PART 1: TROGLITAZONE ANALOGUES AS CYCLIN D1 ABLATIVE AGENTS: THE POTENTIAL DRUGS FOR BREAST CANCER THERAPY PART 2: VITAMIN E AND ITS ANALOGUES INDUCE APOPTOSIS IN PROSTATE CANCER CELLS IN PART THROUGH INHIBITION OF BCL-2/BCL-XL FUNCTIONS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jui-Wen Huang, M.S. ***** The Ohio State University 2005 Dissertation Committee Professor Ching-Shih Chen, Advisor Professor Robert W. Brueggemeier Approved by Professor Charles L. Shapiro Professor Tatiana M. Oberyszyn Advisor College of Pharmacy ABSTRACT Part 1: Troglitazone analogues as cyclin D1 ablative agents: the potential drugs for breast cancer therapy Cyclin D1 overexpression has been implicated in oncogene-induced mammary tumorigenesis as it is detected in over 50% of primary breast carcinomas and is correlated with poor prognosis. It has been reported that troglitazone (TG), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, can induce degradation of cyclin D1 as part of its mechanism for causing cell cycle arrest and growth inhibition in breast cancer cells. In this study, we obtained evidence that the ability of high doses of TG to repress cyclin D1 is independent of PPARγ activation. First, a PPARγ-inactive TG analogue (Δ2-TG) causes cyclin D1 ablation with potency similar to that of TG in MCF-7 cells. Secondly, MDA-MB-231 breast cancer cells, which exhibit higher PPARγ expression, are less sensitive to this TG-induced cyclin D1 down- regulation than MCF-7 cells. In addition, our data also indicate that TG- and Δ2-TG-induced cyclin D1 repression is mediated via proteasome-facilitated proteolysis as it can be inhibited by multiple proteasome inhibitors, including MG132, lactacystin, and epoxomicin, and is preceded by increased ubiquitination. The dissociation of these two pharmacological activities, i.e., PPARγ activation and cyclin D1 ablation, provides a molecular basis to use Δ2-TG as a scaffold to develop a novel class of cyclin D1-ablative agents. Accordingly, a small library of Δ2-TG ii derivatives has been synthesized. Among derivatives in this library, Δ2-TG-28 represents a structurally optimized agent with potency an-order-of-magnitude higher than that of Δ2-TG in cyclin D1 repression and MCF-7 cell growth inhibition. Part 2: Vitamin E and its analogues induce apoptosis in prostate cancer cells in part through inhibition of Bcl-2/Bcl-xL functions Vitamin E and its analogues, such as a-tocopheryl succinate (α-TOS), have been shown to be proapoptotic agents in cancer cells, but the precise mechanism of their antineoplastic activity is not fully elucidated. To investigate the mechanism and the relationships between the structures and apoptosis-inducing activities of vitamin E and α-TOS, a series of vitamin E analogues were synthesized. Among these analogues, including α-tocopherol and α-TOS, compound VEA-7 which has a two-unit isopranyled side chain and an ether linkage at the phenol position exhibits the most potent proapoptotic activity. Fluorescence polarization analysis and immunoprecipitation data confirmed that α-TOS and most apoptosis-inducing vitamin E analogues inhibit the proliferation of PC-3 and LNCaP prostate cancer cells in part by repressing the heterodimerization of Bcl-2/Bcl-XL and Bax. VEA-7, a more potent apoptotic inducer than α-TOS, also displays stronger Bcl-XL binding affinity. In addition, α-TOS and these vitamin E analogues selectively induce apoptosis in malignant prostate cancer cells but not in normal prostate epithelial cells. The synthetic vitamin E analogue, VEA-7, represents a novel apoptogenic agent that may have clinical value in chemotherapeutic strategies for prostate cancer in the future. iii Dedicated to my parents my sister, Rachel in the heaven and especially, my husband, Chung-Wai, for his support and everything iv ACKNOWLEDGMENTS I would like to acknowledge Dr. Chen for his guidance, constant encouragement, support, and for providing excellent working environment for his students. I also would like to express my sincere appreciation for my defense committee Dr. Brueggemeier, Dr. Shapiro, and Dr. Oberyszyn for their advice of my dissertation and research. Thank Dr. Samuel K. Kulp for his patience in correcting my dissertation errors. I would like to thank Dr. DashengWang who synthesized all vitamin E analogues for my second project and Chung-Wai who synthesized most troglitazone and ciglitazone analogues for my cyclin D1 project. Chung-Wai also performed PPARγ binding assay, DNA fragmentation, and competitive fluorescence polarization analysis for this study. Ya-Ting helped me to analyze cell cycle data. Ho-Pi and Yu-Chieh taught me how to do RT-PCR. Jim and Chang-Shi provided a lot of ideas for the two