• 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