“Diet and the Prostate” Potential Targets of Phytochemicals in the Prostate

1. “Diet and the Prostate” Potential Targetsof Phytochemicals in the Prostate Richard S. Gunasekera, Ph.D.University of Houston-Victoria Joint Program in Bioactive Phytochemicals IBT-TAMUS Health Science Center Vegetable & Fruit Improvement Center TAMUK Citrus Centerhttp://www.uhv.edu/asa/faculty/faculty.asp?id=1

2. “It is now essential to focus our attention on identifying the specific dietary factors that either increase or decrease the incidence of prostate cancer.” Donald S. Coffey, Ph.D. Professor of Urology, Pathology, Pharmacology and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD. American Assoc. for Cancer Research Meeting on Prostate Cancer

3. WASHINGTON - Federal Reserve Chairman Alan Greenspan will undergo surgery Tuesday for an enlarged prostate, the Fed said Monday in an announcement that also said tests last week showed no cancer. Greenspan is often spoken of as the second-most-powerful man in Washington because of the impact his words and the Federal Reserve's actions can have on interest rates and the economy in general. Greenspan to Undergo Prostate Surgery By JEANNINE AVERSA, Associated Press Writer

4. Approach to lecture • Rationale to Prevention • Plant-derived Bioactive Molecules (PBAMS) • Review relevant Molecular & Cellular Biochemistry- cell communication • Discuss specific/ potential targets for example PBAMS

5. Rationale for Prevention • Incidence highest in Western countries • Incidence rises in immigrants to Western countries without intermarriage • Equal incidence of benign or “pre-clinical” disease

6. PROSTATE CANCERFROM CHINA TO GEORGIA<2 TO 91/100,000

7. CLINICAL: Japan 5>Hawaii 31>USA 17 White 50BENIGN: Japan 19>Hawaii 50>USA 49 White 87

8. Prostate Cancer • Increasing to epidemic proportions with aging • Second most common cause of death in men; suffering, loss of productivity • Multi-billion $ burden; 2nd to cataract surgery by Medicare • Only 10% diagnosed will be effected. Which 10% is our dilemma?

9. Prostate Abnormality “Cancer” Reaches 50% age 55 100% age 85

10. Biology of the Prostate • Why is it a problem? • Complex endocrine & local control • Prostate disease is progressive • Cell & molecular biology • Local control by growth factors • Targets of bioactive products

12. Complex endocrine & local control

13. Androgen is the Master, But it’s a Balancing Act

14. Progressive... X Marks the Critical Spot

15. CRITICAL POINT FOR INTERVENTION Intervention...

16. BIOACTIVE PLANT DERIVED MOLECULES (PBAMS)

17. METABOLIC CONVERSION OF PRO-PBAMS

21. Approach Continued… I. Phytochemicals [Plant-derived Bioactive Molecules] A. Phytosteroids • B.    Citrus products • C. Bioflavone a II Potential Targets A. Inhibition of Steroid Metabolizing Enzymes •      5 a- Reductase Enzyme •      17 b- Hydroxysteroid dehydrogenase •      Aromatase Enzyme B. Tyrosine specific kinases C. Transcriptional Factors

22. III. Review relevant Molecular & Cellular Biochemistry- Cell Communication • A.         Hormonal Regulation • B.         Enzyme Inhibitors • C.         Growth Factors & Receptors • D.         Signal Transduction IV. Experimental: • In vitro- Cell Culture Studies • Growth factor Studies • Transcriptional factors

24. Potential Targets of: • Plant-derived Bioactive Molecules (PBAMS) • Discuss specific/potential targets for examples: • Phytosteroids • Citrus products • Citrus lycopene • Bioflavone a , curcumin, capsaicin

25. 5 a- Reductase Enzyme 17 b- Hydroxysteroid dehydrogenase Aromatase Enzyme Tyrosine specific kinases Inhibition of Metabolizing Enzymes

27. Hormonal Regulation • Steroid hormones regulate gene expression by binding to intracellular receptors • Hormone-receptor complexes = transcriptional activators that bind to DNA regulatory sequences • Hormones initiate gene expression from target genes

29. Transcriptional Enhancers • Enhancers are DNA regulatory sequences that bind specific transcription factors to activate transcription by RNA polymerase • Enhancers may be located in the 5’ or 3’ direction from a gene and may be found at great distances from a gene

