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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 8595% of non-cancer/cancer disease
Contaminants
Stress
Diet
HUMAN 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)
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
Relative Risk
CARDIOVASCULAR DISEASE
ALL CANCERS
1
1
0.5
0.5
0
0
<1.5 1.5- 3.00- 5.00- 7.002.99 4.99 6.99 8.99
9
<1.5
1.5- 3.00- 5.00- 7.002.99 4.99 6.99 8.99
Vegetables (servings/day)
9
EFFECT OF FRUIT AND VEGETABLE
CONSUMPTION ON LUNG CANCER
Relative Risk
ALL FRUIT/VEGETABLES
(NHS WOMEN)
CRUCIFEROUS VEGETABLES
(NHS WOMEN )
2
2
1
1
0
0
0
2
4
6
Servings/Day
8
2
4
6
Servings/Day
8
EFFECTS OF FRUIT AND VEGETABLE
CONSUMPTION ON LUNG CANCER- NETHERLANDS
COHORT STUDY
Relative Risk
1
All
Veg
Brassica Vegetables
0.5
0
<2
<1
2 to 3
4
8
Servings/month
Cancer Causes & Control 11,101,2000
62,573 Women & 58,279 men
CRUCIFEROUS VEGETABLE INTAKE AND
BLADDER CANCER
1.5
1.5
Cabbage
<1
1
>2
Brussels
Sprouts
Cauliflower
1
Relative Risk
Relative Risk
1
0.5
0
0.5
0
<1.0
1.0-3.0
>4
Servings/Months
<1
1
>2
Servings/Week
JNCI 91,605,1999 (HPFS – MEN)
CRUCIFEROUS VEGETABLE INTAKE AND
CANCER CHEMOPREVENTION
Relative Risk
Non-Hodgkin’s Lymphoma
Prostate Cancer
1
1
0.5
0.5
0
0
<2
2.0-4.0
5 to 6
Serving/Week
Cancer Epid. Biom. Prev
9,477,2000 (NHS)
<8.8 8.9-20 21-36 36-73
>73
Gram/Day
Cancer Epid. Biom. Prev
9,795,2000 (Case-Control Study)
CRUCIFEROUS VEGETABLES AND CANCER –
ANIMAL MODELS
DMBA
100
Basal diet
Basal diet
Brussels sprout
% rats W
tumors
(mammary)
0
0
4
Weeks
19
ANTICARCINOGENIC COMPOUNDS IN
CRUCIFEROUS VEGETABLES
CH2OH
N
H
Glucobrassican
(Indole3-carbinol glucosinolates)
R-N=C=S
Isothiocyanate
(as a glutosinolate)
O
ll
CH3-S-(CH2)4N=C=S
Sulfurophane
CANCER CHEMOPREVENTION BY
INDOLE-3-CARBINOL (I3C) – RAT MAMMARY
Tumors
rat
9
CH2OH
DMBA
DMBA+ 50
mg/Kg/Day
N
DMBA+100
mg/Kg/Day
H
I3C
Anticancer Res
15,709,1995
0
140
DMBA
Days
CANCER CHEMOTHERAPY BY I3C
Inhibition of mammary tumor
growth
CH2OH
N
Inhibition of colon prostate
cancer growth
H
Inhibition of endometrial &
cervical cancer cell growth
ANTICARCINOGENIC ACTIVITIES OF I3C
Activation of ER
stress
Mitochondrial
damage
Inducation of cell
death pathways
CH2OH
N
H
Induction of phase I &II
drug metabolizing
enzymes
Inactivation of cell
cycle kinases
Activation of cell cycle
inhibitory kinases
PROBLEMS FOR DEVELOPMENT OF I3CRELATED DRUGS
CH2OH
5
PH5
N
H2
3 C
4
6
7
H
N1
2
H
N
H
DIM
I3C
ICZ
+ many more (trimers, tetramers…)
ADVANTAGES IN USING DIM AS A MODEL
FOR DEVELOPING ANT CANCER DRUGS
ring
methylene
CH 2
•DIM is readily synthesized from
I3C
•DIM is stable at low pH
N
H
2
•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)
ANTITUMORIGENIC ACTIVITY OF DIM: RAT
MAMMARY TUMOR MODEL
(0.5 - 5.0 mg/kg/2d)*
Tumor Volume (mm3)
1400
Control
0.5
1.0
5.0
1200
1000
800
600
400
200
0
1
3
5
7
9
11
13
15
17
19
21
DAY
* no effect on organ weight/histopathology; no induction of
CYP1A1/A2 (Carcinogenesis 19:1631)
SUBSTITUTED DIMs: STRUCTURE
ACTIVITY RELATIONSHIPS
H2
C
4
3
5
X
6
7
N1
H
X=
2
X
N
H
Cl, Br, CH3, C6H5, Fl, OH, OCH3
at different positions
ANTITUMORIGENIC ACTIVITY OF
SUBSTITUTED DIMs (500 μg/kg)
Tumor Size (mm3)
6000
Control
Control
4000
2,2'-MeDIM
