OBJECTIVETo investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC).

METHODSThe human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-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 western 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.

RESULTSCisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment 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 less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05).

CONCLUSIONPhosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.