleros is and

va ri ous lung di seases including chronic obstru cti ve pulmonary di sease (Borchers et

a l. , 1999) . ln addition, acrolein is found in the vapeurs of overheated cooking oil

where severe human tox ic exposures have occurred (Beauchamp et a l. , 1985). As one

of the tox ic products of endogenous 1 ipid perox idation (Adams and Klaidman, 1993),

acrole in has been im plicated in chronic neurodegenerative di sorders such as

Alzheimer's di sease (Lovell et a l. , 2002; Pugazhenthi et a l. , 2006) . Acrolein is a

metabolic product of the anticancer drug cyc lophosphamide, where it may be

in vo lved in its therapeuti c effect as we il as its tox ic side effects (Schwartz and

Waxman, 2001 ).

Acrolein tox icity has been demonstrated in many di fferent ce llular systems. lt

is known to induce apoptos is in severa! ce l! types such as keratinocytes (Takeuchi et

al. , 200 1 ), neutrophils (F inkelstein et al. , 2005), cultured neurons (Pugazhenthi et al. ,

2006), and Chinese hamster ovary (CHO) ce ll s (Tanel and Averiii-Bates, 2005),

while necros is occurs in others (Luo et al. , 2005; Liu -Snyder et a l. , 2006).

Apoptos is is a phys iolog ica l ce l! dea th process which plays an

important role during development and maintenance of tissue homeostas is (Chandra

et al. , 2000) . The mitochondri al pathway of apoptos is is tri ggered by di fferent

stresses such as growth factor deprivati on, ionizin g radi ati on, corti co-stero ids and

anti cancer drugs . Anti-apoptotic (Bcl-2 and Bel-XL) and pro-apoptotic (Bax, Bad)

members of the Bcl-2 family are thought to control apoptos is by modulating release

of pro-apoptoti c molecules such as cytochrome-c and apoptos is-inducing factor (AJF)

from mitochondri a. Bcl-2-Bax interactions at the outer mi tochondri al membrane are

203

known to pre vent induct ion of the m itochond ria l apoptot ic pathway. T rans locat ion of

Bad to mitochondria is necessary for promoting fo rm at ion of po res cons istin g of Bax­

Bax dimers on the outer mitochondri a l membrane, whi ch may be respo nsible for the

re lease of pro-apoptotic factors (Orreniu s et al., 2003). Bad trans location to the

mitochondria removes the inhibitory effect of Bc l-2 on Bax, by in teract ing with Bcl-2

itse lf. The Bcl-2 fami ly has two types of pro-apoptotic members: the Bax and Bak

subfamily and the BH3-only members such as Bid, Bad , Bim/Bod and PUMA

(Huang and Strasser, 2000) . Studies with gene-targeted cell s indicated that the

presence of Bax or Bak is req uired for many fo rm s of apoptos is (L indsten et a l. ,

2000), and each type of cel! needs at !east one of the anti-apoptotic Bcl-2 fa mily

members to survi ve (Cory and Adams, 2002 ; We i et a l. , 2000). The BH3-only

proteins are activated in response to various apoptot ic stimuli and a re a ble to indu ce

apoptos is through interacting directly w ith Bax and Bak (Wei et a l. , 2000) or binding

to the hyd rophobi e groove of anti-apoptotic members such as Bcl-2 or Bel-xL, thus

removing their inhibitory effect on pro-apoptotic mo lecules such as Bax and Baie

1 nteraction of cytochrome-c and dA TP with apoptos is protease act ivat ing

factor (Apaf-1 ) in the cytosol leads to convers ion of pro-cas pase-9 to active caspase-9

through auto-proteo lyt ic cleavage (Chandra et a l. , 2000). Ca pase-9 subseq uently

activates effector caspases such as caspases-3, 6 and 7 (Sa lvesen and Dixit, 1997).

Effector caspases then c leave cellul ar protein substrates such as polyADP ribose

polymerase (PARP), lamins, ge lso lins, fodr ins and inhibi tor of caspase act ivator

DNase ( ICAD). T he ce l! then exhibits the characteri stic morphological features of

apoptos is such as chromat in condensat ion, cytoske leta l changes, nuclear membrane

breakage, cel! blebbing and fo rmation of apoptot ic bodies, whi ch are th en

phagocytosed by macrophages. E limination of these dying ce ll s avo ids an

intlammatory response in surrounding ti ssues, such as occu rs during necros is.

Acrole in wi ll rapidly bind to and dep lete cellu lar nucleophiles such as

gl utathi one (Heck, 1997). The antioxidant g lu tathione appears to have an important

ro le in the detoxification of acrole in (Finkelstein et a l. , 200 1 ). Ce llular cytotoxic

204

responses to acrolein are likely to depend on in trace llular glutathione leve ls. lt is

li ke ly that increased glutathione leve ls co ul d attenuate tox icity of acrolein . To

increase g lutathione leve ls, the thiol-containing compound , N-acety i-L-cyste ine

(NAC) can be used as a glutathione precursor, since glu tathione itself does not eas ily

penetrate cell s (Roberfroid and Ca lderon, 1995). NAC also increases cysteine poo ls

inside cell s and acts as a thi ol-containing reducing agent (Cotgreave, 1997). Thiol

compounds such as NAC could be potentially useful fo r protecti ng tissues and ce ll s

from acrolein-induced toxicity. Therefore, the objective of thi s study is to investigate

whether NAC can protect ce ll s aga inst acrolein-induced cytotox icity and act ivation of

the m itochondrial pathway of apoptosis.

205

MATERIALS AND METHODS

Cel/ culture

CHO cell s (A uxB I) were grown in monolayer in minimum essential medium­

Alpha (o.-M EM) (G ibco Canada, Burlington, ON) pl us 10% feta l bov ine serum (FBS)

(G ibco Canada) and 1% penicillin (50 units/mL)-streptomycin (50 ~Lg/mL) (F low

Laborato ries, Mississauga, ON), in tissue cul ture flasks (Sarstedt, St Laurent, QC), in

a humidified atmosphere of 5% C02 in a water jacketed incubator at 37°C (Lord­

Fontaine and Averill , 1999) . The ce ll s were grown to near confl uence and were then

incubated fo r 24 h with fres h cul ture medium . Confluent ce ll s were then harvested

using PBS-citrate, washed by centrifugation ( 1 OOOg, 3 min) and resuspended in o.­

MEM for experimental studies .

Pretreatment witlt NAC

Pretreatment of cell s with 1 mM NAC (S igma Chemical Co., St. Louis, MO)

was performed fo r 24 h on confluent cell s in monolayer (Lord-Fontaine and Averill ,

1999). Ce lls were subsequentl y washed three times to remove NAC and then

harvested. Intact cell s were tested fo r ce ll survival, total glutathione levels and

ana lysis of apoptos is. Treatment with NAC did not cau e any loss of ce ll viab ility,

determ ined by the trypan blue exclusion assay. Plat ing effic iency did not decrease

relative to contro l cell s.

Dosage ofglutathione

Tota l g lutathione was determined as previously described (Lord- Fontaine and

Averill , 1999) . The rate of formation of 5-thio-2-nitrobenzo ic acid , which is

proportional to total glutathione concentration, was fo ll owed at 412 nm by an

enzymatic assay using gl utathi one red uctase. Samp le va lues were calculated from a

standard curve of nm ol glutathione versus rate and expressed as nmol/ 106 ce li s.

206

Clonogenic cytotoxicity assay

Cytotoxicity was eva luated as the ab ility of ce l! to proliferate to form

macroscop ic co loni es after a tax ie in ult with acro lein , using a clonogenic ce l!

survival assay (Lord-Fontaine and Averill, 1999). Ce li s ( 1 05/mL) were incubated with

acrole in (A ldrich Chemical Co, Milwaukee, Wl), in a-MEM containing 10% FB for

1 h at 37 °C. Cytotox icity was expressed as the mean number of macroscopic

co loni es (>50 cell s) obtained relative to the mean number of co lonies obtained in the

control.

MoJyJito/ogical analysis of apoptosis

To vi ualize nuclear morphology and chromatin condensation, ce ll s were

incubated with acro lein for 4 h and then apoptot ic ce ll s were tained with 1-loechst

(33258) (0.06 mg/mL) (Tane l and Averi ii -Bates, 2005). Propid ium iodide (P l) (50

~tg/mL) was added to stain necrotic ce ll s. Observati ons were made by fluorescence

microscopy (Carl Ze iss Ltd, Montrea l, QC) and photographs were taken by digital

camera (camera 3CCD, Sony DXC-950P, - mpix lmaging lnc, Miss issauga, ON).

Images were analysed by Northern Eclipse oftware. The fractions of apoptotic and

necrotic ce lls were determined relative to total ce ll s (obta ined using bright field

illumination) . A minimum of 600 ce ll s was counted per di sh.

Determination of ca spa se activity by fluoresceuce spectroscopy

Freshl y harvested cell s (0.5 x 1 06) were incubated with acrolein in a-MEM at

37°C. Cells were lysed by freezing at -20°C for 20 minutes and reaction buffer (20

mM piperazine-N,N'-bis(2-ethanesul fo nic ac id) (P l PES), 100 mM a Cl, 10 mM

dithiothreitol , 1 mM EDTA, 0. 1% 3-[(3-cholamidopropy l)dimethylammonio ]-2-

hydroxy-1-propanesulfonic acid (Cl-l APS) , 10% ucrose, pH 7.2) was added

(Stennicke and a lvesen, 1997) . The kinetic reaction was started after addit ion of the

appropriate ca spa e su bstrate at 3 7°C using a spectrofluori meter ( pectra Max

207

Gemini , Molecular Dev iees, Sunnyva le, CA) (Ta nel and Averiii-Bates, 2005).

Ca pase-7 acti vity was measured by cleavage of the flu orogeni c substrate 1 MCA­

VDQYDGWK(DNP)-NI-b (Ca lbiochem, La .J a ll a, CA) with À.max excitati on at 325

nm and À.max emiss ion at 395 nm . Caspase-8 act ivity was measured by cleavage of

the substrate Z-IETD-AFC to produce trifluorocoum arin (AFC) with À.max exc itati on

at 4 15 nm and À.max emiss ion at 490 nm . Caspase-9 acti vity was measured by

cleavage of Ac-LEHD-AFC to produce AFC.

Subcellular fractionation and immunodetection of Bad, Bax, Bcl-2, cytochrome-c,

caspase-9 and PARP

Fo llowing treatment with acrole in , cells were washed in buffer A ( 1 00 mM

sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4) and resu pended in buffe r B [buffe r A

plus 5% Perco ll , 0.01 % digitonin and a cocktail of protease inhibitors: 10 ~LM

aprotinin, 10 ~LM pepstatin A, 10 ~LM leupeptin, 25 ~M ca l pa in inhibitor I and 1 mM

phenylmethylsul fo nyl flu oride (PMSF)]. After 30 min incubat ion on ice, lysates were

homogenised using a hand patter (Kontes glass CO, Dual! 22, Fisher, QC). Unbroken

ce ll s and nuclei were pell eted by centifugati on at 2500 g fo r 10 min . The supernatant

was centri fuged at 15000 g for 20 min and the re ultant pe ll et was des ignated as the

mitochondrial fracti on. A further centri fugation of the supern ata nt fraction at 1 00 000

g fo r 1 h resulted in a pellet fraction des ignated as the mi crosomal fracti on, whereas

the supernatant was des ignated as the cytoso lic fraction (Samali et al. , 1999) . For

immunodetection of caspase-9 and PARP, who le ce l! lysa tes were used.

Proteins (3 0 ~g) were quantified according to Bradfo rd (Bradfo rd, 1976),

separated using a 15% SDS-polyacrylamide ge l and then transferred to a

polyv inylidene difluoride membrane, as prev iously described (Tanel and Averiii­

Bate , 2005). The blots were probed with the fo llow ing primary antibod ies: anti-Bad,

anti- Bax, anti-Bcl-2, anti-caspase-9, ant i-PARP (Sa nta Cruz Biotechnology, Santa

Cruz, CA) or anti-cytochrome-c (BD Biosc iences Canada, Miss issauga, ON).

Secondary an ti bodies ( 1: 1 000) consisted of horseradi sh perox idase (HRP)-conjugated

- - -----------

208

goat anti-mouse, anti-rabbit and anti-goat lgG (B ioso urce, Camarill o, CA). Proteins

were detected using the ECL plus chemiluminescence kit (Perkin Eimer, Boston, MA)

and protein express ion quantified using a scanning la er densitometer (Molecular

Dynamics, Sunnyva le, CA). Purity of mitochondri al and cytoso lic tl·actions was

verified using antibodi es to cytochrome-c ox idase (Santa Cruz Biotechnology) and

GSTn 1 (Calbiochem), respecti ve ly. Caspase-9 and PARP express ion in whole ce l!

lysates was quantified using JPGEL software, relati ve to ~-tubulin loading controls.

