盐酸埃克替尼和细胞因子诱导的杀伤细胞对肺腺癌细胞的体外杀伤作用研究

Killing effect of icotinib combined with CIK on human lung adenocarcinoma cells in vitro

目的探讨盐酸埃克替尼（icotinib）和细胞因子诱导的杀伤细胞（CIK）对人肺腺癌细胞的体外抑制作用。方法采用CCK－8法检测icotinib和CIK对HCC827和A549细胞的抑制作用，应用Annexin V－FITC/PI双染法检测肿瘤细胞凋亡，流式细胞仪分析icotinib作用CIK表型的变化。结果1.5、3、6、12 μmol/L icotinib对HCC827细胞的抑制率分别为（5.64±0.05）%、（8.62±0.45）%、（14.57±0.65）%和（18.52±0.91）%，对A549细胞的抑制率分别为（1.64±0.48）%、（2.09±0.28）%、（3.69±0.45）%和（4.41±0.58）%。在相同浓度下，icotinib对HCC827细胞的抑制率明显高于A549细胞（P〈0.05）。效靶比为10∶1、20∶1、40∶1时，CIK对HCC827细胞的抑制率分别为（15.17±2.33）%、（42.59±7.18）%和（62.59±8.95）%，对A549细胞的抑制率分别为（16.99±2.81）%、（46.31±1.89）%和（58.24±4.23）%，相同效靶比的CIK对HCC827和A549细胞的抑制率差异无统计学意义（P10∶1＝0.299，P20∶1＝0.318，P40∶1＝0.366）。6 μmol/L icotinib联合CIK后，在10∶1、20∶1、40∶1效靶比下，对HCC827细胞的抑制率分别为（37.07±3.50）%、（76.03±6.55）%和（80.34±10.69）%，对A549细胞的抑制率分别为（25.72±1.41）%、（52.76±3.82）%和（62.26±1.94）%，除6 μmol/L icotinib＋40∶1 CIK组与40∶1 CIK组对A549细胞的抑制率差异无统计学意义外（P＝0.089），其他各联合组对肿瘤细胞的抑制率明显高于icotinib组和相同效靶比下CIK组（P〈0.05），且联合组CI值均〈1。联合组的HCC827和A549细胞凋亡率明显高于icotinib组和空白对照组（P〈0.05），晚期凋亡或坏死细胞比例显著上升，随着CIK效靶比的升高，其抑制作用更强（P〈0.05）。icotinib作用前后的CIK表型表达率差异无统计学意义（P〉0.05）。结论icotinib对表皮生长因子受体（EGFR）突变型肺腺癌细胞更敏感，而EGFR突变状态对CIK细胞的杀伤作用无影响。icotinib联合CIK对肿瘤细胞的抑制作用更强，两者具有协同作用，且icotinib对CIK细胞的表型无影响。

ObjectiveTo explore the inhibitory effect of icotinib combined with cytokine induced killer （CIK） on various human lung adenocarcinoma cell lines in vitro.MethodsThe inhibitory effect of icotinib alone or icotinib combined with CIK on HCC827 and A549 cells was detected by cell counting kit-8（CCK-8）. The apoptosis was detected by flow cytometry via Annexin V/PI staining. The effect of icotinib on CIK phenotype was detected by flow cytometry.ResultsThe inhibitory rates of HCC827 cells treated with 1.5, 3, 6, 12 μmol/L icotinib were （5.64±0.05）%, （8.62±0.45）%, （14.57±0.65）% and （18.52±0.91）%, respectively. The inhibitory rates of A549 cells were （1.64±0.48）%, （2.09±0.28）%, （3.69±0.45）%, （4.41±0.58）%, respectively. At the same concentration, the inhibitory rate of HCC827 cells with icotinib treatment was significantly higher than that of A549 cells （P〈0.05）. When the effector/target ratio was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells co-cultured with CIK were （15.17±2.33）%, （42.59±7.18）%, （62.59±8.95）%, respectively, and the inhibitory rates of A549 were（16.99±2.81）%, （46.31±1.89）%, （58.24±4.23）%, respectively. The inhibitory rate of HCC827 cells co-cultured with CIK was not significantly different from that of A549 cells at the same effector/target ratio （P10∶1=0.299, P20∶1=0.318, P40∶1=0.366）. When the effector/target ratio of CIK combined with 6 μmol/L icotinib was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells were （37.07±3.50）%, （76.03±6.55）%, （80.34±10.69）%, respectively, and the inhibitory rates of A549 cells were（25.72±1.41）%, （52.76±3.82）%, （62.26±1.94）%, respectively. The inhibitory rates of 6 μmol/L icotinib combined with CIK were significantly higher than those of icotinib group and CIK group alone at the same effector/target ratio （P〈0.05）, except for the effector/target ratio at 40︰1 on A549 cells （P=0.089）. Moreover, all of the combination index （CI） of combined group were 〈1 （P〈0.05）. The apoptotic rates of HCC827 and A549 cells induced by icotinib combined with CIK were significantly higher than those of icotinib group and blank control group （P〈0.05）, especially the proportion of late apoptotic or necrotic cells.Increasing effector/target ratio of CIK contributed to stronger inhibition（P〈0.05）. The expressional rate of CIK phenotype with or without icotinib treatment was not significantly different from each other（P〉0.05）.ConclusionsEGFR mutant lung adenocarcinoma cells are more sensitive to icotinib, while the EGFR mutation status has no effect on the killing effect of CIK cells. icotinib combined with CIK has a synergistic effect on the inhibition of tumor growth, and icotinib has no any impact on the phenotype of CIK cells.