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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMÏ CHEMOPREVENTATIVE PROPERTIES OF CRUCIFEROUS VEGETABLE EXTRACTS AND PURIFIED COMPONENTS FOR HUMAN PROSTATE CANCER DISSERTAHON Presented m Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Corey Edison Scott, M.S. ***** The Ohio State University 2001 Dissertation Committee: Professor Steven Schwartz, Adviser Approved by Professor Grady Chism Professor David Min iviser Dr. Steven Clinton Food Scienc^and Nutrition Profa'am Dr. Mark Morse UMl Number: 3031262 UMl* UMl Microform 3031262 Copyright 2002 b/Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthori^ copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT A recent case-control epidemiological study has shown a significant reduction (41%) in the risks of developing prostate cancer among men who consume at least three servings of cruciferous vegetables (e.g. broccoli, cabbage, watercress, brussel sprouts) per week versus those who consume less than one. Cruciferous vegetables are known to contain large amounts of isothiocyanates, which are potent PhaseH enzyme inducers. We are investigating new mechanisms of chemoprevention by cruciferous vegetables aside &om Phase H enzyme manifestation and carcinogen metabolism. This research addresses the effects of extracts and purified components (glucosinolates, isothiocyanates, indoles, etc) from cruciferous vegetables on malignant (PC-3, DU145, and LNCaP(androgen sensitive)) and prostatic epithelial cell growth and isolation and identification of chemopreventative compounds from the cruciferous vegetables. The ultimate goal of this research is to illustrate a means of lowering prostate cancer risks by consumption of cruciferous vegetables and to identify the components that have biological activity that can be linked to chemoprevention for human and animal studies. Both extracts and purified isothiocyanates from cruciferous vegetables inhibit cell proliferation, arrest cell cycle progression, and induce apoptosis in each of the three studied malignant cell lines in a dose dependent manner. The most potent extracts in growth inhibition were the broccoli sprout extract (S phase arrest) and watercress extract (GzM phase arrest), as we ti observed a greater concentratioa of isothiocyanates in these extracts. The most potent isothiocyanates were benzyl isothiocyanate, sulforaphane, and phenethyl isothiocyanate with IC50 values between 2.3 and 9.8 [xM. Purified glucosinolates and indoles were less effective in the growth inhibitory effects with IC 5Q values over 100 pM. Sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate were able to induce apoptosis in each line by 12%, 45%, and 54% respectively after 72 hours. Human prostatic epithelial cells (non-malignant) were much more resistant to the growth inhibitory effects and pro- apoptotic effects of isothiocyanates up to 50 pM concentrations. Insulin like growth factor (IGF-l) was added to AT6.3 (rat prostate) cells and their proliferation increased 3.4-fold. Isothiocyanates sulforaphane, phenethyl, and benzyl isothiocyanate inhibited IGF-l stimulated cell proliferation and induced apoptosis in the cells at concentrations under 5 pf/I. Ultimately, this research shows that compounds from cruciferous vegetables can lower cancer risks by a variety of mechanisms such as proliferation inhibition, induction of apoptosis, and through influencing signal transduction pathways in malignant prostate cells. ui \ Dedicated to my family IV ACKNOWLEDGMENTS I wish to sincerely thank my advisor and mentor Professor Steven Schwartz for introducing me to the study of Food Science. His conscientious attitude, kind spirit, and vast expertise in analytical methods helped to guide me in this task of obtaining a PhJ) in Food Science. E wish to acknowledge Dr. Steven Clinton for his efibrts in establishing the collaboration of Food Science and Medical Oncology. This was sometimes a difficult endeavor, but he was available and critical with all parts of this research. I wish to thank Professor Grady Chism for his technical advice in the area of plant enzymes and Food Science. 1 truly appreciated the kind words of encouragement during my tenure at The Ohio State LTniversity. I thank Professor David Min for his expert advice in essentially all areas of food chemistry. I will forever remember his active participation in my work and helping me become acclimated to food science. I acknowledge Dr. Mark Morse for his expertise in dealing with isothiocyanates chemistry and isothiocyanate metabolism. He served a crucial role in the success of this collaboration of food science and medical oncology. VITA October 14,1973 ......................................Æom-Thomasville, NC 199 5.................................................BA Chemistry, The University ofNorth Carolina at Chapel Hill 199 6......................................................... .Researcher, UNC School of Medicine Cystic Fibrosis Center 1998.......................................................... MS Chemistry, North Carolina Agricultural and Technical State University 1998-present .............................................. USDA Fellow The Ohio State University PUBLICATIONS Research Publications 1. Adebodun, F., Scott, CH., Cunningham, C., Bustamante, PM., Bradshaw, A., Ping, L., Williams, K. Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: Implication in the mechanism of apoptosis. Cell Biochem, Funct, 18: 59-66.2000. 2. Scott, CH., Adebodun. F. ^^C-NMR investigation of protein synthesis during apoptosis in human leukemic cell lines./. Cell Physiol. 181:147-152.1999. 3. Scott, CH., L JT. Abdullah, Davis, C.W. Ca^^ and protein kinase C activation of mucin granule exocytosis in. permeabilized SPOCl cells. Am. J. Physiol. 275 {Cell Physiol. 44):C285-C292.1998 vr FIELDS OF STUDY Major Field: Food Science and Nutrition m TABLE OF CONTENTS Page Abstract.....................................................................................................................ü Dedication ................................................................................................................iv Acknowledgements ....................................................................................................v Vita.____________________________________________________________ vi List of Tables .......................................................................................................... xii List of Figures ......................................................................................................... xii Chapters: L Review of the literature ..................................................................................... I LI Etiology of prostate cancer ........................................................................... I L.LI Ensulin-like growth factor and prostate cancer. .................................... 3 1.1.2 Dietary influences on prostate cancer ...............................................6 1.2 Chemopreventative properties of cruciferous vegetables ................................9 1.3 Cruciferous vegetables and malignant cell culture studies ........................... 23 1.4 Cruciferous vegetables and cancer in animal models ...................................25 1.5 Human and rodent metabolism of glucosinolates and isothiocyanates .......... 37 1.6 Implications for food processing of cruciferous vegetables and. anticancer Properties ....................................................................................................42 1.7 Analysis and detection of phytochemicals from cruciferous vegetab les .........50 1.8 Research objectives .....................................................................................57 References .................................................................................................. 58 2. Cruciferous vegetable extracts inhibit malignant prostate epithelial cell growth and lower the apoptotic threshold ..........................................................
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