Flow cytometry analysis of mitochondrial membrane potentia/ (Aiflm)

Freshly harvested ce ils (1 06) were incubated with acrolein, or the pos itive

contro l p -trifluoromethoxy-phenyl-hydrazone (FCCP), in a -M EM fo r 1 h at 37 oc. To measure /).'fm , ce lls were incubated with rhodamine 123 (800 ng/mL) fo r 5 min

(Tanel and Averiii-Bates, 2005) . PT was added to identi fy dead ce ll s. Cell s were

analysed with a FACScan fl ow cytometer equipped with an argon laser emitting at

488 nm (Becton Dickinson, Oxford , UK). Data was acq uired and analysed using

Lys is II software (Becton Dick inson). The mean flu orescence in tensity of 20,000

cell s was ca lculated fo r each sample. Rhodamine 123 fl uorescence was detected on

the FL 1 detector and Pl fluorescence on the FL2 detector. Fo r each sample, only li ve

cell s were selected, and their mean flu orescence in FL 1 was analysed. Apoptotic

ce ll s, which undergo a decrease in mitochondrial membrane potential, inco rporate

Jess of the rhodamine 123 dye, therefore emi tt ing Jess fluorescence on the FLI

detector. Control measurements of mitochondri al mass were carried using the

flu orescent probe MitoTracker Green (Molecul ar Probes).

Statistical analysis

Stat ist ical di ffe rences between control and treated groups without AC fo r

fi gures 1, 2, 31, 3J, 7D, 9A, 1 OA and 1 OB were determined usin g a one-way ANOV A

209

with a Bonferroni-Holm (a stepwise method) post-test correction fo r multiple

comparisons. An adj ustment was made to limi t the fami lywise error rate (FWE) to

S% by ca lcu lating an adj usted p -value wh ich is a simulated based p-value obtained

from the multivariate t distribution (number of simulat ion = 1000 000) (Westfa ll et

a l. , 1999). Statistical differences between control and treated groups with or without

AC for figu res 1, 2, 31, 31 , 7D, 9A, 1 OA and 1 OB were determined using a two­

tailed unpaired Student's t test. Data for figures 4B, 4C, SB, SC, 6B, 6C, 8B, 8C, 9C,

9D, 1 1 B and 1 J C were analysed using a bilateral 1 test. Va lues are expressed as

means ± SEM. Differences were cons idered stat istica lly significant atp < O.OS.

------------------------ - - --------

2 10

RESULTS

NA C in/ii bits acrolein-ùuluced cytotoxicity and apoptosis

The antioxidant glutath ione appears to have an important role in the

detox ificati on of acrolein (Finke lstein et a l. , 200 1 ). evertheless, the poss ible

protective effect of antioxidants aga inst acrolein-induced apoptos is is not known.

Therefore, we invest igate whether NAC, a precursor of glutathione, can protect ce lls

aga inst acrolein-induced cytotoxicity and apoptos is. 1 nduction of cytotoxicity (loss of

ce llular pro liferat ion) occurred after a 1 h exposure to acrolein concentrations of 50

fm ol/cell and higher (F ig. 1 ). Exposure to 350 jmol/cell induced 5 logarithms (1 05)

of ce l! killi ng. NAC afforded significant protecti on aga inst cytotoxicity of acrolein .

The protective effect was only partial, as acrolein cytotox icity was not complete ly

prevented.

On the basis of the known reactivity of a,~-un saturated aldehydes such as

acrolein with thiols (Heck, 1997), it is li ke ly that acrole in could medi ate its toxic

effects primarily by depleting ce llular glu tathione and/or by modifying protein

cysteine res idues. lndeed, exposure of ce ll s to acrolein caused rapid and severe

depleti on of GSH (Fig. 2) . GSH leve ls were reduced to 36 % of ini tial leve ls, from

2.28 ± 0.08 to 0.82 ± 0.13 nmol/106 ce ll s, by treatment \·Vith 4 fm ol/ce ll of acrolein

during 30 min . We determined whether NAC could prevent the loss of glu tath ione

foll owing exposure to acrolein, by increas ing glu tathione leve ls. Pretreatment of ce li s

with !mM NAC fo r 24 h effectively increased total intracellular glutathione leve ls in

untreated contro l cell s by about 30%, from 2.28 ± 0.08 nmol/1 06 ce lls to 3.22 ± 0. 17

(F ig. 2). However, NAC parti all y decreased the loss of intrace llular glutathione in

ce li s exposed to a low dose of acro lein ( 4 jmol/ce ll ), but not at h igher concentrati ons

(~ 10 f mol/ce ll ) (F ig. 2) . Even thou gh ini tial leve! of glutathione were elevated,

acro lein st ill caused a severe loss of glu tathione in ce l! pretreated with NAC (F ig. 2).

2 11

Given that NAC protected cells aga inst acrolein-induced cytotox icity (F ig. 1 ),

it is poss ible that NAC could tri gger a change in the type of ce l! death from necros is

to apoptos is. Morphological analys is demonstrates that acrolein induces both

apopto is and necros is in CHO cell s (F ig. 3). Apoptos i was revealed by

condensation of chromatin with the flu orescent probe Hoe cht (b lue) whereas

necrosis was revea led by the flu orescent probe Pl (red). Acrolein (50 fm ol/cell)

induced apoptos is and necrosis in 2 1% and 3.5% of ce li s, respective! y (Fig. 3C, 3 L,

3J). A higher concentration of acrolein ( 100 f mol/ce ll ) switched the mode of ce l!

death from apoptos is (8.5%) to necros is (20%) (Fig. 3D, 31, 3J). We evaluated

whether the NAC-induced increase in leve ls of intrace llul ar glutathione could

attenuate ce l! death induced by acro lei n. When NAC-pretreated cell s were exposed to

50 f mol/cell of acrolein (F ig. 3G, 31), the fract ion of apoptot ic cell s decreased from

21 % to 2%. NAC also diminished necrosis in ce li s exposed to 50 and 100 fm ol/ce ll

of acrolein (F ig. 3G, 3H, 3J). Therefore, NAC increased the threshold leve! of

acrolein for induction of apoptosis from 50 to 100 fmol /ce ll , relative to cell s with

native leve ls of glutathione (Fig. 3C, 3G, 3H, 31). Treatment with NAC alone did not

affect control cell s (F ig. 3E versus 3A). Taken together, these results suggest that

NAC is an effect ive compound to inhibit acrolein-induced cytotoxicity as weil a

acro lein-induced apoptos is.

NA C inhibits acrolein-induced translocation ofBad and Bcl-2

We previously reported that acrolein can induce apoptos is by the

mitochondrial pathway in CHO ce li s (Tanel and A veriii-Bates, 2005). However, the

effect of acrolein on proteins in the Bcl-2 fa mily is not known. Acrolei n cou id alter

the balance between pro-apoptotic and anti-apoptoti c Bcl-2 prote ins at the

mitochondri al membrane. Therefore, we examined whether acro lein could induce the

translocation of Bad, Bax and Bcl-2 between the cytosolic and mitochondri al

subcellular compartments. Exposure to acrole in (20 fmol /cell ) fo r 1 h resulted in

2 12

translocation of the pro-apoptotic prote in Bad from the cytoso l to the mitochondri a

(F ig. 4). Translocation of Bad to mitochondri a was parti ally inhibited by NAC (F ig.

4A, 4B). Acro lein also induced translocation of the pro-apoptotic protein Bax from

th e cytosol to the mitochondri a (Fig. 5) . Hovvever, NAC did not inhibit Bax

translocation, but rather NAC a lone promoted Bax translocati on to mitochondria

from the cyto ol (F ig. 5). ln addition, acro le in induced translocati on of the anti­

apoptotic prote in Bcl-2 from mitochondri a (Fi g. 6A, 6B) to the cytoso l (Fig. 6A, 6C).

Thi s was inhibited by NAC. Together, these findin gs indicate that acrole in has a pro­

apoptotic effect at the mitochondrialmembrane. NAC played an anti-apoptoti c role at

the mitochondrial leve! by inhibiting acrolein-induced translocation of Bad to

mitochondri a and by maintaining Bcl-2 leve ls at the mitochondri almembrane.

NA C inhibits acrolein-induced depolarisation f~l mitocflondrial membrane

potentia/

The balance between Bcl-2 proteins at the mitochondria l membrane is often a

determinant in the opening of the permeability transition pore (PTP) and loss of

mitochondrial membrane potenti al (MMP) under apoptoti c st imuli . Since NAC can

exert protective, anti-apoptotic effects at the leve! of the mitochondri al membrane, we

examined whether it could prevent mitochondri a l membrane depolari sati on in

acrolein-treated cell s. Acrolein ( 1 0-20 jmol/ce ll ) caused depolarisati on of MMP (Fig.

7C, 70 ). FCCP was used as a pos itive control fo r MM P depolari ati on (F ig. 7 A, 70).

However pretreatment with NAC afforded a signi fica nt leve! of protection aga inst

acrolein-induced mitochondrial depolari sati on (F ig. 7C, 70). NAC alone caused a

small decrease in MMP (Fig. 7B). To veri fy that changes in rhodamine fluorescence

signal were not due to changes in the total mi tochondri al mass, the effect of acrolein

(20 fm ol/cell ), with and without NAC treatment, wa detennined on the flu orescence

uptake of th e redox-insensitive mitochondria l el y MitoTracker Green (F ig. 7E).

There was no ev idence fo r a shift in flu ore cence in tens ity in ce ll s treated with

2 13

acrolein alone, or acrolein and NAC, confirming th at changes in rhodamine

fluorescence were indeed due to changes in MMP, thus ruling out the possibi li ty that

acro le in and/or NAC affected the mitochondrial mas

A decrease in MMP often leads to the release of cytochrome-c from

mitochondria . lndeed, acro lei n (20 fmol /ce ll ) caused release of cytochrome-c into the

cytoso l (F ig. 8). However, NAC did not appear to inhibit thi s effect, but instead

induced a sma ll increase in cytochrome-c leve ls in both th e mitochondrial and

cytoso lic fract ions.

NAC inhibits acrolein-induced activation of caspase-9

ince NAC inhibited acro lein-induced translocati on of Bad and Bcl-2, as weil

as mitochondrial membrane depolarisation, it is likely that NAC could inhibit post­

mitochondrial events such as caspase-9 activation. Here we show th at exposure to

acro lein (4-50 fmol /cell ) for 1 h activated the initiator caspase-9 (F ig. 9A), the ftrst

downstream caspase induced via the mitochondri al death pathway. Caspase-9

activation by acro le in was totall y prevented by pretreatment with NAC (F ig. 9A). ln

addition , pretreatment with NAC inhibited the cleavage of procaspase-9 (F ig. 9B, 9C,

90 ) and the generat ion of the p35 active cleavage fragment induced by acrolei n (F ig.

90). These ftndings show that NAC can inhibit acrolein-induced apoptos is at the

post-mitochondrial leve!.

NA C inhibits acrolein-induced activation of caspase-7 and caspase-8, as weil as

PARP cleavage

We next determinee! whether NAC could inhibit other events in the apoptotic

cascade. Acrolein induced act ivat ion of the effector caspase-7 (F ig. 1 OA) and initiator

caspase-8 (F ig. 1 OB), and these events were signiftcantly inhibited in ce ll s that had

been pretreated with NAC. Since caspase-7 was inhibited by NAC, we in vest igated

whether NAC could prevent the cleavage of PARP, whi ch is a downstream substrate

2 14

of ca pase-7. PARP is a key nuclear enzyme in vo lved in DNA repair and has a

complex ro le in ce l! death . Acrole in (20 f mol/cell ) caused cleavage of PARP protein ,

from the full-length 116 kDa form to generate the characteri sti c apoptosis-related 85-

kDa cleavage fragment (Fi g. Il A, Il B, Il C). lndeed, NAC inhibited the cleavage of

PARP induced by acrole in (F ig. 11 A, Il B, Il ).

------------------------ --------

215

DISCUSSION

The important new finding of this study is th at NAC, a precursor of the major

in trace llular antioxidant glutathione, is capable of inhibiting acrolein-induced

cytotox icity and apoptosis in proliferating Cl-I O ce ll s. We prov ide new information

on how NAC can inhibit activation of th e mitochondri al pathway of apoptos is by

acrolein.

Recently, we reported that acrolein in duces apoptosi via the mitochondrial

pathway by decreas ing MMP, liberation of cytochrome-c, and acti vati on of caspases-

9 and -7 (Tanel and Averiii-Bates, 2005). This study prov ides new information on the

implication of the mitochondrial patll'vvay in acrolein-induced apoptos is, where Bcl-2

fa mily proteins are critica l regul ators. This i the first study to show that acrolein

stimu lates translocation of the pro-apoptotic Bcl-2 prote ins, Bax and Bad, fro m the

cytoso l to mitochondri a, as weil as the translocat ion of anti-apoptotic prote in Bcl-2

from mitochondria to the cytosol. The translocat ion of pro-apoptotic proteins such as

Bax and Bad to mitochondria is an important step in removing the inhibitory effect of

Bcl-2 on apoptos is at the mitochondrial leve!, thus promoting re lease of cytochrome

c.

Acrole in induced a decrease in MMP a we il as liberat ion of cytochrome-c

from mitochondria into the cytosol. The implication of the PTP in acrolein-induced

apoptos is was already demonstrated through inhibition of nu clear changes by

cyclosporine A (Ta nel and Averiii-Bates, 2005), a blocker of opening of the PTP.

