NOTE TO USERS The original manuscript received by UMI contains pages with slanted pn'nt. Pages were microfilmed as received. This reproduction is the best copy available THEEFFECTS OF PLANT-DERIVED COMPOUNDSON SEXHORMONE RECEPTORS: ~MPLICATIONSFOR HORMONE- DEPENDENT CANCERDEVELOPMENT AND TREATMENT Rachel Stacey Rosenberg A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Nutritional Sciences University of Toronto @Copyright by Rachel Rosenberg 1997 ~ibiiographicServices services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your lile Votre raference Our file Notre réUrence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive pennethnt à la National Librq of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. ~MPLICATIONSFOR HORMONE- DEPENDENT CANCERDEVELOPMENT AND f REATMENT Master of Science, 1997 Rachel Stacey Rosenberg Graduate Department of Nutritional Sciences University of Toronto ABSTRACT Breast and prostate cancers are leading causes of cancer morbidity and mortality of women and men in North America. This risk is significantly higher than in the Japanese population, with epidemiologicaf studies suggesting the difference due to high fiavonoid consumption in the latter group. The purpose was to examine effects of plant-derived compounds on androgen (AR) and progesterone receptors (PR). In vitro studies on BT-474 and T47-D breast cancer cells were performed and 7 synthetic antiprogestins and 45 plant-derived compounds were tested. Prostate-specific antigen (PSA) was rneasured as a marker of receptor activity. The strongest blockers of PR were chlorophylline (87 k 7%), p-carotene (82 f 3%),taxifolin (80 f 5%), and chlorogenic acid (61 -t. 2%). The most potent antagonists of AR were chlorophylline (94 5 3%), hesperetin (79 i 11%), syringic acid (72 + 11 %), and a-tocopherol (65 + 5%). Apigenin had an agonistic effect on these receptors. - --- -- -- ----- ------.-.- Many people have contributed to the inception, planning, and development of this thesis. I would first like to thank Dr David Jenkins for his support, guidance, and patience in al1 aspects of this project and other endeavors. I would also like to thank Dr Eleftherios Diamandis for his teachings and direction, without which 1 would still be struggling with lab technique and data interpretation. Many thanks to Linda Grass for her help in the lab, as well as for Dr Gudrun Borchert for aiding me in assay techniques, and especially to Tina Perera for her administrative, ernotional, and moral support throughout. Thanks to Dr Cyril Kendall and Tom Ransom, for the creation and planning of this project, and to the rest of the group, for their assistance. 1 would like to also thank my family and friends who have provided me with the emotional tools that carried me from day to day. To my parents, thank you for your unconditional love, guidance, and support through the years. Jeff and Jason, thank you for being the brothers I could also count on in times of need. To my grandmother Tillie Binder, I hope that upon reading this, you understand somewhat the work I do, and its importance to me. To Sammy, Tigger, Ashley, Patch, and Smokey, I hope this thesis does not end up being your biting toy, or worse yet, your kitty litter. And last but not least, to Frank, you have been a source of total support and caring. I plan on making the utmost use of your promise to continue in this rote always ... TABLE OF CONTENTS Page .. Abstract .................................................................................... II Acknowledgements ................................................................................... iii List of Abbreviations .................................................................... v i List of Figures and Tables ............................................................ vii Chapter 1 lntroduction and Literature Review ..................................... 1. 1 Introduction ......................................................................... 1.2 Literature Review ................................................................ Demographicç and Epidemiology ............................ Carcinogenesis ........................................................ Treatment Modalities ............................................... Sex Hormones ......................................................... Sex Hormone Receptors .......................................... 1. 2.5. 1 Transactivation Domain: A/B Region .. 1.2.5.2 DNA binding Domain: C Region .......... 1.2.5.3 Hormone binding Domain: E Region ... 1.2.5.4 Mechanism of Action ............................ Plant-derived Compounds ......................................... Components of Various Plant Foods ......................... 1.2.7.1 Soya ..................................................... 1.2.7.2 Coffee ................................................... 1.2.7.3 Tea and Red Wine ................................ 1.2.7.4 Fruits and Vegetables ........................... Methods of Assessing Sex Hormone Activity in Vitro . 1.2.8.1 Breast Cancer Cells Lines ..................... 1.2.8.1 .1 T47-D .......................................... 1.2.8.1.2 BT-474 ........................................ 1.2.8.2 Prostate-Specific Antigen .................... 1.3 Research Hypothesis and Objectives .................................... Chapter 2 Synthetic Antiprogestins ......................................................... 2.1 Materials and Methods ........................................................... 2.1 1 Growth and Maintenance of Cells ............................... 2.1.2 Harvesting of Supernatant .......................................... 2.1.3 Experimental Design ................................................... 2.1 .3.1 Screening for Potential Agonistic Activity 2.1 .3.2 Dose-Response of Agonistic Activity ..... 2.1 .3.3 Assessment of Antagonistic Activity ...... r . I .L) r3n nssay .................................................................. 2.1.5 Statistics ...................................................................... Results ................................................................................... 2.2.1 Screening for Potential Agonistic Activity .................... 2.2.2 Dose-Response of Agonistic Activity ........................... 2.2.3 Assessrnent of Antagonistic Activity ............................ Chapter 3 Plant-derived Cornpounds ...................................................... 3.1 Materials and Methods ........................................................... 3.1.1 Experimental Design ................................................... 3.1 -1-1 Screening for Potential Agonistic Activity 3.1.1.2 Dose-Response of Agonistic Activity ..... 3.1 .1.3 Assessrnent of Antagonistic Activity ...... Results ................................................................................... 3.2.7 Screening for Potential Agonistic Activity .................... 3.2.2 Dose-Response for Agonistic Activity .......................... 3.2.3 Assessment of Antagonistic Activity ............................ Chapter 4 General Discussion and Conclusions .................................... 4.1 General Discussion ................................................................ 4.2 Conclusions ............................................................................ 4.2.1 Future Studies ............................................................. 4.2.2 Summary ..................................................................... Chapter 5 References ............................................................................ Page table 2.1 Structures of Synthetic Antiprogestins .............................. 41 2.2 Agonistic Activity of Synthetic Antiprogestins in the BT 474 and T 47D Breast Cancer Cell Lines ...................................... 42 2.3 Antagonistic Activity of Synthetic Antiprogestins ................. 45 3.1 Plant-derived Compounds ............................................... 47 3.2 Antagonistic Activity of Plant-derived Compounds ............... 54 figure 1.1 Structures of Steroid Hormones ....................................... 1.2 ldealized Hormone Response Elements ........................... f .3 PSA Production ........................................................... 1.4 Structures of Flavonoids ................................................ 1.5 Cell Cycle .................................................................. 2.1 Creation of Triads for Assessrnent of Antagonistic Activity ..... 2.2 Dose-Response of Synthetic Antiprogestins #2, 5. and 9 ...... 3.1 Structures of Plant-derived Compounds ........................... 3.2 Dose-Response of Apigenin .......................................... 3.3 Antiprogesterogenic Activity of Plant-derived