喉癌Hep2细胞系APAF-1表达及对5-Aza-CdR诱导凋亡敏感性的研究

Apoptosis sensitivity induced by 5-Aza-CdR and its association with APAF-1 expression in laryngeal carcinoma cell line Hep2

目的:探讨喉癌中APAF-1基因的表达及其对5-氮杂-2′-脱氧胞苷(5-az-a-2′-deoxycitydine,5-Aza-CdR)诱导的Hep2细胞系凋亡的影响。方法:不同浓度5-Aza-CdR(5×10-8、10-7、2×10-7和3×10-7mol/L)处理体外培养的Hep2细胞后,用流式细胞仪检测处理72h的细胞周期分布和细胞凋亡率变化;通过RT-PCR及免疫组织化学技术检测APAF-1基因在喉癌中的表达情况,并建立APAF-1瞬时过度表达系统,观察在APAF-1过度表达情况下,5-Aza-CdR对Hep2细胞凋亡的影响。结果:10-7mol/L5-Aza-CdR处理Hep2细胞72h后,凋亡率由对照组的1·07%增加到13·96%,且发生G1期细胞周期阻滞;喉鳞癌组织中APAF-1mRNA和蛋白表达都明显下调,分别为16/42、14/31,且未发现APAF-1表达与肿瘤的分化程度具有相关性,P=0·093;在10-7mol/L浓度5-Aza-CdR诱导下,APAF-1过度表达组Hep2细胞系凋亡率由空白对照组的12·46%及空载体转染组的14·74%增高到28·64%,且发生S期细胞周期阻滞。结论:过度表达APAF-1基因可以明显增加Hep2细胞系对去甲基化药物5-Aza-CdR诱导的凋亡敏感性。

OBJECTIVE：To explore the expression of APAF-1 gene in laryngeal carcinoma and its effect on 5-Aza-CdR induced Hep2 cell apoptosis. METHODS： Human laryngeal carcinoma cell line Hep2 was cultured in RPMI1640 and then treated with different concentrations of 5-Aza-CdR （5×10^-8,10^-7,2×10^-7 and 3×10^-7 mol/L）. The alteration of apoptosis and cell cycle profiles were assayed by flow cytometry （FACS）. The expression of APAF-1 gene in laryngeal carcinoma was detected by RT-PCR and immunohistochemistry staining. The over-expressed APAF-1 system was established by instant transfection and the effect of 5-Aza-CdR on APAF-1 gene over-expressed Hep2 cells apoptosis was detected by FACS. RESULTS： In comparison with control, the Hep-2 cells apoptosis increased significantly from 1.07% to 13.96% after 10^-7 mol/L 5-Aza-CdR treated for 72 h and cells occurred G1 stage block. RT-PCR and Western blot analysis showed that APAF-1 mRNA and pratein down-regulated significantly, that was 16/42, 14/31 respectively. Meanwhile, there was no correlation of APAF-1 gene expression with the differentiation of laryngeal carcinoma （P=0.093）. With over-expression of APAF-1 gene, 10^-7 mol/L 5-Aza-CdR significantly increased the Hep2 cell apoptosis and blocked the cells to S stages. The apoptosis of pcDNA3.1-APAF-1 transfection group increased from 12.46% and 14.74% to 28.64% compared with the PBS and pcDNA3.1 transfection groups. CONCLUSION： Over-expression of APAF-1 gene can significantly enhance the sensitivity of de-methylation agent 5-Aza-CdR induced Hep2 cell apoptosis.