Inhibition Of Autophagy Increases The Effect Of DDP In DDP-resistant I-10 Testicular Cancer Cells

Objective:1.Observe the change of cisplatin(DDP)-induced autophagy fluxs2.Clarify the role of autophagy in mediating cellular resistance to DDP-induced cytotoxicity.Methods:1.The expression of LC3?p62?Atg5 and Atg7 were assessed by western blotting in I-10 and I-10/DDP.2.The autophagic flux was detected by mCherry-GFP-LC3 B transfection.3.Autophagosomes were detected under transmission electron microscopy(TEM).4.Cell surviving fraction was measured by MTT assay.5.Cell death rates was assessed by PI staining assay.6.Cloning efficiency was detect by Colony-forming assay.7.For gene silencing,cells were transfected with shRNA.8.Statistical analysis was performed using SPSS 16.0.Data are presented as mean and standard deviation(SD).Statistics were analyzed using Student's t-test when only two groups were present or assessed by one way analysis of variance(ANOVA)and SNK-q tests when more than two groups were compared.Statistical significant was defined as P < 0.05.Results:1.Treatment with DDP led to autophagy in DDP-resistant testicular cancer cell I-10/DDP but not in parent testicular cancer cell I-10.The LC3-II levels in I-10/DDP cells was increased in response to DDP treatment(P<0.001),however,the p62 levels was was decreased(P<0.05).No significant change was observed in parental DDP-sensitive I-10 cells.Transmission electron microscopy(TEM)-based analysis revealed increased formation of autophagosomes in DDP-treated I-10/DDP cells but not in DDP-treated I-10 cells.Confocal fluorescent microscopy-based analysis revealed the number of autophagosomes and autolysosomes were increased in DDP-treated I-10/DDP cells.2.CQ inhibited DDP-induced autophagy fluxs.Transmission electron microscopy(TEM)-based analysis revealed increased formation of double-membrane vesicles(autophagosomes)in CQ+DDP group compared with DDP group.Confocal fluorescent microscopy-based analysis revealed the number of autophagosomes and autolysosomes were increased in CQ+DDP group compared with DDP group.3.CQ enhanced the DDP-induce antitumor effect in I-10/DDP cells.Cell survival fraction was decreased in CQ+DDP group compared with DDP group(P<0.05).Cell death rates was increased in CQ+DDP group compared with DDP group(P<0.001).The colony formation ability was decreased in CQ+DDP group compared with DDP group(P<0.05).4.Knockdown of atg5 or atg7 specifically inhibited DDP-induced autophagy fluxs.Transmission electron microscopy(TEM)-based analysis revealed decreased formation of double-membrane vesicles(autophagosomes)in Atg5+DDP or Atg7+DDP group compared with NC+DDP group.Confocal fluorescent microscopy-based analysis revealed the number of autophagosomes and autolysosomes were decreased in Atg5+DDP or Atg7+DDP group compared with NC+DDP group.5.Knockdown of atg5 or atg7 enhanced the DDP-induced antitumor effect in I-10/DDP cells.Cell survival fraction was decreased in Atg5+DDP or Atg7+DDP group compared with NC+DDP group(P<0.001,P<0.001).Cell death rates was increased in Atg5+DDP or Atg7+DDP group compared with NC+DDP group(P<0.01,P<0.001).The colony formation ability was decreased in Atg5+DDP or Atg7+DDP group compared with NC+DDP group(P<0.01,P<0.01).Conclusion:1.DDP induces autophagic flux in I-10/DDP cells.2.Inhibition of autophagy enhances the DDP-induced antitumor effect in I-10/DDP cells.