下调Pannexin2通道能增强顺铂诱导睾丸癌(I-10)细胞凋亡

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

目的探讨Pannexin2(Panx-2)蛋白在睾丸癌细胞中的表达水平以及干扰Panx-2表达对顺铂诱导睾丸癌(I-10)细胞凋亡的影响。方法免疫印迹法Panx-2蛋白在睾丸癌细胞中的表达水平;以睾丸癌I-10细胞株为研究对象,实验分为转染试剂对照组(mork组)、阴性对照质粒组(NC组)、干扰Panx-2组(shRNA1质粒组和shRNA2质粒组),采用免疫印迹法验证Panx-2的表达;在转染细胞中添加16μmol/L的顺铂诱导细胞死亡,通过MTT法分别检测细胞在24、48、72 h的存活率,集落克隆法检测细胞的集落形成能力;在顺铂(16μmol/L)处理8 h后,采用AnnexinV/PI双染法检测细胞的早期凋亡;在顺铂(16μmol/L)作用24 h后,免疫印迹法检测细胞凋亡相关蛋白caspase-3、Bcl-2和Bax的表达水平。结果与I-10细胞和Tcam-2细胞相比,I-10/DDP细胞(P<0.001)和Tcam-2/DDP细胞(P<0.01)中Panx-2的表达显著增加;干扰Panx-2基因后,免疫印迹结果显示,与NC组相比,shRNA1和shRNA2组中Panx-2的表达量下降(P<0.05);MTT法显示shRNA1和shRNA2组细胞的存活率均低于NC组(P<0.001);在含顺铂培养基的培养下,集落克隆法显示shRNA1和shRNA2组细胞的集落形成能力低于NC组(P<0.001);AnnexinV/PI双染法显示shRNA1和shRNA2组的早期凋亡率(P<0.01)均低于NC组;免疫印迹结果显示,与NC组相比,shRNA1和shRNA2组中凋亡相关蛋白caspase-3表达升高(P<0.05),Bcl-2表达降低(P<0.01),Bax表达升高(P<0.05)。结论下调Pannexin2通道能增强顺铂诱导睾丸癌(I-10)细胞凋亡。

Objective To investigate the effect of down-regulation of pannexin 2(Panx-2)channels on cisplatin-induced apoptosis in I-10 cells.Methods 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(16μmol/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 16μmol/L cisplatin,AnnexinV/PI double staining was used to detect the early apoptosis of the cells.After 24 h of treatment with 16μmol/L cisplatin,the cells were examined for expressions of caspase-3,Bcl-2 and Bax using Western blotting.Results 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).Conclusion Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.