Role Of Pannexin1 Channels In DDP-Resistant I-10 Testicular Cancer Cells

Objective: 1.To detect expression of pannexin1(Panx-1)in I-10 and I-10/DDP.2.To observe the role of ATP/IP3 pathway mediated by pannexin1 channels on cisplatin-induced apoptosis.3.To investigate the potential mechanisms of cisplatin-resistance.Methods: 1.Total expression of Panx-1 was assessed by western blotting in I-10 and I-10/DDP.2.The expression of Panx-1 on membrane was assessed by immunofluorescence in I-10 and I-10/DDP cells.3.Cell surviving fraction was measured by MTT assay.4.Early-stage apoptosis was assessed by FITC-Annexin V/PI staining assay.5.Late-stage apoptosis was assessed by Hoechst 33258 staining method.6.The extracellular ATP concentration was measured using luminescence assay.7.Intracellular IP3 was determined using ELISA.8.For gene silencing and overexpression,cells were transfected with sh RNA.9.Statistical analysis was performed using SPSS 16.0.Data indicate mean ± standard deviation(SD).Differences among groups were determined with one-way ANOVA.Statistical significant was defined as P<0.05.Results: 1.I-10/DDP cells show decreased expression of Panx-1.Total expression level of Panx-1 in I-10/DDP cells was decreased compared with I-10 cells,P<0.01.The expression of Panx-1 on membrane in I-10/DDP cells was dramaticlly decreased compared with I-10 cells.2.CBX(Panx-1 inhibitor)decreased cytotoxicity of cisplatin.Cell survival fraction was increased by CBX compared with DDP groups,P<0.01.3.CBX(Panx-1 inhibitor)decreased cisplatin-induced apoptosis.Early-stage apoptosis was decreased by CBX compared with DDP groups,P<0.001.Late-stage apoptosis was decreased by CBX compared with DDP groups,P<0.01.4.CBX(Panx-1 inhibitor)inhibited ATP release and IP3 level.ATP concentration was decreased by CBX compared with DDP groups,P<0.05.IP3 level was decreased by CBX compared with DDP groups,P<0.05.5.Panx-1 overexpression increased cisplatin-induced apoptosis and enhanced ATP release and IP3 level.Cell survival fraction was decreased by Panx-1 overexpression compared with DDP groups,P<0.001.Early-stage apoptosis was increased by Panx-1 overexpression compared with DDP groups,P<0.01.Late-stage apoptosis was increased by CBX compared with DDP groups,P<0.001.ATP concentration was increased by Panx-1 overexpression compared with DDP groups,P<0.01.IP3 level was increased by Panx-1 overexpression compared with DDP groups,P<0.01.6.Panx-1 knockdown decreased cisplatin-induced apoptosis and inhibited ATP release and IP3 level.Cell survival fraction was increased by Panx-1 knockdown compared with DDP groups,P<0.01.Early-stage apoptosis was decreased by Panx-1 knockdown compared with DDP groups,P<0.01.Late-stage apoptosis was decreased by Panx-1 knockdown compared with DDP groups,P<0.001.ATP concentration was decreased by Panx-1 knockdown compared with DDP groups,P<0.05.IP3 level was decreased by Panx-1 knockdown compared with DDP groups,P<0.01.7.Apyrase and xestospongin C decreased cisplatin-induced apoptosis.ATP concentration was decreased by apyrase compared with DDP groups,P<0.05.IP3 level was decreased by apyrase compared with DDP groups,P<0.01.Cell survival fraction was increased by apyrase or xestospongin C compared with DDP groups,P<0.01.Early-stage apoptosis was decreased by apyrase or xestospongin C compared with DDP groups,P<0.001.Late-stage apoptosis was decreased by apyrase or xestospongin C compared with DDP groups,P<0.01.Conclusions: 1.The expression level of Panx-1 is decreased accompany with resistance of I-10 testicular cancer cells to cisplatin.2.ATP/IP3 pathway mediated by Panx-1 channels participates in cisplatin-induced apoptosis in testicular cancer.3.Up-regulating Panx-1 channels can amplify the anti-tumor effect of cisplatin in I-10 testicular cancer cells by enhancing the ATP/IP3 pathway.