GENETICS,ENVIRONMENT & HUMAN DISEASE

1. 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

2. 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… )

3. 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

4. FRUIT AND EFFECTS OF VERGETABLE CONSUMPTION ON CARDIOVASCULAR DISEASE & CANCER CARDIOVASCULAR DISEASE ALL CANCERS Relative Risk Vegetables (servings/day)

5. EFFECT OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER ALL FRUIT/VEGETABLES (NHS WOMEN) CRUCIFEROUS VEGETABLES (NHS WOMEN ) Relative Risk Servings/Day Servings/Day

6. EFFECTS OF FRUIT AND VEGETABLE CONSUMPTION ON LUNG CANCER- NETHERLANDS COHORT STUDY All Veg Brassica Vegetables Relative Risk Servings/month Cancer Causes & Control 11,101,2000 62,573 Women & 58,279 men

7. CRUCIFEROUS VEGETABLE INTAKE AND BLADDER CANCER Relative Risk Relative Risk Servings/Week Servings/Months JNCI 91,605,1999 (HPFS – MEN)

8. CRUCIFEROUS VEGETABLE INTAKE AND CANCER CHEMOPREVENTION Non-Hodgkin’s Lymphoma Prostate Cancer Relative Risk Gram/Day Serving/Week Cancer Epid. Biom. Prev 9,795,2000 (Case-Control Study) Cancer Epid. Biom. Prev 9,477,2000 (NHS)

9. CRUCIFEROUS VEGETABLES AND CANCER – ANIMAL MODELS DMBA Basal diet Basal diet Brussels sprout % rats W tumors (mammary) 0 4 19 Weeks

10. O ll CH3-S-(CH2)4N=C=S Sulfurophane ANTICARCINOGENIC COMPOUNDS IN CRUCIFEROUS VEGETABLES Glucobrassican (Indole3-carbinol glucosinolates) R-N=C=S Isothiocyanate (as a glutosinolate)

11. CANCER CHEMOPREVENTION BY INDOLE-3-CARBINOL (I3C) – RAT MAMMARY Tumors rat I3C Anticancer Res 15,709,1995 140 Days DMBA

12. CANCER CHEMOTHERAPY BY I3C Inhibition of mammary tumor growth Inhibition of colon prostate cancer growth Inhibition of endometrial & cervical cancer cell growth

13. ANTICARCINOGENIC ACTIVITIES OF I3C Mitochondrial damage Activation of ER stress Inducation of cell death pathways Induction of phase I &II drug metabolizing enzymes Inactivation of cell cycle kinases Activation of cell cycle inhibitory kinases

14. HN HN HN HN HN PROBLEMS FOR DEVELOPMENT OF I3C-RELATED DRUGS PH5 Acid environ. of gut (pH < 2) DIM I3C ICZ + many more (trimers, tetramers…)

15. CH 2 N H ADVANTAGES IN USING DIM AS A MODEL FOR DEVELOPING ANT CANCER DRUGS ring methylene • DIM is readily synthesized from I3C • DIM is stable at low pH • Ring and methylene-substituted DIMs can be synthesized 2 DIM has many of the same properties as I3C but is more potent in vitro (cells) & in vivo (animals)

16. ANTITUMORIGENIC ACTIVITY OF DIM: RAT MAMMARY TUMOR MODEL(0.5 - 5.0 mg/kg/2d)* 1 4 0 0 C o n t r o l 1 2 0 0 0 . 5 1 . 0 1 0 0 0 5 . 0 8 0 0 Tumor Volume (mm3) 6 0 0 4 0 0 2 0 0 0 1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 DAY * no effect on organ weight/histopathology; no induction of CYP1A1/A2 (Carcinogenesis 19:1631)

17. SUBSTITUTED DIMs: STRUCTURE ACTIVITY RELATIONSHIPS X X X = Cl, Br, CH3, C6H5, Fl, OH, OCH3 at different positions

18. ANTITUMORIGENIC ACTIVITY OF SUBSTITUTED DIMs (500 μg/kg) Control 4000 5,5'-BrDIM 3000 Tumor Size (mm3) 2000 * * 1000 * 0 1 5 9 13 17 21 Days Days

