激活表皮生长因子受体增强MKN45细胞对MET抑制剂耐受性的机制研究

摘要

目的 探讨产生MET抑制剂耐药性的分子机制.方法 MKN45细胞培养好以后,使用MET特异性抑制剂PHA-665752 (250 nmol)或多西环素(DOXY,1μg/ml)诱导的shRNA系统使MET基因沉默,计算不同处理组(未处理组、EGF组、Gefitinib组和EGF+ Gefitinib组)MKN45细胞的存活率,同时通过Western-Bolt方法检测表皮生长因子受体(EGFR)、MET、AKT和MAPK的磷酸化水平.结果 PHA-665752或多西环素(DOXY)处理后,抑制MET基因表达,导致细胞存活率严重下降(P＜ 0.001),同时EGFR、MET、AKT和MAPK的磷酸化水平亦严重下降.然而,加入EGF后,细胞存活状况得到明显改善(P＜0.001或P＜0.01),并且AKT和MAPK的磷酸化水平明显上升.结论 激活表皮生长因子受体EGFR增强MKN45细胞对MET抑制剂的耐受性可能是AKT/MAPK信号通路发挥作用的.%Objective To investigate the molecular mechanism that could cause resistance to MET inhibition.Methods The prepared MKN45 cells treated with MET inhibitor,PHA665752,or doxycycline (DOXY) inducing MET silence.Then the cell viability and the phosphorylation levels of EGFR,MET,AKT and MAPK by Western-Blotting in different treated groups,including untreated,EGF treated,Gefitinib treated and EGF + Gefitinib treated group,were evaluated.Results The prepared MKN45 cells treated with PHA or DOXY,inducing MET silence,resulted in strong impairment of cell survival (P ＜ 0.001) and the phosphorylation of MET,EGFR,AKT and MAPK.However,stimulation with EGF restored cell survival (P ＜ 0.001 orP ＜ 0.01) and activation of AKT and MAPK.Conclusions The resistance to MET inhibition induced by EGFR activation may be due to its ability to activate the AKT/MAPK signaling pathways.