Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells

Abstract

Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.

Keywords: Phosphorylation.; Triptolide, Enzalutamide, Castration-resistant prostate cancer, Androgen receptor.

Figure 1

TPL inhibits the transactivation activity of AR. (A) TPL inhibits ligand-dependent transactivation activity of AR. LNCaP cells were transfected with a PSA-luciferase reporter and treated with TPL and R1881 under serum-free conditions for 24 h. (B) and (C) Transactivation assays of the AR-NTD were performed in LNCaP cells cotransfected with p5x3 Gal4UAS-TATA-luciferase and AR-NTD-Gal4 DBD. Enzalutamide (Enza) or TPL was added 1 h before incubation with FSK or IL-6 for 24 h. (D), (E) and (F) Western blots showing the levels of AR and AR-NTD in cell extracts from (A), (B) and (C), respectively. (G) and (H) TPL inhibits expression of endogenous target genes of AR and AR-Vs. (G) LNCaP cells were pretreated with TPL then incubated for 24 h with R1881. (H) 22Rv1 cells were treated with TPL for 24 h. mRNA levels of AR and AR-V target genes were measured by qRT-PCR and normalized to β-actin mRNA. Bars represent the mean ± SD. (I) and (J) TPL reduces the protein levels of PSA in LNCaP (I) and 22Rv1 (J) cells. The results are represented as means ±SD of 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

TPL reduces the recruitment of AR to the PSA promoter by inhibiting phosphorylation of AR at S515. (A) Effect of TPL on the levels of pAR S515 and related proteins in PCa cells. LNCaP and C4-2/AR-V7 cells were pretreated with TPL or BS-181 for 1 h, and then incubated with R1881 for 4 h. Cell lysates were subjected to western blotting analysis with the indicated antibodies and the protein levels of AR and pAR S515 were quantified (B). (C) Effect of the phosphorylation status of AR on TPL activity. 293T cells were transiently co-transfected with pGL3.PSA-Luc and pcDNA3.1-AR/WT, or -AR/S515E, and then pretreated with TPL for 1 h prior to incubation with R1881 for 24 h. Luciferase activity was then measured. (D) and (E) Effect of TPL on the binding of AR or AR-V7 to the promoter of PSA. LNCaP (D) and C4-2/AR-V7 (E) cells were pretreated with TPL prior to the addition of R1881 for 6 h. ChIP assays were performed with rabbit or mouse IgG, anti-AR antibody, anti-CDK7 antibody, anti-XPB antibody, anti-Flag antibody or anti-RNA pol II S5 antibody. The results are represented as means ±SD of 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3

Knockdown of XPB mimics the effect of TPL on AR activity in PCa cells. (A) Western blotting analysis of AR phosphorylation and several related proteins in C4-2 cells with XPB depletion. C4-2 cells were infected with lentivirus carrying control shRNA or XPB shRNA. The transduced cells were lysed and subjected to western blotting analysis with the indicated antibodies. (B) Effect of XPB depletion on the AR transactivation activity. 293T or C4-2 cells with stable knockdown of XPB were transiently co-transfected with pGL3.PSA-Luc and pcDNA3.1-AR/WT or -AR/S515E plasmids for 24 h. Luciferase activity was then measured. (C) Effect of XPB depletion on the DNA-binding activity of AR and RNA pol II. C4-2 cells with stable knockdown of XPB were lysed and subjected to ChIP assays with control IgG, anti-AR antibody and anti-RNA pol II S5 antibody. The results are represented as means ±SD of 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Low doses of TPL block the proliferation of CRPC cells in vitro. (A) LNCaP cells were treated with 6.25 nM TPL for 1 h before the addition of R1881 (1 nM) for 3 days. Cell viability was measured by MTT. (B) Number of colonies formed by CRPC cells (22Rv1 and C4-2) following incubation with different concentrations of TPL. (C) C4-2/Luci and C4-2/AR-V7 cells were treated with different concentrations of TPL for 4 days. Cell viability was measured by MTT. (D) Number of colonies formed by C4-2/Luci and C4-2/AR-V7 cells after treatment with TPL. (E) C4-2/Luci and C4-2/AR-V7 cells were cultured in 8% CSS 1640 medium for 24 h, followed by treatment with 6.25 nM TPL or 20 μM enzalutamide for 72 h. PSA was then detected by ELISA. The results are represented as means ±SD of 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

TPL shows a synergistic anti-cancer effect with enzalutamide on PCa cells in vitro. (A) C4-2R cells were cultured in medium containing 8% FBS and treated with different concentrations of TPL for 72 h. MTT assays were then carried out. (B) 22Rv1 or (C) C4-2R cells were treated with 6.25 nM TPL with or without 20 μM enzalutamide. MTT assays were carried out after 96 h. (D) Number of colonies formed by 22Rv1 or C4-2R cells treated with individual or combined TPL and enzalutamide. (E) Western blotting was performed to detect apoptotic marker proteins in 22Rv1 or C4-2R cells treated with individual or combined TPL and enzalutamide. (F) 22Rv1 and C4-2R cells were treated with individual or combined TPL and enzalutamide at various concentrations for 48 h and then subjected to SRB assay. The Combination Index of TPL and enzalutamide in the two cell lines was calculated by the Chou-Talalay method. Open triangles indicate high combination concentrations of TPL and enzalutamide (≥60 nM for TPL and ≥200 μM for enzalutamide); black dots indicate low combination concentrations of TPL and enzalutamide (<60 nM for TPL and <200 μM for enzalutamide). The results are represented as means ±SD of 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6

TPL enhances the anti-cancer effects of enzalutamide in vivo. Mice bearing 22Rv1 xenografts were treated with vehicle control, enzalutamide, TPL, or enzalutamide + TPL for 3 weeks. (A) Tumor volumes in the different groups (volumes were measured twice every week). (B) Photographs of xenograft tumors harvested at day 21. (C) Weight of the xenograft tumors in the different groups. (D) Serum PSA level of the different groups. (E) Hematoxylin and eosin (H&E) staining and IHC staining of Ki67, cleaved-Caspase-3 (c-Caspase-3) and AR of representative sections of xenograft tumors. (F) and (G) Quantification of c-Caspase-3 staining (apoptotic index) and Ki-67 staining (proliferative index) in tumor sections. The results are represented as means ±SD. Scale bars represent 125 μm in all micrographs. *, p < 0.05; **, p < 0.01; ***, p < 0.001.