19. MCF-7 MDA-MB-231 INHIBITION OF BREAST CANCER CELL GROWTH BY DIM AND 5,5’-DIBROMODIM A B

20. X X 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

21. H C R X N R = R = H 2 METHYLENE-SUBSTITUTED DIMs (C-DIMs) DIM-C-Ph DIM-C- PhX OMP C-DIM did not bind the Ah receptor however they exhibited anti-estrogenic activity

22. 200000 Me2SO 180000 1 mM 160000 5 mM 140000 10 mM 120000 Cells / Well 100000 80000 60000 40000 20000 0 1 3 5 7 Days C-DIMS INHIBIT BREAST CANCER CELL GROWTH DIM-C-pPhC6H5

23. C-DIMS INHIBIT RATMAMMARY TUMOR GROWTH 1600 Control 1400 DIM-C-pPhC6H5 1mg/kg/2d 1200 1000 Tumor volume (mm^3) 800 600 400 200 0 0 20 Treatment (days)

24. GAL4 RE GAL4 RE C-DIMS ACTIVATE PPARγ Screening receptors that bind lipophilic compounds MCF-7 Cells 10 PPARγ GAL4 R R P GAL4 Fold Induction RE 5 R = RAR, RXR AhR, PPARα or PPARγ P = Arnt or RXR 1 10 1 10 DMSO PGJ2 (μM) DIM-C-pPhCF3 (μM)

25. C-SUBSTITUTED DIMS AS PPARγ AGONISTS - SARs 0.8 Gal4Luc / pM-PPARg 0.7 0.6 0.5 Normalized Gal4Luc activity (U) 0.4 0.3 0.2 0.1 0 C 10 20 10 20 10 20 10 20 40 80 P C Cig PGJ2 O M (μM)

26. C-SUBSTITUTED DIMs AS PPARγ AGONISTS – SARs 2 Gal4Luc / pM-PPARg 1.8 1.6 1.4 1.2 Normalized Gal4Luc activity (U) 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 H OH C6H5 CN CH3 (μM)

27. C 2 MOST ACTIVE C-SUBSTITUTED DIMs AS PPARγ AGONISTS* DIM-C-pPhX DIM-C-pPhCF3 (X=CF3, #1) DIM-C-pPhtBu (X=tBu, #4) DIM-C-pPhC6H5 (X=C6H5, #9) * First generation agonists

28. GROWTH INHIBITORY PATHWAYS OF PPAR IN CANCER CELLS * • Induction of cdk inhibitors p21 and p27 • Downregulation of cyclin D1 • Induction of apoptosis • G0/G1  S phase block Ligand CoR RXR PPARγ PPRE * Mechanisms not well understood

29. 2 C H X N H 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

30. 2 Corn Oil Corn Oil 2000 2000 DIM-C-pPhC6H5(X=C6H5) DIM-C-pPhOCH3(X=OCH3) C H X N H 21 21 Day Day INHIBITION OF TUMOR GROWTH BY C-DIMs DMBA-INDUCED MAMMARY TUMORS

31. RXR PR RXR HETERODIMERS OTHER NRs AS POTENTIAL TARGETSFOR C-DIMs T3R FXR COUP OR OR CAR RAR HNF4 PXR/SXR VDR RXR LXR PPARs GCNF EcR NGFI-B TLX DIMERIC ORPHANRECEPTORS • receptors with known ligands (endogenous or synthetic) • orphan receptors with no known ligands (except RXR)

32. A/B C D E F Nur77 Zn Zn NGF1-B: AN ORPHAN RECEPTOR FAMILY OF STRUCTURALLY RELATED PROTEINS* 27% 92% 67% Nurr1 21% 91% 64% Nor1 * Initially identified after treatment of PC12 cells with NGF

33. ApoptosisInducer translocation Nur77 (cytosolic) Nur77 bcl2 Nur77 (nuclear) Apoptosis INDUCTION OF APOPTOSIS IN CANCER CELL LINES: ROLE OF Nur77 – TRANSLOCATION PATHWAY Mitochondria * Cell, 2004; Cancer Res, 2003

34. Panc-28 253JB-V-33 MiaPaCa-2 HCT-15 SW480 LNCaP Panc-1 MCF-7 HT-29 DLD1 RKO KU7 Nur77 N.S IS Nur77 WIDELY EXPRESSED IN CANCER CELL LINES?

