Xeno-oestrogens and phyto-oestrogens are alternative ligands for the androgen receptor

The androgen receptor （AR） plays a critical role in prostate cancer development and progression. This study aimed to use a computerized docking approach to examine the interactions between the human AR and phytooestrogens （genistein, daidzein, and flavone） and xeno-oestrogens （bisphenol A, 4-nonylphenol, dichlorodiphenyl trichloroethane [DDT], diethylstilbestrol [DES]）. The predicted three-dimensional structure of AR and androgens was established using X-ray diffraction. The binding of four xeno-oestrogens and three phyto-oestrogens to AR was analysed. The steroids estradiol and dihydrotestosterone （DHT） were used as positive controls and thyroxine as negative control. All the ligands shared the same binding site except for thyroxine. The endogenous hormones DHT and 17β-oestradiol showed the strongest binding with the lowest affinity energy （〈 -10 kcal mol-1）. All three phyto- oestrogens and two xeno-oestrogens （bisphenol A and DES） showed strong binding to AR. The affinities offlavone, genistein, and daidzein were between -8.8 and -8.5 kcal mol 1, while that of bisphenol A was -8.1 kcal mol-l and DES -8.3 kcal mol-1. Another two xeno-oestrogens, 4-nonylphenol and DDT, although they fit within the binding domain of AR, showed weak affinity （-6.4 and -6.7 kcal mol 1, respectively）. The phyto-oestrogens genistein, daidzein and flavone, and the xeno-oestrogens bisphenol A and DES can be regarded as androgenic effectors. The xenooestrogens DDT and 4-nonylphenol bind only weakly to AR.

Xeno-oestrogens Bisphenol A and Diethylstilbestrol Selectively Activating Androgen Receptor Mediated AREs-TATA Reporter System

We cloned the three androgen response elements（AREs, including AREI, AREII, and AREIII ） with a core transactivation TATA element of the prostate-specific antigen（PSA） promoter into pGL2 basic vector to create an artificial pGL2/AREs-TATA reporter system, which was applied to evaluating the effects of different xeno- oestrogens[bisphenol A（BPA）, 4-nonylphenol（4-NP）, dichlorodiphenyl trichloroethane（DDT） or diethylstilbestrol （DES）] on androgen receptor（AR） abnormal activation to regulate PSA expression and cell proliferation. In all the three AREs, AREIII-TATA displayed as a major element responsive to AR-mediated DHT stimulation of PSA promoter. Therefore, pGL2/AREIII-TATA reporter was adopted to analyze the activation capacity of AR activated by four different xeno-oestrogens. The activation of pGL2/AREIII-TATA reporter by each xeno-oestrogen was analyzed in two different cell lines, one was HEK293T（Human Embryonic Kidney 293T） cell line, and the other was AR stably expressed DU145 cell line, which was produced by infecting AR with pLenti-puro-AR into the prostate cancer DU145 cells and that were scanned with puromycin and tested by AR antibody. In both the two cell lines, BPA or DES significantly induced AR-mediated transcriptional activity of AREIII-TATA reporter, whereas DDT or 4-nonylphenol did not. Moreover, AR-mediated cell proliferation in response to each of four xeno-oestrogens was measured in MTT assays in both HEK293T cell or AR stably expressed DUI45 cell lines. BPA or DES, as an AR inducer, exhibited an enhanced effect in cell proliferation, rather than the effect of DDT or 4-NP, in both cell lines. Finally, we demonstrated that BPA or DES stimulated PSA expression and enhanced the recruitment of AR onto the PSA promoter, resulting in stronger binding to AREIII sites. Taken together, four xeno-oestrogens were identified to have different activities on AR. BPA and DES are demonstrated to be androgenic effectors in the regulation of PSA activation or cell proliferation.