目的 探讨死亡相关蛋白激酶(DAPK)基因启动子区域甲基化对非小细胞肺癌(NSC LC)细胞株中表皮生长因子酪氨酸激酶抑制剂(EGFR-TKI)敏感性的影响.方法 采用不同浓度吉非替尼作用2株EGFR外显子19缺失突变型(H 1650、PC9)和2株EGFR野生型(A549、H520) NSCLC细胞.5-氮杂-2’-脱氧胞苷(5-aza-CdR)去甲基化处理,用CCK-8法检测细胞增殖率,流式细胞技术检测细胞凋亡率,荧光定量PCR法检测DAPK mRNA表达情况,甲基化特异性PCR(MSP)法检测DAPK启动子区甲基化状态.结果 吉非替尼对4种细胞株均存在不同程度的增殖抑制作用,呈浓度依赖性.5-aza-CdR去甲基化后,H1650细胞及H520细胞对吉非替尼的敏感性较前增强(P＜0.05)；流式细胞仪检测显示,H1650、H520细胞凋亡率上升较明显(P＜0.05).未经5-aza-CdR处理的细胞株中,吉非替尼敏感型的PC9、A549细胞株存在DAPK mRNA高表达,其基因启动子处于非甲基化状态；吉非替尼耐药型的H1650、H520细胞株DAPK mRNA表达水平较低,DAPK启动子处于高甲基化状态.5-aza-CdR去除H1650及H520细胞的DAPK基因启动子区甲基化后,DAPK基因表达及对吉非替尼的敏感性较前显著升高(P＜0.05)；而吉非替尼敏感的PC9及A549细胞株经5-aza-CdR处理后未见明显改变(P＞0.05).结论 抑癌基因DAPK启动子区域的高甲基化能够下调DAPK基因的表达,从而降低NSCLC对吉非替尼的敏感性.对DAPK基因进行甲基化状态检测,可能为临床上预测吉非替尼疗效提供新的手段.%Objective To explore the relationship between death associated protein kinase(DAPK) promoter methylation and epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI) resistance in human lung cancer cell lines.Methods Two EGFR-mutation lung cancer cells(H1650,PC9) and two wild type lung cancer cells(A549,H520) were treated with 5-aza-CdR(1 μmol/L) plus gefitinib at different concentrations.The cell proliferation was determined by CCK-8 assay.Apoptosis of cells was observed using flow cytometry.The mRNA expression level of DAPK was detected by RT-PCR.The methylation of DAPK gene promoter region was examined by methylation-specific PCR.Results Gefitinib had proliferative inhibition on the 4 lung cancer cell lines at different extent in a dose dependent manner.CCK-8 assay showed that inhibitive effect of H1650 and H520 cells after 5-asa-CdR demethylation was significantly stronger than before(P ＜0.05).Flow cytometry results showed that the apoptosis rate of H1650 and H520 cells treated with 5-aza-CdR was significantly higher than that of them untreated with 5-aza-CdR(P ＜0.05).In lung cancer cell lines without the processing of 5-aza-CdR,the gefitinib sensitive PC9 and A549 cell lines displayed high expression of DAPK mRNA,with the gene promoter in a non-methylation state,while in drug-resistant type(H1650 and H520 cell lines),the DAPK mRNA expression level was low with DAPK promoter in high methylation state.Removing the methylation of DAPK gene promoter region in H1650 and H520 cell lines by 5-aza-CdR,DAPK gene expression and gefitinib sensitivity in H1650 and H520 cell lines apparently increased compared to the previous(P ＜0.05),but the drug sensitivity in PC9 and A549 cell lines did not change (P ＞ 0.05).Conclusion High promoter methylation of tumor suppressor gene DAPK can lead to down-regulation of DAPK gene expression,and reduce the sensitivity of gefitinib on NSCLC.It may provide a new method for predicting clinical curative effect to develop DAPK gene methylation state detection.
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