Although the mechani sms are not complete ly un derstood, it i considered that

cytochrome c can be released from mitochondri a by severa! mechani sms, including

the PTP, as weil as through channels on the membrane involving Bax-Bax

interactions (Orrenius et al. , 2003). At the post-mi tochondri al leve!, liberation of

cytochrome-c leads to activation of caspase-9 and -7. Caspase-7 is an effector caspase

that cleaves many substrates such as PARP. everal studi es have reportee! that

-------------------

2 16

acro lei n can inhibi t caspase-3 act ivity (Tanel and A veriii-Bates, 2005 ; Fi nkelste in et

a l. , 200 1 ), which could occur by direct alkylation of its cysteine residue by acrolein.

The cellular effects ofacro lein are believed to be due primarily toits ab ility to

react rapidly with red uced thiols such as GS H (Heci(, 1997). GSH plays a key role in

ce llular reductive processes and detoxification of harmful oxidative spec ies and

va ri ous xenob iotics (Meister and Anderson, 1983) . GS H redox status is criti ca l fo r a

va ri ety of biological process including transcriptional act ivat ion of var ious genes,

regulation of cell proliferation, infl ammation and apoptos is. An important

consequence of severe glutathione depletion caused by taxie compounds such as

acro lein is the resulting perturbation of the cellular redox balance between pro­

oxidants and antioxidants in favor of a pro-ox idant tate. T hi would leave the ce l!

vulnerab le to damage, and poss ibly death, induced by norm al leve ls of ce llular

oxidants (02.-, 1-1 20 2) or mild exposure to other taxie compounds (Luo et al. , 2005) .

The present study demonstrates that acrolein causes glutathi one depletion at

much IO\-ver concentrations (4 fmollcell) than th ose that induce cytotox icity and

apoptos is (30 to 50 fmollcell) . Thi s indicates that glutathione depletion is an earlier

event than cytotoxic ity, evaluated by Joss of cellular proliferation. There was also a

correlat ion between acrolein-induced changes in GSH leve ls and inhibition of ce l!

proliferation, in A549 lung carcinoma ce ll s (Horton et al. , 1997).

Thi s is the first study that links GS H to induction of apoptos is by acrolein ,

si nce pretreatment with NAC, a glutathi one precursor, inhibited acrolein-induced

apo ptos is. Acrole in concentrati ons (20 fmol /ce ll ) that complete ly dep leted GS H

levels co incided with those required to induce ea rly apoptotic events, such as

translocation of Bax and Bad, mitochondrial membrane depolarisation, liberation of

cytochrome-c and activati on of caspase-9. Furthermore, thi s is the first study to report

protection against acrolei n-induced apoptosis with a thi ol anti oxidant. However, it

was reported that sul fur compounds (thiamine, NA C. a corbi e acid) (Sprince, 1985),

a -tocopherol and ascorbic ac id (Nardini et al. , 2002) as we il as the nucleophili c drug

~-- -- --

2 17

hydralaz ine (Kaminskas et al. , 2004) could affo rd protecti on aga inst acrolein-induced

cytotox icity. lndeed, NAC inhibited acrolein -induced apopto is, at bath th e

mitochondri al and post-mi tochondri al leve! . Caspase-9 acti vati on, which is

consideree! as an important indicator fo r activa ti on of the mitochondrial pathway of

apoptos is, was tota lly prevented by NAC. NAC inhi bited acrole in-induced activati on

of caspase-7, PARP cleavage and chromatin condensati on, whi ch are downstream

events from bath caspase-9 and -8. Total in hibi tion of caspa e-9 acti vati on and partial

inhibition of caspase-8 act ivation suggests that the mi tochondrial pathway is

primarily invo lved in protecti ve mechani sms in vo lving A . Caspase-8 act ivati on

mediates apoptotic signalling via death receptor pathways ( aikumar et al. , 1999).

At the mi tochondrial levet, NAC inhi bited the translocation of Bad to

mi tochondria as weil as translocation of Bcl-2 to cytoso l, st imulated by acrolein

exposure. NAC mainta ined anti-apoptotic Bcl-2 prote in at the mitochondrial

membrane. Therefore, NAC changed the ba lance between pro- and anti-apoptoti c

Bcl-2 prote ins at the mitochondri al membrane in l'"avor of an anti -apoptot ic state,

contirmed by protecti on against the acrolein-induced decrease in MMP. Surpri singly,

NAC did not appear to protect ce lls aga inst acrolein-incluced Bax translocation to the

mitochond ria. However, it is li kely that the ant i-apoptoti c effect of NAC may

predominate by increas ing Bcl-2 . Even though NAC does not prevent Bax

translocation to mitochondria, NAC ensures in creased leve ls of Bcl-2 at the

mitochondrial membrane. Bcl-2 binds to Bax in rder to prevent its pro-apoptotic

effects.

Surpri singly, NAC did not protect ce l! against acro lein-induced cytochrome­

c release from mitochondria to the cytoso l. 1-l owever, it was reported that the

1 iberat ion of cytochrome-c persists in neutroph il s exposed to acrolein even though

apoptoti c fea tures such as caspase-9 and -8 acti vati on, and externali sati on of

phosphatidylserine were inhibited by acro lein (F in kel tein et al. , 2005). Thi s suggests

that cytochrome-c release is not necessaril y a hallmark in acro le in-induced apoptos is.

Moreover glutathione depleti on caused cytochro me-c release even in the absence of

2 18

apoptos is (G hibelli et al. , 1999). Thi s aga in suggests that cytochrome-c release is not

necessarily a terminal event leading to apoptos is, but could be the consequence of a

redox di sequilibrium arising from in creased mi tochondri a l generation of reacti ve

oxygen spec ies that, under some circumstances, may be assoc iated with apoptos is

(G hibelli et al. , 1999). The mechani sms in vo lved in cytochrome-c release during

apoptos is are not completely understood and appea r to in vo lve multiple and distinct

path ways, in vo lving Bax-Bax dimers, t-Bid , the MPT and ox idati on of cardi olipin

(Orrenius et al, 2006). Further work is required to characterize the mechanisms

in vo lved in the mitochondri al regulation of apoptos is.

Depletion of GS H and other cellular thiols has been correlated with apoptos is

in a number of ce ll types (Aoshiba et al. , 1999; Sato et al. , 1995). lt was suggested

that the effects of acrolein might be the resul t of redox changes of cri tica l protein

cysteine res idues secondary to GSH depleti on or to direct ox idation/a lkylati on of

prote in thio l res idues that may occLu· along with or subsequent to the loss of GS H

(F in ke lstein et al. , 2001 ). However, th e relat ionshi p between GS H depleti on and

apoptosis is not clear and GSH depletion itse lf may not be suffi cient to tri gger

apoptos is. ln fact, it has been proposed that apoptos is could in vo lve effl ux of reduced

GS H from the ce l! (G hibelli et a l. , 1995). Thu s, depleti on of ce llular GSH may be a

consequence rather than a cause ofapoptos is.

Du ring recent years, many compounds have been te ted as precursors of GSH

or as inducers of the enzymes related to its synthes is (A nderson, 1997). 1 ncreas ing

GS H leve ls could be of great interest in des igning new th erapeutic strateg ies aga inst

environmenta ll y toxic compounds, such as acrolein , and aga inst th e side effects of

many therapeuti c agents. NAC has proven to be a useful compound fo r increas ing

GS H synthes is. The protective role ofNAC aga inst acro lein-induced cytotox icity and

apoptos is could be explained by its ability to st imulate synthes is of glutathione, to

increase ce llular thiol poo ls or to act as a thiol-containing reducing agent. Bes ides

increas ing ce llular thiols, NAC can act as an anti ox id ant by directly reducing reactive

oxygen spec ies uch as OH·, 1-120 2 and HOC 1. AC reduced protein carbonyls

2 19

induced by acrole in , markers for lipid peroxidati on, in rats deve loping acute hepatic

injury (Kitamura et a l. , 2005). An advantage of using AC as a protective agent is

that it already has approval for clini ca l use. ln tàct, NAC is used clinically to treat

overd ose of the hepatotoxic drug acetaminophen (Perry and Shannon, 1998).

Admini stering the cyste ine delivery compound NAC to human subj ects appears to be

safer than administering cysteine itse lf, s ince cyste ine has been reported to have taxie

effects on the central nervous system (Dizdar et al. , 2000) .

On the bas is of these results, we conclude that AC appears to have a

benefi ciai effect in protecting cell s aga inst acrolein-induced apoptos is and inhibition

of ce ll pro li feration. These findings also suggest that intrace llular GSH levels may

play an important ro le in ce llular toxicity of acrolein , and in particul ar, the induction

of apoptos is and cytotoxicity. These findin gs may also be relevant to our

understanding of the toxicity of environmental exposures to low doses of acrolein as

we il as the norm al tissue tox icity of cyc lophospham ide, wh ich genera tes acrolein as

one of its metabolites.

Acknowledgments: Fi nancial support was obtained from NSERC (Natural Sciences

and Engineerin g Research Council of Canada) and CIHR (Canadian Institu tes of

Hea lth Research) (DAB). AT was the rec ipient of the Bourse Francine Beaudoin­

Denizeau from the Fondation UQAM.

- -------------

220

FIGURE LEGENDS

Fig. 1: NAC protects cells against auolein cytotoxicity. C l-IO cell s (1 05/mL) , with

or without pretreatment with NAC, were exposed to acrolein during 1 h at 37°C in 1

mL of a-MEM containing 10% FBS. Acrolei n was then removed, and ce ll s were

incubated to a llow the formation of macroscopic co loni es. The control va lue

represents 105 ce lis and this was normal ized to re present 1 00% ce l! su rvival. Data

represent means and SEM from five independent experiments performed with

multiple est imat ions per point. P<O.OS (*) or P<O.OO 1 (***) indicates a statist ically

significant difference between treatment with acro lein and the untreated contro l.

P<O.OS (~) or P <O.Oül (~~~) indicates a statist ica lly significant difference between

cells exposed to acro lein, with and without NAC. When not shown, error bars lie

within the symbols.

Fig. 2: Acrolein causes severe depletion of intracellular glutathione levels: effect

of NAC. CHO ce lis (Sx 106 /mL), with or without pretreatment with NAC, were

incubated for 30 min with acro lei n in a-MEM contai ning 10% FBS. Leve ls of

glutathione were measured in cellular extracts. Data repre ent means and SEM from

five independent experiments performed with multiple est imations per point. P<0.001

(***) indicates a statist ically significant difference between treatment with acro lein

and the untreated control. P<O.OS (~) or P<O.OO 1 CH~) indicates a statistically

significant difference between ce lls exposed to acrolein , with and without NAC.

Fig. 3: Morphological analysis of apoptosis and necrosis in cells following

exposure to acrolein: protective effect of NAC. Cell s (0.3 x 1 06) were seeded and

cul tured fo r 2 days to near confl uence in tissue cul ture di shes containing a-M EM and

10% FBS at 37°C. Cells, that were pretreated with (E, F, G, H) or without (A, B, C,

D) NAC, were incubated with acrole in : (A, E) 0, (8 , F) 30, ( , G) 50 and (D, H) 100

fmo l/cell , for 4 h. The fract ions of (1) apoptotic (Hoech t) and (J) necrotic (Pl) cells

22 1

are g iven re lative to tota l ce lls (magnifi cation 320x). Data represent means and SEM

from five independent experiments performed with multipl e est imat ions per point.

P<O.OS (*) or P<O.OOJ (***) indicates a statisticall y ign ifi cant difference between

treatment with acrolein and the untreated control. P<O.OS (~) , P<0.01 (~~) or

P<O.OO 1 (~~~) indicates a stat istically significant difference between ce ll s exposed to

acro lei n, with and without NAC.

F ig. 4: Acrolein-induced translocation of Bad to the mitochondria is inhibited by

NAC. CHO ce ll s ( 106 /mL), with or without pretreatment with NAC, were incubated

fo r 1 h with acrole in (20 jmol/cell). For immunodetection of (A) mitochondrial and

cytoso lic Bad (25 lilla), anti-cytochrome ox id ase and anti-GSTn 1 were used as a

load ing controls fo r cytoso lic and mitochondri al fract ions, respectively. A

representative gel is shawn from five independent experim ents. Densitometric

analyses of express ion of (B) mitochondria l and (C) cytoso l ic Bad are re lative to the

untreated control. Data represent means and SEM from five independent experiments

perfonned with multiple est imations per point. P<O.OS (*) indicates a statistically

signifi cant di ffe rence between treatment with acrole in and the control. P<O.OS (~)

indicates a statistica lly significant di ffe rence between cell s exposed to acrolein, w ith

and without NAC.

Fig. 5: Acrolein promotes translocation of Bax to mitochondr·ia. CHO cell s (1 06

/mL) , with or without pretreatment with NAC, were incubated fo r 1 h with acrolein

(20 jmol/cell ). For immunodetecti on of (A) mitochondria l and cytoso lic Bax (23

kDa), anti-GSTn 1 and anti-cytochrome ox idase were used as loading contra is for

cytoso li c and mitochondri al fractions , re pective ly. A representati ve gel is shown

from five independent experiments. Densitometric analyses of express ion of (B)

mitochondrial and (C) cytoso lic Bax are relative to the untreated control. Data

represent means and SEM from fi ve independent experiments performed with

222

multiple estimations per point. P<0.05 (*) indicates a statist ica lly significant

diffe rence between treatment with acrolein and the control. P<0.05 (~ ) or P<O.O 1

(~~) indicates a stati stically significant difference between cell s exposed to acrole in,

with and without NAC.