projects and assisted me to complete some part of experiment. Kuen-Feng donated his hypostasis to my both projects. Nicole aided for immunohistochemitry experiment. Kathy Brooks and Kelli Ballouz also provide a lot of assist to make graduate studies going smoothly. Colleagues and friends: Julie, Qiang, Joe, Dr. Hung, Leo, Ping-Hui, Yeng-Jeng and his family, Shih-Jiuan, Wen and Li-Shu, Jian, Dennis, Erica, Po-Hsien, Yukao, and Ma are all nice people and gives a lot of help in the daily life. v VITA September 1992- June 1996 B. S. Chemistry National Chung-Hsin University, Tai-Chung, Taiwan September 1996- June 1998 M. S. Chemistry (Organic) National Taiwan University, Taipei, Taiwan September 2000-May 2001 Graduate student for Ph.D program, Chemistry State University of New York at Stony Brook, Stony Brook, NY August 2001-present Graduate Teaching and Research Assistant, Pharmacy The Ohio State University, Columbus, OH PUBLICATIONS 1. J. -W. Huang, C. -W. Shiau, Y. -T. Yang, S. K. Kulp, K.- F. Chen, R. W. Brueggemeier, C. L. Shapiro, and C. -S Chen (2005) “Peroxisome Proliferator-Activated Receptor γ-Independent Ablation of Cyclin D1 by Thiazolidinediones and Their Derivatives in Breast Cancer Cells.” Molecular Pharmacology, 67, 1342-1348. vi 2. C. -W. Shiau, C. –C. Yang, S. K. Kulp, K.- F. Chen, C. –S. Chen, J. -W. Huang, and C. -S Chen (2005) “Thiazolidenediones Mediate Apoptosis in Prostate Cancer Cells, in part, through the Inhibition of Bcl-xL/Bcl-2 Functions Independently of PPAR gamma.” Cancer Research, 65, 1561-1569 3. J. –X. Zhu, J. –W. Huang, P. –H. Tseng, Y. –T. Yang, J. Fowble, C. –W. Shiau, Y. –J. Shaw, S. K. Kulp, C –S. Chen (2004) “From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-pliosphoinositide-dependent protein kinase-1 inhibitors.” Cancer Research, 64 , 4309-4318. 4. J. -W. Huang, C. –D. Chen, M. –K. Leung (1999) “Magnesium Bromide promoted Barbier Type Intramolecular Cyclization of Halo-Substituted Acetals, Ketals and Orthoesters.” Tetrahedron letters, 40, 8647-8650. 5. C. –D. Chen, J. -W. Huang, M. –K. Leung, H. –H. Li (1998) “S,S-dimethyl dithiocarbonate: A Novel Carbonyl Dication Synthon in the synthesis of ketones.” Tetrahedron, 54, 9067-9078. FIELDS OF STUDY Major Field: Pharmacy Specification: Medicinal Chemistry vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii Dedication ………………………………………………………………………………..iv Acknowledgements ……………………………………………………………………….v Vita …………………………………………………………...…………………………..vi List of tables ………………………………………………………….……………..….xii List of figures………………………………………………………………………...…xiii List of schemes ………………………………………………………………………..xvii Abbreviations…………………………………………………………………………xviii Chapters: 1. Introduction……………………………………………………………………………1 1.1 Overview of cyclin D1…………………………………………………………….1 1.2 Cyclin D1 and cancer……………………………………………………………...2 1.3 Peroxisome proliferator-activated receptor γ and thiazolidinediones……………..6 1.4 TZDs as Anti-tumor Regents……………………………………………………...9 2. Discovery and synthesis of thiazolidinedione derivatives without PPARγ activities ………………………………………………………………………………………..19 2.1 The discovery of TZD derivatives without PPARγ activation…………………..19 2.2 Synthesis of TZD compounds and their derivatives……………………………..20 viii 3. Thiazolidinedione and derivatives down-regulate cyclin D1………………………..28 3.1 Effect of TZDs on cyclin D1 down-regulation is independent of PPARγ.............28 3.2 TG and Δ2-TG facilitate proteasome-mediated proteolysis of cyclin D1……….30 3.3 Investigation of the mechanism of TG and Δ2-TG-mediated cyclin D1 degradation……………………………………………………………………..31 4. Development of novel Δ2-TG-derived cyclin D1-ablative agents………………..…47 4.1 Modification of Δ2-TG……………………………………………………….….47 4.2 Structure-activity relationship (SAR) study of Δ2-TG analogues……………….48 4.3 Bioactivities of STG-28………………………………………………………….50 5. Conclusions and future directions …………………………………………………64 5.1 Down-regulation of cyclin D1 by TG and Its Analogues Is PPARγ- Independent……..……………………………….……………………………….64 5.2 Development of cyclin D1 ablative agents………………………………………65 5.3 Future directions………………………………...……………………………….66 6. Experimental methods and material for part 1 ………………………………………69 6.1 Reagents…...……………………………………………………………………..69 6.2 Cell culture……………………………………………………………………….70 6.3 Cell viability analysis………………………………………………………….....70 6.4 Analysis of PPARγ activation……………………………………………………70 6.5 Western blot analysis…………………………………………………………….71 6.6 Coimmunoprecipitation/western blot…………………………………………….72 6.7 Reverse transcriptase (RT)-PCR analysis of mRNA transcripts of cyclin D1 gene ix ………………………………………………………………………………………..73 6.8 Cell cycle analysis………………………………………………………………..73 6.9 siRNA transfection procedure……………………………………………………74