【摘要】 目的探讨抑制MET对于RAS突变结肠癌的体内体外抗肿瘤作用。方法选取4种常见RAS突变型结肠癌细胞(HCT-116、DLD-1、Lovo和HCT-15),采用si RNA敲除MET蛋白表达,或者加入特异性MET抑制剂PHA-665752,采用MTT及集落形成实验观察抑制MET对结肠癌细胞增殖抑制的作用,应用Western Blotting检测PHA-665752对MET/AKT/ERK信号通路的作用。另外,应用HCT-116细胞系构建裸鼠皮下成瘤模型,给予PHA-665752腹腔注射,观察MET抑制剂对体内肿瘤的抑制作用。结果 RAS突变的4种结肠癌细胞系均有MET蛋白表达。应用si RNA敲除MET蛋白表达对HCT-116和Lovo细胞的增殖抑制作用分别为19.6±4.5%和27.8±5.8%,而应用MET特异性抑制剂PHA-665752对两种细胞的增殖抑制作用呈剂量依赖性,细胞克隆形成实验证实PHA-665752可有效抑制细胞克隆形成。另外,应用HCT-116细胞构建裸鼠皮下成瘤模型,给予PHA-665752单药证实MET抑制剂可明显抑制HCT-116细胞的皮下移植瘤(第四周肿瘤体积:PHA-665752组为300±72 mm~3,对照组为608±59 mm~3,t=5.731,P=0.005)。Western Blotting证实应用PHA-665752预处理能明显抑制HGF激活的p-MET、p-AKT和p-ERK。结论抑制MET对于RAS突变型结肠癌具有抗肿瘤作用,靶向MET可能成为RAS突变型结肠癌的有效治疗。更多还原

【Abstract】 Objective To explore The effect of MET inhibition on RAS mutant colon cancer in vivo and in vitro experiments. Methods Four kinds of common RAS mutant colon cancer cells(HCT-116, DLD-1, Lovo and HCT-15) were selected in this study. The MTT assay and colony formation assay were used to testify the growth inhibitory effect of MET inhibition by si RNA and MET inhibitor PHA-665752, and Western Blotting was used to test the effect of PHA-665752 on MET/AKT/ERK signalling. HCT-116 cells were used to construct the subcutaneous tumor model in nude mice, and PHA-665752 was injected intraperitoneally to observe the inhibitory effect in vivo. Results The four kinds of RAS mutant colon cancer cells all possessed MET expression. The growth inhibitory rate of MET inhibition by si RNA in HCT-116 and Lovo cells was 19.6±4.5% and 27.8±5.8%, respectively. The inhibitory effect of MET specific inhibitor PHA-665752 in HCT-116 and Lovo cells was dose dependent, and the colony formation assay confirmed that PHA-665752 could effectively inhibit the formation of cell clones. Furthermore, tumor growth with PHA-665752 was analyzed in vivo. HCT-116 xenografts showed antitumor response when treated with PHA-665752(the tumor volume in the fourth week: 300±72 mm~3 for PHA-665752 arms and 608±59 mm~3 for the control arms, P=0.005). Western Blotting showed pretreatment with PHA-665752 significantly abrogated p-MET, p-AKT and p-ERK stimulated by HGF. Conclusions MET inhibition exhibited antitumor effect in RAS mutant colon cancer cells, suggesting targeting MET might become a potential targeted therapy.更多还原