35. Nur77 Nur77E/F GAL4DBD Nur77 Nur77 GAL4DBD GAL4-RE -luc GAL4-RE -luc   -luc NuRE C-SUBSTITUTED DIMs: ACTIVATION OF Nur77 TRANSACTIVATION ASSAYS

36. GAL4-Nur77/pGAL4 * 250 200 150 Fold Induction * 100 * * 50 * * * * * * 0 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 BY C-DIMs STRUCTURE-ACTIVITY RELATIONSHIPS Panc-28Cells

37. GAL4-Nur77 (EF)/pGAL4 80 * 60 * Fold Induction 40 * * * 20 * 0 5 10 15 5 10 15 5 10 15 5 10 15 C CF3 OCH3 H OH ACTIVATION OF Nur77 LBD (E/F DOMAIN) BY C-DIMs (AF1-INDEPENDENT)

38. DMSO (IgG) DMSO (anti-Nur77) DIM-C-pPhCF3 (anti-Nur77) DIM-C-pPhOCH3 (IgG) DIM-C-pPhOCH3 (anti-Nur77) DIM-C-Ph (anti-Nur77) SUBCELLULAR LOCATION OF LIGAND-ACTIVATED Nur77

39. 120 10 mM 1 mM DMSO 5 mM 100 80 60 % Cell Survival 40 20 0 DIM-C-Ph DIM-C-pPhOH DIM-C-pPhOCH3 DIM-C-pPhCF3 LIGAND ACTIVATED Nur77 – PANC-28 CELL SURVIVAL

40. DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph DIM-C-pPhOH DMSO 10 mM 20 mM 10 mM 20 mM 10 mM 20 mM 20 mM PARP 112kDa PARP 85kDa Bax N.S LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PANC-28 CELLS

41. DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph DMSO 10 mM 20 mM 10 mM 20 mM 10 mM 20 mM PARP 112kDa LNCap PARP 85kDa PARP 112kDa MiaPaCa-2 PARP 85kDa PARP 112kDa MCF-7 PARP 85kDa LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PROSTATE, BREAST AND PANCREATIC CANCER CELLS

42. INDUCTION OF APOPTOSIS Stimuli (FasLTRAIL…) Death Receptors Extrinsic Caspase 9 Caspase 8 Nucleus Bax Cytochrome C Caspase 3 VDAC Apoptotic Substrates Caspase Independent Intrinsic (mitochondrial) VDAC Bcl-2

43. * * * * DIM-C-pPhCF3 DIM-C-pPhOCH3 DIM-C-Ph DIM-C-pPhOH * DMSO 10 mM 20 mM 10 mM 20 mM 10 mM 20 mM 20 mM * TRAIL N.S Nur77 AGONISTS ACTIVATE EXTRINSIC APOPTOTIC PATHWAYS – INDUCTION OF TRAIL* (PANC 28) *also observed in thymocytes overexpressing Nur77

44. APOPTOSIS- Parp cleavage- nuclear condensation- increased annexin staining- decreased cell survival C-DIMs ProapoptoticGenes(TRAIL…) + + + Nur77 RE Nur77 AGONISTS: A NEW CLASS OF ANTICANCER DRUGS THAT INDUCE APOPTOSIS

45. X CHR CH 2 N N 2 2 H H X CH 2 N 2 H DEVELOPMENT OF I3C/DIM-DERIVED COMPOUNDS FOR CANCER CHEMOTHERAPY Potent anticancer drugs Interact with AhR/AR Mitochondrial toxicity I3C Ring DIMs Potent anticancer drugs Interact with PPARY, Nur77 & other receptors Induce other cell death pathways (mitochondrial toxicity) DIM C-DIMs