5000
5,5'-BrDIM
3000
4000
3000
2000
2000
* *
*
1000
1000
0
0
1
5
9
13
Days
17
21
1
5
9
13
Days
17
21
INHIBITION OF BREAST CANCER CELL
GROWTH BY DIM AND 5,5’-DIBROMODIM
A
B
MCF-7
MDA-MB-231
4
3.5
3
2.5
2
1.5
1
0.5
0
5,5'-diBrDIM
DMSO
5µM
10µM
20µM
0
5
Cell number (x10 )
5
Cell number (x10 )
5,5'-diBrDIM
2
4
2.5
DMSO
5µM
10µM
20µM
2
1.5
1
0.5
0
6
0
2
Time (Days)
DMSO
5µM
10µM
20µM
0
5
2
4
Time (Days)
6
4
6
DIM
Cell number (x10 )
5
Cell number (x10 )
DIM
3.5
3
2.5
2
1.5
1
0.5
0
4
Time (Days)
6
DMSO
5µM
10µM
20µM
2.5
2
1.5
1
0.5
0
0
2
Time (Days)
COMPARATIVE ANTICARCINOGENIC
ACTIVITIES OF DIM AND RING-SUBSTITUTED
DIMs
3
5
X
X
H2
C
4
3
5
6
7
N1
H
2
H2
C
4
N
H
• Growth inhibitory
• AhR agonist
(antiestrogen)
• Modulates cell cycle genes
• Decreased MMP (+)
• Antiandrogen
6
7
N1
H
2
N
H
• Growth inhibitory
• AhR agonist
(antiestrogen)
• Modulates cell cycle genes
• Decreased MMP(+++)
• Antiandrogen/androgen
METHYLENE-SUBSTITUTED DIMs (C-DIMs)
H
C
N
H
R
R=
DIM-C-Ph
X
2
R=
DIM-C-
O
M
P
PhX
C-DIM did not bind the Ah receptor however
they exhibited anti-estrogenic activity
C-DIMS INHIBIT BREAST CANCER CELL
GROWTH
DIM-C-pPhC6H5
200000
Me2SO
Cells / Well
180000
1 mM
160000
5 mM
140000
10 mM
120000
100000
80000
60000
40000
20000
0
1
3
5
Days
7
Tumor volume (mm^3)
C-DIMS INHIBIT RATMAMMARY TUMOR
GROWTH
1600
Control
1400
DIM-C-pPhC6H5
1mg/kg/2d
1200
1000
800
600
400
200
0
0
20
Treatment (days)
C-DIMS ACTIVATE PPARγ
Screening receptors that
bind lipophilic compounds
MCF-7 Cells
10
PPARγ
R
GAL4
R P
GAL4 RE
RE
Fold Induction
GAL4
GAL4 RE
5
R = RAR, RXR AhR, PPARα or
PPARγ
P = Arnt or RXR
DMSO
1
10
PGJ2 (μM)
1
10
DIM-C-pPhCF3
(μM)
Normalized Gal4Luc activity (U)
C-SUBSTITUTED DIMS AS PPARγ
AGONISTS - SARs
0.8
Gal4Luc / pM-PPARg
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
C
C
40
Cig
80
10
20
PGJ2
10
20
O
10
20
10
20
P
M
CF3
DIM -C
(μM)
Normalized Gal4Luc activity (U)
C-SUBSTITUTED DIMs AS PPARγ
AGONISTS – SARs
2
Gal4Luc / pM-PPARg
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
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
DIM -C
X
H
(μM)
OH
C6H5
CN
CH3
MOST ACTIVE C-SUBSTITUTED DIMs
AS PPARγ AGONISTS*
H
C
X
N
H
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
PPARg IN CANCER CELLS
*
• Induction of cdk
inhibitors p21 and p27
Ligand
CoR
PPARγ RXR
PPRE
• 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 PPARg
CH
X
N
H
2
• 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 PPARg, RAR, RXR, AhR
INHIBITION OF TUMOR GROWTH BY C-DIMs
DMBA-INDUCED MAMMARY TUMORS
CH
X
N
H
2000
Corn Oil
DIM-C-pPhC6H5
(X=C6H5)
Day
2
2000
21
Corn Oil
DIM-C-pPhOCH3
(X=OCH3)
Day
21
OTHER NRs AS POTENTIAL TARGETS
FOR C-DIMs
RXR
PR
T3R
RAR
VDR
PPARs
EcR
FXR
CAR
PXR/SXR
LXR
OR OR
COUP
HNF4
RXR
GCNF
NGFI-B
TLX
RXR HETERODIMERS
DIMERIC ORPHAN
RECEPTORS
• 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*
A/B
C
D
E
F
Nur77
Zn
Zn
27%
92%
67%
21%
91%
64%
Nurr1
Nor1
* Initially identified after treatment of PC12 cells with NGF
INDUCTION OF APOPTOSIS IN CANCER
CELL LINES: ROLE OF Nur77 –
TRANSLOCATION PATHWAY
Apoptosis
Inducer
Nur77 (cytosolic)
Nur77 bcl2
Nur77
(nuclear)
Mitochondria
* Cell, 2004; Cancer Res, 2003
Apoptosis
IS Nur77 WIDELY EXPRESSED IN CANCER
CELL LINES?
