Study On The Mechanism Of Triptolide Suppress The Proliferation Of Prostate Cancer Cells

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.),become a hot pot in the field of drug research,for it could suppress inflammatory,cystogenesis and immunosuppression,especially its anti-cancer activity.The present study found that TPL has antitumor activity in prostate cancer cells.However,the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown.Therefore,we used CRPC cells as the research model to explore the antitumor activity and mechanism of action of TPL,and we get several results listed in following:We performed transcriptional reporter assay and demonstrated that TPL can inhibit the ligand-dependent and ligand-independent transactivation activity of AR-FL and AR variants,and the effect is not due to a reduction in the their protein level.We carried out realtime PCR and Western blot to examined the mRNA levels of endogenous target genes of AR-FL(PSA,FKBP5,TMPRSS2)and AR-V(UBE2C and AKT1).The results showed that TPL suppressed the expression of AR-FL and AR-Vs target genes in 22Rv1 cells in a dose-dependent manner,without changing the AR mRNA level.The results were validated by western blotting for AR and PSA protein levels.we used selective CDK7 inhibitor BS-181 and performed ChIP assays to explore the mechanism of TPL on pAR S515.Our results found that TPL decreased the level of AR phosphorylated at Ser515(pAR S515),suppresses AR-mediated transcriptional activation by inhibiting AR binding and RNA pol II recruitment to target gene promoters.Depletion of XPB by RNA interference has a similar effect to TPL on the level of p AR S515.Therefore,These results indicate that TPL suppresses CDK7-mediated phosphorylation of AR at Ser515 through XPB.using a variety of CRPC cell model to detect cytotoxicity of low dose TPL.Our results showed that TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs and induces the apoptosis of CRPC.We performed the MTT assay,clony formation assay and western bloting to examine the combined effect of TPL and enzalutamide in vitro.The results showed that Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival,colony formation ability and induce the cleaved of PARP and caspase 3.We also found TPL enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects in vivo.Taken together,the 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.