c-Met抑制剂AMG-102增强喉鳞癌细胞放射敏感性的机制  

Effect of c-Met inhibitor AMG-102 on radiosensitivity in laryngeal squamous carcinoma cells

作　　者：曹峰[1] 吕欣 董凯峰[3] 范才 张建军 陈坤 马博敬 侯春立 张翠红 Cao Feng;Lyu Xin;Dong Kaifeng;Fan Cai;Zhang Jianjun;Chen Kun;Ma Bojing;Hou Chunli;Zhang Cuihong(Department of Radiation Oncology,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,China;Department of Medical Services,the Central Hospital of Baixiang County,Xingtai 055450,China;Department of Otolaryngology,the First Hospital of Hebei Medical University,Shijiazhuang 050031,China;Department of Radiation Oncology,the 980th Hospital of the Joint Logistic Support Unit of the People′s Liberation Army of China,Shijiazhuang 050082,China;Department of Otolaryngology Head and Neck Surgery,the 980th Hospital of the Joint Logistic Support Unit of the People′s Liberation Army of China,Shijiazhuang 050082,China;Department of Oncology,the 980th Hospital of the Joint Logistic Support Unit of the People′s Liberation Army of China,Shijiazhuang 050082,China)

机构地区：[1]河北医科大学第四医院放疗科,石家庄050011 [2]邢台市柏乡县中心医院医务科,055450 [3]河北医科大学第一医院耳鼻喉科,石家庄050031 [4]解放军联勤保障部队第九八〇医院放疗科,石家庄050082 [5]解放军联勤保障部队第九八〇医院耳鼻喉头颈外科,石家庄050082 [6]解放军联勤保障部队第九八〇医院肿瘤科,石家庄050082

出　　处：《中华肿瘤杂志》2019年第12期909-917,共9页Chinese Journal of Oncology

基　　金：河北省卫生厅青年科技课题(20160201、20150382)。

摘　　要：目的探讨c-Met抑制剂AMG-102对喉鳞癌细胞的增殖抑制和放射增敏作用。方法采用四甲基偶氮唑蓝(MTT)法检测AMG-102对喉鳞癌细胞株Hep-2和KBV200的增殖抑制作用。采用克隆形成实验分析AMG-102对Hep-2和KBV200细胞的放射增敏作用。采用流式细胞术检测Hep-2和KBV200细胞的凋亡情况。采用Western blot法检测细胞中c-Met/p-Met、凋亡蛋白cleaved caspase 3及下游信号通路蛋白Akt/p-Akt和Erk/p-Erk的表达。利用RNA干扰技术下调细胞中c-Met表达,转染c-Met过表达质粒使细胞中c-Met过表达,分析Hep-2和KBV200细胞对AMG-102的敏感性变化。结果与KBV200细胞比较,Hep-2细胞对AMG-102更加敏感,半数抑制浓度(IC50)分别为14和9μmol/L。Hep-2细胞中c-Met和p-Met蛋白的相对表达量分别为194.48±0.57和177.76±1.53,均明显高于KBV200细胞(分别为171.24±1.00和115.37±0.56,均P<0.001)。AMG-102作用于肝细胞生长因子(HGF)处理后的KBV200细胞,可使其p-Met蛋白的相对表达水平明显降低(P<0.001)。与二甲基亚砜(DMSO)组比较,AMG-102联合照射可明显增加Hep-2细胞的放射敏感性(SER=1.28,P<0.001);而AMG-102对KBV200细胞的放射敏感性影响很小(SER=1.18,P=0.002)。与4 Gy照射组和5μmol/L AMG-102处理组比较,4 Gy照射+5μmol/L AMG-102处理组Hep-2细胞的凋亡率明显增加,cleaved caspase-3蛋白的表达水平明显升高。而各处理组KBV200细胞的凋亡率和cleaved caspase-3蛋白的表达水平无明显变化。与DMSO处理组比较,4 Gy照射组、5μmol/L AMG-102处理组和4 Gy照射+5μmol/L AMG-102处理组Hep-2细胞中p-Met、p-Akt和p-Erk蛋白的表达水平下降,其中4 Gy照射+5μmol/L AMG-102处理组中p-Met、p-Akt和p-Erk蛋白的表达水平比4 Gy照射组和5μmol/L AMG-102处理组更低。但在KBV200细胞中,各处理组的p-Met、p-Akt和p-Erk蛋白表达水平均未明显降低。照射前及照射后30 min、1 h、4 h、8 h、24 h Hep-2细胞中p-Met蛋白的相对表达水平分别为99.89±0.61�Objective To investigate the effect of c-Met inhibitor AMG-102 on proliferation and radiosensitivity in laryngeal squamous carcinoma cells.Methods The effects of AMG-102 on proliferation and radiosensitivity of laryngeal squamous carcinoma cell lines Hep-2 and KBV200 were detected by 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide(MTT)assay and colony formation assay,respectively.The apoptosis of Hep-2 and KBV200 cells was detected by flow cytometry.The expression levels of c-Met,phospho-Met(p-Met),cleaved caspase-3 and Akt/p-Akt,Erk/p-Erk were detected by Western blot.Specific small interfering RNA targeting c-Met or plasmid of c-Met were transfected into Hep-2 and KBV200 cells to investigate the cell sensitivity to AMG-102.Results Compared with KBV200 cells,Hep-2 cells were more sensitive to AMG-102 with IC50 of 14 and 9μmol/L,respectively.The relative expression levels of c-Met and p-Met proteins in Hep-2 cells were 194.48±0.57 and 177.76±1.53,respectively,which were significantly higher than those in KBV200 cells(171.24±1.00 and 115.37±0.56,respectively,P<0.001 for both).Exogenous hepatocyte growth factor(HGF)was added to increase the expression level of p-Met protein in KBV200 cells.The results showed that AMG-102 significantly reduced the expression of p-Met in KBV200 cells treated with HGF(P<0.001).Compared with the dimethyl sulfoxide(DMSO)group,AMG-102 treatment combined with radiotherapy significantly increased the radiosensitivity of Hep-2 cells(SER=1.28,P<0.001).However,AMG-102 had little effect on the radiosensitivity of KBV200 cells(SER=1.18,P=0.002).Compared with the 4 Gy radiotherapy alone group and the 5μmol/L of AMG-102 alone treatment group,the apoptosis rate of Hep-2 cells in the combined treatment group was significantly increased.Meanwhile,the expression level of cleaved caspase-3 protein was also markedly increased.However,there were no significant changes in the apoptotic rate and cleaved caspase-3 expression in each treatment group of KBV200 cells.Compared with DMSO t

关 键 词：喉鳞状细胞癌 C-MET AMG-102 增殖 凋亡 放射增敏 

分 类 号：R73[医药卫生—肿瘤]