Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells

Abstract

To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells. The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) via Lipofectamine2000, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 μmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 μmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 μmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting. The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP (P < 0.001) cells and Tcam-2/DDP (P < 0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression (P < 0.05) and significantly reduced cell surviving fraction (P < 0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups (P < 0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group (P < 0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions (P < 0.05) and significantly decreased the expression of Bcl-2 (P < 0.01). Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.