Dual activation of estrogen receptor a and aryl hydrocarbon receptor by the prenylflavone, icaritin restrict breast cancer cell growth and destabilize estrogen receptor a protein

DUAL ACTIVATION OF ESTROGEN RECEPTOR α AND ARYL HYDROCARBON RECEPTOR BY THE PRENYLFLAVONE, ICARITIN RESTRICT BREAST CANCER GELL GROWTH AND DESTABILIZE ESTROGEN RECEPTOR α PROTEIN TIONG CHI TZE B.Sc (Hons), NTU A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ii    ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to my supervisor Prof Yong Eu Leong and my co-supervisor Dr Li Jun for their invaluable supervision, support and guidance throughout the course of this endeavor I am grateful to the ex-postdoctoral fellow in the laboratory, Dr Shen Ping for her encouragement, technical help and critical comments My gratitude goes to Wilson Wong, for helping me in the microarray data analysis I am greatly appreciative of all the laboratory members (Vanessa, Faisal, Eileen, Dr Shijun, Gaik Hong and Seok Eng) who have generously extended their warm friendship and assistance Their presence made the laboratory an enjoyable place to work in Many special thanks to my family members and friends for their constant support and encouragement Above all, to God be the glory for His guidance and providence iii    TABLE OF CONTENTS ACKNOWLEDGEMENTS ii  TABLE OF CONTENTS iii  SUMMARY vi  LIST OF TABLES viii  LIST OF FIGURES ix  ABBREVIATIONS x  CHAPTER 1  1.1  INTRODUCTION 1  Breast cancer 1  1.1.1 Risk factors for breast cancer   2  1.2  Estrogens 4  1.2.1 Physiological roles of estrogens  . 4  1.2.2 Metabolism of estrogens   8  1.2.3 Estrogens and mammary gland   8  1.3  Estrogen receptors 9  1.3.1 Structure of ERα and ERβ  . 9  1.3.2 ER signaling   11  1.3.3 Co-activators and co-repressors of ER   11  1.3.4 Proteasome-dependent degradation of ERα protein  . 12  1.4  Selective estrogen receptor modulators (SERMs) and treatment of breast cancer 14  1.4.1 Breast cancer treatments   14  1.4.2 Selective estrogen receptor modulators (SERMs)   16  1.5  Aryl hydrocarbon receptor 18  1.5.1 Structure and function of AhR  . 18  1.5.2 AhR signal transduction pathway   18  1.5.3 Cross-talk of AhR with ERs   21  1.5.4 Ubiquitin ligase activity of AhR and degradation of ERα  . 22  1.6 Phytoestrogens and breast cancer 24  1.6.1 Classification of phytoestrogens   24  1.6.2 Functional similarity of phytoestrogens and estrogens   24  1.6.3 Phytoestrogens and risk of breast cancer  . 25  iv    1.6.4 Potential mechanism for stimulatory effect of phytoestrogens on breast cancer   26  1.6.5 Potential mechanism for inhibitory effect of phytoestrogen on breast cancer . 27  1.7  Herba Epimedii flavonoids and cancer cell proliferation 30  1.7.1 Traditional use of Herba Epimedii   30  1.7.2 Modern use of Herba Epimedii   30  1.7.3 Chemical constituents of Herba Epimedii  . 31  1.7.4 Geographical distribution of Herba Epimedii in China   32  1.7.5 Epimedium flavonoid and cancer cell proliferation  . 32  1.8  Icaritin and cancer cell proliferation 36  1.8.1 Icaritin and cancer cell proliferation   36  1.8.2 Bioavailability of icaritin  . 39  1.9  Combinatorial effects of estradiol and phytoestrogen on cancer cell risk 42  1.10  Objectives 47  CHAPTER 2  2.1  MATERIALS and METHODS 48  Materials 48  2.1.1 Cell culture media, supplements, trypsin and antibiotics  . 48  2.1.2 Compounds and antibodies   49  2.1.3 Assay systems   50  2.1.4 Equipments   50  2.1.5 Probes for real-time PCR  . 51  2.1.6 siRNAs   51  2.2  Mammalian cell culture 52  2.2.1 Dextran-coated charcoal treated FBS preparation   52  2.2.2 MCF-7 cell line   53  2.2.3 MDA-MB-231 cell line   53  2.2.4 ERα stable cell line   53  2.3  Human breast cancer cell proliferation assay 54  2.4  Plasmid DNA purification and nucleofection 55  2.4.1 Plasmid DNA purification  . 55  2.4.2 Plasmid DNA nucleofection   56  2.5  Reporter gene assay 57  2.5.1 ER responsive reporter assay   57  2.5.2 AhR responsive reporter assay  . 57  2.6  Real-time PCR experiment 58  v    2.6.1 Total RNA extraction  . 58  2.6.2 cDNA synthesis  . 58  2.6.3 Real-time PCR  . 59  2.7  Western blotting 60  2.7.1 Cellular protein extraction  . 