Fig. 6: NAC inhibits acrolein-induced translocation of Bcl-2 to the cytosol. CI-10

cells ( 106 /mL), with or without pretreatment with NAC, were incubated for 1 h with

acrolein (20 fm ol/ce ll ). For immunodetection of (A) mitochondri al and cytoso lic Bcl-

2 (26 kDa), anti-GSTn 1 and anti-cytochrome ox idase were used as load ing con trois

for cytoso li c and mitochondrial fract ions, respectively. A representative ge l is shown

from five independent experiments. Densitometric analyses of express ion of (8)

mitochondrial and (C) cytosol ic Bcl-2 are relative to the untreated control. Data

represent means and SEM from five independent experiments perfonned with

multiple estimations per point. P<O.O 1 (**) in dicates a stat istically signifi cant

difference between treatment with acrole in and the control. ?<0.05 (~) indicates a

tati stica lly significant difference between ce ll s exposed to acmle in, with and without

NAC.

Fig.7: NAC inhibits acrolein-induced depolarization of the mitochondrial

membrane. CHO ce li s ( 1 06/mL) were treated with (A) 5 !-LM FCCP for l h, or (B)

NAC (24 h) alone for 24 h, or (C) acrolein (20 fmo l/ce ll ) fo r 1 h (with or without

NAC pretreatment), re lative to untreated control ce ll s (red). Ce ll s were then analysed

by fl ow cytometry for rhodamine 123 fluorescence in channel FL 1. (D) Data

represent means and SEM of re lative flu orescence intensity of rhodamine from eight

independent experiments. The absolute data value fo r the untreated contro l ce ll s from

8 experiments was 38±2 (mean ± S.E.M) relative fluorescence units. The control

va lue was des ignated as 100%, and data fo r treated ce l! were normali zed to th is

va lue. P<0.05 (*)or P <O.OOl (***) indicates a stat istica ll y significant difference

223

between treatment with acro lei n or FCCP and the untreated contro l. P<0.05 (~)or

P<O.O 1 (~~) indicates a stat istically significant di fference between ce ll s exposed to

acro le in, with and without NAC. (E) Contra is for mi tochondri almass were stained

with the redox-insensitive li ving dye MitoTracker Green and analyzed in the FL-1

channel of a flow cytometer. Control (red), acrolein-treated (bl ack), NAC-treated

(green) and acrolein-NAC-pretreated (blue) ce ll s were compared in a hi stogram

overl ay .

F ig. 8: Acrolein-induced liberation of cytochrome-c to the cytosol is not affected

by NAC. CHO ce lis ( 106 /mL), with or without pretreatment with NAC, were

incubated for 1 h with acrolein (20 f mol/cell ). For im munodetection of (A)

mitochondria l and cytoso lic cytochrome-c ( 15 k.Da) , anti -GSTn 1 and anti­

cytochrome oxidase were used as load ing contra is fo r cytoso li c and mi tochondri al

fract ions, respective ly. A representative gel is shawn from ftve independent

experiments. Densitometric analyses of exp ression of (B) mi tochondria l and (C)

cytoso li c cytochrome-c are relative to the untreated control. Data represent means and

SEM fro m ftve independent experiments perfo rmed with multi ple estimations per

point. P<O.O 1 (**) or P<O.OO 1 (***) indicates a stat ist icall y signiftcant difference

between treatment with acrolei n and the control. P<0.05 (~) indicates a stati stically

signift cant difference between cells exposed to acrolein , with and without NAC.

F ig. 9: Activation of initia tor caspase-9 by acrolein is inhibited by NAC. (A) CHO

cel! (O.S x 1 06/mL), with or without pretreatment with NAC, were incubated with

acrole in for 1 h. Caspase-9 activity in ce l! lysates was expressed relative to the

untreated control, designated as 1. Data rep resent means and SEM. from eight

independent experiments performed with multiple est imations per poi nt. (B) Ce ll s

( 1 06/mL), with or without pre-treatment with AC, were incubated with acro lein (20

224

fm ol/ce ll ) for 1 h. For immunodetection of procaspase-9 and its cleavage fragment

(35 kDa), P-tubulin was used as a loading control. Densitometric analyses of

express1on of (C) procaspase-9 and (D) the cleavage fragment are relati ve to the

untreated control. Data represent means and SEM from fo ur independent experiments

perfo rmed with multiple estimati ons per point. P<0.05 (*)or P<O.OO 1 (***) indicates

a tati stically significant difference between treatm ent with acrolein and the control.

P<O.Ol (~~)or P<O.OOI (H~) indicates a stati stically significant difference between

ce ll s exposed to acro lein , with and without A .

Fig. 10: NAC inhibits activation of effecto•· caspase-7 and initiat01· caspase-8 by

anolein. (A) CHO cells (0.5x 1 06/mL), with or without pretreatment with NAC, were

incubated with acrolein for (A) 2 h (caspase-7) or (8 ) 1 h (caspase-8) in a-M EM with

10% F8S. Activities of caspases were expressed relative to the untreated control s,

des ignated as 1. Data represent means and SEM from seven (caspase-7) or nine

(caspase-8) independent experiments perform ed with multiple estimations per point.

P<0.05 (*) or P<O.OOI (***) indicates a sta ti st ica ll y signi fica nt di ffe rence between

treatm ent with acrole in and the control. P<0.05 (~), P<O.O 1 (~~) or P<O.OO 1 (~H)

indicates a statistically significant di ffe rence between ce ll s ex posed to acrolein, with

and without NAC.

Fig. 11: NAC inhibits acrolein-induced cleavage of PARP. (A) Ce lls ( 106/inL),

with or without pretreatment with NAC, were incubated with acrole in (20 jmol/ce ll )

fo r 1 h. For immunodetection of PARP ( 11 6 kDa) and its cleavage fragment (85

kDa), P-tubulin was used as a loading control. Densitometric analyses of express ion

of (8 ) PA R.P ( 1 16 kDa) and (C) the cleavage fragment (85 kDa) are relative to the

untreated control. Data represent means and SEM from five independent experiments

performed with multiple estimations per poin t. P<0.05 (*)or P<O.OO 1 (***) indicates

a stati stica lly significant di fference between treatment with acrolein and the control.

225

P<O.OS (~) or P<O.OO 1 (~~~) indicates a stati sticall y signifi cant difference between

cell s exposed to acrolei n, with and without NAC.

Fig. 12: Proposed schema for NAC-induced inhibition of acrolein-induced

apoptosis. NAC, which increased intrace llular glu tathione leve ls, afforded protection

against acrolein-induced apoptosis. NAC dimini shed apoptos is by inhibiting acrolein­

induced activation of the mitochondrial death pathway. NAC inhibited acrolein­

induced Bad translocation to the mitochondri a, as weil as Bcl-2 translocation from

mitochondria to the cytosol. NAC had no effect on Bax translocation to the

mitochondria and cytochrome-c liberation to the cytoso l. NAC inhibited

depolarisation of mitochondrial membrane potential, procaspase-9 processing,

caspase-9, -7 and -8 activat ion, as we il as PARP cleavage induced by acro lein.

Acro lein causes procaspase-3 cleavage, but caspase-3 activity is immediately

inhibited and caspase-7 is responsible for downstream cleavage events. Inhibition of

acro lei n-induced apoptosis using NAC was confinned morphologica lly by

condensation of nuclear chromatin. Apaf-1: apoptosis protease activati ng facto r-! ; t­

b id : truncated bid, CAD: caspase act ivated DNase; Cyt-c: cytoch rome-c; FADD: Fas

associated death domain ; Fas: fibroblast-associated; ICA D: inhibitor of caspase

activated DNase; NAC: N-acety lcystei ne; PARP: poly ADP-ribose polymerase.

226

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Meister A and Anderson ME ( 1983) Glutathione. An nu Re v Bioehem 52:71 1-760.

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23 0

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23 1

Westfa ll PH, Tobias RD, Rom D, Russel D and Hochberg Y ( 1999) Multiple

comparisons and Multiple Tests Us ing the SAS System, Cary, NC : SAS lnstitute

lnc.: 4 16.

Figure 1: NAC protects cells aga inst auolein cytotoxicity

-~ 10 0 -ra 1 > ·-> 1..

::::1 0,1 U) --~ 0,01

-+-+ NAC

-+-- NAC

0,001+---~--~--~~--~--~--~~--

0 50 1 00 150 200 250 300 350 400

Acrolein ( fmol/ cell)

232

233

Figure 2: Acrolein causes severe depletion of in t t·acellula t· glutathione levels:

effect ofNAC

4

3,5 4>4>4>

......... l NAC: 30%

D -NAC - 3 QJ Qi rzl +NAC c u 0 ID 2,5 ·- 0 .c ~ '1"4

2 ta ........ ~ ë :::J - E 1,5 (,!) c ........ 1

0,5

0 0 4 10 20 40

Acrolein ( fmol/ cell)

234

F igure 3: Morphological analysis of apoptosis and necrosis in cells following

exposure to acrolein: protective effect ofNAC

E . · ... ' ·.· . . .

F .

. . .. . ~ . -. ·'' ": -· ...

. . . . . .

1 J

25 25

~ ...

20 D- NAC

Il) 20 Il)

Il) c Il) ~+ NAC 0 0

~15 loo u 15

0 QJ

a. z < ..... 010

0 10 ~

~ 0

0 5 5

0 0

0 30 50 100 0 30 50 100

Acrolein (fmol/cell)

----------------------------------------------------- -

23 5

Figure 4: Acrolein-induced tra nslocation of Bad to th e mitochondria is inhibited

by NAC

A Acrolein + +

NAC M ito Bad - 1

+ +

Cyto Bad -

B c Qi

2, o - NAC 10 > Qi ~= 2 > -c e ~ :=-75 n:l ... Ill l5 "C 0

1, n:l ~ _u Ill l5 n:l 0

•t: .... .~~50 "C ~ - 0 C::;::; ~ ~ 0~

.s::. .. .s- 25 g ~ 0, >-~

()

:E 0 0

0 20 0 20 Acrolein {fmol/cell)

---- ---------------

Figure 5: Acrolein promotes translocation of Bax to mitochondria

A Acrolein + +

B

Q) > Q)~

-"0

~~ Ill 8

1,4

ca .s o, ·;: Q)

"C > r::: :.-::; 0 o.!! ' ..r::: Q)

u!E. 0

:'!:::: :::!:

NAC ~------------~+ ____ ~+~ Mito Bax ----.1

~============~ Cyto Bax----. L~------------------------'

0 20

c

Q)

>

10

~ î 75 11:1 .... Ill §

(.)

~ b 50 0 ~ en~ 0 .... >- 25 ()

Acrolein {fmol/cell) 0

D- NAC

IHNAC

20

236

237

Figure 6: NAC inhibits acrolein-induced t.-anslocation of Bcl-2 to the cytosol

A + - +

+ +

Mito Bcl-2---+

Cyto Bcl-2--

B c 100

Qj 0 - NAC 'P Qj > > =- 1,6 C1l C1l 0 N :=; 75 -.::.

1 0 N t: - ... 1 0 1,2 (.) ... - (,) m g (.) 0 - (,)

m ... liS - 50 (.) ~ ·;:: 0

~ ~ 0,8 -c ?f!.. =- (/) Qi 0 0 0::

..t: 25 ..... ->. (.) (..) 0,4

0 ~

::!E 0 0

0 20 0 20 Acrolein (!mol/ ce li)

238

Figure 7: NAC inbibits acrolein-induced depolarization of the mitochondrial

membrane

D

(]) ~ c ·v; . E c Q) ....,

ro .~ '"0 Q)

0 > ..c; ~ 10

Qj 0::: ~

A / Control

r~. t --.•

1,2 ~ $$ Iii FCCP

0,8

a - NAC r- ri-!I +NAC *;f ••• r+

0,6

0,4

0,2 Il

0 FCCP 0 10 20

Acrolein ( fmoljcell)

B

E

FU.....,.

• Control D Acrolein

D NAC

c

Figure 8. Acrolein-induced liberation of cytochrome-c to th e cytosol is not

affected by NAC

A Acrolein + +

NAC + +

Mito cyt-c----+

Cyto cyt-c----+

B c Q) 125 Q) 9 ••• > > ~ Q) 0 0 :::::- 100

... .... 1 0 0 r::: - ... ..!. 0

>-'E 0 6 0 0 >- 0

0 75 0 .... --n:s 0 0 (1)

> .i: ~ ~

0 <Il "C~ 50 Qi t: rn 0:: 3 0 0 -..!:: 25 >-0 u 0 ~

~ 0 0 0 20 0 20

Acrolein ( fmol/ ce li)

239

Figure 9: Activation of initia tor caspase-9 by acrolein is inhibited by NAC

A

1,6 0 - NAC ... • ••

~ 1,4 ~ + NAC

·s; 1,2 t~ ta 1

0'1 1 0,8 cu Ill ta 0,6 Q. Ill ta 0,4 u

0,2

0

0 4 10 20 30 Acrolein (fmol/cell)

B Acrolein + +

NAC .-~~------------~+ ______ ~+~-. Procas pase-9 ____,. 1

Cleaved fragment ____,.

