Wang Meng, Zhang Lianmin, Zhao Xiaoliang, Liu Jun, Chen Yulong, Wang Changli
Department of Lung Cancer, Cancer Institute & Hospital, Tianjin Medical University, Key Laboratory of Cancer Prevention and Therapy Tianjin, Tianjin 300060, China.
Department of Lung Cancer, Cancer Institute & Hospital, Tianjin Medical University, Key Laboratory of Cancer Prevention and Therapy Tianjin, Tianjin 300060, China. Email:
Zhonghua Zhong Liu Za Zhi. 2014 Sep;36(9):651-6.
OBJECTIVE: The aim of this study was to investigate the effects of combination of icotinib and cetuximab on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC, and provide experimental evidence for rational treatment of NSCLC. METHODS: The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4, 5-dimethylthiazol-2-yl)- 5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. The expression of molecular markers of tumor proliferation PCNA and Ki-67 protein was further examined by immunohistochemistry, and the expression of EGFR-signaling-related proteins in tissue sections taken from H1975 tumor xenografts was assessed by Western blot assay. Sensitivity to EGFR inhibitors was detected in human H1975 tumor xenograft in nude mice. RESULTS: The in vitro experiment showed that the proliferative ability of H1975 cells was inhibited in a dose-dependent manner, along with the increasing doses of cetuximab and icotinib, and the combination of cetuximab with icotinib resulted in a more pronounced growth inhibition of the H1975 cells. The apoptosis rate of H1975 cells after treatment with 0.5 µmol/L icotinib and 1 µg/ml cetuximab was (22.03 ± 2.41)% and that after treatment with 5 µmol/L icotinib and 10 µg/ml cetuximab was (42.75 ± 2.49)%, both were significantly higher than that after treatment with the same dose of icotinib or cetuximab alone (P < 0.05). The nude mouse experiment showed that the transplanted tumor was growing to (614.5 ± 10.8) mm(3) in the blank control group and to (611.2 ± 8.7) mm(3) at 28 days after icotinib treatment, but (30.8 ± 2.0) mm(3) in the cetuximab treatment group and 0 mm(3) in the cetuximab combined with icotinib group. There was a significantly decreased expression of Ki-67 and PCNA proteins and down-regulation of phosphorylation of EGFR signaling-related proteins in the cetuximab combined with icotinib group. CONCLUSIONS: The combination of icotinib with cetuximab can exert synergistic inhibitory effect on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC H1975 cells, interrupts the EGFR-downstream signaling pathway, and enhances the anticancer activity of chemotherapeutic drugs. Our results provide further experimental evidence for the clinical studies of combination of icotinib with cetuximab in the treatment of NSCLC patients associated with secondary drug resistance caused by T790M mutation of EGFR.
目的：本研究旨在探讨埃克替尼与西妥昔单抗联合应用对非小细胞肺癌（NSCLC）中表皮生长因子受体（EGFR）T790M突变所致获得性耐药的影响，为NSCLC的合理治疗提供实验依据。 方法：采用3-(4,5-二甲基噻唑-2)-5-二苯基四氮唑溴盐（MTT）法、膜联蛋白V染色法及蛋白质免疫印迹法评估这两种药物对细胞增殖、凋亡及EGFR相关信号传导的影响。通过免疫组化进一步检测肿瘤增殖分子标志物增殖细胞核抗原（PCNA）和Ki-67蛋白的表达，并采用蛋白质免疫印迹法评估取自H1975肿瘤异种移植瘤组织切片中EGFR信号相关蛋白的表达。在裸鼠人H1975肿瘤异种移植瘤中检测对EGFR抑制剂的敏感性。 结果：体外实验表明，随着西妥昔单抗和埃克替尼剂量的增加，H1975细胞的增殖能力呈剂量依赖性抑制，且西妥昔单抗与埃克替尼联合应用对H1975细胞的生长抑制作用更明显。0.5 μmol/L埃克替尼与1 μg/ml西妥昔单抗处理后H1975细胞的凋亡率为(22.03±2.41)%，5 μmol/L埃克替尼与10 μg/ml西妥昔单抗处理后凋亡率为(42.75±2.49)%，两者均显著高于相同剂量埃克替尼或西妥昔单抗单独处理后的凋亡率（P<0.05）。裸鼠实验显示，空白对照组移植瘤在28天时生长至(614.5±10.8)mm³，埃克替尼处理组为(611.2±8.7)mm³，而西妥昔单抗处理组为(30.8±2.0)mm³，西妥昔单抗联合埃克替尼组为0 mm³。西妥昔单抗联合埃克替尼组中Ki-67和PCNA蛋白表达显著降低，EGFR信号相关蛋白磷酸化水平下调。 结论：埃克替尼与西妥昔单抗联合应用可对NSCLC H1975细胞中EGFR T790M突变所致获得性耐药发挥协同抑制作用，阻断EGFR下游信号通路，增强化疗药物的抗癌活性。我们的结果为埃克替尼与西妥昔单抗联合治疗EGFR T790M突变所致继发性耐药的NSCLC患者的临床研究提供了进一步的实验依据。
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