PTEN依赖其磷酸酶活性诱导肝癌细胞发生失巢凋亡机制的探讨

PTEN induces anoikis through its phosphatase activity in hepatocellular carcinoma cells

目的研究抑癌基因PTEN对人肝癌SMMC7721细胞失巢凋亡的影响并探讨其可能的机制。方法将携有野生型PTEN基因及无磷酸活性的突变型C124A PTEN基因的真核表达载体转染SMMC7721细胞,利用Western印迹杂交法,检测PTEN蛋白表达与蛋白激酶B(PKB/Akt)、焦点黏附激酶(FAK)磷酸化水平的变化,并应用流式细胞仪技术和激光共聚焦显微镜技术,分析各种细胞在黏附与失黏附状态下的凋亡。结果与对照细胞相比,转染野生型PTEN的SMMC7721细胞中,FAK和Akt的磷酸化水平分别降低了65.2%和89.1%,且失巢凋亡率由9.5%增至31.3%;而转染C124A PTEN的SMMC7721细胞中,FAK和Akt的磷酸化水平及失巢凋亡率均无明显变化。结论抑癌基因PTEN可依赖其磷酸酶活性抑制FAK和Akt的磷酸化,并诱导肝癌细胞发生失巢调亡。

Objective To investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of hepatocellular carcinoma SMMC-7721 cells. Methods SMMC-7721 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN (C124A-PTEN) in vitro; The PTEN expression and the phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) were detected by Western blotting;Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells. Results Compared with the control, PTEN expression in the cells transfected with wild-type PTEN increased to 248%, while the phosphorylation level of FAK and Akt decreased 65.2% and 89.1%, respectively; and the anoikis percentage increased from 9.5% to 31.3%. In the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the anoikis percentage had obviously changed, although the PTEN expression enhanced dramatically in comparison with the control. Conclusion Through its phosphatase activity, tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt, and induce anoikis in hepatocellular carcinoma cells.