The effects of novel anti-androgens on androgen receptor action and expression.

5340

Androgens play an important role in prostate cancer (PC) development and progression through activation of the androgen receptor (AR). Alterations in the AR, including increased expression, mutation, and amplification have been highlighted as potential mechanisms for PC progression and resistance to treatment with androgen antagonists. Recent studies increasingly show a continuing role of the AR, even in hormone-refractory PC. Current PC therapies include androgen ablation through chemical or surgical castration, as well as AR blocking agents. Compounds that inhibit androgen biosynthesis, and antagonize/downregulate the AR, may be more effective than current agents. We have previously identified VN/85-1, VN/108-1, VN/124-1, VN/125-1, VNLG/14-1 and VNLG/21-1 as novel inhibitors of 17α-hydroxylyase/C_17,20-lyase, a key enzyme in androgen biosynthesis. The purpose of this study was to further evaluate the interactions of these compounds with the AR. Compound-AR interactions were evaluated in vitro using competitive binding, transcriptional activation, and western blot techniques. The human prostatic cancer cell lines LAPC4, LNCaP, and PC3 transiently transfected with a mutated AR (PC3-T575A, kindly provided by Dr. Lamb and Dr. Marcelli, Baylor, TX), were utilized. LAPC4 cells express wild-type AR, while LNCaP and PC3-T575A cells express mutated forms of the AR in the ligand and DNA binding domains, respectively. Competitive binding studies revealed binding to the AR with IC₅₀ values ranging from 248 to 4300nM. The binding affinities of all compounds were similar for the wild-type and T575A receptors, whereas binding to the LNCaP receptor was generally decreased. Inhibitors were further assessed to determine if they were AR antagonists in the presence and absence of the AR ligand dihydrotestosterone (DHT). A luciferase assay system (Promega) was used with the Probasin luciferase reporter construct ARR2-Luc. All compounds showed inhibition of DHT induced transcriptional activation with a potency similar to or greater than the anti-androgen bicalutamide at 1μM or higher. VNLG/21-1 and VNLG/14-1 inhibited >60% at 1μM concentration, and >90% at 10μM. None of the compounds exhibited agonistic properties in the absence of DHT. Western blot analysis of protein expression shows strong down regulation of the AR after 24 hour treatment with 10μM VN/85-1, VN/108-1, VN/124-1, and VN/125-1. The results indicate that these compounds, with activities to reduce androgen synthesis and action, may be useful in the treatment of PC.