西妥昔单抗联合照射对结直肠癌CL187细胞的抑制作用及机制探讨

Inhibitory effect of combination of cetuximab and irradiation on colorectal carcinoma CL187 cells

目的研究西妥昔单抗（C225）联合外照射对结直肠癌CL187细胞生物学效应的影响，并探讨相关分子机制。方法结直肠癌CL187细胞分单纯照射组和C225处理的联合照射组，受6MVX射线照射0、4和8Gy后24和48h，用四甲基偶氮唑盐（MTT）比色法测吸光度（A）值，比较两组细胞死亡率的差异。利用克隆形成实验比较两组细胞增殖能力的差异。采用流式细胞仪，检测细胞周期和细胞凋亡。蛋白免疫印迹法分析两组细胞DNA—PKcs、Ku70和Ku80蛋白表达量的变化。结果联合照射组较单纯照射组死亡细胞比例增加（t=-6．14、-6．53，P〈0．05），细胞克隆形成能力下降。C225增强了射线对细胞的杀伤作用，放射增敏比SER为1．38。联合照射组G0／G1期细胞阻滞增加（t=-4．64，P〈0．05），细胞凋亡比例增加（t=-9．16，P〈0．05），DNA修复相关蛋白DNA—PKcs、Ku70和Ku80蛋白表达量减少。结论西妥昔单抗增强照射对结直肠癌CL187细胞的杀伤作用，可能是通过影响细胞周期、细胞凋亡和DNA损伤修复基因实现的。

Objective To investigate the combination effect of cetuximab and irradiation on colorectal carcinoma CL187 cell line and underlying molecular mechanism. Methods CL187 cells with or without cetuximab treatment were irradiated by 0, 4 and 8 Gy X-rays, then cell death percentage was determined by MTT 24 and 48 h post-irradiation. Clone forming assay was used to evaluate the cell reproliferation ability. Cell cycle distribution, apoptosis, and necrosis were analyzed by flow eytometry. Western blot was used to detect the protein expressions of DNA-PKcs, Ku70 and KuS0. Results The cetuximab enhanced the percentage of radiation-induced cell death, while descreased the cloning formation capacity and increased radiosenvtivity （t = - 6. 14, - 6.53, P 〈 0.05）. The SER of cetuximab on CL187 cell line approached to 1.38. In addition, cetuximab also increased radiation-induced G0/G1 phase arrest （t= -4.64, P〈0.05） and the percentage ofapoptosis and necrosis （t = -9.16, P〈0.05）, but it descreased the expression levels of DNA-PKcs, KuT0 and KuS0 proteins. Conclusions The cetuximab treatment might enhance the inhibitory effect of irradiation on colorectal carcinoma CL187 cell line by influencing cell cycle distribution, cell apoptosis, and the expression of DNA repair proteins.