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GENETICS,ENVIRONMENT & HUMAN DISEASE. Genetic factors associated with critical gene mutations/deletion may be responsible for 5-15% of non-cancer & cancer diseases Lifestyle/environment accounts for 85-95% of non-cancer/cancer disease. Contaminants. Stress. Diet. HUMAN DIET & DISEASE. - PowerPoint PPT Presentation
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• Genetic factors associated with critical gene mutations/deletion may be responsible for 5-15% of non-cancer & cancer diseases
• Lifestyle/environment accounts for 85-95% of non-cancer/cancer disease
GENETICS,ENVIRONMENT & HUMAN DISEASE
GENETICS,ENVIRONMENT & HUMAN DISEASE
Contaminants Stress Diet
HUMAN DIET & DISEASEHUMAN DIET & DISEASE
• A “balanced” diet of nutrients promotes health
• Vegetables & fruit are generally thought to be health promoting
• Foods leading to obesity are thought to be harmful
• Many other factors influence effects of food on health (e.g. age, genetics, smoking, occupation, alcohol… )
FRUIT/VEGETABLE INTAKE AND RISK FOR CHRONIC DISEASE (JNCI 96, 1577, 2004)
FRUIT/VEGETABLE INTAKE AND RISK FOR CHRONIC DISEASE (JNCI 96, 1577, 2004)
Prospective study on two major groups a) Nurses Health Study (NHS est 1976) 121,700 b) Health Professionals Followup Study (HPFS) (est1980) 51,529 male dentist, optometrists….
Biannual Questionnaires on :• Medical history• Food consumption• Other individual characteristics/behaviors
FRUIT AND EFFECTS OF VERGETABLE CONSUMPTION ON CARDIOVASCULAR DISEASE &
CANCER
FRUIT AND EFFECTS OF VERGETABLE CONSUMPTION ON CARDIOVASCULAR DISEASE &
CANCER
CARDIOVASCULAR DISEASE
0
0.5
1
<1.5 1.5-2.99
3.00-4.99
5.00-6.99
7.00-8.99
9
Rel
ativ
e R
isk
0
0.5
1
<1.5 1.5-2.99
3.00-4.99
5.00-6.99
7.00-8.99
9
ALL CANCERS
Vegetables (servings/day)
EFFECT OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER
EFFECT OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER
0
1
2
0 2 4 6 8
ALL FRUIT/VEGETABLES (NHS WOMEN)
Rel
ativ
e R
isk
0
1
2
2 4 6 8
Servings/Day
CRUCIFEROUS VEGETABLES (NHS WOMEN )
Servings/Day
EFFECTS OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER- NETHERLANDS
COHORT STUDY
EFFECTS OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER- NETHERLANDS
COHORT STUDY
Cancer Causes & Control 11,101,2000 62,573 Women & 58,279 men
Servings/month
Rel
ativ
e R
isk
0
0.5
1
<2 <1 2 to 3 4 8
All Veg
Brassica Vegetables
CRUCIFEROUS VEGETABLE INTAKE AND BLADDER CANCER
CRUCIFEROUS VEGETABLE INTAKE AND BLADDER CANCER
