Inhibiting autophagy by silencing ATG5 and ATG7 enhances inhibitory effect of DDP on DDP-resistant I-10 testicular cancer cells

Abstract

To observe the changes in autophagy of cisplatin-resistant I-10 testicular cancer cells (I-10/DDP cells) in response to cisplatin treatment and the effect of silencing ATG5 and ATG7 on autophagy and proliferation of cisplatin-treated cells. I-10/DDP cells treated with 15 μmol/L cisplatin for 12 h were examined for expressions of LC3 and p62 by Western blotting and for autophagy level through transmission electron microscopy and mCherry-GFP-LC3B. I-10/DDP cells were transfected with short hairpin RNAs shRNA-ATG5 or shRNA-ATG7 via Lipfectamine2000, the empty vector (NC group), or Lipfectamine2000 alone (blank control group), and the cellular expressions of ATG5 and ATG7 were detected with Western blotting. The transfected cells were treated with 15 μmol/L cisplatin for 12 h, after that the expressions of LC3 and p62 were detected with Western blotting. Transmission electron microscopy and mCherry-GFP-LC3B were used to detect autophagy level in the cells. MTT assay and colony-forming assay were performed to assess the cell survival fraction and colony formation ability of the treated cells, respectively. After cisplatin treatmert, the expression level of LC3 II increased significantly (P < 0.001), the expression level of p62 decreased (P < 0.05), and the number of autophagosomes increased in I-10/DDP cells. The cells transfected with shRNA-ATG5 or shRNA-ATG7 showed significantly decreased expressions of ATG5 or ATG7 (P=0.005 or P < 0.001). Cisplatin treatment of the transfected cells obviously reduced the cellular expression of LC3 II (P < 0.001), increased the expression of p62 (P < 0.001), and decreased the number of autophagosomes, cell survival fraction and colony formation ability of the cells (P < 0.001). Silencing ATG5 and ATG7 inhibits cisplatin-mediated autophagy and enhances the inhibitory effect of cisplatin on inhibiting cell proliferation.