~================~

c ~ ëii c Ql -c

0>

cl> Vl

"' Q. Vl

"' (,)

0 ... Il.

f3 -tu bu li ne ____,. l._ _______________ z• ___ · _:1 ______ __J

120

100

80

60

40

20

0 0

D

~ :ii -.~

LI) M Q. -c Ql

1,5

E o,5 Cl

"' ... u...

~ ~

20 0 Acrolein (fmol/cell)

50

20

240

24 1

Figure 10: NAC inhibits activation of effector caspase-7 a nd initia tor caspase-8

by acrolein

A

B 2

1,8

1,6

~ 1,4 ·:; ·.g 1,2

ra 00

1

1

~ 0,8 ra Q. 0,6 Ul ra u 0,4

0,2

0 15 25 30 50 100

Acrolein (fmol/cell)

...

0 5 15 30 50 100

Acrolein (/mol/ cell)

242

Figut·e 11: NAC inhibits acrolein-induced cleavage ofPARP

A Acrolein + + NAC + +

PARP-----+

1 Cleaved PARP -----+ -

~-tubuline -----+

1

B 120 c ~ 1,5

>-100 :'=

(/) 1:

>- Cil ..... :!:: 80 1: (/) 1: ..... Cil 1:

..... Cil 1: 60 E a.. Cl

"' a:: .... <( 40 ....

0,5 a.. "C

Cil >

20 "' Cil

ë3 0 0

0 20 0 20

Acrolein (!mol/ cell)

243

Figure 12: Proposed schema for NAC-induced inhibition of acrolein-induced

a po ptosis

NAC-----" Apoptosis induced by acrolein

NAC NAC NAC

NAC

----+ Stimulation

- ·- ·- ·- ·+No effect

CHAPITRE III

CONCLUSION

L 'acroléine est la plus électrophile des aldéhydes cx,f3-insaturés, extrêmement

toxique et elle est très présente dans l ' environnement. Elle est utili sée comme

herbicide et générée par la fumée de cigare tte, la cuisson d' huile, le métabolisme de

certains agents anticancéreux, lors de la peroxidation des lipides et après expos ition

aux rayons UV. L'acro léine est impliquée dans di fférentes maladies respiratoires

chroniques (bronchite, asthme) (Hogg 200 1) et neurodégénératives (A lzheimer) (Arlt

et al. , 2002) ainsi que l ' athérosclérose (Biswa l et al. , 2002). La peau est auss i une des

cibles de l ' acro léine, puisque la molécule est fo rm ée de façon endogène suite aux

irradiat ions des lipides par les rayons ultrav iolets du soleil et la quantité d' acroléine

formée peut être mesurée par immunohistochimie. Dans la peau, l 'acroléine peut

causer une hypersensibilité non-immunolog ique, une inflammation, le développement

de cancer, le vieillissement, une cytotoxicité et une génotox icité.

Cette étude montre pour la première fo is que l ' inducti on de 1 apoptose par

l ' acro léine passe par la vo ie mitochondriale et par la vo ie du récepteur de mort Fas et

que cette induction est MAPK-dépendante et caspase-dépendante.

3.1. Induction de l'apoptose par l'acroléine selon la littérature

La majorité des données de la littérature concernant la tox icité de l ' acroléine

porte sur sa nature électrophilique et son effet tox ique in vivo et in vitro . En ce qui

concerne les mécanismes d' activation de l ' apoptose par l 'acroléine, peu de recherches

ont été effectuées. Ces études ont montré plutôt que l 'acrol éine induit l ' apoptose par

détection des évènements tardifs (fragmentation de l ' ADN), sans déterminer les

mécanismes par lesquels l 'acroléine induit l ' apoptose. L ' étude des mécanismes

d' acti vat ion de la cascade apoptotique constitue la contribution importante et

ori gi nale de cette étude. Dans la littérature, on rapporte une induction de l ' apoptose

245

par 1 'acrolé ine et ceci est démontré par la fragmentation de 1 ' ADN au ni veau des

macrophages humains (Li et al. , 1997 et 1999) et des kératinocytes humains

(Takeuchi et al., 200 1), par l' extern alisat ion des phosphat idylsér ines (PS) chez les

ce llules épithé li ales branchiales humaines HB E 1 (Nardini et al , 2002). En plus,

l' apoptose éta it révé lée par la coloration nucléaire et le c li vage de la caspase-3 chez

les ce llules endothéli ales HUV EC (Misonou et al. , 2005) et par l'act ivité de la

caspase-3 chez les cellules tubulaires proximales humains (Schwerdt et al, 2005) .

Cependant, d' autres études démontrent que l' acro léine inhibe l' apoptose en faveu r de

la nécrose et cec i en abaissant l' activité enzymatique des caspases-3 , -8 et -9 chez les

lymphocytes B provenant de souri s (Kern et Kehrer, 2002). De plus, dans les cellules

neuronales PCI2, l'acro léine inhibe l' activ ité de la caspase-3 et la fragmentation de

l' ADN , et rend les cellules perméables à l' iod ure de propidium (L iu-Snyder et al.,

2006; Luo et al., 2005). En plus, l' acroléine inhibe l' apoptose chez les neutrophiles

humains en supprimant 1 'activité de la caspase-3, la frag mentat ion de 1 'ADN et

l'externali sati on des PS (Finkelstein et al. , 200 1). Mai s, une récente étude par la

même éq uipe a démontré que l' acroléine indui t l' apoptose à tàible concentration chez

les neutrophiles humains (< IO!J.M) et ce ll e-ci est révé lée par l' induction de l' activité

des caspases-3 , -8 et -9, 1 'external isation des PS et la 1 ibération du cytochrome-c.

Mais à plus forte dose (> IO!J.M) l'acro léine a induit la nécrose, une baisse de

l' activ ité des caspases -8, -9 et -3 ainsi qu'une diminu tion de l' externali sat ion des

PS. Pourtant la libérat ion du cytochrome-c est restée forte à ces deux conditions

(F inkelstei n et al. , 2005). Ces résultats va ri ab les de l' act ivat ion ou l' inhibition de

l' apoptose par l' acroléine peuvent être dus aux différences dans les processus

biochimiques impliqués dans l' induction de la mort ce llula ire dans ces différents

types cellulaires, qui incluent des cellules primaires non-pro liférat ives, des ce llules

immunitaires et des li gnées cancéreuses, ain si que le temps et la dose d ' acroléine

ad mini strée. En plus, différentes méthodologies ont été utili sées dans ces études,

comme des temps d 'expos ition à l' acro léine beaucoup plus long, de 24 h à 48 h,

246

comparat ivement à des courtes expositions de l'o rdre de 30 min à 4h . Dans quelques

études incluant la présente, les cellules éta ient incubées dans un milieu contenant du

sérum (Hamilton et al., 1997); pourtant dans d' autres études, l' acroléine éta it incubée

dans un milieu sans sérum (Takeuchi et al., 200 1; Na rdini el al. , 2002 ; Finkelste in et

al., 2001 ; Kern et Kehrer, 2002). L' acroléine réagit avec les protéines du sérum

diminuant ai nsi sa concentrat ion 1 ibre di sponibl e. Cec i peut ralentir ou modifier le

processus toxique induisant l'apoptose. La nécrose semble la forme de mort cellulaire

prédominante quand les ce llules sont exposées à l' acroléine dans un mili eu sans

sérum (Kern et Kehrer, 2002). Dans cette étude. !"acroléi ne induisa it la nécrose mais

à plus fo rte dose d' acro léine.

Alors, on remarque que les deux principales voies de l'activat ion de

l' apo ptose, la vo ie mitochondriale et la vo ie de récepteurs, n' ont pas été investi guées

en profond eur jusqu 'à présent ainsi que le rôle de la vo ie de signali sation des MAPK

dans l'activation de la cascade apoptotique par l' acrolé ine.

Cependant, de récentes études ont démontré que d' autres aldéhydes peuvent

induire l' apoptose. En fait, le 4-hydroxynonénal, un marqueur de la maladi e

d'Alzheimer, induit l' activat ion des caspases -2 , -3, -8 et -9, le c livage de PARP et la

libérat ion du cytochrome-c (J i et al. , 200 1). En plus, le 4-hydroxynonénal stimule la

vo ie de signalisation des MAPKs et ceci par la phos phorylat ion de c-jun (Pugazhenthi

et a l. , 2006; Bruckner et Estus, 2002) et par l' act ivat ion de .JNK (Castello et al. ,

2005). Cependant, une nouvelle étude sur des fibroblastes a démontré que l' apoptose

induite par l' aldéhyde 4-hydroxynonénal est JNK-dépendante (K utuk et al. , 2006).

D'autre part, la p53 qui joue un rôle majeur dans l"apopto e es t surexprimée sui vant

une expos ition à l' hydroxynonénal chez une li gnée de neurob lastomes (Laurora et a l. ,

2005) . Enfin, l' induction de l' apoptose pa r la vo ie mi tochondriale par le

cinnamaldéhyde est p38 et JNK-dépendantes (Wu et a l. , 2006).

247

3.2. Les mécanismes d'action de l'apoptose induite par l'acroléine

3.2.1. Induction de la voie mitochondriale de l'apoptose par

l'acroléine

Lors de la présente étude, des tests de cytotox icité ont démontré que l'ac roléine est un

composé très toxique pour les cellules CHO pui squ ' un e concentrati on de 200

fm ole/ce llule éliminait 95% des ce llules après une heure d'expos ition. On a observé

que l'acrolé ine induit l'apoptose (condensati on de la chromatine après quatre heures

d 'expos ition) et la nécrose à petite dose (3 0 f mole/ce llule), mais une élevation de la

concentrati on à 100 fm ole/cellule entraîne la nécrose plutôt qu e l' apoptose. Notre

in vesti gation sur les mécanismes d' activati on de l'apoptose a montré que l'acrolé ine

st imule la vo ie mitochondria le en causant la ba isse du potenti el membranaire

mitochondrial, la libération du cytochrome-c dans le cytoso l, la translocation des

proté ines pro-apoptotiques Bax et Bad vers la membrane mitochondriale, la

translocati on de la proté ine anti-apoptotique Bcl-2 vers le cytoso l, a in si qu ' une

acti vation de la caspase-9 initiatrice (F igure 3. 1 ). Cette cascade enzymatique a

entraîné le cli vage de di fférents substrats des cas pases dont l' inhibi teur ICA D et la

protéine PARP, l'externa li sation des phosphat idylsérines et l' apparition de la

condensati on de la chromatine (F igure 3. 1). Les caspases -3 et -6 n' éta ient pas

acti vées mais plutôt inhibées et ceci pourra it probablement résulter d' une li a ison

directe au s ite actif par l' addition de Michael pui sque les caspases sont des cysté ines

protéases qui sont régulées suite à un stress oxydati f par modification de leur thi o l du

site acti f (Chandra et al. , 2000; Mannick et al., 1999). Il éta it rapporté que les cellules

du carcinome du sein MCF7, qui sont dépourvues de la caspase-3, peuvent subir

l'apoptose par l' in termédiaire de la caspase-7 effectrice (Degterev et al., 2003). En

effet, l' apoptose induite par l' ac roléine chez les ce llules CHO semble être médiée par

la caspase-7 effectri ce (F igure 3. 1). En plus de la caspase-3 , la caspase-9 est capable

de cl iv er directement la caspase-7 et entraîner son act ivati on (S iee et al., 1999). Dans

cette étude, le clivage de I' ICAD semble être médié par la caspase-7 (Wolf et al.,

-- ------- --------------

248

1999; Houde et al. , 2004). C'est souvent difficile de distinguer entre l' implication de

la caspase-3 ou -7 dans l'apoptose puisque les deux partagent plusieurs substrats en

commun (PARP, lCAD) et le peptide Ac-DEVD-AMC (Cohen, 1997).

ACROLÉINE

/

ACROLÉINE+ ! GsH __. APOPTOSE

ACROLÉINE

Inhibition

-----+ Stimulation -·-·-·-·-.. Pas d'effet

Figure 3.1 : Schéma représentant les voies de signalisation dans l' induction de l'apoptose par l'acroléine : inhibition par le NAC.

