nm23-H1基因转染前后人舌鳞状细胞癌Tca8113细胞中蛋白激酶C转位的变化

Changes of PKC-θ translocation before and after transfection with nm23-H1 gene in human tongue squamous cell carcinoma Tca8113 cell lines

目的研究nm23-H1基因转染诱导人舌鳞状细胞癌(以下简称舌鳞癌)Tca8113细胞凋亡过程中蛋白激酶C-θ(PKC-θ)的作用。方法采用脂质体转染法将真核表达载体pAdEasy-nm23-H1转染人舌鳞癌Tca8113细胞株。应用PKC-θ活化抑制剂Calphostin C和DMSO预处理细胞30min后,常规培养24h,倒置显微镜观察细胞形态的变化;流式细胞仪检测细胞凋亡率;Westernblot分析Tca8113细胞中PKC-θ裂解活化状况;酶标仪(ELISA)检测Caspase-3的相对活性。结果人舌鳞癌Tca8113细胞胞浆中及核周存在PKC-θ的表达,nm23-H1基因转染后PKC-θ从胞浆、核周转位到细胞核内表达。nm23-H1基因转染可导致PKC-θ的裂解活化,形成功能性的催化片段,而CalphostinC抑制转染诱导的PKC-θ裂解活化。nm23-H1基因转染后,Calphostin C预处理组和DMSO预处理组Tca8113的凋亡率、Caspase-3的活性增加,两组间有明显差异(P<0.05)。结论 nm23-H1诱导细胞凋亡过程中有PKC-θ的裂解活化,PKC-θ是nm23-H1诱导舌鳞癌Tca8113细胞株凋亡过程中的参与者,PKC-θ激活Caspase-3诱导细胞凋亡是nm23-H1基因诱导肿瘤细胞凋亡机制之一。

Objective To study the role of protein kinase C-θ （PKC-θ） in apoptosis in human tongue squamous cell carcinoma TcaSll3 cell lines after transfected by nm23-H1 gene. Methods Tca8113 ceils were transfected with pAdEasy-nm23-H1 by lipofection. The cells were pretreated with Calphostin C, a PKC-θ specific suppressor and DMSO for 30 minutes, the morphologic changes of the cells were observed under inverted microscope after routine culture of 24 hours. Apoptotic ratio was detected by flow cytometry. Activation and cleavage of PKC-θ were examin-ed by Western-blotting. Relative activities of Caspase-3 were studied with ELISA. Results The expression of PKC-θ was found translocating from cytoplasm and perinucleus to nucleus in Tca8113 ceils after transfected with nm23-H1 gene. Gene transfection of nm23-H1 could induce PKC-g activation and formed functional catalytic fragment, but Calphostin C played a suppressive role. The relative activity of Caspase-3 and apoptotic ratio were increased in both Calphostin C pretreated and DMSO pretreated group after transfected with nm23-H1 gene, and the differences were significant statistically （P 〈 0.05）. Conclusions The results of this study suggest that activation and cleavage of PKC-0, which were induced by gene transfection of nm23-H1, played a role in the process of Tca8113 cells apoptosis. Activation of Caspase-3 by PKC-θ maybe one of the mechanism of tumor cells apoptosis induced by nm23-H1.