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

Abstract

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 17ß-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 1 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. 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 ERa induced by estradiol/icaritin combination, indicating the central role of icaritin on AhR-mediated receptor stability. In contrast, knockdown of AhR gene did not restore estradiol-mediated degradation of ERa, 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 estradiol-stimulated MCF-7 cell proliferation via AhR-mediated proteasomal degradation of ERa 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.