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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.

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Presentation on theme: "Genetic factors associated with critical gene mutations/deletion may be responsible for 5-15% of non-cancer & cancer diseases Lifestyle/environment accounts."— Presentation transcript:

1 Genetic factors associated with critical gene mutations/deletion may be responsible for 5-15% of non-cancer & cancer diseases Lifestyle/environment accounts for % of non-cancer/cancer disease GENETICS,ENVIRONMENT & HUMAN DISEASE ContaminantsStressDiet

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 Relative Risk ALL CANCERS Vegetables (servings/day)

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

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

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

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

9 CRUCIFEROUS VEGETABLES AND CANCER – ANIMAL MODELS Basal diet Brussels sprout DMBA % rats W tumors (mammary) Weeks 0419

10 ANTICARCINOGENIC COMPOUNDS IN CRUCIFEROUS VEGETABLES R-N=C=S Isothiocyanate (as a glutosinolate) CH 3 -S-(CH 2 ) 4 N=C=S Sulfurophane ll O Glucobrassican (Indole3-carbinol glucosinolates)

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

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 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

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

15 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 CH 2 H 2 ring methylene

16 Control DAY Tumor Volume (mm3) * no effect on organ weight/histopathology; no induction of CYP1A1/A2 (Carcinogenesis 19:1631) ANTITUMORIGENIC ACTIVITY OF DIM: RAT MAMMARY TUMOR MODEL ( mg/kg/2d)*

17 XX X = Cl, Br, CH 3, C 6 H 5, Fl, OH, OCH 3 at different positions SUBSTITUTED DIMs: STRUCTURE ACTIVITY RELATIONSHIPS

18 * * * Days Tumor Size (mm 3 ) ANTITUMORIGENIC ACTIVITY OF SUBSTITUTED DIMs (500 μg/kg) Control 5,5'-BrDIM

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

20 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 X X

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

22 C-DIMS INHIBIT BREAST CANCER CELL GROWTH DIM-C-pPhC 6 H 5

23 C-DIMS INHIBIT RATMAMMARY TUMOR GROWTH Treatment (days) Tumor volume (mm^3) Control DIM-C-pPhC 6 H 5 1mg/kg/2d 0

24 C-DIMS ACTIVATE PPAR γ Screening receptors that bind lipophilic compounds GAL4 R GAL4 RE RE RP R = RAR, RXR AhR, PPARα or PPARγ P = Arnt or RXR 5 10 Fold Induction DMSO PGJ2 (μM)DIM-C-pPhCF 3 (μM) 1101 MCF-7 Cells GAL4 RE PPARγ GAL4

25 C-SUBSTITUTED DIMS AS PPARγ AGONISTS - SARs Gal4Luc / pM-PPAR  C CCigPGJ2OM P Normalized Gal4Luc activity (U) (μM)

26 C-SUBSTITUTED DIMs AS PPARγ AGONISTS – SARs Gal4Luc / pM-PPAR  C CF3BrFtBuOCH3N(CH3)2HOHC6H5CNCH3 Normalized Gal4Luc activity (U) (μM)

27 MOST ACTIVE C-SUBSTITUTED DIMs AS PPARγ AGONISTS* C 2 DIM-C-pPhX DIM-C-pPhCF 3 (X=CF 3, #1) DIM-C-pPhtBu (X=tBu, #4) DIM-C-pPhC 6 H 5 (X=C 6 H 5, #9) * First generation agonists

28 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

29 C-DIMs WHICH INHIBIT TUMOR/CELL GROWTH BUT EXHIBIT LOW ACTIVATION OF PPAR  DIM-C-pPhOCH 3 (X=OCH 3 ) 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

30 INHIBITION OF TUMOR GROWTH BY C-DIMs DMBA-INDUCED MAMMARY TUMORS Day Corn Oil DIM-C-pPhC 6 H 5 (X=C 6 H 5 ) Day Corn Oil DIM-C-pPhOCH 3 (X=OCH 3 ) N H CH X 2

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

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

33 INDUCTION OF APOPTOSIS IN CANCER CELL LINES: ROLE OF Nur77 – TRANSLOCATION PATHWAY Nur77bcl2 Nur77 (cytosolic) Apoptosis Nur77 (nuclear) translocation Apoptosis Inducer * Cell, 2004; Cancer Res, 2003 Mitochondria

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

35 C-SUBSTITUTED DIMs: ACTIVATION OF Nur77 Nur77 GAL4 DBD GAL4-RE -luc Nur77 E/F GAL4 DBD GAL4-RE -luc Nur77  -luc Nur77 NuRE TRANSACTIVATION ASSAYS

36 ACTIVATION OF Nur77 BY C-DIMs STRUCTURE-ACTIVITY RELATIONSHIPS * * * * * * * * * * GAL4-Nur77/pGAL CCF 3 BrFtBuOCH 3 N(CH 3 ) 2 HOHC6H5C6H5 CNCH 3 Fold Induction Panc-28 Cells

37 ACTIVATION OF Nur77 LBD (E/F DOMAIN) BY C-DIMs (AF1-INDEPENDENT)

38 SUBCELLULAR LOCATION OF LIGAND- ACTIVATED Nur77 DMSO (anti-Nur77) DMSO (IgG) DIM-C-pPhOCH 3 (IgG) DIM-C-pPhCF 3 (anti-Nur77) DIM-C-Ph (anti-Nur77) DIM-C-pPhOCH 3 (anti-Nur77)

39 LIGAND ACTIVATED Nur77 – PANC-28 CELL SURVIVAL 1  M 5  M DMSO 10  M DIM-C-pPhCF 3 DIM-C-pPhOCH 3 DIM-C-PhDIM-C-pPhOH % Cell Survival

40 LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PANC-28 CELLS DIM-C-pPhCF 3 DIM-C-pPhOCH 3 DIM-C-PhDIM-C-pPhOH DMSO 10  M20  M10  M20  M10  M20  M PARP 112kDa PARP 85kDa N.S Bax

41 LIGAND ACTIVATED Nur77: INDUCTION OF PARP CLEAVAGE IN PROSTATE, BREAST AND PANCREATIC CANCER CELLS DIM-C-pPhCF 3 DIM-C-pPhOCH 3 DIM-C-Ph DMSO 10  M20  M10  M20  M10  M20  M PARP 112kDa PARP 85kDa PARP 112kDa PARP 85kDa PARP 112kDa PARP 85kDa LNCap MiaPaCa-2 MCF-7

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

43 Nur77 AGONISTS ACTIVATE EXTRINSIC APOPTOTIC PATHWAYS – INDUCTION OF TRAIL* (PANC 28) DIM-C-pPhCF 3 DIM-C-pPhOCH 3 DIM-C-PhDIM-C-pPhOH DMSO 10  M20  M10  M20  M10  M20  M TRAIL N.S * * * * * * *also observed in thymocytes overexpressing Nur77

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

45 DEVELOPMENT OF I3C/DIM-DERIVED COMPOUNDS FOR CANCER CHEMOTHERAPY Ring DIMs C-DIMs DIM I3C Potent 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


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