KonKonííččkovkováá R, VaR, Vaňňkovkováá K, VanK, Vanííkovkováá J, J, Zelenka J, Zelenka J, SubhanovSubhanováá I, MuchovI, Muchováá L, L,

ZadinovZadinováá M, M, VVíítek Ltek L

11stst FacultyFaculty of of MedicineMedicine, , Charles University in PragueCharles University in Prague

Czech Czech RepublicRepublic

Effects of plant tetrapyrroliccompounds on pancreatic

cancer and heme oxygenaseactivity

Effects of plant tetrapyrroliccompounds on pancreatic

cancer and heme oxygenaseactivity

Tetrapyrroles in blue-green/red algaeTetrapyrroles in blue-green/red algae

Spirulina platensis(Cyanophyta, phycocyanins)3,5 billion years old!

Tetrapyrroles in red algae(Rhodophyta - Cyanidium caldarium, Porphyridium cruentum): phycoerythrins

Tetrapyrroles in red algae(Rhodophyta - Cyanidium caldarium, Porphyridium cruentum): phycoerythrins

Chlorophyllin

Phycoerythrobilin

Phycocyanobilin

AnticancerAnticancer effectseffects of bilirubin, of bilirubin, phycocyanobilinphycocyanobilin andand chlorophyllschlorophylls

Keshavan P et al. Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization. Int J Cancer 2004;112:433-45.

Ollinger R et al. Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle2007;6:3078-85.

Jiraskova A at al. Association of serum bilirubin and promoter variations in HMOX1 andUGT1A1 genes with sporadic colorectal cancer. Int J Cancer 2011 [Epub]

Ismail MF et al. Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina. Int J Biol Sci 2009;5:377-87.

Castro DJ et al. Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacentalcarcinogenesis. Carcinogenesis 2009;30:315

Balder et al. Heme and chlorophyll intake and risk of colorectal cancer in the Netherlands cohort study. Cancer Epidemiol Biomarkers Prev 2006;15:717-25.

HypothesesHypothesesDoes Spirulina platensis have antiproliferativeeffects on human pancreatic cancer?Can algeal tetrapyrroles account for these antiproliferative effects?Do algeal tetrapyrroles affect HMOX activity in a way analogous to metaloporphyrins?

Drummond and Kappas. Prevention of neonatal hyperbilirubinemia by tin protoporphyrin IX, a potent competitive inhibitor of heme oxidation. PNAS 1981;78:6466-70.

HypothesesHypothesesDoes Spirulina platensis have antiproliferativeeffects on human pancreatic cancer?Can algeal tetrapyrroles account for these antiproliferative effects?Do algeal tetrapyrroles affect HMOX activity in a way analogous to metaloporphyrins?Do algeal tertrapyrroles effect mitochondrialmembrane potential analogously to bilirubin?

MethodsMethodsIn vitro studies on pancreatic cancer cell lines PaTu-8902, BxPC-3, MIA PaCa-2 exposed to various tetrapyrroliccompounds.

Commercial tetrapyrroles used except for phycocyanobilinwhich was isolated from freeze-dried S. platensis (Martin Bauer, GmbH, Germany, isolation method adopted from Smith A.G., Witty M.: Heme, chlorophyll, and bilins - Method and protocols.

Humana Press 2002) and verified by MS and NMR spectra

HMOX activity (measured as CO production by GC) andHMOX1/BLVRA mRNA expressions were determined

The effect of tetrapyrroles on mitochondrial membranepotential determined by fluorescent dye (JC-1)

In vivo studies on athymic mice xenotransplanted with humanpancreatic cancer treated with S. platensis (PaTu-8902)

Effect of S. platensis extract on viability of human pancreatic cancer cell lines

Effect of S. platensis extract on viability of human pancreatic cancer cell lines

Vliv extraktu ze Spiruliny platensis na viabilitu nádorových linií karcinomu pankreatu

0

20

40

60

80

100

120

140

0,08 0,16 0,31 0,63 1,25 2,50koncentrace g/L

viab

ilita

buněk

v %

PA-TU-8902

MIA PaCa-2

BxPC-3

*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT eseje

*

*

*

*

*

**

*

*p<0.05, Kruskal-Wallis test with post-hoc analysisViability determined by MTT test

concentration [g/L]

Cell viability

[%]

*

*

Effect of oral intake of S. platensison pancreatic ca growth

Effect of oral intake of S. platensison pancreatic ca growth

0,15

0,25

0,35

0,45

0,55

0,65

0,75

0,85

0 4 7 10 14 17 20days

Tum

or s

ize

(cm

3 )

placebo

Spirulina

Data analyzed by RM ANOVA on Ranks with post-hoc testing

12 athymic mice (CD1 strain) sc. transplanted with PaTu-8902 pancreatic ca cell line6 mice treated daily with S. platensis (0.5 g/kg for 17 days)

Spirulina-treated placebo-treated

Are plant tetrapyrroles responsible forobserved antiproliferative effects?

