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目的探讨佛波酯(TPA)诱导胃癌细胞系凋亡过程中蛋白激酶B(PKB)的作用。方法通过BrdU处理,流式细胞术检测以及DAPI染色,荧光显微镜观察分析TPA对胃癌BGC-823细胞的影响;免疫印迹法检测TPA对PKB蛋白表达水平、磷酸化的影响;核浆分离获得核浆蛋白,用于免疫印迹法检测TPA对胃癌细胞内PKB和其Ser 473位点磷酸化的核浆表达影响,以及激光扫描共焦显微镜观察TPA是否改变胃癌细胞内PKB的分布。结果TPA诱导BGC-823细胞凋亡;TPA下调BGC-823细胞内PKB蛋白表达,并且与TPA作用时间和TPA浓度呈正相关,与PP2A降解作用无关;TPA抑制PKB的Ser 473位点磷酸化,而对其Thr 308位点磷酸化没有明显影响;TPA下调细胞核内PKB的表达以及Ser 473位点的磷酸化;TPA并不改变PKB在胃癌细胞内的分布。结论PKB的抑制作用可能参与TPA诱导胃癌细胞凋亡过程;由TPA诱导的胃癌细胞凋亡可能部分是由于细胞核内PKB蛋白表达和Ser 473位点磷酸化下降所致。
Objective To investigate the role of phorbol ester (TPA) in the induction of protein kinase B (PKB) during gastric cancer cell apoptosis. Methods BrdU treatment, flow cytometry and DAPI staining were used to observe the effect of TPA on gastric cancer BGC-823 cells by fluorescent microscopy. The effect of TPA on PKB protein expression and phosphorylation was detected by Western blotting. Protein was used to detect the effect of TPA on the expression of PKB and its phosphorylated Ser 473 phosphorylation in gastric cancer cells by laser scanning confocal microscopy and observe whether TPA changes the distribution of PKB in gastric cancer cells. Results TPA induced the apoptosis of BGC-823 cells. TPA down-regulated the expression of PKB protein in BGC-823 cells, which was positively correlated with the time of TPA and the concentration of TPA. TPA inhibited the phosphorylation of PKB at Ser 473 Had no significant effect on Thr308 phosphorylation; TPA down-regulated the expression of PKB and the phosphorylation of Ser 473; TPA did not change the distribution of PKB in gastric cancer cells. Conclusions The inhibitory effect of PKB may be involved in the apoptosis of gastric cancer cells induced by TPA. The apoptosis of gastric cancer cells induced by TPA may be partly due to the decrease of PKB protein and the decrease of Ser 473 phosphorylation in the nucleus.