真核起始因子2α的磷酸化抑制顺铂介导的肝癌细胞凋亡

Phosphorylation of eukaryotic initiation factor 2-alpha mhibits cisplatin-mediated apoptosis of hepatocellular carcinoma cells

目的探讨真核起始因子2α（eW2α）的磷酸化对顺铂诱导肝癌细胞凋亡的影响。方法将肝癌细胞随机分为对照组和实验组，在用突变载体eIF2αS51A转染和小分子抑制剂salubrinal改变实验组细胞在顺铂作用条件下eIF2cL磷酸化的基础上，用流式细胞和Westerm blot分别检测细胞凋亡的百分率和细胞凋亡的分子标志物。多组间比较采用方差分析，两组间比较采用t检验。结果顺铂诱导肝癌细胞eIF2α发生51位丝氨酸磷酸化。在突变载体转染条件下，顺铂（10ug／ml）诱导对照组和实验组SMMC-7721细胞在24h凋亡的平均百分率分别为21．7％±1．5％和50．7％±2．1％（t=19．454，P〈0．05）；顺铂诱导对照组和实验组HepG2细胞在24h凋亡的百分率分别为21．0％±1．0％和57．3％±2．1％（t=27．250，P〈0．05）。在抑制剂作用条件下，顺铂（15μg／ml）诱导对照组和实验组SMMC～7721细胞在36h凋亡的百分率分别为50．3％±2．5％和16．3％±2．1％（t=18．031，P〈0．05）；顺铂诱导对照组和实验组HepG2细胞在36h凋亡的百分率分别为42．0%±2．6%和12．0%±2．％（t=15．667，P〈0．05）。同时，Westem blot检测显示eIF2α的磷酸化抑制顺铂诱导的凋亡蛋白抗多聚ADP-核糖聚合酶的剪切。结论eIF2α的磷酸化在肝癌细胞抵抗顺铂介导的凋亡中起重要作用。

Objective To investigate whether the phosphor.clarion （functionally inhibitive） of eukaryotic initiation factor 2-alpha （eIF2-α） affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma （HCC）. Methods The human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone （controls; 24 h） or in combination with pre-Wansfeetion of a dominant-negative elF2-α mutant （eIF2αS51A） or pre-exposure to an elF2-ct-specific phosphatase inhibitor （salubrinal） to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and westem blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test. Results Cisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment （10 mg/ml） induced significant apoptosis in the eIF2ctS51A pre-transfected SMMC-7721 （control： 21.7 ± 1.5% vs. 50.7 ± 2.1%, t = 19.454,P 〈 0.05） and HepG2 （21.0 ± 1.0% vs. 57.3 ± 2.1%, t = 27.250,P 〈 0.05）. Salubrinal pre-lreatment significantly inhibited the cisplatin （15 mg/ml）-induced apoptosis in SMMC- 7721 （control： 50.3 ± 2.5% vs. 16.3 ± 2.1%, t= 18.031,P 〈 0.05） and HepG2 （42.0 ± 2.6% vs. 12.0 ± 2.0%, t = 15.667,P 〈 0.05）. Conclusion Phosphorylation of eIF2-α may act to inhibit cisplatin-induced apoptosis of HCC.