60  2.7.2 SDS-polyacrylamide gel electrophoresis  . 60  2.7.3 Western blot detection and analysis  . 61  2.8  Competitive ligand binding assay 62  2.8.1 ERα competitive ligand binding assay   62  2.8.2 AhR competitive ligand binding assay   62  2.9  AhR knockdown 64  2.10  Global gene expression profiling 65  2.11  Statistical analysis 67  CHAPTER 3  3.1  RESULTS 68  Icaritin as a phytoestrogen 68  3.1.1 Icaritin induced a dose-dependent stimulatory/inhibitory effect on MCF-7 proliferation   69  3.1.2 Icaritin bound directly to ERα   72  3.2  Combinatorial effect of estradiol and icaritin 74  3.2.1 Estradiol/icaritin in combination exerted lower proliferative effect than estradiol alone   74  3.2.2 Estradiol/icaritin in combination induced lower GREB1 gene expression compared to either ligand alone   76  3.2.3 Estradiol/icaritin in combination induced lower ER-regulated promoter activity compared to either ligand alone   78  3.3  Icaritin as an AhR agonist 81  3.3.1 Icaritin induced CYP1A1 gene expression   81  3.3.2 Icaritin induced AhR-regulated promoter activity   87  3.3.3 Icaritin bound directly to AhR  . 89  3.4  Estradiol/icaritin in combination destabilized ERα protein more than either ligand alone 91  3.5  AhR knockdown reversed icaritin modulation of estradiol stimulated MCF-7 cell proliferation 95  CHAPTER 4  DISCUSSION 97  BIBLIOGRAPHY 107  vi    SUMMARY Hormone replacement therapy (HRT) is usually prescribed to postmenopausal women suffering from menopausal symptoms such as hot flushes, vaginal atrophy, reduced sexual function and depression However, according to Women Health Initiative from United States National Institute of Health (US NIH), the use of HRT is associated with 26% increase in breast cancer risk On the other hand, epidemiological evidence suggests that phytoestrogen might be beneficial for alleviating menopausal symptoms without increasing the risk of breast cancer As such, we have chosen to study icaritin, a phytoestrogen derived from Herba Epimedii Herba Epimedii is a medicinal herbal plant traditionally prescribed for improving bone health, amongst other indications Since 17β‐estradiol (estradiol) is naturally present in the body even after menopause, the combinatorial effect estradiol and icaritin has relevance to the use of these compounds in subjects at risk of or suffering from breast cancer Hence, as the initial step, we performed in vitro study to test the combinatorial effects icaritin and estradiol on MCF-7 breast cancer cell Icaritin increased MCF-7 breast cancer cell proliferation at doses less than 10 µM However, the combination of µM of icaritin with 100 pM estradiol inhibited the cell growth induced by estradiol Estradiol/icaritin combination also induced lower estrogen receptor (ER)-regulated promoter activity and decreased GREB1 (growth regulation by estrogen in breast cancer 1) mRNA level compared to either ligand alone vii    As we were interested to investigate the molecular basis for this effect, we used a bioinformatics approach to fish for genes that are differentially regulated by icaritin Microarray analyses directed our attention to CYP1A1 gene, an AhR-regulated gene Independent quantitative real-time PCR experiments confirmed that icaritin induced CYP1A1 and the AhR-regulated XRE promoter, linking icaritin to the AhR-regulated signaling pathways Knockdown of AhR blocked the profound degradation of ERα induced by estradiol/icaritin combination, indicating the central role of icaritin on AhRmediated receptor stability In contrast, knockdown of AhR gene did not restore estradiol-mediated degradation of ERα, consistent with the fact that estradiol is not a ligand for AhR AhR knockdown blocked suppressive effects of icaritin on estradiol-stimulated breast cancer cell proliferation and GREB1 gene expression Our study indicates that icaritin can modulate estradiolstimulated MCF-7 cell proliferation via AhR-mediated proteasomal degradation of ERα to decrease GREB1 mRNA level In conclusion, the concurrent use of icaritin with estradiol reduces estradiol-stimulated MCF-7 cell growth in vitro via activation