尼妥珠单抗对肺癌A549细胞系的体外放射增敏效应

Nimotuzumab enhances the radiosensitivity of lung cancer A549 cells in vitro

目的研究尼妥珠单抗对肺腺癌A549细胞系的放射增敏作用及潜在的分子机制。方法体外培养人肺腺癌A549细胞系,克隆形成实验测定尼妥珠单抗和辐射联合作用于A549细胞系的细胞存活率,并根据数学模型拟合细胞存活曲线,求出放射增敏比(SER)。其次,采用流式细胞术检测尼妥珠单抗联合2 Gy、8 GyX线照射后的细胞周期变化。最后,Western blot检测尼妥珠单抗与放射联合作用后细胞EGFR磷酸化水平的差异,再检测A549细胞接受辐射后p27、AKT及其磷酸化形式在不同时间点的表达情况,与单纯放射组比较表达的差异。结果克隆形成实验显示尼妥珠单抗显著增加了A549细胞的放射敏感性,放射增敏比(SER)为1.36。尼妥珠单抗使细胞周期阻滞于G_1/S期,而辐射后细胞周期阻滞于G_2/M期,二者联合作用后S期比例较单纯放射组细胞显著减少。尼妥珠单抗与放射联合作用后,辐射诱导的EGFR磷酸化被抑制,AKT在放射后各时间点Thr308位点磷酸化也受到显著抑制,p27蛋白在放射后各时间点表达水平均显著下降,而尼妥珠单抗作用后p27表达明显增加,再行照射后仍有较高表达。结论尼妥珠单抗对体外培养的肺腺癌细胞系具有放射增敏作用,其机制在于通过对EGFR功能的抑制,抑制了其下游信号通路中AKT的激活,而p27的表达增加,可能与细胞周期分布发生改变相关。

Objective To investigate the potential molecular mechanism of nimotuzumab in its capacity of enhancing lung cancer cell radiosensitivity in vitro.Methods Lung cancer A549 cells were pretreated with or without nimotuzumab for 24 h before exposure to radiation.Clonogenic survival assay was performed and the single- hit muti- target model was applied to evaluate the radiosensitivity related parameters.Then cell cycle distribution by flow ctyometry after different doses of radiation were analyzed.EGFR activation was examined,the levels of AKT with its phosphorylaed form and that of p27 in A549 cells at different time point were detected by Western blot.Results Pretreatment with nimotuzumab reduced clonogenic survival after radiation and the sensitivity enhancing ratio was 1.36.The combination of nimotuzumab and radiation treatment led to a significant S phase reduction.EGFR activation was greatly inhibited after pretreating with nimotuzumab,despite it could be activated by radiation.The levels of AKT did not alter between nimotuzumab combined with radiation group and radiation group,but the radiation- induced phosphorylation of AKT were greatly inhibited in the group pretreated with nimotuzumab.On the contrary,the suppressed p27 expression by radiation was substantially elevated after pretreatment with nimotuzumab.Conclusion Our results revealed that the possible mechanism of nimotuzumab enhancing the lung cancer cell radiosensitivity was that nimotuzumab inhibited the radiation- induced activation of EGFR,which directly inhibited the activation of its downstreaming AKT.On the other hand,nimotuzumab increased the expression of p27,which is associated with the redistribution of cell cycle.