ABCG2在奥希替尼耐药的非小细胞肺癌中的作用及机制研究

The role and mechanism of ABCG2 in osimertinib-resistant non-small cell lung cancer

目的:探索ABCG2在非小细胞肺癌细胞对奥希替尼耐药中的作用机制及逆转其耐药的方法。方法:选取A549、NCI-H1299、NCI-H1975细胞并诱导其对奥希替尼产生耐药;通过慢病毒转染使A549、NCI-H1299产生T790M基因突变,并诱导其对奥希替尼耐药;通过CCK-8法检测细胞对奥希替尼的IC_(50)值;通过高效液相色谱法检测细胞内奥希替尼浓度;Western-blot检测细胞内EGFR、p-EGFR、ABCG2、AKT、p-AKT、PI3K、GSK、p-ERK和ERK等蛋白表达;基因测序检测18、19、20和21号外显子是否发生突变;通过质粒转染调控肺癌细胞中ABCG2的表达并检测细胞对奥希替尼的敏感性变化。结果:通过CCK-8法检测,验证耐药系数大于10,证明耐药细胞构建成功。奥希替尼耐药细胞中ABCG2表达增加,且细胞内奥希替尼浓度降低。使用过表达质粒上调了肺癌细胞中ABCG2表达后,发现肺癌细胞对奥希替尼的敏感性明显降低;相反,下调耐药细胞中ABCG2表达后细胞对奥希替尼的敏感性恢复。抑制MAPK通路及PI3K/AKT通路能明显抑制ABCG2的表达。结论:ABCG2表达增高是导致非小细胞肺癌对奥希替尼耐药的重要原因之一;ABCG2的表达与MAPK及PI3K/AKT信号通路的激活相关。

Objective:To explore the role of ABCG2 in NSCLC(non-small cell lung cancer) cells of drug resistance and the methods to reverse drug resistance.Methods:We chosed A549,NCI-H1299,NCI-1975 cells and induced them resistant to osimertinib, transfected lentiviral into A549,NCI-H1299 to make them have the T790M gene mutation, and induced them resistant to osimertinib.The IC_(50) value of osimertinib was detected by CCK-8 method.Intracellular osimertinib concentration detected by high performance liquid chromatography.Western-blot detection of intracellular EGFR,p-EGFR,ABCG2,AKT,p-AKT,PI3K,GSK,p-ERK and ERK protein expression.Gene sequencing detected mutations in exons of 18,19,20 and 21.The expression of ABCG2 in lung cancer cells was regulated by plasmid transfection and the sensitivity of cells to osimertinib was detected.Results:It was verified that the resistance coefficient was greater than 10 by CCK-8 method, it was proved that the construction of resistant cells was successful.The expression of ABCG2(ATP binding cassette subfamily G member 2) in osimertinib-resistant cells was increased and intracellular osimertinib concentration was decreased.It was found that the sensitivity of lung cancer cells to osimertinib decreased significantly after upregulating ABCG2 expression in lung cancer cells.Instead, the sensitivity to osimertinib was recovered after down-regulating the expression of ABCG2 in drug-resistant cells.The inhibition of both MAPK pathway and PI3K/AKT pathway can significantly inhibit the expression of ABCG2.Conclusion:The increase of ABCG2 expression is one of the important reasons for the resistance of non-small cell lung cancer to osimertinib.ABCG2 expression is related to MAPK and PI3K/AKT signaling pathway.