ABT-737逆转EGFR T790M突变肺腺癌细胞EGFR-TKIs耐药的机制研究

Mechanism of ABT-737 reversing EGFR-TKIs resistance in EGFR T790M mutant lung adenocarcinoma cells

目的通过细胞和在体研究探讨ABT-737联合吉非替尼对EGFR T790M突变肺腺癌细胞EGFR-TKIs耐药的逆转机制。方法利用MTT和FCM法检测ABT-737联合吉非替尼对RPC-9细胞的增殖和凋亡的影响,以RT-PCR检测细胞内Bim、Bak、Caspase-3 mRNA表达水平。以皮下异位移植法建立EGFR T790M突变肺腺癌裸鼠模型,对各组瘤体组织进行组织病理学检查、基因测序法检测、Real-time PCR和免疫组织化学法分析Bim、Bak、Caspase-3 mRNA及蛋白表达水平。结果 ABT-737联合吉非替尼对RPC-9细胞有生长抑制作用和凋亡作用,且在ABT-737浓度4μmol/L范围内呈浓度依赖性,最大抑制率为(54.113±2.986)%,最大凋亡率为(55.042±3.151)%,差异均有统计学意义(P<0.05);Bim、Bak及Caspase-3 mRNA表达水平随着ABT-737浓度的升高而增加(P<0.05)。各组瘤体组织病理均为腺癌;ABT-737联合吉非替尼灌胃组裸鼠瘤体体积较其他组小(P<0.05),Bim、Bak、Caspase-3 mRNA和蛋白表达水平较其他组显著增高(P<0.05)。结论 ABT-737能增强吉非替尼促细胞凋亡作用,使耐药细胞重新发生凋亡。

Objective To investigate the reversal mechanism of ABT-737 combined with gefitinib in reversing EGFRTKIs resistance of EGFR T790M mutant lung adenocarcinoma cells by cell and in vivo studies. Methods The effect of ABT-737 combined with gefitinib on proliferation and apoptosis of RPC-9 cells were detected by MTT and FCM. The expression levels of Bim, Bak and Caspase-3 mRNA were detected by RT-PCR. The nude mouse model of EGFR T790M mutant lung adenocarcinoma was established by subcutaneous ectopic transplantation. The expression levels of Bim, Bak and Caspase-3 mRNA and protein were analyzed by histopathological examination, gene sequencing detection, Real-time PCR and immunohistochemical analysis. Results ABT-737 combined with gefitinib had growth inhibi tion and apoptosis effects on RPC-9 cells, and was concentration-dependent in the range of 4 μmol/L of ABT-737. The maximum inhibition rate was(54.113±2.986)%. The maximum apoptotic rate was(55.042±3.151)%, and the difference was statistically significant(P<0.05). The expression levels of Bim, Bak and Caspase-3 mRNA increased with the in crease of ABT-737 concentration(P<0.05). The histopathology of each group was adenocarcinoma. The volume of tumor in nude mice of ABT-737 combined with gefitinib intragastrical administration group was smaller than that of other groups(P<0.05), and the expression levels of Bim, Bak and Caspase-3 mRNA and protein in this group were significantly higher than those of the other groups(P<0.05). Conclusion ABT-737 can enhance the apoptosis of gefitinib and make the resistant cells re-apoptosis.