Study On Cell Model And Mechanism Of Bipolar Androgen Therapy For Castration-resistant Prostate Cancer

Background Bipolar androgen therapy reduces PSA levels and slows the growth of castration-resistant prostate cancer by regulating androgen levels between castration to superphysiological concentration in the body by using antiandrogen drugs and hyperphysiological androgen.The precise mechanism of this method has not been fully elucidated.Objective This research used the suitable cell model for bipolar androgen treatment,aims to observe the effects of superphysiological androgen on the proliferation,cell cycle,apoptosis,pyroptosis,cell senescence and DNA damage of enzalutamide-resistant prostate cancer cells during bipolar androgen therapy in vitro.To further reveal the mechanism of the effect of superphysiological androgen on enzalutamide-resistant prostate cancer cells in bipolar androgen therapy,and to establish a theoretical foundation for more accurate treatment of castration-resistant prostate cancer in the future.Methods(1)Enzalutamide-sensitive C4-2 and C4-2B cells were invoked as prototypes,the enzalutamide-resistant C4-2 and C4-2B cells were obtained by increasing the concentration of DHT,for the bipolar androgen treatment cell model.MTT assays,cell colony assays were utilized to detect the response of enzalutamide-resistance and enzalutamide-sensitive C4-2 and C4-2B,PC-3,LNCa P and 22RV-1 cells to superphysiological androgen.(2)Enzalutamide-resistant and enzalutamide-sensitive C4-2 and C4-2B cells were treated with solvent control group(EtOH)and superphysiological dose androgen group(DHT 10 nM)respectively,and the cell cycle changes after 6,12,24 and 48 hours of the above treatments were detected by FLOW cytometry using PI staining.The above cells were collected 48 hours after the superphysiological androgen and the proteins were extracted for detecting the expression changes of the key molecular proteins involved in cell cycle by Western blotting.(3)Enzalutamide-resistant and enzalutamide-sensitive C4-2and C4-2B cells were treated with EtOH and DHT 10 nM respectively.Annexinv-PI double staining were utilized to detect the apoptosis of cells at 0,72 and 96 h after the above treatments on flow cytometry.(4)After 48 hours of treatment with the solvent control group(EtOH)and superphysiological androgen group(DHT 10 nM),the cells were collected and the proteins were extracted.The changes of molecular markers of cell apoptosis,pyroptosis,cell senescence and DNA damage were detected by Western blotting.(5)The above cells were collected from the solvent control group(ETOH)and the superphysiological androgen group(DHT 100 nM)for 48 h,and the RNA was extracted by Trizol.After reverse transcription,PCR was performed to observe the changes in the expression of cell cycle related genes,and to screen the genes involved in the effects of superphysiological androgen.(6)In the enzalutamide-resistant and enzalutamide-sensitive prostate cancer cell C4-2 and C4-2B,the relevant genes were disrupted respectively,to further observe the effect of superphysiological androgen on the cell colony assays formation ability and cell cycle.Results(1)In the bipolar androgen therapy cell model,10 nM or higher DHT inhibited the growth of enzalutamide-resistant prostate cancer cells.(2)In the above cell model treated with superphysiological androgen,there was no significant change in the cell cycle of enzalutamide-sensitive prostate cancer cells after the treatment,but the cell cycle of enzalutamide-resistant prostate cancer cells was blocked in G1 phase after the treatment.However,the expression of the key molecular proteins involved in cell cycle regulation did not change significantly.(3)No apoptosis was observed in enzalutamide-sensitive and enzalutamide-resistant C4-2B cells treated with superphysiological dose of androgen.(4)Western blotting showed no significant changes in the molecular markers of a cell apoptosis,pyroptosis,cell senescence and DNA damage in the above cell model treated with superphysiological androgen.(5)The gene GNMT expression detected by PCR was significantly increased in enzalutamide-resistant prostate cancer cells,and the protein expression of GNMT was significantly increased in the cells treated with high dose of androgen by Western blotting.(6)After the GNMT gene knockdown,the inhibitory effect of superphysiological androgen on enzalutamide-resistant prostate cells was weakened,and we did not find significant cell cycle arrest.Conclusion(1)Superphysiological androgen can induce the G1 phase arrest of the cell cycle in enzalutamide-resistant prostate cancer cells,thereby inhibiting the growth of enzalutamide-resistant prostate cancer cells.(2)Superphysiological dose of androgen can up-regulate the expression of GNMT gene,leading to enzalutamide-resistant prostate cancer cell cycle arrest.