249

Nous avons observé que l'acroléine cli ve la procaspase-3 (2-50 fm ole/ce llule)

mais inhibe très rapidement (< 5 min) son act ivité enzymatique. Le c livage de la

procaspase-3 résulte probablement de l' act ion enzymat ique directe de la caspase-9

act ive (Lawen, 2003). En plus, l' utilisat ion d' un inhibiteur spéc ifique de la ca pase-3

n' a pas emmené une baisse de l' apo pto e induite par l' ac rolé ine. L' inhibition

enzymatique peut être due à un e alkylation directe du site actif par l'acrolé ine, ce qui

est en accord avec d' autres études (Finkelstein et al. , 200 1). La baisse de l' activité de

la caspase-3 se produit chez les neutrophiles après 2 à 8 h d ' expos ition à 10 ).lM

d ' acrolé ine (F inkelstein et al. , 2001). De même, les act ivités des caspases -3, -8 et -9

étaient inhibées après 30 min d 'expos ition à l' acrolé ine (5 to 40 ).lM) chez les

lym phocytes murins (Kern et Kehrer, 2002) . Les cas pases -7 et -9 ont aussi c li vées

et act ivées par l' acro léine dans les cellules Cl-IO mai il emble qu 'ell es so nt moins

suscepti bles que la caspase-3 à l' inhibition par l'ac roléi ne. L' inhibition des caspases-

7 et -9 se produit éga lement, mais seul ement après 3 h et 2 h d' expos iti on,

respectivement, puisqu ' elles possèdent toutes les deux un résidu cystéine

nucléophilique dans leur site actif qui peut être alky lé par l' acrolé ine (Finkelstein et

al. , 200 1 ). L' act ivati on des caspases par l'ac roléine semble être le rés ultat d ' un net

éq uilibre entre deux processus, so it l'act ivat ion et l'inhibition des enzymes. Bien que

ces caspases puissent être inhibées, il reste un e act ivité caspase nene mais minimale

pour induire le clivage de I' ICAD et la cond ensation de la chromat ine (Figure 3.1 ).

La différence dans l' inhibition de ces caspases peut s' exp liquer aussi par le fait que

les caspases ne se ressemblent pas toutes au nivea u du site actif mais plutôt au reste

de la structure, ce qui exp liquerait la limitat ion de l' accès et les différences des

substrats (Degterev et al., 2003).

Récemment, on a rappo1té que le c li vage protéolytique n' est pas suffi sant pour

l' act ivat ion de la caspase-9 initiatrice (Boatri ght et Salvesen, 2003) . La dimérisation

plutôt que le cli vage sem ble le plus important dans la fo rm ation du s ite actif et

l' act ivation de la caspase-9. Des changements confo rmat ionnels identiques pour

250

former le s ite actif ont été observés pour les caspases -9 et -7, même si elles sont

act ivées par des mécanismes distincts. D'autre part, un clivage interdomaine par les

caspases initi atrices est nécessa ire pour 1 ' act ivat ion des caspases effec trices -3 et -7

(Boatright et Salvesen, 2003).

L' activation des caspases -9 et -7 éta it confirmée par le c livage de leurs

fo rmes pro-enzymes et la génération des fragments appropri és, mais surtout par

l' augmentation de l' activité enzymatique. De plus, le rôle de la caspase-9 dans

l' induction de l' apoptose par l' acro lé ine était confirmé en utili sant un inhibiteur de

son activ ité. Jusqu 'à présent, il n' y a pas d' inhibiteur spéc ifique disponible pour la

caspase-7.

L' implication du pore de transition de perméabilité (PTP) et du relargage du

cytochrome-c dans l' apoptose induite par l'acrolé ine éta ient confirmés en util isant la

cyclospori ne A, un inhibiteur du pore PTP. Bien que le mécani sme du relargage du

cytochrome-c n'est pas complètement connu , i 1 est considéré que le cytochrome-c est

1 ibéré via les pores PTP et les canaux formés par les in teract ions des proté ines pro­

apoptotiques Bax (Orrenius et al., 2003). L' inhibition de l' apoptose par la

cyclosporine A su pporte un rôle pour les pores PTP et la 1 ibérat ion du cytochrome-c

dans l'apoptose induite par l'acroléine. L' inhibition partielle par la cyclosporine A

suggère que l'acroléine peut induire l' apoptose par des mécani smes alternatifs à la

vo ie mitochondri ale et que le cytochrome-c peut être re largué par plusieurs

mécanismes.

3.2.2. Induction de la voie des récepteurs de mort par l'acroléine

Une autre nouvelle contribution de cette étude est que l' expos ition des cellules

prolifératives CHO à l' acro léine st imule la voie du récepteur de mort Fas (F igure

3. 1 ). Cette affi rmation est confirmée par 1 ' augmentation de 1 ' expression du 1 igand

Fas, la translocation de l'adapteur de domaine de mort de Fas (FADO) à la membrane

plasmique et l' act ivation de la caspase-8 initi atri ce. Le Kp7-6, un antagoniste du

25 1

récepteur Fas et 1 ou Z-IETD-FMK, un inhibiteur de la caspase-8, bloquaient les

évènements apoptot iques en aval de la caspase-8, comme l ' act ivat ion de la caspase-7

exécutrice et la condensation de la chromatine nucléa ire. L ' acroléine induit une

transducti on croisée entre la voie de signali sat ion des récepteurs de mort et celle de la

mitochondrie en clivant la protéine Bid en sa fo rme tronquée t-bid qui se transloque à

la membrane mitochondriale pour st imuler cette vo ie. L 'inhibition du récepteur Fas

ou de la caspase-8 inhibait partiellement 1 'act ivat ion de la caspase-9, un évènement

post-mitochondria l, par l 'acroléine. Ces résultats démontrent que l ' acro léine active la

vo ie du récepteur Fas en amont de la voie mitochondriale. La caspase-9 demeurait

partiellement act ive malgré 1' inhibition du récepteur Fas et de la caspase-8, suggérant

que l ' acroléine peut induire la vo ie mitochondriale indépendamment de la vo ie des

récepteurs. En plus, le Z-LEHD-FMK, un inhibi teur de la caspase-9, inhibait

partiellement la condensation de la chromatine indui te par l ' acroléine. Ces résultats

suggèrent que les deux voies sont act ivées indépendamment et que la vo ie des

récepteurs peut stimu ler la voie mitochondriale par l ' intermédiaire de t-bid.

Chez les neutrophiles, de fa ibles doses d' acro léine activa ient la caspase-8 et

au contraire, de fot1es doses inhibaient son act iv ité (F inkelstein et al., 2005). En plus,

l ' acroléine inhibait l 'activité de la caspase-8 chez les lymphocytes B murins (Kern et

Kehrer, 2002). Dans ces études, l'exposition à !"acroléine était courte mais la

détection de 1 'activité se faisait de 6 à 24 h après 1 'expos ition. Après une longue

incubati on, il est possible que le type de mort ce llulaire change de l ' apoptose en

nécrose. Il a été rapporté que les composés tox iques induisent l ' apoptose à des fa ibles

doses et la nécrose à des conditions plus évères et que le type de mort dépend du

type ce llulaire (Orrenius et Zhivotovsky, 2006).

252

3.2.3. Implication de la voie de signalisation des MAPK dans

l'apoptose induite par l'acroléine

La vo ie de récepteur de mort Fas converge aussi à l' acti vat ion de la

MAPKKK AS K 1 par l' intermédiaire de l' adapteur Daxx. Ensui te, AS K 1 act ive les

vo ies MA PKs, JNK et p38 qui sont impliquées da ns la transcription des gènes des

protéines apoptotiques dont Bax, Bim, le 1 igand Fas et le récepteur Fas.

L'acroléine induit l'apoptose par l' act iva ti on du récepteur du facteur de

croissance épidermale (EGFR) qui active la vo ie des MAPKs chez les kératinocytes

humains (Takeuchi et al., 2001) . Menti onnons ici que l' activati on des MAPKs peut

prendre di fférentes vo ies de signali sati on et a insi emmener l' apoptose dans le cas des

kérat inocytes humains (Takeuchi et al., 200 1) et 1 'inhibition de l' apoptose chez les

neutrophiles humains (Finkelstein et al. , 200 1 ). Toutefo is le lien entre l' apoptose et

l' activation des MAPK n'était pas défi ni dans ce deux études. La présente étude

montre pour la première fois que l' apoptose induite par l' acroléin e est MAPK­

dépendante. On a démontré par des inhibi te urs pharm aco log iques, que l'acroléine

acti ve les vo ies de signali sation ASK 1/p38 et ERK, indui ant plusieurs

caractéristiques de 1 ' a po ptose dont 1 'acti vat ion des caspases-9 et -7, le c livage de

I' ICA D et la condensati on de la chromatine.

Trois di fférentes MA P kinases, p38, JNK et ERK sont impliquées dans

l' apoptose. Bien qu 'e lles jouent un rôle important dans la signali sati on de l'apoptose,

leurs rôles dans l' induction de l' apoptose par l'acroléine demeurent inconnus. Cette

étude démontre que l'acroléine active la c-jun (F igure 3. 1 ), un substrat de JNK, et

l' inhibition de l'activité de JNK par le SP6001 25, change le mode de mort par

apoptose en une mort totale par nécro e. Ceci confi rme que la JNK est impliquée

dans une vo ie de survie contre l' agress ion par acroléin e. En plus, le SP600 125

inhi ba it l'acti vation des caspases -7 et -9, et le cli vage de I' ICA D. Ces observati ons

ont des ré ultats attendus puisque l' acti vat ion des caspases n' est pas un e

caractéri stique de la nécrose. Il était rapporté que J K e t impliquée dans l' apoptose

253

des ce llules du carcinome pulmonaire (Chuang el aL. , 2000) et ce lle des macrophages

murins (Kim et Sharma, 2004) mais elle n' était pas impliquée dans l'apoptose des

ce llules neuronal es HT4 (Rockwell el aL., 2004) où p38 jouait le rô le maj eur.

Cepend ant, JNK est importante dans le déve loppement et la urvie des macrophages

(Himes et aL. , 2006). Ainsi, la fo ncti on de JNK dans l' apoptose est contradictoire.

Par contre, la p38 MA PK est impliquée da ns l' in duction de l' apoptose par

l'acroléine. L' incubation des ce llules Cl-IO avec l' acroléine induisait la

phosphorylation de la p38 (F igure 3. 1) et un prétra itement avec le SB2035 80, un

inhibiteur de la p38, inhibait l' apoptose induite par l' acroléine. L' inhibiteur

SB203580 inhibait l' activation des caspases-7 et -9, le c li vage de l' fCA D et la

condensation de la chromatine induites par l' acroléine. Bien que les stimuli de stress

acti vent la p38 et induisent l'apoptose dan des modèles de dommages cellula ires, ce

n' est pas le cas chez tous ces modèles. L' acti vation de la p38 éta it requi se pour

l'apoptose induite par le cadmium (Ga lan et al., 2000), la soustraction des facteurs

trophiques (Kummer et al. , 1997), et l' ischémi e (Mackay et Mochly-Rosen, 1999),

pourtant l' incapac ité de SB203580 à prévenir l'apoptose induite par les UV (Franklin

et al., 1998) et la S-nitrosoglutathion (Call sen et Brune, 1999) prouve la fa ible

corrélation entre p38 et l' apoptose. D'autres résul ta ts contradi cto ires sont obtenus

chez des ce llules neuronales. Dans une étud e récente dans les ce llules neuronales

HT4, l' inhibition de la p38, et non de JN K, bl oq uait 1 apo ptose induite par le

cadmium (Rockwe ll et aL. , 2004), des résul tats qui concordent avec notre étude où

une inhibition de la p38 bloquait l' apoptose induite par l' acrolé ine. Une te lle

anomalie suggère l' existence d' une grande di ffé rence dans la régulati on de la réponse

face au stress dépendamment du type cellulaire et de l' agent admini stré.

En plus des facteurs de croissance, de cytokines, et des mitogènes, ERK est

auss i activé par des agents cytotoxiques. Dans notre étude, ERK semble être

impliquée dans l'apoptose induite par l'acro lé ine. Acro léine induit la phosphorylation

de ERK, et l' inhibition de son activité par le UO 126 inhibait l' acti va tion des caspases-

7 et -9, le clivage de l'TCAD et la condensation de la chromatine par l' acroléine. En

254

plus, un autre inhibiteur de ERK, le PD98059, inhibait l'acti vati on des caspases-7 et-

9, et le clivage de I' ICAD sans toutefois avo ir un effet sur la condensation de la

chromatine, une des étapes fin ales dans l' induction de 1 ' apoptose. PD98059 et UO 126

sont toutes les deux des inhibiteurs de MEK qui sont utiles pour étudier les

mécani smes biologiques et surtout ceux impliqués dans l' activati on des MAPKs. Il s

sont des inhibiteurs non-compétiti fs des ubstrats de M EK, 1 'A TP et ERK

respectivement (Favata et al., 1998). Elles ont des mécani smes d ' action différents

(Dudley et al. 1995; Favata et al. 1998), UO 126 est in inhib iteur spécifique de

l' activité de MEKl et 2 tandis que le PD98059 est un inhibiteur de l'activation de

MEK 1. Ainsi le UOJ 26 est un plus puissant inhibiteur qu e le PD98059 (Aless i et al. ,

1995 ; Dud ley et al., 1995 ; Favata et al., 1998) . L' activati on de la MAPK ERK peut

inhiber l'apoptose (C huang et al., 2000), l' acti ver (Lee el al., 2006; Ruffels et al.,

2004; Yang G. et al. , 2004) ou même n'avo ir aucun effet (Ga lan et al., 2000). En

plus, le rôle de ERK est di fférent d' un agent toxique à 1 autre da ns le même type

ce llulaire. Par exemple, chez les ce llules de neurob lastomes humains SH-SY5Y,

l' apoptose induite par le peroxyded' hydrogène était inhibée par un inhibiteur de ERK

(Ruffels et al. , 2004) qui n' avait pas d'effet sur l' apoptose induite par le cadmium

(Kim et al. , 2005). Ceci suggère que les agresseurs n' induisent pas tous les mêmes

vo ies de signalisati on.

