人肺腺癌吉非替尼耐药细胞系A549/GR的建立及小白菊内酯逆转耐药机制研究

Establishment of the gefitinib-resistant A549/GR cell line of humam lung adenocarcinoma and investigation of the mechanism of parthenolide in reversing drug resistance

目的建立人肺腺癌吉非替尼耐药细胞系A549/GR,研究其耐药机制,探讨小白菊内酯(PAR)逆转A549/GR耐药的机制。方法以吉非替尼为诱导剂,人肺腺癌细胞系A549为诱导对象,采用大剂量冲击和逐步增加剂量相结合的方法,诱导建立人肺腺癌吉非替尼耐药细胞系A549/GR。采用细胞毒实验(MTT)法测定细胞耐药指数,免疫印迹法(Western blotting)检测细胞耐药相关蛋白表达水平,进一步通过MTT法检测PAR对A549/GR的逆转倍数,Annexin V-FITC双标法检测PAR的促凋亡作用,Western blotting检测细胞凋亡相关蛋白表达水平。结果所建立的人肺腺癌吉非替尼耐药细胞系A549/GR耐药指数为18.7。A549/GR细胞中MDR和ABCG2耐药相关蛋白表达水平较A549升高。MTT结果显示PAR对耐药细胞A549/GR的逆转倍数为8.66。Annexin V-FITC凋亡检测结果显示PAR能有效促进A549/GR细胞凋亡,Western blotting检测显示PAR能有效降低survivin、Bcl-2两种抑制凋亡相关蛋白的表达。结论成功建立了人肺腺癌吉非替尼耐药细胞系A549/GR;PAR通过促进A549/GR细胞凋亡有效逆转其耐药。

Objective To establish a gefitinib resistant cell line A549/GR of human lung adenocarcinoma and investigate the mechanism of drug resistance.Then,to find a reversal agent parthenolide to reverse A549/GR drug resistance.Methods The gefitinib resistant cell line A549/GR was established by a method of repeated treatment with high dose of gefitinib for a short period and followed by low but gradually increasing concentrations of the drug.The drug resistance index was determined by MTT assay,and the expression levels of drug resistant related protein MDR and ABCG2 were detected by the Western blotting assay.The reversal factor of parthnolide in A549/GR cells was measured by MTT assays.The cell apoptosis was detected by AnnexinV FITC staining and the apoptosis related proteins surviving and Bcl-2 were detected by Western blotting analysis.Results The resistance index of A549/GR to gefitinib was 18.7.The expression levels of the drug resistance-related protein MDR and ABCG2 were obviously increased in A549/GR cells.Parthenolide effectively reversed drug resistance of A549/GR,with a reversal factor 8.66.Importantly,parthenolide potently induced A549/GR cell apoptosis and inhibited expression of anti-apoptotic protein surviving and Bcl-2.Conclusion A drug-resistant cell line A549/GR expressing typical drug-resistant proteins was established.Parthenolide can reverse the drug-resistant cells by inducing apoptosis of A549/GR cells.