Background Hepatocyte growth factor(HGF) can induce non-small cell lung cancer(NSCLC) cells exerting resistance to epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)in vitro,suggesting that HGF induced c-Met phosphorylation may be associated with the mechanism of erlotinib resistance in sensitive NSCLC.Objective The study aimed to demonstrate that c-Met inhibitor SU11274 can reverse the erlotinib resistance induced by HGF in sensitive NSCLC cells in nude mice.Methods This study was conducted from March 2014 to January 2015.Two cell lines including H292 and human embryonic fibroblast MRC-5 were chosen.The HGF produced by MRC-5 cells in cell culture supernatants was quantitated by enzyme-linked immunosorbent assay(ELISA).The proliferation of H292 cells was measured by using MTT assay and the expressions of c-Met and its downstream signaling proteins were examined by Western blotting.We selected forty-nine 6-week old nude mice and equally divided them into 7 groups,group C (control group),group H,group HS,group HD,group E,group HE,group HES,treated with H292 cell suspension,HGF,HGF+SU11274,HGF+1% DMSO,erlotinib,HGF+erlotinib,HGF+erlotinib+SU11274,respectively.The tumor growth was observed and the growth curve was drawn.The tumor weight was recorded.The expressions of c-Met and downstream signaling proteins in tumor tissues were determined via immunohistochemistry.Results ELISA assay found that the HGF level in the culture supernatant of MRC-5 cells was (1 262±90) pg/ml.MTT assay showed that in the mixture of H292 and MRC-5 cells culture supernatant with erlotinib concentration of IC50,the proliferation of H292 cells was 97.07%.The expressions of p-Met,p-STAT3,p-AKT and p-ERK1/2 in the culture supernatant of H292 with MRC-5 cells were higher than those in the H292 cell culture supernatant(P<0.05).Intervention method and duration exerted significant interaction effects on the tumor volume(P<0.001).Intervention method produced main effect on the tumor volume(P<0.001).Intervention duration had main effect on the tumor volume(P<0.001).The tumor volume was smaller in HES group than in HE group measured on the 7th,11th,14th,18th,21st and 25th days of intervention (P<0.05).The tumor mass differed significantly between the groups(F=39.120,P<0.001).HES group had less tumor mass than HE group did(P<0.05).Significant differences in the expressions of c-Met,p-Met,p-STAT3,p-AKT and p-ERK1/2 were found between the groups(P<0.05).The expressions of c-Met,p-Met,and p-STAT3 proteins were lower in HES group than those in HE group(P<0.05).Conclusion HGF secreted by MRC-5 induces erlotinib resistance in H292 cells in nude mice.c-Met inhibitor SU11274 reverses the resistance of HGF-induced sensitive NSCLC cells to erlotinib in vivo.The mechanism may be related to the inhibition of HGF-induced activation of c-Met and its signaling pathway proteins.%背景 肝细胞生长因子(HGF)能够在体外诱导敏感非小细胞肺癌(NSCLC)细胞形成对表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)耐药,敏感NSCLC对厄洛替尼的耐药机制可能与HGF刺激c-Met磷酸化有关.目的 旨在研究c-Met抑制剂SU11274在逆转HGF诱导的裸鼠体内敏感NSCLC对厄洛替尼耐药作用及其机制.方法 于2014年3月-2015年1月选择NSCLC细胞株H292及人胚肺成纤维细胞(MRC-5),通过酶联免疫吸附(ELISA)法检测MRC-5培养上清液中HGF水平,MTT法检测细胞增殖情况,应用Western blotting法检测细胞中c-Met及其下游通道蛋白的变化.建立H292细胞的裸鼠移植瘤模型,设对照组(C组)、HGF处理组(H组)、HGF+SU11274处理组(HS组)、HGF+1%二甲基亚砜(DMSO)处理组(HD组)、厄洛替尼处理组(E组)、HGF+厄洛替尼处理组(HE组)、HGF+厄洛替尼+SU11274处理组(HES组),观察肿瘤生长情况,测量肿瘤体积,称取肿瘤质量.免疫组织化学法检测裸鼠移植瘤组织中c-Met及其下游通道蛋白的变化.结果 ELISA法检测结果显示,MRC-5培养上清液中HGF水平为(1 262±90)pg/ml.MTT法检测结果显示,H292与MRC-5培养上清液混合培养后,H292在厄洛替尼IC50时的增殖率为97.07%.H292 MRC-5阳性细胞p-Met、p-STAT3、p-AKT、p-ERK1/2表达水平高于MRC-5阴性细胞(P<0.05).处理方法与时间对肿瘤体积存在交互作用(P<0.001);处理方法对肿瘤体积主效应显著(P<0.001);时间对肿瘤体积主效应显著(P<0.001).其中HES组第7、11、14、18、21、25天肿瘤体积小于HE组(P<0.05).各组肿瘤质量比较,差异有统计学意义(F=39.120,P<0.001);其中HES组肿瘤质量小于HE组(P<0.05).各组c-Met、p-Met、p-STAT3、p-AKT、p-ERK1/2表达水平比较,差异均有统计学意义(P<0.05);其中HES组c-Met、p-Met、p-STAT3表达水平低于HE组(P<0.05).结论 MRC-5分泌的HGF能够在裸鼠体内诱导敏感NSCLC细胞株H292对厄洛替尼产生耐药,c-Met抑制剂SU11274可逆转HGF诱导敏感NSCLC细胞株H292对厄洛替尼耐药,其机制可能与抑制了HGF活化的c-Met及其下游通道蛋白相关.
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