自噬在表皮生长因子受体酪氨酸激酶抑制剂耐药中的作用

Role of autophagy in EGFR-TKI resistance of non-small cell lung cancer cells

目的探讨自噬在表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinases inhibitor,EGFR-TKI)耐药中的作用。方法浓度递增筛选法诱导非小细胞肺癌PC9细胞株对erlotinib耐药,Western blot检测自噬相关分子LC3B、P62、Beclin1的表达,观察自噬抑制剂氯喹(chloroquine,CQ)PC9及其耐药细胞株敏感性的影响,Western blot检测P-ERKl/2、P-AKT蛋白的表达情况。结果成功诱导对erlotinib耐药的细胞株PER,其IC50为(9.6±2.1)μmol/L,是亲本PC-9细胞的192倍,且自噬相关分子LC3B、P62、Beclin1的表达明显增高。CQ可显著提高erlotinib对PC9和PER细胞的敏感性。P-ERKl/2、P-AKT在耐药PER细胞株中持续活化,联合给予自噬抑制剂CQ则可部分恢复erlotinib对P-ERKl/2、P-AKT信号的抑制作用。结论自噬参与了EGFR-TKI耐药的发生,联合自噬抑制剂和EGFR-TKI有望成为克服耐药的新策略。

Objective Investigate the role of autophagy in the epidermal growth factor receptor tyrosine kinase inhibitor （EGFR-TKI） resistance in non-small cell lung cancer （NSCLC）. Methods Stepwise erlotinib selection was used to establish the EGFR-TKI-resistant cell line PER from human NSCLC cell line PC9. Autophagy marker LC3B, P62, and Beclin1 were detected by Western blotting. The influence of autophagy inhibitor chloroquine （CQ） on the drug sensitivity of the PER and PC9 cells was tested. Cell viability was measured by MTT assay and the expression of P-ERKl/2 and P-AKT were measured by Western blotting. Results The resistant cell line PER had IC50 value of 9.6±2.1 μmol/L to erlotinib, 192 times higher than that of the PC9 cells （0.05±0.02 μmol/L）. The expression of LC3B, P62 and Beclin1 in the PER cells was obviously higher than in the PC9 cells. Cytotoxicity induced by erlotinib was greatly enhanced after autophagy inhibition with CQ. The persist activation of P-ERKl/2 and P-AKT in the PER cells could be inhibited by treatment with CQ. Conclusion Autophagy may be involved in EGFR-TKI resistance of NSCLC. Autophagy inhibition thus represents a novel strategy to potentiate erlotinib efficacy in NSCLC treatment.