EGFR-T790M突变所致吉非替尼耐药肺腺癌细胞化疗药物敏感性变化的研究

The research on the change of chemosensitivity of gefitinib-resistant lung adenocarcinoma cell caused by EGFR-T790M mutation

背景与目的:EGFR-TKI治疗NSCLC失败后,化疗仍可取得一定的治疗效果,是可选择的治疗方案之一。核苷酸还原酶(ribonucleotide reductase,RR)、胸苷酸合成酶(thymidylate synthase,TS)、核苷酸切除修复交叉互补基因1(excision repair cross complementstion group 1,ERCC1)、3型β微管蛋白(β-tubulin-Ⅲ,TUBB3)分别与吉西他滨、培美曲塞、铂类药物及微管类药物的化疗药物敏感性存在相关性,可以通过这些分子标志物的表达水平来预测化疗药物的敏感性。RRMI、TS、ERCC1和TUBB3高表达患者化疗药物的敏感性降低,低表达患者化疗药物敏感性增高。本研究拟探讨EGFR-T790M突变所致吉非替尼耐药肺腺癌细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞化疗药物敏感性的变化。方法:通过MTT法检测PC9及PC9/GR细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的IC50,探讨其对上述药物的化疗敏感性。采用液相芯片法,检测PC9及PC9/GR细胞ERCC1 m RNA、RRM1 m RNA、TUBB3 m RNA和TS m RNA的表达水平。通过蛋白质印迹法(Western blot)检测PC9及PC9/GR细胞ERCC1、RRM1、TUBB3和TS蛋白的表达水平。结果:与PC9细胞株相比较,PC9/GR细胞株对吉非替尼、顺铂、吉西他滨和培美曲塞的IC50明显增高(P<0.05);对长春瑞滨、紫杉醇和多西他赛的IC50明显降低(P<0.05)。PC9/GR细胞对吉非替尼、顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的耐药指数分别为70、1.56、1.61、0.34、0.39、0.14和1.71。与PC9细胞株m RNA的表达量相比较,PC9/GR细胞株ERCC1 m RNA、RRM1 m RNA和TS m RNA的表达量明显增高(P<0.05),TUBB3的m RNA的表达量明显降低,差异均有统计学意义(P<0.05)。与PC9细胞株蛋白的表达量相比较,PC9/GR细胞株ERCC1、RRM1和TS的蛋白表达量明显增高,TUBB3蛋白的表达量明显降低,差异均有统计学意义(P<0.05)。结论:肺腺癌细胞株发生EGFR-T790M突变后对化疗药物敏感性发生变化,对顺铂、吉西他滨和培美曲塞的敏感性降低,对长春瑞滨、紫杉醇和多西他赛的敏感性增高;其化疗药物敏感性发生变化的原因可能与肺腺癌细胞株发生EGFR-T790M突变后ERCC1 m RNA、RRM1 m RNA、TS m RNA及其蛋白表达量发生变化相关。

Background and purpose：Chemotherapy is an alternative treatment option, which could still get a therapeutic effect, when the EGFR-TKI treatment of non-small cell lung cancer failed. Studies have shown that RR, TYMS, ERCC1 and TUBB3 have respectively relationship with chemosensitivity of gemcitabine, pemetrexed, platinum-based drugs and microtubule-based chemotherapy drugs.The expression levels of these molecular markers can predict the sensitivity of these chemotherapy drugs. The patients with RRMI, TS, ERCC1 and TUBB3 higher expression have reduced chemosensitivity, and lower expression have increased sensitivity. The purpose of this study was to explore the sensitivity of tumor cell lines with acquired resistance to geiftinib caused byEGFR-T790M mutation to cisplatin, gemcitabine, pemetrexed, vinorelbine, paclitaxel and docetaxel.Methods：MTT assay was used to detect the IC50 values of cisplatin, gemcitabine, vinorelbine, paclitaxel and docetaxel, pemetrexed to PC9 and PC9/GR cells, and to explore the chemosensitivity of lung adenocarcinoma cells to these chemotherapy drugs; Luminex method was used respectively to detect the expression levels of ERCC1 mRNA, TUBB3 mRNA, TS mRNA, and RRM1 mRNA in PC9 and PC9/GR cells. Western blot was used to detect the protein expression levels of ERCC1, TUBB3, TS and RRM1 in PC9 and PC9/GR cells.Results： The IC50 values of cisplatin, gemcitabine and pemetrexed to PC9/GR cells were signiifcantly higher than those to PC9 cells （P〈0.05）, while the IC50 values of vinorelbine, paclitaxe and docetaxel to PC9/GR cells were signiifcantly decreased （P〈0.05）. Luminex method showed the expressions of ERCC1 mRNA, TS mRNA and RRM1 mRNA in PC9/GR cells were signiifcantly increased than those in PC9 cells （P〈0.05）, while the expression of TUBB3 mRNA was signiifcantly decreased （P〈0.05）. Western blot method showed the expressions of TUBB3, TS and RRM1 protein in PC9/GR cells were signiifcantly increased than those in PC9 cells （P〈0.05）, while TUBB3 protein expression in PC9/GR cells was signiifcantly decreased （P〈0.05）. Western blot method analysis result showed that the expressions of TUBB3, TS and RRM1 protein in PC9/GR cells were significantly increased than those in PC9 cells （P〈0.05）, while TUBB3 protein expression in PC9/GR cells was signiifcantly decreased （P〈0.05）. Conclusion：The chemosensitivity of lung adenocarcinoma with EGFR-T790M mutation is changed. It has decreased sensitivity to cisplatin, gemcitabine, pemetrexed and increased sensitivity to vinorelbine, paclitaxel and docetaxel. The reason of the change of chemosensitivity of geiftinib-resistant lung adenocarcinoma cell maybe related to the changes of ERCC1 mRNA, RRM1 mRNA and TS mRNA and their protein expressions.