0
0.5
1
1.5
<1 1 >2
<1
1
>2
0
0.5
1
1.5
<1.0 1.0-3.0 >4
Cabbage
BrusselsSproutsCauliflower
Servings/Months Servings/Week
JNCI 91,605,1999 (HPFS – MEN)
Rel
ativ
e R
isk
Rel
ativ
e R
isk
CRUCIFEROUS VEGETABLE INTAKE AND CANCER CHEMOPREVENTION
CRUCIFEROUS VEGETABLE INTAKE AND CANCER CHEMOPREVENTION
0
0.5
1
<2 2.0-4.0 5 to 60
0.5
1
<8.8 8.9-20 21-36 36-73 >73
Rel
ativ
e R
isk
Cancer Epid. Biom. Prev9,477,2000 (NHS)
Cancer Epid. Biom. Prev9,795,2000 (Case-Control Study)
Serving/Week Gram/Day
Non-Hodgkin’s Lymphoma Prostate Cancer
CRUCIFEROUS VEGETABLES AND CANCER – ANIMAL MODELS
CRUCIFEROUS VEGETABLES AND CANCER – ANIMAL MODELS
0
100
Basal diet
Basal diet
Brussels sprout
DMBA
% rats W tumors
(mammary)
Weeks0 4 19
ANTICARCINOGENIC COMPOUNDS IN CRUCIFEROUS VEGETABLES
ANTICARCINOGENIC COMPOUNDS IN CRUCIFEROUS VEGETABLES
R-N=C=SIsothiocyanate
(as a glutosinolate)
CH3-S-(CH2)4N=C=SSulfurophane
llO
Glucobrassican(Indole3-carbinol glucosinolates)
N
CH2OH
H
CANCER CHEMOPREVENTION BY INDOLE-3-CARBINOL (I3C) – RAT MAMMARY
CANCER CHEMOPREVENTION BY INDOLE-3-CARBINOL (I3C) – RAT MAMMARY
0
9
DMBA
DMBA+ 50mg/Kg/Day
DMBA+100mg/Kg/Day
Tu
mo
rsra
t
DMBA Days140
Anticancer Res 15,709,1995
I3C
N
CH2OH
H
CANCER CHEMOTHERAPY BY I3CCANCER CHEMOTHERAPY BY I3C
Inhibition of mammary tumor growth
Inhibition of colon prostate cancer growth
Inhibition of endometrial & cervical cancer cell growth
N
CH2OH
H
ANTICARCINOGENIC ACTIVITIES OF I3C
ANTICARCINOGENIC ACTIVITIES OF I3C
Activation of ER stress
Inactivation of cell cycle kinases
Induction of phase I &II drug metabolizing
enzymes
Activation of cell cycle inhibitory kinases
Inducation of cell death pathways
Mitochondrial damage
N
CH2OH
H
PROBLEMS FOR DEVELOPMENT OF I3C-RELATED DRUGS
PROBLEMS FOR DEVELOPMENT OF I3C-RELATED DRUGS
N
H2 C
N
H H1
2
34
5
6
7N
CH2OH
H
HN
HN
HN H
NHN
+ many more (trimers, tetramers…)
DIM
Acid environ. of
gut (pH < 2)
I3C
ICZ
PH5
ADVANTAGES IN USING DIM AS A MODEL FOR DEVELOPING ANT CANCER DRUGS
ADVANTAGES IN USING DIM AS A MODEL FOR DEVELOPING ANT CANCER DRUGS
•DIM is readily synthesized from I3C
•DIM is stable at low pH
•Ring and methylene-substituted DIMs can be synthesized
DIM has many of the same properties as I3C but is more potent in vitro (cells) & in vivo (animals)
N
CH2
H 2
ring methylene
0
200
400
600
800
1000
1200
1400
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1
Control0.51.05.0
DAY
Tu
mo
r V
olu
me
(mm
3)
* no effect on organ weight/histopathology; no induction of CYP1A1/A2 (Carcinogenesis 19:1631)
