Abstract 943: Lupeol, a novel androgen receptor inhibitor acts as a double-edged sword: Competitive binding as well as transcriptional inhibition

Introduction: Androgen receptor (AR) has emerged as a potential molecular target for therapeutic agents due to its crucial role in the pathogenesis of prostate cancer (CaP) of both androgen-dependent (AD-CaP) as well as androgen-independent (AI-CaP) type. Upon activation by androgen or by constitutive activation, AR triggers the induction of signaling pathways which help in the proliferation and survival of CaP cells. Dismal outcome of conventional therapy has necessitated a need to identify agents those could target AR in both types of patients with AD-CaP and AI-CaP disease. Objectives: We determined the mechanism-based efficacy of Lupeol triterpene as a potent AR inhibitor for both AD-CaP and AI-CaP. Methods and Results: We selected a series of cells representing (i) AD-CaP (LAPC4-wild type AR; LNCaP-mutant/functional AR), (ii) transition state from AD-CaP to AI-CaP (22Rv1-mutant/functional AR) and (iii) AI-CaP (LNCaP-derived C4-2b cells-mutant/functional AR). By employing MTT, 3[H]thymidine uptake and soft−agar assays, we show that Lupeol (10−50 μM/L) significantly inhibits the growth and proliferation of LAPC4, LNCaP, 22Rv1 and C4-2b cells. Androgen is known to induce AR and AR-responsive genes such as PSA in CaP cells. By employing RT-PCR, immunoblot, reporter and ChiP assays, Lupeol was observed to (1) reduce the growth-inducing effects of R1881, an androgen analogue, in LNCaP and LAPC4 cells, (2) inhibit the activation of AR and PSA, and (3) decrease the occupancy of AR on the ARE sequence of PSA gene in all CaP cells. To understand the mechanism underlying AR inhibition, we asked if Lupeol acts as a (A) competitive inhibitor of androgen or (B) transcriptional/translational inhibitor of AR. By employing drug-receptor interaction in silico and cell-based radio-ligand competitive binding assays, we show that Lupeol (1) binds to AR at its ligand-binding site and (2) antagonistically binds to both mutant and wild type AR in CaP cells. Interestingly, Lupeol was observed to reduce mRNA and protein levels of AR and PSA in all CaP cells. Lupeol exhibited more AR inhibitory potential than Curcumin, Lycopene, EGCG and Fisetin. Lupeol was also observed to sensitize CaP cells for Bicalutamide treatment. Finally, intraperitoneal administration of Lupeol (40mg/Kg; 3 days/week) inhibited the growth of LNCaP and C4-2b cell-originated tumors and reduced serum-PSA levels in xenograft mouse models. Immunohitochemical analysis of tumors showed a decreased AR and PSA levels in Lupeol treated mice. Conclusion: These data show that due to its dual role as a competitive ligand-binding inhibitor and a transcriptional inhibitor, Lupeol qualifies as a potent AR inhibitor. We suggest that Lupeol alone, or as an adjuvant to chemotherapeutic agents could be developed as a novel therapeutic agent to treat human CaP patients with hormone-sensitive as well as hormone-refractory disease type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 943. doi:10.1158/1538-7445.AM2011-943