3.2.4. Induction de la voie de survie AKT pat· l'acroléine

Afin de vérifi er le li en entre l'acrolé in e et les vo ie de survie ce llulaire, on a

entrepri s des expériences sur l' AKT (protéine kinase B ou PKB) qui est une protéine

activée en aval des récepteurs de facteurs de croissance. AKT est la plus caractéri sée

des kinases qui sont connues de promouvoir la survi e ce llulaire et de jouer un rôle

majeur dans le métabolisme du gluco e. AKT est act ivée en réponse à plusieurs

facte urs de croissance, à des hormones ainsi qu'à des agresseurs exogènes (Datta et

al. , 1999; Law lor et Aless i, 200 1; Ta lapatra et Thompson, 200 1; Downward , 1998;

255

Brazil et Hemmings, 2001). Les facte urs de cro issance et les hormones qui act ivent la

voie AKT inc luent le facteur de cro issance ressemblant à l' acro léine (IGF), le facteu r

de cro issance ép idermale (EGF), le facteur de croissance des fibrob lastes (bFGF),

1' insuline et l' interleukine 6 (Datta et al., 1999 ; Ta lapatra et Thompson, 200 1;

Downward , 1998). Dans nos cellules, l' AKT joue un rôle dans la survie cellul aire

contre l' agress ion par l'acroléine. En effet, l' acrolé ine phosphory le l' AKT et le

prétra itement avec des inhibiteurs pharmaco logiques de l' AKT, qui sont le

L Y294002 et le Wortmannin, changea it la mort ce llulaire de 1 ' apoptose en nécrose

tota le. Ces deux inhibiteurs agissent sur la vo ie de urvie cellula ire de la

phosphoinositide 3-kinase (PJ3 -K) et il s sont très ut il es dans 1 'étude des mécanismes

biologiques impliquant la voie PI3-K (S tein et Waterfïeld , 2000; Fruman , 1998). Le

LY294002 et le Wortmannin diminuaient s ignifï cativement l'activation des caspases-

7 et -9 et le c livage de l' lCAD par l'acro léine, des résultats attendus dans des

cond itions nécrotiques.

Parmi les protéines kinases kinases kinases activées par des mitogènes

(MAPKKK), la kinase régul atri ce du signal d ' apoptose 1 (ASK I) est activée en

réponse à des stresseurs cytotoxiques te ls que les EO R, le FasL, le H20 2 et le TNF-a,

et induit l'activation de JNK et p38 , et l' apoptose (To biume et al. , 2001 ). Le

mécani sme de tox icité de l' acroléine implique la générati on du stress oxydatif.

L'acroléine génère des EORs (Luo et Shi , 2004 et 2005) et des radicaux libres, et des

anti oxydants comme le glutathione et l' hydra laz ine rédui sent sa tox ic ité (N unoshiba

et Yamamoto, 1999; Burcham el al., 2000). Comme attendu, no résultats démontrent

que 1 ' acro léine entraîne 1 'augmentation de 1 'express ion de ASK 1 (F igure 3.1) et la

phosphorylation de la p38 et du substrat de JNK, c-jun , à des concentrations

apoptotiq ues.

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3.2.5. Phosphorylation de la p53 par l'acroléine

Un nouvel aspect de cette étude démontre la phosphory lat ion de la p53 aux

sérines 15 et 46 par l 'acroléine (F igure 3. 1) qui est un agent endommageant de l ' ADN

(Esterbauer et al., 199 1 ). Les sérines 15 et 46 de la p5 3 sont phosphory lées en

réponse aux dommages à l 'ADN (Cnman et al. , 1998 ; Bulavin el al., 1999). La

phophory lat ion de la sérine 15 réduit la liaison de l ' oncogène mdm2 à la p53 in v itro

(Shieh el al., 1997) pour inhiber sa dégradation par la mdm2 (Haupt el al. , 1997).

Effectivement, l 'acro léine induit la phosphory lati on de la p53 à la sérin e 15, et

comme attendu ce lle de la sérine 46 qui est la cible de la p38 (B ulav in et al., 1999)

qu 'on a démontré son activation. Parmi les évènements méd iés par la p53 , il y a

l 'apoptose et l 'arrêt du cyc le ce llulaire. La p53, qui est un facteur de transcription

induit la transcription des protéines proapoptotiques comme Bax et le ligand Fas, et

supprime celle des antiapoptotiques comme le Bc l-2 (B ras el al., 2005). D 'ai lleurs,

nous avons démontré que l 'acroléine induit une augmentation de Bax et une ba isse de

Bcl-2 à la membrane mitochondriale ai nsi que l ' augmentation de l 'express ion du

ligand Fas, tous des changements régulés par la p53. A insi, la présente étude

démontre que p38 et ERK sont des molécules clés dans l ' induct ion de l ' apoptose par

l 'acroléine et que AKT et JNK sont des voies de surv ie empruntée par la ce llule pour

contrer sa toxicité cellulaire.

3.2.6. L'antioxydant N-acétylcystéine inhibe l'apoptose induite par

l'acroléine

Puisque l ' acroléine est un toxique omniprésent dans notre environnement, on

a étudié l ' effet protecteur des antioxydants sur les ce llules afi n de pouvoir traiter les

gens qui y sont exposés. On a démontré que le g lutathion qui est parmi les plus

importants antioxydants intracellulaires, joue un rôle important dans la détoxification

de l 'acroléine. Ains i, le NAC, un précurseur du glutathion, a inhibé la toxicité induite

par l 'acroléine dans les cellules proli férat ives Cl-1 0. L·acro léine se lie rap idement aux

25 7

molécules nucléophiliques comme l'antioxydant glu tathi on, qui est impl iqué dans la

défense ce llulaire, et emmène une diminution de a concentrati on intracellulaire

(Heck, 1997; Kehrer et Biswal, 2000). De plus, ell e inact ive l'enzyme glutathione

rédu ctase qui regénère la fo rme réduite du glutathi one (GS H) par le cyc le rédox du

glutathione. La baisse de la forme réduite du GS H rend la ce llule plus vulnérable aux

dommages causés par le stress oxydatif. Nos résultats montrent une baisse accrue du

GS H intracellulaire avec des faibles concentrati ons de l'ordre de 10 fm ole/cellule.

Une expos ition des cellules CHO à un e concentrat ion non-l éth ale d ' acro léine (4

f mole/ce llule) diminue le glutathione intrace llulaire de 55% da ns 30 min. Cette

baisse est plus rapide et dramatique par rapport à ce lle observée chez les cellules

CHO exposées au H20 2 pendant l heure (25%) (Lord- Fonta ine et Averill-Bates,

2002) . Puisque les concentrations d ' acroléine qui abaissent le glutathion

intrace llula ire sont beaucoup plus fa ibles que celles qui induisent la cytotox icité, ce la

suggère que la baisse du glutathi on est une étape importante qui précède les

condit ions cytotoxiques. Dans les cellules du carcin ome pulmonaire A5 49, il y ava it

une fo rte corrélati on entre la baisse du g lutathi on et l' inhibi tion de la proli fé ration

ce llula ire (Horton et al., 1997).

Le NAC, qui augmente le nt veau intrace llula ire de glutathion, offre une

protecti on contre la cytotoxicité, la nécrose et l'apoptose (F igure 3. 1) engendrées par

l' acroléine. Le GSH joue un rôle important dans la détox ift cati on ce llulaire des

espèces oxydatives tels que le H20 2 et les hydroperoxydes lipidiques (Meister et

Anderson, 1983) et la protecti on des ce llules épithé li ales pulm onaires de l' oxydati on

et de l' infl ammation. La balance oxyda-réductrice du glutathion est cri tique pour

plusieurs fonctions cellulaires et des changements indui ts pa r l' acrolé ine semblent

affecter plusieurs vo ies de signali sation. Cec i inclu t l' activati on de plusieurs gènes de

transcripti on, la régulation de la proli fé rat ion ce llulaire et l' apoptose (Horton et al. ,

1997, 1999; Ramu et al. , 1996; Rahman et MacNee, 1999, Rahman et al., 1999;

Arr igo, 1999; Bojes et al., 1999).

258

En plus, nous avons apporté de nouve lles informations sur les mécanismes de

protection par le NAC contre l ' apoptose induite par l ' acroléine. Les concentrations

d' acroléine qui abaissent le glutathion co ïn cident avec ce lles qui induisent les

évènements précoces de l ' apoptose comme la translocat ion du Bax et Bad, la

dépolarisation de la membrane mitochondriale, la libérat ion du cytochrome-c et

l ' act ivati on de la caspase-9 (F igure 3.1 ). Aucune autre étude n' a rapporté une

protecti on contre l ' apoptose induite par l ' acroléine par n' importe quel antioxydant.

La protection de l ' apoptose par le NAC a été confirmée morphologiquement par

1' inhibition de la condensation de la chromatine, un évènement tardif de 1 'apoptose,

induite par l 'acroléine. Un prétraitement avec NA inhibait l ' induction de la voie

mitochondriale par l ' acroléine puisque le cli vage de la caspase-9 et son activat ion ont

été totalement inhibés. L 'activation de la caspase-9 e t considérée comme un

indicateur de la vo ie mitochondriale (Chandra el al. , 2000). NAC inhibait la

translocation du Bad vers la mitochondrie ainsi que la translocation du Bcl-2 au

cytosol induite par l 'acroléine. NAC inhibait l ' act ivat ion de la caspase-7 etTectri ce et

ce lle de la caspase-8 initiatrice ainsi que le cli vage de PARP par l 'acroléine.

L ' inhibition totale de la caspase-9 et l ' inhibi tion partielle de la caspase-8

suggèrent que la vo ie mitochondriale est la première cible du mécanisme du

protection par le NAC puisque l ' activation de la caspase-8 méd ie l ' apoptose par la

vo ie du récepteur Fas (Saikumar et al., 1999). A in i, no résul tats suggèrent que le

GSH joue un rôle important dans la toxicité et la modulation de l ' apoptose induite par

l ' acroléine.

La baisse du glutathi on et d' autres thiols ce llulaires corrèlent avec l 'apoptose

dans plusieurs types cellulaires (Aoshiba el al. , 1999 ; Sato el al. 1995). En effet,

l ' apoptose induite par l ' acroléine chez les neutrophiles co ïncide avec la baisse du

glutathion intrace llulaire (Finkelstein el al. , 2001 ). Il éta it uggéré que les effets de

1 ' acroléine sont probablement dues aux changements d'oxydoréduction dans des

molécules connexes au GSH (F inkelste in et al., 200 1 ) . Cependant, la relat ion entre la

bai sse du glutathion et l 'apoptose n'est pas encore claire et la bai sse du GSH toute

_______________________________ _____________ _j

259

seule peut ne pas être suffisante pour induire l 'apoptose. En effet, il était proposé que

l ' apoptose impliquerait une sorti e du GSH intracellulaire (G hibelli el al., 1995 ; Yan

den Dobbelsteen et al. , 1996). Ainsi , la diminution du G H est plutôt une

conséquence et non une cause de l ' apoptose.

Un rôle pour le stress oxydatif dans l'apoptose intrigue de plus en plus de

chercheurs. Pour plusieurs années, on a pensé qu ' une exposition directe des ce llules

au peroxyded ' hydrogène ou à des quinone induit seulement la nécrose, mais de plus

récentes études démontrent que ces agents à des plus faibles doses peuvent induire

l ' apoptose (Hampton et Orrenius, 1997). En plus, de plu en plus de chercheurs

suggèrent que la production des espèce réactives de l ' oxygène (EOR) fa it partie

intégrante de 1 ' a po ptose et que ces espèces sont déterminantes dans la tox icité

assoc iée à la radiation ionisante et aux médicaments chimiothérapeutiques (Zamzami

et al., 1995; M acho et al. , 1997). Puisque l ' acroléi ne génère un stress oxydatif en

augmentant la production des EORs et que le AC inhibe l ' induct ion de l 'apoptose

par le NAC, on pense que l ' apoptose induite par l 'acroléine est médié par le stress

oxydatif. La capacité de plusieurs antioxydants ce llulaires com me la cata lase et le

AC à bloquer l ' apoptose induite par diver agents autres que des oxydants auss i

confirme le rôle central du stress oxydati f dans l 'apoptose (B uttke et Sandstrom,

1994). Réc iproquement, on a assigné une fonction antioxydante à la famille anti­

apoptotique Bcl-2 et à la protéine du baculov irus p35 (Jacobson, 1996; Sah, 1999),

démontrant que la génération des EOR est probablement une condition prérequise

pour démarrer l 'apoptose. Cependant, d' autres études ont démontré ques les radicaux

libres peuvent atténuer l ' apoptose (Hampton et Orrenius. 1998).

Durant les récentes années, plusieurs précurseurs du GSH ou inducteurs de ses

enzymes de synthèse ont été développés (Anderson, 1997) . L ' augmentati on du GSH

est d' un grand intérêt dans le développement de nouvelles tratégies thérapeutiques

contre les composés tox iques de l 'environemment, comme l ' acroléine, et les effets

secondaires de plusieurs médicaments tel que 1 'hépatotox icité de 1 'acétaminophène.