ANTITUMORIGENIC ACTIVITY OF DIM: RAT MAMMARY TUMOR MODEL
(0.5 - 5.0 mg/kg/2d)*
ANTITUMORIGENIC ACTIVITY OF DIM: RAT MAMMARY TUMOR MODEL
(0.5 - 5.0 mg/kg/2d)*
N
H2 C
N
H H1
2
34
5
6
7
X X
X = Cl, Br, CH3, C6H5, Fl, OH, OCH3
at different positions
SUBSTITUTED DIMs: STRUCTURE ACTIVITY RELATIONSHIPS
SUBSTITUTED DIMs: STRUCTURE ACTIVITY RELATIONSHIPS
0
1000
2000
3000
4000
5000
6000
1 5 9 13 17 21
Control
2,2'-MeDIM
** *
Days Days
Tu
mo
r S
ize
(mm
3 )ANTITUMORIGENIC ACTIVITY OF SUBSTITUTED DIMs (500 μg/kg)
ANTITUMORIGENIC ACTIVITY OF SUBSTITUTED DIMs (500 μg/kg)
0
1000
2000
3000
4000
1 5 9 13 17 21
Control
5,5'-BrDIM
INHIBITION OF BREAST CANCER CELL GROWTH BY DIM AND 5,5’-DIBROMODIM
INHIBITION OF BREAST CANCER CELL GROWTH BY DIM AND 5,5’-DIBROMODIM
5,5'-diBrDIM
0
0.5
1
1.5
2
2.5
0 2 4 6
Time (Days)
Ce
ll n
um
be
r (x
10
5) DMSO
5µM10µM20µM
5,5'-diBrDIM
00.5
11.5
22.5
33.5
4
0 2 4 6
Time (Days)
Ce
ll n
um
be
r (x
10
5) DMSO
5µM10µM20µM
DIM
00.5
11.5
22.5
33.5
0 2 4 6
Time (Days)
Ce
ll n
um
be
r (x
10
5) DMSO5µM10µM20µM
DIM
0
0.5
1
1.5
2
2.5
0 2 4 6
Time (Days)
Ce
ll n
um
be
r (x
10
5) DMSO
5µM10µM20µM
MCF-7 MDA-MB-231A B
COMPARATIVE ANTICARCINOGENIC ACTIVITIES OF DIM AND RING-SUBSTITUTED
DIMs
COMPARATIVE ANTICARCINOGENIC ACTIVITIES OF DIM AND RING-SUBSTITUTED
DIMs
• Growth inhibitory• AhR agonist (antiestrogen)• Modulates cell cycle genes• Decreased MMP (+)• Antiandrogen
• Growth inhibitory • AhR agonist (antiestrogen)• Modulates cell cycle genes• Decreased MMP(+++)• Antiandrogen/androgen
N
H2 C
N
H H1
2
34
5
6
7 N
H2 C
N
H H1
2
34
5
6
7
X X
METHYLENE-SUBSTITUTED DIMs (C-DIMs)METHYLENE-SUBSTITUTED DIMs (C-DIMs)
R =
X
R =
DIM-C-Ph
DIM-C- PhXOMP
2
N
H
H
CR
C-DIM did not bind the Ah receptor however they exhibited anti-estrogenic activity
C-DIMS INHIBIT BREAST CANCER CELL GROWTH
C-DIMS INHIBIT BREAST CANCER CELL GROWTH
020000
40000
60000
80000
100000
120000
140000
160000
180000
200000
1 3 5 7
Days
Cel
ls /
Wel
l
Me2SO
1 M
5 M
10 M
DIM-C-pPhC6H5
C-DIMS INHIBIT RATMAMMARY TUMOR GROWTH
C-DIMS INHIBIT RATMAMMARY TUMOR GROWTH
0
200
400
600
800
1000
1200
1400
1600
20
Treatment (days)
Tu
mo
r v
olu
me
(mm
^3
) Control
DIM-C-pPhC6H5
1mg/kg/2d
0
C-DIMS ACTIVATE PPARγC-DIMS ACTIVATE PPARγ
Screening receptors that bind lipophilic compounds
GAL4R
GAL4 RE RE
R P
R = RAR, RXR AhR, PPARα or PPARγ
P = Arnt or RXR
5
10
Fo
ld I
nd
uct
ion
DMSO
PGJ2 (μM) DIM-C-pPhCF3 (μM)
1 10 1 10
MCF-7 Cells
GAL4 RE
PPARγ
GAL4
C-SUBSTITUTED DIMS AS PPARγ AGONISTS - SARs
