MET抑制剂PHA665752对非小细胞肺癌细胞吉非替尼耐药的影响

Effect of MET Inhibitor PHA665752 on Gefitinib Resistance in Non-small Cell Lung Cancer Cells

目的探讨MET抑制剂PHA665752对非小细胞肺癌(NSCLC)细胞株对吉非替尼敏感和耐药的影响。方法根据实验需要将细胞分为对照组、吉非替尼组(Gef组)、PHA665752组(PHA组)、Gef+PHA组,采用对吉非替尼敏感的NSCLC细胞系Hcc827和PC9分别诱导建立吉非替尼继发耐药细胞系Hcc827-GR、PC9-GR。比较耐药株与敏感株MET表达水平变化,自噬相关蛋白LC-3、ATG5、ATG7表达的变化。比较MET抑制剂PHA665752对Hcc827-GR、PC9-GR细胞自噬相关蛋白表达的影响,并测定PHA665752联合吉非替尼对耐药逆转的效果。结果 Hcc827-GR、PC9-GR两株耐药细胞对吉非替尼的药物敏感性明显降低,MET蛋白在Hcc827和PC9耐药株较敏感株表达上调(P<0.05);自噬信号蛋白LC-3、ATG5、ATG7在Hcc827和PC9耐药株较敏感株表达上调(P<0.05);Gef组、PHA组较对照组细胞数量下降约20%,Gef+PHA组较对照组细胞数量下降约60%;联合应用MET抑制剂PHA665752,耐药细胞株对吉非替尼重新敏感,MET抑制剂PHA 665752下调自噬信号蛋白LC-3、ATG5、ATG7在Hcc827和PC9耐药株表达(P<0.05)。结论 MET在NSCLC细胞继发耐药的过程中表达上调,同时伴随着自噬通路的激活;MET能够通过抑制自噬通路逆转NSCLC细胞对吉非替尼的耐药,联合MET抑制剂可能是基于EGFR-TKI治疗NSCLC的新策略。

Objective To investigate the effect of MET inhibitor PHA665752 on the expressions of non-small cell lung cancer （NSCLC） cell lines sensitive and resistant to gefitinib. Methods The cell lines were divided into control group,gefitinib group （Gef group）,PHA665752 group （PHA group）,and Gef＋PHA group.Effects of gefitinib was analyzed on Hcc827,Hcc827-GR,PC9 and PC9-GR cell lines.MET and autophagy related molecules LC-3,ATG5 and ATG7 expression were analyzed in gefitinib-resistant cell lines compared to sensitive cell lines.Furthermore,the influence of MET inhibitor PHA 665752 on gefitinib resistance in NSCLCs and autophagy signaling were detected by CCK8 and Western blotting. Results Growth inhibition of gefitinib in Hcc827-GR and PC9-GR was significantly lower than in Hcc827 and PC9 cell lines（P〈0.05）.MET and autophagy related molecules LC-3,ATG5 and ATG7 expression were up-regulated in gefitinib-resistant cell lines compared to sensitive cell lines（P〈0.05）.Compared with the control group,the number of cells in the Gef group,PHA group,and Gef＋ PHA group was reduced by 20%,20%,and 60%,respectively.MET inhibitor PHA665752 down-regulated LC-3,ATG5 and ATG7 protein expression in gefitinib-resistant cell lines（P〈0.05）. Conclusion Elevated expression of MET reveals important functions of MET signaling and autophagy signaling in NSCLC TKIs-resistance development.MET inhibitor PHA665752 reverses gefitinib resistance in NSCLC cells via negatively regulating autophagy.