对TRAIL耐药的肝癌细胞caspase-8基因甲基化状态及其mRNA表达

Methylation status and mRNA expression of caspase-8 in human hepatocellular carcinoma cells resistant to tumor necrosis factor-related apoptosis-inducing ligand

目的研究肝癌细胞株HepG2和SMMC 7721对肿瘤坏死因子相关与凋亡诱配体(tumor necrosis factor-relat-ed apoptosis-induc ing ligand,TRAIL)的敏感性与caspase-8基因启动子区5′CpG岛的甲基化状态和mRNA表达的关系及5′-氮-2′-杂脱氧胞苷(5-Aza-CdR)对TRAIL抗癌活性的影响。方法采用甲基化特异性PCR(m ethylation-spec ific PCR m eth-od,MSP)方法检测caspase-8基因5′CpG岛甲基化状态;采用RT-PCR方法检测caspase-8 mRNA表达;采用吖啶橙/溴化乙锭(AO/EB)染色方法检测细胞凋亡。结果2株肝癌细胞均属TRAIL耐药细胞,caspase-8基因5′CpG岛均为非甲基化状态;5-Aza-CdR即不能增加caspase-8 mRNA表达,亦不能提高肝癌细胞对TRAIL的敏感性。结论肝癌细胞对TRAIL的敏感性与caspase-8基因的甲基化状态及mRNA表达无关,5-Aza-CdR不能提高肝癌细胞对TRAIL的敏感性。

Objective To study the association of methylation status of C5 of the cytosine in the CpG dinucleotide of caspase-8 promoter and expression of caspase-8 mRNA with the resistance to tumor necrosis factorrelated apoptosis-inducing ligand （TRAIL） in human hepatocellular carcinoma cell lines, and to evaluate the effect of demethylation agent 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the resistance to TRAIL of human hepatocellular carcinoma cell lines. Methods Methylation status of caspase-8 promoter was measured with methylation-specific PCR method （MSP）. Expression of caspase-8 mRNA was detected with RT-PCR. Apoptosis induced by TRAIL was observed by Acridine Orange/Ethidium Bromide （AO/EB） staining. Results Unmethylated status of caspase-8 promoter was found in both HepG2 and SMMC 7721 hepatocellular carcinoma cells. 5-Aza-CdR neither up-regulated caspase-8 mRNA expression nor increased the sensitivity of hepatocellular carcinoma ceils to TRAIL. Conclusion caspase-8 promoter methylation status and caspase-8 mRNA expression are not related to the resistance to TRAIL. 5-Aza-CdR can not increase the sensitivity of hepatocellular carcinoma ceils to TRAIL.