Are plant tetrapyrroles responsible forobserved antiproliferative effects?

Vliv fykokyanobilinu na viabilitu nádorových linií karcinomu pankreatu

0

20

40

60

80

100

120

140

31,3 62,5 125 250 500

koncentrace μmol/L

viab

ilita

buněk

v %

PA-TU-8902MIA PaCa-2

BxPC-3

*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT eseje

*

*

*

*

Vliv bilirubinu na viabilitu nádorových linií karcinomu pankreatu

0

20

40

60

80

100

120

5 10 50 100koncentrace μmol/L

viab

ilita

bun

ěk v

%

PA-TU-8902

MIA PaCa-2

BxPC-3

*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT

**

* **

**

**

Phycocyanobilin Effect of chlorophyllin on pancreatic cell lines viability

020406080

100120140

31 63 125 250 500

concentration μmol/L

cell

viab

ility

%

PA-TU-8902MIA PaCa-2BxPC-3

*p<0.05, Kruskal-Wallis comparison w ith post-hoc testing, viability w as determinated by MTT assay

***

***

p<0.05, Kruskal-Wallis test with post-hoc analysis, viability determined by MTT test

Chlorophyllin

concentration [μmol/L] concentration [μmol/L]

concentration [μmol/L]

Cell viability

[%]

Cell viability

[%]

Cell viability

[%]

BilirubinB

Plant tetrapyrroles and heme oxygenasePlant tetrapyrroles and heme oxygenasePaTu‐8902 HMOX activity 

100% 167% 100% 92% 48% 94%0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

200%

control  1 hemin 30 uM

control UCB 10uM

chlorophyllin30uM

PCB30uM

HO activity (%

 of control)

2

HMOX1 mRNA expression

HMOX activity measured as CO production by GC-reduction gasdetector (pmol CO/hr/mg protein), qPCR analyses performed on Applied Biosystems ViiA7instrument

Inhibitory effects of chlorophyllinobserved also in other pancreatic(MiaPaCa-2) and prostate ca cells(PC3) BLVRA expression was not affected by any of used substances

UCB: unconjugated bilirubin; PCB: phycocyanobilin

Plant tetrapyrroles and mitochondrial membrane potentialPlant tetrapyrroles and mitochondrial membrane potential12 hr incubation ofhepatoblastoma andpancreatic ca cellswith therapeuticsAddition of JC-1 fluorescent dye(Invitrogen, 20´)Determination ofMMP as a relativeproportion ofprecipitated and freedye

0.74PCB (10 uM)

1.29PCB (5 uM)

2.91PCB (1 uM)

3.29PCB (0.5 uM)

6.30controlPaTu‐8902

3.54S. platensis extrakt (0.3 mg/ml)

3.55S. platensis extract(0.1 mg/ml)

4.14S. platensis extract(0.03 mg/ml)

0.55chlorophyllin (30 uM)

1.34chlorophyllin (3 uM)

5.65controlHepG2

MMPInterventionCell line

MMP: mitochondrial membrane potential; PCB: phycocyanobilin

ConclusionsConclusionsOral administration of Spirulina platensis has potentantitumor effect on experimental human pancreatic cancerIn vitro, both chlorophylls and phycocyanobilindemonstrated potent antiproliferative effects on pancreatic cancer cellsChlorophylls substantially modulated HMOX activity in cancer cells, which might contribute to their putativeantiproliferative actionChlorophylls, as well as phycocyanobilin at lowconcentrations inhibited mitochondrial membranepotential, which might also account for their anticancereffects

AcknowledgementsAcknowledgements

This work has been supported by grants No. LH11030 given by Czech Ministry of Education, and GAUK-52210, GAUK-4389/2011 and SVV–2011–262513 givenby the Charles University in Prague