Nur77
N.S
C-SUBSTITUTED DIMs: ACTIVATION OF Nur77
Nur77
Nur77
E/F
GAL4
DBD
GAL4
DBD
GAL4-RE
-luc
GAL4-RE
Nur77 Nur77
-luc
 
NuRE
TRANSACTIVATION ASSAYS
-luc
ACTIVATION OF Nur77 BY C-DIMs
STRUCTURE-ACTIVITY RELATIONSHIPS
GAL4-Nur77/pGAL4
H
Fold Induction
250
200
Panc-28
Cells
150
N
H
*
100
50
*
X
C
*
*
*
0
2
*
* *
*
*
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
ACTIVATION OF Nur77 LBD (E/F DOMAIN) BY
C-DIMs (AF1-INDEPENDENT)
Fold Induction
GAL4-Nur77 (EF)/pGAL4
H
X
C
80
*
60
N
*
40
*
20
H
2
*
*
*
0
5
C
10
CF3
15
5
10
OCH3
15
5
10
H
15
5
10
OH
15
SUBCELLULAR LOCATION OF LIGANDACTIVATED Nur77
DMSO
(IgG)
DIM-C-pPhOCH3
(IgG)
DMSO
(anti-Nur77)
DIM-C-pPhOCH3
(anti-Nur77)
DIM-C-pPhCF3
(anti-Nur77)
DIM-C-Ph
(anti-Nur77)
LIGAND ACTIVATED Nur77 – PANC-28
CELL SURVIVAL
120
DMSO
1 mM
5 mM
10 mM
% Cell Survival
100
80
60
40
20
0
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph
DIM-C-pPhOH
LIGAND ACTIVATED Nur77: INDUCTION OF
PARP CLEAVAGE IN PANC-28 CELLS
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph
DMSO 10 mM
PARP 112kDa
PARP 85kDa
Bax
N.S
20 mM 10 mM 20 mM
10 mM 20 mM
DIM-C-pPhOH
20 mM
LIGAND ACTIVATED Nur77: INDUCTION OF PARP
CLEAVAGE IN PROSTATE, BREAST AND
PANCREATIC CANCER CELLS
DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph
DMSO
PARP 112kDa
LNCap
PARP 85kDa
MiaPaCa-2
PARP 112kDa
PARP 85kDa
PARP 112kDa
MCF-7
PARP 85kDa
10 mM
20 mM 10 mM 20 mM
10 mM 20 mM
INDUCTION OF APOPTOSIS
(FasL
TRAIL…)
Stimuli
Death
Receptors
Extrinsic
Caspase 9
Bax
Caspase 8
Nucleus
Cytochrome C
Caspase 3
VDAC
Caspase
Independent
VDAC
Bcl-2
Intrinsic
(mitochondrial)
Apoptotic
Substrates
O.D Units
Nur77 AGONISTS ACTIVATE EXTRINSIC
APOPTOTIC PATHWAYS – INDUCTION OF TRAIL*
(PANC 28)
160
140
120
100
80
60
40
20
0
*
*
*
*
*
*
DIM-C-pPhCF3 DIM-C-pPhOCH3
DMSO 10 mM
20 mM 10 mM 20 mM
DIM-C-Ph DIM-C-pPhOH
10 mM
20 mM
TRAIL
N.S
*also observed in thymocytes overexpressing Nur77
20 mM
Nur77 AGONISTS: A NEW CLASS OF
ANTICANCER DRUGS THAT INDUCE APOPTOSIS
C-DIMs
+++
Nur77
RE
Proapoptotic
Genes
(TRAIL…)
APOPTOSIS
- Parp cleavage
- nuclear
condensation
- increased annexin
staining
- decreased cell
survival
DEVELOPMENT OF I3C/DIM-DERIVED
COMPOUNDS FOR CANCER CHEMOTHERAPY
X
CH 2
I3C
• Interact with AhR/AR
N
H
X
CH 2
N
H
• Potent anticancer drugs
2
Ring DIMs
• Mitochondrial toxicity
• Potent anticancer drugs
2
DIM
CHR
N
H
C-DIMs
• Interact with PPARY, Nur77
& other receptors
• Induce other cell death
2
pathways (mitochondrial
toxicity)