Le NA diminue les protéines conj uguées à l 'acroléine, un marqueur de la

260

perox idati on lipidique, chez les rats développant des dommages hépatiques

chroniques (Kitamura et al. , 2005). En plu , le NAC supprimait l 'apoptose induite par

des agresseurs tox iques ou par le facteur de nécrose tumorale (TN F-a) (Cotgreave,

1997). Le NAC est utilisé pour ses propriétés chimioprotectrices contre les effets

secondaires de plusieurs médicaments (Mclellan et al. , 1995 ; Gilli ssen et al. , 1997).

Le NAC est utili sé cliniquement pour traiter les overdoses de l 'acétaminophène

(Perry et Shannon, 1998) . En outre, le NAC peut agi r directement comme un

antioxydant en réduisant les espèces réacti ves de l 'oxygène comme le radical hydroxy

(OH"), le péroxyde d' hydrogène (H20 2) et l ' ac ide hypochloreux (HOC l ) .

A dministrant le précurseur de la cystéine, le AC, est plus sCrr pour les humains que

la cysté ine elle-même qui a montré des effets toxiques pour le système nerveux

central (Dizdar et al., 2000). En plus, le GSH ne peut pas être administré car il

n' entrera pas dans la cellule à cause de son vo lume.

3.2.7. Pertinence de l'acroléine dans le traitement des cancers

De nos jours la thérapie anticancéreuse par inducti on cl ' apoptose prend de pl us

en plus d' intérêt. Cette étude v ise à comprendre le mécanisme de mort cellulaire

induit par l ' acroléine ainsi que l ' impact de la modulat ion du glutathion intracellulaire

sur la toxicité de cet aldéhyde. Nous somme intére és à étudier l ' acroléine parce

qu 'elle est produite par l ' oxydation des polyamines (Agostinelli et al. , 1996). Il est

déjà connu que les tumeurs contiennent des concentrations élevées en polyamines

(Bachrach et Heimer, 1989), qui sont des molécules nécessa ires à la proliférati on

ce llulaire (Heby et Persson, 2003). A insi, en administrant une enzyme de la famille

des amines oxydases, l ' amine oxydase du sérum du bovin (BSAO, EC 1.4.3.6), les

polyamines seront oxydés pour produire de l ' acroléine et du peroxyded ' hydrogène

(A iarcon, 1970; Tabor et Tabor, 1984). Ce fa isant, la taille de la tumeur diminuera

puisqu 'on la pri ve des polyamines et on la met en présence de produits apoptogènes

(Lord- Fontaine et al., 200 1), qui sont le peroxyded ' hydrogène et l ' acroléine. La

26 1

BSAO a été immobilizée avec succès dans des hydroge ls de po lyéthy lène glyco l

biocompatibles, ce qui peut être utile pour la tabilité et la li vraison de l ' enzyme dans

des tumeurs in vivo (De mers et al. , 200 1 ). La BSAO immobi 1 izée était injectée in

vivo dans des tumeurs de mélanome chez la souris et elle a diminué la croissance

tumorale (Averiii-Bates et al., 2005) . Nous avous caractéri sé le type de mort

ce llulaire, qui était l 'apoptose, révélée par la condensati on de la chromatine, chez les

ce llules tumorales (test de Hoescht) (Averiii-Bates et al. , 2005). En ce qui me

concerne, j ' ai parti cipé à cette étude in vivo en confirmant l ' apoptose à l ' aide du test

de l 'annex ine V 1 propid ium iodide et cec i par cyrométri e de flu x (Averiii- Bates et

al ., 2005). Les résultats de cette étude suggèrent que l ' enzyme immobili sée possède

un potentiel anticancéreux en clinique, pui sque les produi ts générés sont relachés

graduellement ce qui stimule l ' apoptose des ce llules plutôt que la nécrose. A insi, les

résul tats de mon proj et de doctorat pourront aider au déve loppement de cette nouvelle

thérapie anticancéreuse dont les mécanismes sont investigués en détails afin

d ' opt imiser le traitement.

En conclusion, nos résultats ont permi s une meilleure compréhension de la

tox icité de l ' acroléine, un des produits maj eurs de l 'oxydati on des polyamines

impliqués dans la régulation de la proli férati on ce llulaire et la croissance des tumeurs.

En plus, cette étude a permi s une explication poss ible de l ' acti v ité pharmaco log ique

du cyc lophosphamide, un agent anticancéreux, qui est métaboli sé en acroléine et en

un mélange de phosphoramides, ce qui ouvre la vo ie au déve loppement de

1 ' enzymothérapi e par la BSAO pour 1 utter contre les cancers. D 'autre part, les

ré ultats de ce proj et envisagent la po sib i lité d" ut ili er le composés qui augmentent

le niveau de GSH dont le N AC dans des cas de tox icité à l 'acroléine, molécule qui

prend de l 'ampleur dans la recherche sur la maladie cl ' A lzheimer.

En perspective, il sera important et ori ginal de démontrer si l ' acroléine peut

induire l 'apoptose in vivo et cec i sur des ti ssu pulmonaires des rats Sprague-Dawley

sui te à une expos ition par inhalation. L 'étude se fera par vo ie respirato ire puisque

norm alement les gens sont exposés à l ' acroléine par l 'air comme la cuisson d' huile,

262

l' émiss ion des moteurs et les feux de forêt. Différentes études in vivo se sont

intéressées au métabo li sme de l' acroléine et son etTet tox ique sans avo ir étudier la

mort cellul ai re par apoptose (Morri s et al. , 2003; Penn et al., 2001 ; Borchers et al. ,

1999; Linhart et al. , 1996). Les rats seront exposés à diffé rentes concentrat ions en

acro lé ine suivi e des étapes de prélèvement de poumons et du lavage

bonchoalvéolaires . Afin d' invest iguer l' apoptose induite in vivo par inhalation de

l' acroléine, on utili sera un homogénat de poum ons. Les analyses à utiliser seront : la

mi croscop ie de fluorescence pour confirmer la présence des noyaux apoptotiques à

1 'a id e du co lorant Hoescht, le test du TU EL pour démontrer la fragmentation de

1 ' ADN (Barazzone et al., 1998), la tluorimétrie et l' immunobuvardage pour étudier

l' act ivité et le clivage des caspases -7, -8 et -9. Ensuite, par HPLC on effectuera le

dosage de l' hydroxyproline qui démontre le dépôt de co ll agène qui est un indicateur

de fibrose (Hagimoto et al., 1997). La fibrose se ca ractéri se par une surpopulation de

fibroblastes au niveau des poumons en cas de tox icité pulmonaire (Hagimoto et al.,

1997). Enfin, la microscopie de flu orescence pour étudi er l' activité de la phagocytose

qui est un mécanisme de défense majeure des poum ons et ceci sur les différents types

ce llula ires du système immunitaire obtenu par lavage bonchoa lvéo la ire (Kuronuma

et al. , 2004) . Au niveau des poumons les macrophages sont responsables d ' attaquer et

de di gérer les bactéries et mi croorgani smes qui entrent par la vo ie respirato ire. Ainsi,

une act ivité phagocytose dérégulée représente un danger pour la santé humaine.

Enfin, nos résultats suggèrent que l' acrolé ine induit l' apoptose par la voie de

récepteur de mort Fas et par la voie mitochondriale et que ces mécanismes sont

MAPK- et caspases-dépendant. En plus, l' antioxydant NAC peut être utilisé dans le

traitement d ' une toxicité à l'acroléine et po ur aba isser les effets econdaires

indési rabl es de l' agent anticancéreux, le cyc lophosphamide.

--- - --·-- - ·-- ----

ANNEXE

Avai lable online at www.scienced irect.com

$CII!NCE@OIAI!CT "' Blochemlcal Pharmacoloov

An Li - lumoral c ll ·c1 o i' nat i ve ~ t ml il1l mobi l i;:cd bm inc -.,c rul1l

am ine o,\ ida'i ' in a lll OU · ' mc l;uw m<. t mod ·1

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p1 111 tP .uH ttlllllPI 11 <-'.t llllo.'il h \\llh n:ll t\1..'' aml tlllllll 'htlt Jt:d B"i 0 Tht.· :.·n;~nll' '':t' imnwbihtnl tn a f)('J} I l" th~ll·nl' ~J:~..·pJl tPF<~ J

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w a -.. tn.k ck·d 11l to th~ lll llW I. th <.·•e ' ' ,t, a m;uk<:d d ~~..· feaw of 7{ 1 1 f th:: tlll ll1 1 g•uwth . Th 1 ~ wa:- t0mp;.~ te d w ft h a d;: rea:-{· n! ont) J ~ '< l'f

lU lill' • ~ ll.f.' when the ~ =ti l h.· amou ru (li n:Lt i\i• BSAO \\.:l' achlll ll b tt rt.>d. Th~ t~ p~ nf <."rll J ca 1h wa-. analy~e<l t n tU II II' ' ' th al were trc:u("(l \ \ i th

II;Ui\· f\( Îr!'llllll l' i lrl<. l nS;\0 . \Vhcn Hi ll lVI' W<.'"I'C lr{:a l t~d wilh imml>h ih7J.'tl OSAO. thCi l' "'"' indliCIIÎ'<Il of il hig.h leveJ of apoptl'<,i ... ~ :u nu nd

71y; l ... ·pmp:u<.·d 11 1 h.·" th:u1 lil l"{ \\'i th the n~ll ih·l'll /~ ltll' Apnptnt i.: t:dl dL~ :uh \\ a ' H"C~:-l~d h ~ nu:;k·ar · l u nm;u i n l.·ntHk·n,at it ,nu ,ia~

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dm: 10. 1 Cl 1 iliJ.h<: p.ll):b .O :!.O~ S

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du~.· t~ t.uu lJ inhihil lli\.H1tmoll :tn cdl prnli!'~;nJtioll anU lhal

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11 1 p•1l~ .tlltllll"' 111 lunmt \'l'r .. u:- rh•111tal ti:-.\lh.'' H 1. h•\Î t'

NH,(CH~),NH(CH,) .1 NH(CH2J,NH2 + 20, + 2H,O

(spermtne) l r amtne oxtdase

CHO(CH7),NH(CH).NH(CH7),CHO • 2NH, • 2H,O? (d•aldehyde) T

oo1a o•tllinaliM l 1 caral<lso and GS GPx

NH2(CH2),NH2 + 2CH2=CHCHO

{pulroscine) (ocroloin) ~

GSH!GST

h~ 1 R~~l:t1nn .. c llC't:\( i()( lit: t' ll/\ IT'lft ll ~ ox•d.:~ tiO il 01 1h<: pol)anun~

..:pcrnune by BS--\r). ~JX'Illl inc uo~.krr· ' ' OX Idt!lt\ ummJ\ti ("l(t •n the prc'i<'llC'C()! o>.)f't' l'! und \1. :lh.'r 1 01~)(111 h\"~il" f>SCtl p.:rn."(td~· t li ~0:!. :' llHlWtll l\

otX! th< un'( t~bk ;\mtnooh.k h\'dc tnl ( l'nl~dn.IK" 1 v • .v· bt~t.1 prop t<Hkl ldc

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ci ulliil:.lll) 11 IO Î(ulll t\.I'C'\ ICII l t!tlJ plll!("h~l lk' Ji ~()~ l' d~ tO.\ tii.:J h) l~\0

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g lul<t lhir~t \t" 1CiSII •. c:ittl l )lCd h) gluwthioclt j"\:"H>>.tÙ!IIoC fG I'\}. t\(' 10 10 h dciO:\ÎfiOO h\' COOJU_g.nt.on \VIth GSH. cata lpcd h\' ~1 Ul3Ùlinn.c .\ l ron,f~.-r :'K.:

1GS'n

264

prc•dm·b , uch '" H ~( J , and <tllll·hyc.k'C 1 tllu ld l>t' ~ènenned in ~ itu h~ Lkli \t'1 Îll ~ d l llllll.: p,'( idi.l :-.::-- inlP tuntu r:-. h) int.luL'L'

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" lu . il ha> bcc-n wei l t·,tabli. hl·d thal illllnub ili l.ati.m "r t:l l /. ~ lill"~ :-- uch a :.. a:-.pa ra ~ i na,c:.. in tc } pl d )' tll t..T ÎL" tll ~t l ri~· c'\

, uch '" PEG ine lt·:,;·c!- thëi r Slntc·tu ra l , lah ili ty nnd func ­tiun ~tl ~tL' ti\ iti c:- in 'i tn1 ~ 1 ll d i 11 \ i vu (2S 1. hm11nh ilt t.\ tl iun l ) r

8. () int~) ~~ hiUCtl!llpalÎhJc ll l~tll'Ï\ lllaÙC ol' lt(n·int \t~ nuu

r.lbumin und PEG wu;. rqH>rtcd n:ccnt l) 12~ 1 :llld t h~

\..'fllynt t !-.howcd :t high npcrational ,mhilit ) rc lmivc LO

H\ nati' · lonn. Th r ·fore. hoth na ti ve :ttlu llllllh>lulilxJ [3." t\0 ,~. i Il lx: L\)Jitpat ·U in 'i' o in tcnH' nftht-ir ~uHÎlUilll)r

": lfll.' {.H.:~ a~:n n:- t tilt~~.: ll tc lan\) IIHI~.

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