Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

Abstract

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC(50) 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.

Figure 1.

A) Predicted model for EGCG binding to AR-LBD. Left panel: EGCG binds to AR-LBD in in silico molecular docking studies, using AutoDock software and 2PNU.pdb as the starting receptor. Different domains of the AR are distinguished by color. Right panel: enlarged view of boxed area in left panel, with putative binding sites in the model structure of the AR. Predicted distances of hydrogen bonds (Å) are given next to the bonds. B) EGCG competitively interacts with AR-LBD and decreases its interdomain interaction. EGCG competes with the high-affinity androgen Fluormone AL Red to physically interact with AR-LBD. Data are presented as averages ± se of two sample wells. Inset: structure of EGCG. C) AR N-C-interaction assay was performed in CV1 cells as described in Materials and Methods. Graphs represent fold of hormone induction compared with value for the non-hormone-treated group, which was set as 1. *^aP < 0.01 vs. AR.N or AR.C group; *^bP < 0.01 vs. AR.N+AR.C group; 1-way ANOVA followed by Tukey's HSD test. D) Effect of EGCG on AR protein turnover in LNCaP cells. Cells were treated with 40 μM EGCG and 50 μg/ml cycloheximide for the indicated time periods. AR protein levels were determined by Western blot analysis with specific antibody against AR and normalized to β-actin as loading control. E) LNCaP cells were exposed to indicated concentrations of EGCG for 48 h with or without 5 μM of MG132. AR protein levels were determined by Western blot analysis with specific antibody against AR and normalized to β-actin as loading control.

Figure 2.

EGCG inhibits hotspot AR mutant T877A-mediated transactivation. A) CV1 cells were transfected with pSG5-T877A mutant of AR (2 μg), MMTV-luc reporter (1 μg), and Renilla luc (50 ng). Graphs represent fold of hormone induction compared with value for the non-hormone-treated group, which was set as 1. *^aP < 0.01 vs. control group; *^bP < 0.01, **^bP < 0.05 vs. R1881 group; 1-way ANOVA followed by Tukey's HSD test. B) LNCaP cells were transfected with MMTV-Luc reporter and were treated 24 h post-transfection with or without 1 nM R1881 and with indicated concenctration of EGCG. Graphs represent fold of hormone induction compared with value for the non-hormone-treated group, which was set as 1. *^aP < 0.01 vs. R1881 control group; 1-way ANOVA followed by Tukey's HSD test. C) LNCaP cells were transfected with PSA promoter containing reporter and were treated 24 h post-transfection with or without 1 nM R1881 and with indicated concentration of EGCG and casodex. *^aP < 0.01 vs. control group; *^bP < 0.01vs. R1881 group; 1-way ANOVA followed by Tukey's HSD test.

Figure 3.

EGCG antagonizes AR-positive PCa cell growth. A, B) C4-2 (A) and 22Rν1 cells (B) were treated with indicated concentrations of EGCG for 48 h, and cell viability was determined by MTT assay, as detailed in Materials and Methods. *^aP < 0.01 vs. control group; *^bP < 0.01vs. 20 μM EGCG group; 1-way ANOVA followed by Tukey's HSD test. C) EGCG modulated the expression of miRNAs considered to be important in PCa. Relative expression of miRNA-21 and -330 in tumor xenograft tissues isolated from animals treated with EGCG. Real-time PCR was performed for the 2 miRNAs, and fold change was calculated.

Figure 4.

EGCG decreases AR protein expression and its nuclear shuttling in vivo. Localization of AR in androgen-independent 22Rν1 tumor xenograft in nude mice. Photomicrographs (×300) represent immunohistochemical staining (A) and immunofluorescence staining (B) for AR in tumor xenograft.

Figure 5.

Model depicting the effect of EGCG on AR-mediated signaling. EGCG could compete with natural AR agonist DHT to physically interact with the expressed AR protein. Once bound, it decreased the interdomain interaction of AR, leading to a decrease in AR protein expression and, hence, a decrease in AR transactivation functions. This resulted in decreased expression of AR target genes and thus might negatively influence the growth of PCa cells in vitro and in vivo.