肺癌细胞DAPK基因启动子甲基化与吉非替尼敏感性的相关性研究  

Relationship between DAPK promoter methylation and gefitinib resistance in lung cancer cell lines

作　　者：吴魏芹[1] 张晓媛[1] 徐海凤[2] 徐静[1] 沈捷[2] 卢凯华[1] 

机构地区：[1]南京医科大学第一附属医院肿瘤科,南京210029 [2]南京医科大学第一附属医院内分泌科,210029

出　　处：《临床肿瘤学杂志》2013年第6期481-485,共5页Chinese Clinical Oncology

摘　　要：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(NSCLC)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(H1650、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 EG- FR-mutation lung cancer cells（ H1650, PC9 ） and two wild type lung cancer cells （A549, H520） were treated with 5-aza-CdR（ I p, mol/ L） plus gefitinib at different concentrations. The cell proliferation was determined by CCK-8 assay. Apoptosis of cells was observed u- sing 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-aza-CdR demethylation was significantly stronger than before （P 〈 0. 05 ）. Flow cytometry results showed that the apoptosis rate of H1650 and H520 cells trea- ted 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 theprevious （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 ge- fitinib on NSCLC. It may provide a new method for pred

关 键 词：死亡相关蛋白激酶 表皮生长因子受体 吉非替尼 DNA甲基化 非小细胞肺癌 细胞株 

分 类 号：R734.2[医药卫生—肿瘤]