Pharmacological Mechanism Of Cepharanthine Hydrochloride In Prostate Cancer

Background and Objective: Cepharanthine is an alkaloid extracted from Stephania Cepharantha Hayata.It is reported that Cepharanthine has a variety of pharmacological activities including anti-inflammatory,anti-cancer,anti-malaria,anti-viral and so on.Its activity has been validated in many tumor models,but not in prostate cancer.Previous studies,we found that the Cepharanthine can degrade the Androgen Receptor(AR),and AR signaling pathway is closely related to prostate cancer development.The efficacy of Cepharanthine in the treatment of prostate cancer is worth exploring.G protein-coupled estrogen receptor 30(GPR30)is our validated drug target in liver cancer.In liver cancer models,we found that CH regulates Akt phosphorylation by targeting GPR30,and in prostate cancer models,we also validated this drug target.Therefore,a salt-forming compound of Cepharanthine,Cepharanthine hydrochloride(CH),was used to explore its inhibitory effect and AR degradation effect in prostate cancer,so as to provide a new drug choice for the treatment of prostate cancer.At the same time,we hope to provides a new theoretical basis for the clinical application of Cepharanthine.Methods and Results: MTT was used to detect the toxicity of CH.Transwell migration and invasion assay and clone formation assay were used to verify the effects of CH on the migration,invasion and clone formation ability of prostate cancer cells.The effects of CH on cell cycle and apoptosis were detected by flow cytometry combined with Annexin V/PI double staining.Western Blotting,QPCR,co-IP,immunofluorescence and nucleoplasm separation experiments were used to verify the degradation effect,degradation pathway and target of CH on AR.The tumor xenotransplantation experiments in mice were used to verify the anti-prostate cancer effect of CH and the effect of AR degradation in vivo.Results:The toxicity of CH to LNCa P cells with high expression of AR was higher than that of prostate cancer cells without AR expression.CH inhibits migration,invasion and clonal formation of prostate cancer cells.Moreover,AR and its splice AR-V7 can be degraded at the transcriptional,and at protein levels through ubiquitination-proteasome pathway mediated by E3 ligase MDM2 to degrade AR and AR-V7.It was further verified that CH regulates the degradation of AR by targeting GPR30.In vivo experiment,the expressions of AR,AR-V7 and GPR30 in the animal tissues treated with CH were significantly down-regulated,but the anti-tumor phenotypic effect was not significant in vivo.Experimental conclusion and significance: We found that CH has obvious inhibitory effect on prostate cancer cells with high expression of AR,AR and AR-V7 could be degraded in two prostate cancer cells.Further investigation into the degradation pathway of AR showed that CH degraded AR through the ubiquitin proteasome pathway mediated by E3 ligase MDM2 through GPR30 mediated.Phenotypic results of in vivo experiments showed that the anti-cancer effect of CH was not significant,but the expressions of AR,AR-V7 and GPR30 in animal tissues were significantly down-regulated,indicating that CH has a certain effect on anti-cancer.On the basis of the above research results,we confirmed that CH can significantly inhibit prostate cancer and degrade AR and its spliceosome AR-V7 in vitro,and for the first time proved that GPR30 can act as the upstream of AR to regulate AR expression.The results of our study provide new targets and drug options for the treatment of prostate cancer,and also enrich the theoretical basis of pharmacological activity of Cepharanthine.