Cancer Chemoprevention by Sulforaphane, a Bioactive Compound from Broccoli/Broccoli Sprouts DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yanyan Li Graduate Program in Food Science and Nutrition The Ohio State University 2011 Dissertation Committee: Dr. Steven Schwartz, Advisor Dr. Duxin Sun, Co-advisor Dr. Steven Clinton Dr. Hua Wang Dr. Earl Harrison Copyrighted by Yanyan Li 2011 Abstract Sulforaphane, a bioactive compound from broccoli and broccoli sprouts, possess potent cancer chemopreventive activity. In the current studies, we have revealed a novel molecular target of sulforaphane in pancreatic cancer, evaluated the effect of sulforaphane on breast cancer stem cells, and compared different broccoli sprout preparations for delivery of sulforaphane for future chemoprevention studies. We showed that heat shock protein 90 (Hsp90), a molecular chaperone regulating the maturation of a wide range of oncogenic proteins, as a novel target of sulforaphane. Different from traditional Hsp90 inhibitors that block ATP binding to Hsp90, sulforaphane disrupted Hsp90-p50 Cdc37 interaction, induced Hsp90 client degradation, and inhibited pancreatic cancer in vitro and in vivo . We traced its activity to a novel interaction site of Hsp90. Proteolytic fingerprinting and LC-MS revealed sulforaphane interaction with Hsp90 N-terminus and p50 Cdc37 central domain. LC-MS tryptic peptide mapping and NMR spectra of full-length Hsp90 identified a covalent sulforaphane adduct in sheet 2 and the adjacent loop in Hsp90 N-terminal domain. Furthermore, we investigated the combination efficacy of sulforaphane and 17-allylamino 17- demethoxygeldanamycin (17-AAG) in pancreatic cancer. 17-AAG, an Hsp90 inhibitor that blocks ATP binding to Hsp90, has been evaluated in clinical trials; however, hepatotoxicity limits its application as a single agent. Our data indicated that ii sulforaphane potentiated the efficacy of 17-AAG through enhanced abrogation of Hsp90 function, while lowered the dose-limiting toxicity of 17-AAG. Concomitant use of sulforaphane and 17-AAG synergistically down-regulated Hsp90 client proteins. New evidence has shown the existence of cancer stem cells (CSCs) in breast cancer. Targeting CSCs may reduce cancer recurrence. Our data showed that sulforaphane inhibited breast CSCs and down-regulated Wnt/ β-catenin self-renewal pathway. Sulforaphane (1-5 µM) decreased aldehyde dehydrogenase-positive cell population by 65%-80% in human breast cancer cells, and reduced the size and number of mammospheres by 8-125-fold and 45%-75%, respectively, as evidenced by Aldefluor and mammosphere formation assays. Daily injection with 50 mg/kg sulforaphane for two weeks eliminated breast CSCs in nonobese diabetic/severe combined immune-deficient (NOD/SCID) xenograft mice, thereby abrogating tumor growth after re-implantation of primary tumor cells into the secondary mice. Western blotting and reporter assay showed that sulforaphane down-regulated Wnt/ β-catenin pathway. All these studies support the development of broccoli sprout preparations for chemoprevention studies. Therefore, we developed three preparations, compared their ability to deliver sulforaphane in vivo , and evaluated the pharmacokinetics and tissue distribution after oral administration. The sulforaphane-rich preparation generated by two-step procedure contained the highest amount of sulforaphane, 11 and 5 times higher than the freeze-dried sprouts with and without plant enzymes, respectively; and produced the greatest plasma response among all the three preparations, with the peak plasma concentration of sulforaphane 6 and 2.3 times higher, and the AUC 7.9 and 2.2 times iii higher, compared to the other two preparations. Consumption of 2.5 mg/g body weight of the sulforaphane-rich preparation resulted in rapid absorption and distribution, achieving high levels of sulforaphane and its glutathione conjugate in plasma and tissues. This study provides a broccoli sprout preparation that can serve as a good source of sulforaphane for further evaluation of chemopreventive efficacy. iv Dedication Dedicated to my family v Acknowledgements I would like to express my deepest gratitude to my advisors, Dr. Steven Schwartz and Dr. Duxin Sun for their invaluable guidance, inspiration, and support throughout my graduate years. Their conscientious attitude and willingness to explore new avenues made this research possible. Their