Wang J, Wang W, Guo Y, Jing S W, Shang K, Miao M C, Wang J, Wu Y J, Liu L N, Yu J M
Department of Radiation Oncology, Tianjin Medical University Cancer Institute & Hospital, Tianjin 300070, China (Present Unit: Department of Radiation Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China); Department of Radiation Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China.
Department of Radiation Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China.
Zhonghua Zhong Liu Za Zhi. 2016 Oct 23;38(10):732-738. doi: 10.3760/cma.j.issn.0253-3766.2016.10.004.
To investigate the effects of nimotuzumab on radiosensitivity of ECA-109 and TE-13 esophageal carcinoma cell lines and explore its possible mechanism. The ECA-109 and TE-13 cells were divided into control group, irradiation group, medicine group, and combined group (irradiation + medicine). In the combined group, ECA-109 and TE-13 cells were treated with nimotuzumab for 24 h before irradiation, and the cells were collected 2 h after irradiation. The radiosensitizing effects of nimotuzumab on ECA-109 and TE-13 cells were evaluated by clone formation assay. Cell apoptosis was detected by flow cytometry. Western blotting was used to evaluate the expression of EGFR, p-EGFR, DNA-PKcs, p-DNA-PKcs and γH2AX. The values of D (quasithreshold dose), D(mean lethal dose)and SF (surviving fraction at 2 Gy) of ECA-109 and TE-13 cells in the combined group were significantly lower than those of the radiation group (for ECA-109 cells, 1.11 vs. 1.72, 1.40 vs. 2.14, 0.42 vs. 0.66, respectively; for TE-13 cells, 0.41 vs. 0.46, 0.43 vs. 0.65, 0.40 vs. 0.71, respectively (all <0.05). The sensitivity enhancement ratio (SER) of ECA-109 and TE-13 cells were 1.35 and 1.43, respectively. Flow cytometry showed that the apoptosis rate of ECA-109 and TE-13 cells in the combined group were significantly higher than those of the radiation group [for ECA-109 cells, (41.31±1.52)% vs. (9.54±0.52)%; for TE-13 cells, (46.28±0.28)% vs. (11.32±0.31)%, both <0.01]. Western blotting showed that the expression levels of EGFR and DNA-PKcs were not significantly different in all groups (all >0.05). Compared with those of the control group, p-EGFR and p-DNA-PKcs of the radiation group were significantly higher in both cell lines (<0.05), and the γH2AX levels in the radiation group and medicine group were significantly higher than that of the control group (<0.05). Compared with those of the radiation group and medicine group, p-EGFR and p-DNA-PKcs protein expression in the combined group were decreased significantly (<0.05), while γH2AX protein expression was significantly increased (<0.05). Nimotuzumab can enhance the radiosensitivity of esophageal cancer ECA-109 and TE-13 cells. The potential mechanism may be related to the inhibition of EGFR phosphorylation and down-regulation of DNA damage repair proteins. The radiosensitizing effect of nimotuzumab is greater on poorly differentiated esophageal cancer cells.
探讨尼妥珠单抗对人食管癌ECA - 109和TE - 13细胞系放射敏感性的影响并探究其可能机制。将ECA - 109和TE - 13细胞分为对照组、照射组、药物组和联合组(照射 + 药物)。联合组中,ECA - 109和TE - 13细胞在照射前用尼妥珠单抗处理24小时,照射后2小时收集细胞。通过克隆形成试验评估尼妥珠单抗对ECA - 109和TE - 13细胞的放射增敏作用。采用流式细胞术检测细胞凋亡情况。运用蛋白质免疫印迹法评估表皮生长因子受体(EGFR)、磷酸化表皮生长因子受体(p - EGFR)、DNA - 依赖蛋白激酶催化亚基(DNA - PKcs)、磷酸化DNA - 依赖蛋白激酶催化亚基(p - DNA - PKcs)及γH2AX的表达。联合组中ECA - 109和TE - 13细胞的D(准阈剂量)、D(平均致死剂量)和SF(2 Gy时的存活分数)值均显著低于照射组(ECA - 109细胞分别为1.11 vs. 1.72、1.40 vs. 2.14、0.42 vs. 0.66;TE - 13细胞分别为0.41 vs. 0.46、0.43 vs. 0.65、0.40 vs. 0.71,均<0.05)。ECA - 109和TE - 13细胞的增敏比(SER)分别为1.35和1.43。流式细胞术显示,联合组中ECA - 109和TE - 13细胞的凋亡率显著高于照射组[ECA - 109细胞为(41.31±1.52)% vs. (9.54±0.52)%;TE - 13细胞为(46.28±0.28)% vs. (11.32±0.31)%,均<0.01]。蛋白质免疫印迹法显示,各组EGFR和DNA - PKcs的表达水平差异均无统计学意义(均>0.05)。与对照组相比,照射组两种细胞系中p - EGFR和p - DNA - PKcs均显著升高(<0.05),照射组和药物组的γH2AX水平均显著高于对照组(<0.05)。与照射组和药物组相比,联合组中p - EGFR和p - DNA - PKcs蛋白表达显著降低(<0.05),而γH2AX蛋白表达显著升高(<0.05)。尼妥珠单抗可增强人食管癌ECA - 109和TE - 13细胞的放射敏感性。其潜在机制可能与抑制EGFR磷酸化及下调DNA损伤修复蛋白有关。尼妥珠单抗对低分化食管癌细胞的放射增敏作用更强。