C-SUBSTITUTED DIMS AS PPARγ AGONISTS - SARs
Gal4Luc / pM-PPAR
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
C 40 80 10 20 10 20 10 20 10 20
C Cig PGJ2 O M P
No
rmal
ized
Gal
4Lu
c a
cti
vit
y (U
)
DIM-C
CF3
(μM)
C-SUBSTITUTED DIMs AS PPARγ AGONISTS – SARs
C-SUBSTITUTED DIMs AS PPARγ AGONISTS – SARs
DIM-C X
Gal4Luc / pM-PPAR
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
C 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20
CF3 Br F tBu OCH3 N(CH3)2 H OH C6H5 CN CH3
No
rmal
ized
Gal
4Lu
c a
cti
vit
y (U
)
(μM)
MOST ACTIVE C-SUBSTITUTED DIMs AS PPARγ AGONISTS*
MOST ACTIVE C-SUBSTITUTED DIMs AS PPARγ AGONISTS*
N
H
H
XC
2
DIM-C-pPhX
DIM-C-pPhCF3 (X=CF3, #1)
DIM-C-pPhtBu (X=tBu, #4)
DIM-C-pPhC6H5 (X=C6H5, #9)* First generation agonists
GROWTH INHIBITORY PATHWAYS OF PPAR IN CANCER CELLS
GROWTH INHIBITORY PATHWAYS OF PPAR IN CANCER CELLS
PPARγ
PPRE
RXR
CoR
Ligand
• Induction of cdk inhibitors p21 and p27
• Downregulation of cyclin D1
• Induction of apoptosis
• G0/G1 S phase block
*
* Mechanisms not well understood
C-DIMs WHICH INHIBIT TUMOR/CELL GROWTH BUT EXHIBIT LOW ACTIVATION OF PPAR
C-DIMs WHICH INHIBIT TUMOR/CELL GROWTH BUT EXHIBIT LOW ACTIVATION OF PPAR
• DIM-C-pPhOCH3 (X=OCH3) and DIM-C-Ph (X=H) inhibit growth of multiple cancer cell lines
• Both compounds also block DMBA-induced mammary tumor growth in vivo
• Minimal activation of PPAR, RAR, RXR, AhR
N
H
CH X
2
INHIBITION OF TUMOR GROWTH BY C-DIMs DMBA-INDUCED MAMMARY TUMORS
2000
21Day
Corn OilDIM-C-pPhC6H5
(X=C6H5)
2000
21Day
Corn OilDIM-C-pPhOCH3
(X=OCH3)
N
H
CH X
2
OTHER NRs AS POTENTIAL TARGETSFOR C-DIMs
OTHER NRs AS POTENTIAL TARGETSFOR C-DIMs
RXR PR
RXR HETERODIMERS
OR OR
DIMERIC ORPHANRECEPTORS
COUPHNF4RXRGCNFNGFI-BTLX
T3RRARVDRPPARsEcR
FXRCARPXR/SXRLXR
• receptors with known ligands (endogenous or synthetic)
• orphan receptors with no known ligands (except RXR)
NGF1-B: AN ORPHAN RECEPTOR FAMILY OF STRUCTURALLY RELATED PROTEINS*
NGF1-B: AN ORPHAN RECEPTOR FAMILY OF STRUCTURALLY RELATED PROTEINS*
A/B C D E F
ZnZn
Nur77
27% 92% 67%
Nurr1
21% 91% 64%
Nor1
* Initially identified after treatment of PC12 cells with NGF
INDUCTION OF APOPTOSIS IN CANCER CELL LINES: ROLE OF Nur77 –
TRANSLOCATION PATHWAY
INDUCTION OF APOPTOSIS IN CANCER CELL LINES: ROLE OF Nur77 –
TRANSLOCATION PATHWAY
Nur77 bcl2
Nur77 (cytosolic)
Apoptosis
Nur77(nuclear)
translocationApoptosis
Inducer
* Cell, 2004; Cancer Res, 2003
Mitochondria
IS Nur77 WIDELY EXPRESSED IN CANCER CELL LINES?
IS Nur77 WIDELY EXPRESSED IN CANCER CELL LINES?