immeasurable support and every effort for my unusual situation are the best thing that could have happened to me and my family. I wish to thank my dissertation committee members, Dr. Steven Clinton, Dr. Earl Harrison and Dr. Hua Wang for their valuable inputs and encouragement. I wish to thank Dr. Yael Vodovotz for her continuous support and help. I thank Dr. Max Wicha (University of Michigan) for his precious time and resources, and Dr. Hasan Korkaya and Dr. Suling Liu for their help in the project of breast cancer stem cells. I thank Dr. Stefan Rüdiger (Utrecht University, Netherlands), Dr. Kate Carroll (The Scripps Research Institute) and Dr. Young Ho Seo for their help in the study of Hsp90. Their expertise in NMR and LC-MS made this project possible. I am grateful to everyone in the Sun lab and Schwartz lab for their help, valuable inputs, and friendship. I owe special thanks to my husband, for his immeasurable love, steadfast support and encouragement through this entire process. vi Vita 2005................................................................B.S. Biology, Nanjing Normal University 2005 to 2006 .................................................Fellowship, The Ohio State Biochemistry Program, The Ohio State University 2006 to present ..............................................Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Publications 1. Yanyan Li , Max S. Wicha, Steven J. Schwartz, and Duxin Sun. Implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds. J Nutr Biochem, 2010 Feb 3. Epub ahead of print. 2. Yanyan Li , Tao Zhang, Hasan Korkaya, Suling Liu, Hsiu-Fang Lee, Bryan Newman, Yanke Yu, Shawn G. Clouthier, Steven J. Schwartz, Max S. Wicha, and Duxin Sun. Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells. Clin Cancer Res, 2010; 16(9); 2580-90. 3. Yanyan Li , Tao Zhang, Yiqun Jiang, Hsiu-Fang Lee, Steven J. Schwartz, and Duxin Sun. (-)-Epigallocatechin-3-gallate inhibits Hsp90 function by impairing Hsp90 association with co-chaperones in pancreatic cancer cell line Mia Paca-2. Mol Pharm, 2009; 6(4):1152-9. 4. Yanyan Li , Tao Zhang, Steven J. Schwartz, and Duxin Sun. New developments in Hsp90 inhibitors as anti-cancer therapeutics: mechanisms, clinical perspective and more potential. Drug Resist Updates, 2009; 12(1): 17-27. 5. Tao Zhang, Yanyan Li , Zhenkun Zhu, Mancang Gu, Bryan Newman, and Duxin vii 6. Sun. MEK inhibition potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. Mol Pharm, 2010; 7(5): 1576-1584. 7. Yiqun Jiang, Denzil Bernard, Yanke Yu, Tao Zhang, Yanyan Li , Shaomeng Wang, Xueqi Fu, and Duxin Sun. Split renilla luciferase protein-fragment-assisted complementation (SRL-PFAC) to characterize Hsp90/Cdc37 complex and identify critical residues in protein-protein interactions. J Biol Chem, 2010; 285(27): 21023- 36. 8. Yanke Yu, Adel Hamza, Tao Zhang, Mancang Gu, Peng Zou, Bryan Newman, Yanyan Li , A.A. Leslie Gunatilaka, Chang-Guo Zhan, and Duxin Sun. Withaferin A targets heat shock protein 90 in pancreatic cancer cells. Biochem Pharmacol, 2010; 79(4): 542-51. 9. Tao Zhang, Yanyan Li , Yanke Yu, Peng Zou, Yiqun Jiang, and Duxin Sun. Characterization of celastrol to inhibit Hsp90 and Cdc37 interaction. J Biol Chem, 2009; 284(51): 35381-9. Fields of Study Major Field: Food Science and Nutrition viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgements ............................................................................................................ vi Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xvi List of Figures ................................................................................................................. xvii Chapter 1: Literature Review .............................................................................................. 1 1.1 Cruciferous Vegetables, Glucosinolates and Isothiocyanates ................................... 1 1.1.1 Conversion of glucosinolates to isothiocyanates ................................................ 1 1.1.2 Loss of glucosinolates and isothiocyanates during cooking ............................... 2 1.2 Chemopreventive Activity of Sulforaphane .............................................................. 2 1.2.1 Inhibition of Phase 1 enzymes and induction of Phase 2 enzymes .................... 4 1.2.2 Induction of apoptosis and cell cycle arrest ........................................................ 5 1.2.3 Inhibition of