The androgen receptor(AR) signaling activated by dihydrotestosterone(DHT) plays critical roles in prostate cancer development and progression. Phytoestrogens, which are diphenolic compounds with estrogen and anti-estrogen effects, can bind to estrogen receptors. However, their function on AR signaling has not been fully elucidated. In this study, dual-luciferase reporter assay, immunobloting, docking system test, MTT assay, immunofluorescence and chromatin immunoprecipitation(ChIP) assays were employed to examine the potential effects of three phytoestrogens(genistein, daidzein, flavone) on DHT-activated prostate specific antigen(PSA) activation, cell proliferation and AR transactivation in lymph node carcinoma of prostate(LNCaP) cells. Phytoestrogens were detected to down-regulate DHT-activated AR-mediated PSA promoter transactivation by dual-luciferase reporter system. Furthermore, three phytoestrogens, especially genistein, were demonstrated to significantly decrease AR-activated PSA protein expression by Western blotting analysis. MTT experiment proves that phytoestrogens, especially genistein, remarkably inhibits the DHT-induced cell proliferation in LNCaP cells. To provide reasonable explanations for experimental phenomena mentioned above, we did docking system test and detected phytoestrogens to share the same AR-binding site with DHT. To further prove the competition between phytoestrogen and DHT on AR binding, we examined the effects of phytoestrogens on DHT-activated AR nuclear translocation and immunofluorescence analysis which confirms that phytoestrogens, especially genistein, inhibit DHT-activated androgen receptor nuclear translocation. Results from ChIP show that phytoestrogens down-regulate DHT-induces AR binding to the androgen response elements(AREs, including AREI, AREII, and AREIII) in PSA promoter. Genistein remarkably down-regulates AR, binding to the AREI located in -250― -39 bp and AREIII in -4170― -3978 bp in the presence of DHT. In general, three phytoestrogens were identified to inhibit DHT-AR binding by competitively binding to AR and inhibit AR-mediated transactivation. And genistein shows the strongest effects among three phytoestrogens. Our findings confirm that phytoestrogens are AR antagonist in the regulation of AR-related PSA activation and cell proliferation, which provides valuable insights into the treatment of prostate cancer.
