The Molec?lar Mechanism Of Polyphyllin ? And Enzalutamide On Inhibiting Castration-resistant Prostate Cancer Cell Growth

Objecetive:The castration-resistant prostate cancer PC3 and DU145 cells were treated with Polyphyllin I and Enzalutamide to discuss the inhibition of cell growth and the potential molecular mechanism in vitro and vivo.Methods:The first part:1.The cell viability was measured using MTT assay to detect the proliferation of PC3 and DU145 cells on inhibition of Polyphyllin I,silence of LncRNA HOTAIR and overexpression of HOTAIR,EZH2 and DNMT1.2.The cell cycle of PC3 and DU145 treated with Polyphyllin I was detected by flow cytometry.3.The migration ability was analyzed using a wound-healing assay and the invasion ability was detected using Transwell(Boyden Chamber)assay in PC3 and DU145 cells treated with Polyphyllin I.4.Transient transfection assay was used to carry out overexpression or silence of EZH2 and DNMT1 protein and HOTAIR.Western-blot analysis was performed to measure the EZH2 and DNMT1 protein expression level and quantitative real-time PCR was performed to examine the expression level of HOTAIR in PC3 and DU145.5.The promoter activity of EZH2 was detected by Dual-luciferase reporter gene assay in PC3 and DU145 cells.6.Establishment of Tumor-bearing nude mice model: control group,Polyphyllin I group were set up to examine the size and weight differences of the tumor removed from the nude mice between groups,using Western-blot analysis and quantitative real-time PCR to measure expression level of EZH2,DNMT1 and HOTAIR from the tumor.The second part:1.The cell viability was measured using MTT assay to detect the the proliferation of PC3 and DU145 on inhibition of Polyphyllin I and Enzalutamide and overexpression of MUC1.2.The cell cycle of PC3 and DU145 treated with Polyphyllin I,Enzalutamide alone and combined administration was detected by flow cytometry.3.Transient transfection assay was used to carry out overexpression or silence of NF-?B/P65,MUC1 protein and HOTAIR.Western-blot analysis was performed to measure the NF-?B/P65/P50,MUC1 protein expression level and quantitative real-time PCR was performed to examine the expression level of HOTAIR in PC3 and DU145.4.The promoter activity of MUC1 was detected by Dual-luciferase reporter gene assay in PC3 and DU145 cells.5.Establishment of Tumor-bearing nude mice model: control group,Polyphyllin I group,Enzalutamide group,Polyphyllin I+ Enzalutamide group were set up to examine the size and weight differences of the tumor removed from the nude mice between groups,using Western-blot analysis and quantitative real-time PCR to measure expression level of NF-?B/P65/P50,MUC1 and HOTAIR from the tumor.Res?lts:The first part:1.MTT assay:Polyphyllin I can inhibit the growth of PC3 and DU145 cells in a dose and time dependent;Silence of HOTAIR can decrease cell viability and overexpression of HOTAIR,EZH2 and DNMT1 can reverse PPI-inhibited cell proliferation of PC3 and DU145.2.Cell cycle assay: Polyphyllin I can induce cell cycle arrest at S phase in PC3 and DU145.3.Cell migration and invasion assay: Polyphyllin I can markedly delay wound closure and decrease the invasion ability of PC3 and DU145.4.Western-blot and Real-Time-PCR : Polyphyllin I can decrease the expression level of HOTAIR,EZH2,DNMT1.Transient transfection assay shows that HOTAIR silence can decrease the protein expression of EZH2 and DNMT1;Overexpression of HOTAIR can overcome the PPI-decreased protein expression level of EZH2 and DNMT1;Overexpression of EZH2 can reverse Polyphyllin I-decreased expression level of DNMT1;Overexpressed DNMT1 can overcome PPI-decreased expression level of EZH2.5.The promoter activity of EZH2 was inhibited by PPI and overexpressed HOTAIR reversed PPI-decreased promoter activity of EZH2 in PC3 and DU145.6.Intraperitoneal injection of PPI can effectively inhibit tumor growth and expression of HOTAIR,EZH2,DNMT1 in vivo.Difference compared to the control group was statistically significant.The second part:1.MTT assay:Overexpression of MUC1 can reverse PPI-inhibited cell proliferation of PC3 and DU145;Combination of Polyphyllin I and Enzalutamide shows a stronger effect of inhibition on cell growth than Polyphyllin I and Enzalutamide treated alone.2.Cell cycle assay: Combination of Polyphyllin I and Enzalutamide shows a stronger effect of cell cycle arrest at S phase than Polyphyllin I and Enzalutamide treated alone in PC3 and DU145.3.Western-blot and Real-Time-PCR : Polyphyllin I can decrease the expression level of NF-?B/P65,MUC1 and HOTAIR,but has no effect on P50 protein expression.Combination of Polyphyllin I and Enzalutamide can decrease the expression level of NF-?B/P65,MUC1 and HOTAIR more than Polyphyllin I and Enzalutamide treated alone in PC3 and DU145.Transient transfection assay shows that HOTAIR silence can decrease the protein expression of MUC1 but has no effect on NF-?B/P65 and NF-?B/P65 protein expression;overexpression of NF-?B/P65 can overcome the Polyphyllin I-decreased protein expression level of MUC1 and LncRNA HOTAIR,however,overexpression of MUC1 and HOTAIR can not reverse Polyphyllin I-decreased expression level of NF-?B/P65;Overexpressed HOTAIR can overcome Polyphyllin I-decreased expression level of MUC1 and overexpressed MUC1 can not reverse Polyphyllin I-decreased expression of HOTAIR.4.The promoter activity of MUC1 was inhibited by PPI and overexpressed HOTAIR reversed PPI-decreased promoter activity of MUC1 in PC3 and DU145;Combination of Polyphyllin I and Enzalutamide performed a stronger inhibition on promoter activity of MUC1 than Polyphyllin I and Enzalutamide treated alone.5.The vivo experiment showed that combination group can decrease the tumour size and weight as well as expression level of P65,HOTAIR,MUC1 more significantly than Polyphyllin I and Enzalutamide treated alone,but there was no effect on P50.Conclusions:1.PPI can inhibit the cell growth,migration and invasion of PC3 and DU145,also induced the cell cycle arrest at S phase.2.PPI inhibit the cell growth by reg?lating HOTAIR/DNMT1/EZH2 and NF-?B/HOTAIR/MUC1 signaling pathway.3.PPI combined Enzalutamide had a synergy in the inhibition of CRPC cell growth.4.PPI can inhibit the tumor growth in nude mice,and the mechanism involved HOTAIR/DNMT1/EZH2 and NF-?B/HOTAIR/MUC1 signaling pathway reg?lation.PPI can inhibit the growth of PC3 and DU145 cells in vivo and vitro.The mechanism may be significantly related to the reg?lation of HOTAIR/DNMT1/EZH2 and NF-?B/HOTAIR/MUC1 pathway.What's more Enzalutamide can enhance the effect of PPI.The experiment provides a new target and a potential drug combination for further clinical application of CRPC treatment.