吉非替尼获得性耐药细胞株对不同化疗药物的敏感性分析

Sensitivity of two cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs

目的探讨吉非替尼获得性耐药细胞株对不同化疗药物的敏感性，为分子靶向治疗失败的患者选择化疗方案提供临床前的依据。方法体外培养人肺腺癌细胞株PC9和吉非替尼获得性耐药株PC9／G，采用二苯基溴化四氮唑蓝（MTT）法测定PC9和PC9／G细胞对不同药物的敏感性以及细胞的增殖抑制率；采用流式细胞仪检测药物对PC9和PC9／G细胞凋亡的影响及P-170蛋白的表达；采用基因芯片技术分析PC9和PC9／G细胞的表达基因谱差异；采用Western blot法检测PC9和PC9／G细胞中总Akt、磷酸化Akt和整合素B1的表达。结果MTY和凋亡检测的结果表明，与PC9细胞相比，吉非替尼获得性耐药细胞株PC9／G对顺铂的耐药指数为5．4，细胞凋亡减少，联合LY294002后对顺铂的敏感性显著增加（P〈0．05）。PC9／G细胞对多西紫杉醇较PC9细胞更为敏感，培美曲塞对两株细胞的IC50及凋亡影响的差异无统计学意义（P〉0．05）。PC9／G细胞中P-170蛋白的表达水平为5．32，与PC9细胞（7．18）比较，差异无统计学意义（P〉0．05）。基因芯片分析显示，PC9／G细胞中，整合素B1及DNA修复基因的表达上调，有丝分裂期基因表达下调。PC9／G细胞中总Akt、磷酸化Akt乃整合素B1的蛋白表达水平分别为1．32、1．82和1．59，PC9细胞中总Akt、磷酸化Akt及整合素B1的蛋白表达水平分别为0．83、0．87和0．57。结论在吉非替尼获得性耐药细胞株中存在P13K的表达上调及激活、整合素B1及DNA修复基因的表达上调，其表达上调与顺铂耐药相关；表皮生长因子受体-酪氨酸激酶抑制剂（EGFR-TKI）治疗失败的患者在选择化疗方案时应避免使用铂类药物，可选择给予多西紫杉醇或培美曲塞治疗。

Objective To explore the sensitivity of tumor cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs and provide preclinical basis of available chemotherapy regimens after failure of molecular targeted therapy. Methods Human lung adenocarcinoma cell lines PC9 and PCg/G with acquired resistance to gefitinib were cultured in vitro. The sensitivity to chemotherapeutic drugs and inhibition rate of cell proliferation was determined by MTT assay. Effects of drugs on apoptosis and expression of P-170 were determined by flow cytometry. Difference of gene expression profile between PC9 and PCg/G cells was analyzed by DNA microarray. Western blot was used to test the expression of Akt, phospho-Akt and integrin β1. Results The resistance index of PCg/G cells to cisplatin was about 5.4-fold compared with that of PC9 cells. LY294002 may significantly elevate the sensitivity of PCg/G cells to cisplatin （P 〈 0. 05 ）. PCg/G cells were more sensitive to docetaxel than PC9 cells. No significant difference of sensitivity to pemetrexed was found between these two cell lines. Expression level of P-170 in PCg/G cells was lower than that in PC9 cells. In PCg/G cells, the expression of integrin β1 and DNA healing gene was high and expression of gene during mitosis was low. The level of expression of Akt, phospho-Akt and integrin β1 in PCg/G cells was higher than that in PC9 cells： Conclusion In PCg/G cells, a cell line with acquired resistance to gefitinib, over-expression of PI3 K, integrin and DNA restoration gene and continuous activation of PI3K is found to be correlated with resistance to cisplatin. Docetaxel or pemetrexed is a more reasonable choice than cisplatin for treatment of NSCLC patients who failed to respond to EGFR-TKI.