30. Testosterone 5 a- Reductase 5 a- DHT DHT-AR complex and HREs Modulates gene expression DHT is necessary for prostate growth

32. Inhibition of Enzyme Activity by Phyto-estrogens

33. Inhibition of Enzyme Activity by Phyto-estrogens

34. Genistein in GSF homogenates is a Non-competitive Inhibitor Does not bind to active site but other site May not form ES or, ES will not dissociate E + I = EI or ES + I = ESI 5 a- Reductase Enzyme Inhibitors

35. Aromatase Enzyme Inhibition of Steroid Metabolizing Enzymes • Enterolactone in GSF homogenates is • -competitive Inhibitor • -Binds to active site • -Competes with substrate • E + I = EI

37. Aromatase Enzyme Inhibition of Enzymes • Tyrosine specific kinases

38. Growth Factors & ReceptorsSignal Transduction • Inhibit. of tyr kinases (kinase effectors) other PBAMS: PA & SR… • DHT “switching on” proliferation, “orchestrating” intermediary factors- that are directly involved.

39. Experimental Results: Enhancement of Chemotherapuetic Activity • Bioflavone a: • - Non traditional, non alternative ! • Lycopene, Capsaicin, Curcumin: • - Differential effects • Citrus Pectin & other HSPG mimetics: - Effects on growth factors

40. Experimental: LnCap, PC3, DTE, AT3,

41. The effects of Capsaicin alone and cocktails of Capsaicin with Bioflavone α on LnCap Cells ( p<0.0002) Enhancement of the Chemotherapeutic Activity of Curcumin and Capsaicin by Bioflavone on Human Prostate Cancer Cells. Siva G. Somasundaram, Desiree Arrambide, and Richard. S. Gunasekera.Journal of Nutrition 182: 255s- 342

42. R E S U L T S The effects of Curcumin alone and Cocktails of Curcumin with Bioflavone α on LnCap Cells ( p<0.001)

43. R E S U L T S Using the TransAM AP-1 cJun ELISA kit, the phospho-cJun content of the bound AP-1 was determined in a colorimetric reaction using a primary and secondary antibody. Spectrophotom-etric data from the assay is seen in yellow and correlates to the level of cJun activity. The red is the OD ratio (1-cocktail/ control) which correlates to the increase of apoptosis to that of the control.

44. Results of the cocktails containing 2 uM Camptothecin with increasing concentrations of Bioflavone α after a 48 hour incubation in malignant LnCAp (human prostate) cells Fourth Hour First Hour Gunasekera R.S, Arrambide, D., and Somasundaram,S. (2004) Bioflavone a Increases Chemotherapeutic Activity of Antitumor Drug Campothecin, Journal of Nutrition 182: 255s- 342

45. Comparison of the results after a 48 hour incubation of a 2 uM concentration of camptothecin alone and the 10 uM concentration of Bioflavone α alone to the cocktail containing 2 uM Camptothecin with 10 uM Bioflavone α. R E S U L T S

46. Time Curve comparisons: AT3 vs DTE Gunasekera, McKeehan, Patil, et al. (2007) Lycopene and lutein inhibit proliferation in rat prostate carcinoma cells. Nutr & Cancer. 2007;58(2):171-7. .

47. Modified Citrus Pectin • MCP- galactose rich • Saturate galactose binding sites of cancer cell linkers • Inhibit aggregation & adhesion

48. Inhibitory effect of pectin (from lamella of lemon) viewed by FGF/FGFR crosslinking and autoradiography. Citrus Pectin Inhibitory effect of pectin on FGF-1–FGFR1 binding in the presence of various concentrations of heparin. Gunasekera, R.S., Kan, M., Liu, Y., McKeehan W. L., H. Ahmad, and B.S Patil (2001) Activity of Citrus Pectin and other Heparan Sulfate Mimetics at the FGFR Complex, Journal of Nutrition, 131, 3127S-3150S.

50. SUMMARY • Prostate disease has a rationale for prevention. For both BPH and prostate cancer there arepoints of intervention (targets) at the molecular level. • Several PBAM may have potential preventative and therapeutic effects on prostate disease according to results shown. • Results confirm that citrus product have inhibitory effects on prostate cancer cell proliferation in vitro. • Results have also shown that PBAMs such as Bioflavone α may offer a synergistic affect to chemotherapeutic agents thereby enhancing their affects to inhibit prostate cancer cell proliferation.