西妥昔单抗对人食管癌EC9706细胞株的放射增敏作用及其机制

Radiation sensitizing effect of cetuximab in human esophageal carcinoma EC9706 cells

目的探讨西妥昔单抗（Cetuximab）对人食管癌EC9706细胞株的放射增敏作用及其机制。方法EC9706细胞培养于DMEM／F12培养液中，实验分为对照组、药物组、放疗组、联合组（Cetuximab＋放疗）。药物干预细胞24h后，采用四甲基偶氮唑盐（MTT）比色法测定细胞抑制率；采用流式细胞术测定细胞周期分布的变化；细胞免疫组织化学方法从形态上观察各组表皮生长因子受体（EGFR）、磷脂酰肌醇-3-激酶催化亚单位α（PIK3CA）蛋白的表达，采用Westernblot检测EGFR、PIK3CA蛋白的表达。结果Cetuximab抑制EC9706细胞增殖，呈浓度依赖性，当2μg／L浓度Cetuximab作用24h，其抑制率为65．25％；流式细胞技术显示药物组G0／G1期细胞比例为（45．41±2．42）％，与对照组差异有统计学意义（P〈0．05），放疗组和联合组G2／M期细胞比例增加为（28．36±2．68）％、（49．22±1．74）％，与对照组比较差异有统计学意义（P〈0．05），药物组、放疗组和联合组S期比例下降为（39．20±1．45）％、（31．08±2．56）％、（14．33±2．15）％，与对照组比较差异有统计学意义（P〈0．05）；细胞免疫化学法和Westernblot检测EC9706细胞EGFR和PIK3CA蛋白表达结果显示药物组放疗组均表达下降（P〈0．05），联合组下降更为显著（P〈0．01）。结论Cetuximab对人食管癌EC9706细胞株具有放射增敏作用，可能通过下调EGFR和PIK3CA表达、G2／M期阻滞来增加EC9706细胞的放射敏感性。

Objective To evaluate the effect and mechanism of cetuximab for radiosensitization of human esophageal carcinoma EC9706 cell line. Methods Four groups were set up： combined group （ cetuximab ＋ radiotherapy）, drug group, radiotherapy group, control group. The distribution of cell cycle of the treated human esophageal carcinoma EC9706 cells was measured by flow cytometry （FCM）. The ex- pression of epidermal growth factor receptor （EGFR） and phosphatidylinositol 3 - kinase catalytic alpha （PIK3CA） protein of EC9706 cells in different groups was detected by immunocytochemistry and Western blotting. Results Cetuximab could suppress the growth of EC9706 cells in a dose - dependent response, and the inhibitive rate was 65.25% by 2 μg/L cetuximab for 24 h. As compared with the control group, the proportion of G0/G1 cells was （45. 41 ± 2.42 ） % in the drug group （ P 〈 0. 05 ）, and that of GJM cells in the radiotherapy group and the combined group was （ 28.36 ± 2. 68 ） % , and （ 49.22 ± 1.74 ） % , respectively （ P 〈 0. 05 ）. The proportion of S cells in the drug group, the radiotherapy group and the com- bined group was （39.20±1.45）%, （31.08 ±2.56）%, and （14.33 ±2.15）%, respectively （P〈 0.05 ）. The expression of EGFR and PIK3CA protein was significantly decreased in human esophageal car- cinoma EC9706 ceils by immunocytochemistry and Western blotting, most obviously in the the combined group （P 〈0. 05 ）. Conclusion Cetuximab plays a role in the radiation sensitivity in human esophageal carcinoma EC9706 cells probably by downregulating the expression of EGFR and PIK3CA protein, and in-ducing G2/M ceils arrest.