of AhR-E3 ubiquitin ligase pathway Based on the in vitro study, icaritin might be further developed as a drug to be co-administered with HRT to reduce breast cancer risk caused by HRT viii    LIST OF TABLES Table 1.1 Reference intervals for estrone and estradiol in adult males, pre- and postmenopausal females 6  Table 1.2 Serum estrone and estradiol levels in postmenopausal women receiving HRT every other day and every day 6  Table 1.3 Randomized controlled trials to test the effect of estrogen plus progestin therapy in postmenopausal hormone therapy and recurrence of breast cancer 7  Table 1.4 Classes of phytoestrogens 24  Table 1.5 Major and minor flavonoid in Herba Epimedii species 32  Table 1.6 Summary of Herba Epimedii extracts and flavonoid and its effects on cancer growth 34  Table 1.7 Summary of effect of icaritin on cancer growth 38  Table 1.8 Concentration-time profiles of Herba Epimedii prenylflavonoids 41  Table 1.9 Summary of effects of phytoestrogen and estradiol combination on cancer growth 45    Table 3.1 List of genes that are differentially regulated by icaritin treatment compared to estradiol and 4-hydroxytamoxifen 84  ix    LIST OF FIGURES  Figure 1.1 Schematic drawing of steroid hormone receptors 10  Figure 1.2 Ligand activated signal transduction of AhR 20  Figure 1.3 Different modes of the AhR signaling pathways 23  Figure 1.4 Structures of icariin and its derivatives 40    Figure 3.1 Effect of icaritin on MCF-7 or MDA cell proliferation 71  Figure 3.2 Competitive binding of icaritin to ERα 73  Figure 3.3 Effect of estradiol and icaritin combination on MCF-7 cell proliferation 75  Figure 3.4 Effect of estradiol and icaritin on GREB1 mRNA expression 77  Figure 3.5 Effect of icaritin on ERα reporter gene assay 80  Figure 3.6 Hierarchical clustering of differentially expressed gene after icaritin treatment 83  Figure 3.7 Effect of icaritin on CYP1A1 gene expression 86  Figure 3.8 Effect of icaritin on XRE reporter gene assay 88  Figure 3.9 Competitive binding of icaritin to AhR 90  Figure 3.10 Western blotting for ERα protein stability 94  Figure 3.11 Effect of AhR knockdown on MCF-7 cell proliferation and GREB1 expression 96  Figure 4.1 Crosstalk between icaritin and estradiol signaling pathways through AhR-directed proteasomal degradation 106  x    ABBREVIATIONS AhR AhRR ARNT AR CI CO2 CT-FBS CYP1A1 DBD DMSO DNA dNTP E1 Estradiol E3 EB ER ERα ERβ EMEM ERE FBS g GEN GR GREB1 HRE HRT HSP ICT kb kDA l LB LUC M µg µl mg mM mRNA N-CoR NLS NR nM ng aryl hydrocarbon receptor aryl hydrocarbon receptor repressor AhR nuclear translocator androgen receptor confidence interval carbon dioxide charcoal treated-fetal bovine serum Cytochrome P450, family 1, subfamily A, polypeptide DNA binding domain dimethyl sulfoxide deoxyribonucleic acid deoxynucleotide triphosphates estrone 17β-estradiol estriol Epimedium brevicornum estrogen receptor estrogen receptor alpha estrogen receptor beta Eagle’s minimum essential medium estrogen response element fetal bovine serum gram genistein glucocorticoid receptor growth regulation by estrogen in breast cancer hormone response elements hormone replacement therapy heat shock protein icaritin kilobases kilodalton litre Luria-Bertani luciferase molar microgram microlitre milligram millimolar messenger ribonucleic acid nuclear receptor co-repressor nuclear localisation signal nuclear receptor nanomolar nanogram 109 combinatorial libraries on aromatase, estrogen biosynthesis, and metabolism." 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Despite the availability of therapies targeting estrogen and growth factor signaling pathways, the incidence and mortality of breast cancers have not decline at the same rate as other major causes of. .. 36 1.8 Icaritin and cancer cell proliferation 1.8.1 Icaritin and cancer cell proliferation Herba Epimedii is officially listed in China as an herb that prevents the growth of cancer and has been... therapy in postmenopausal hormone therapy and recurrence of breast cancer Table shows the risk of breast cancer recurrence in breast cancer survivors after hormone replacement therapy [Figure adapted