Pan
c-28
Pan
c-1
Mia
PaC
a-2
LNC
aP
MC
F-7
RK
O
DLD
1
SW
480
HT-
29
HC
T-15
KU
7
253J
B-V
-33
Nur77
N.S
C-SUBSTITUTED DIMs: ACTIVATION OF Nur77C-SUBSTITUTED DIMs: ACTIVATION OF Nur77
Nur77
GAL4DBD
GAL4-RE -luc
Nur77E/F
GAL4DBD
GAL4-RE -luc
Nur77
-luc
Nur77
NuRE
TRANSACTIVATION ASSAYS
ACTIVATION OF Nur77 BY C-DIMs STRUCTURE-ACTIVITY RELATIONSHIPS
ACTIVATION OF Nur77 BY C-DIMs STRUCTURE-ACTIVITY RELATIONSHIPS
*
*
* * *
*
*
*
* *
GAL4-Nur77/pGAL4
0
50
100
150
200
250
10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20
C CF3 Br F tBu OCH3 N(CH3)2 H OH C6H5 CN CH3
Fo
ld I
nd
uct
ion Panc-28
Cells
ACTIVATION OF Nur77 LBD (E/F DOMAIN) BY C-DIMs (AF1-INDEPENDENT)
ACTIVATION OF Nur77 LBD (E/F DOMAIN) BY C-DIMs (AF1-INDEPENDENT)
*
*
*
*
**
GAL4-Nur77 (EF)/pGAL4
0
20
40
60
80
5 10 15 5 10 15 5 10 15 5 10 15
C CF3 OCH3 H OH
Fo
ld In
du
ctio
n
SUBCELLULAR LOCATION OF LIGAND-ACTIVATED Nur77
SUBCELLULAR LOCATION OF LIGAND-ACTIVATED Nur77
DMSO(anti-Nur77)
DMSO(IgG)
DIM-C-pPhOCH3
(IgG)
DIM-C-pPhCF3
(anti-Nur77)
DIM-C-Ph(anti-Nur77)
DIM-C-pPhOCH3
(anti-Nur77)
LIGAND ACTIVATED Nur77 – PANC-28 CELL SURVIVAL
LIGAND ACTIVATED Nur77 – PANC-28 CELL SURVIVAL
1 M 5 MDMSO 10 M
DIM-C-pPhCF3 DIM-C-pPhOCH3DIM-C-Ph DIM-C-pPhOH
0
20
40
60
80
100
120
% C
ell
Su
rviv
al
LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PANC-28 CELLS
LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PANC-28 CELLS
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph DIM-C-pPhOH
DMSO 10 M 20 M 10 M 20 M10 M 20 M20 M
PARP 112kDa
PARP 85kDa
N.S
Bax
LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PROSTATE, BREAST AND
PANCREATIC CANCER CELLS
LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PROSTATE, BREAST AND
PANCREATIC CANCER CELLS
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph
DMSO 10 M 20 M 10 M 20 M10 M20 M
PARP 112kDa
PARP 85kDa
PARP 112kDa
PARP 85kDa
PARP 112kDa
PARP 85kDa
LNCap
MiaPaCa-2
MCF-7
INDUCTION OF APOPTOSISINDUCTION OF APOPTOSIS
Stimuli
Extrinsic
Death Receptors
Caspase 9
Cytochrome C
Nucleus
Intrinsic (mitochondrial)
VDAC
VDACBcl-2
Bax
Caspase 3
Caspase 8
Apoptotic SubstratesCaspase
Independent
(FasLTRAIL…)
Nur77 AGONISTS ACTIVATE EXTRINSIC APOPTOTIC PATHWAYS – INDUCTION OF
TRAIL* (PANC 28)
Nur77 AGONISTS ACTIVATE EXTRINSIC APOPTOTIC PATHWAYS – INDUCTION OF
TRAIL* (PANC 28)
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph DIM-C-pPhOH
DMSO 10 M 20 M 10 M 20 M10 M 20 M20 M
TRAIL
N.S
0
20
40
60
80
100
120
140
160
O.D
Uni
ts
**
*
*
*
*
*also observed in thymocytes overexpressing Nur77
Nur77 AGONISTS: A NEW CLASS OF ANTICANCER DRUGS THAT INDUCE APOPTOSIS
Nur77 AGONISTS: A NEW CLASS OF ANTICANCER DRUGS THAT INDUCE APOPTOSIS
Nur77
RE
C-DIMs
+ + + ProapoptoticGenes
(TRAIL…)
APOPTOSIS- Parp cleavage- nuclear condensation- increased annexin staining- decreased cell survival
DEVELOPMENT OF I3C/DIM-DERIVED COMPOUNDS FOR CANCER CHEMOTHERAPY
DEVELOPMENT OF I3C/DIM-DERIVED COMPOUNDS FOR CANCER CHEMOTHERAPY
Ring DIMs
C-DIMs
DIM
I3CPotent anticancer drugs
Interact with AhR/AR
Mitochondrial toxicity
Potent anticancer drugs
Interact with PPARY, Nur77 & other receptors
Induce other cell death pathways (mitochondrial toxicity)
•
•
•
•
•
• N
CH 2
H
X
2
N
CHR
H 2
N
CH 2
H
X
2