辐照联合表皮生长因子受体酪氨酸激酶抑制剂对中心体调节激酶(Aurora-A)高表达的非小细胞肺癌细胞系增殖的影响

张英; 张乐天; 李素君; 王竞

doi:10.3969/j.issn.2095-5227.2022.04.020

辐照联合表皮生长因子受体酪氨酸激酶抑制剂对中心体调节激酶(Aurora-A)高表达的非小细胞肺癌细胞系增殖的影响

Radiation combined epidermal growth factor receptor tyrosine kinase inhibitor inhibits the proliferation of non-small cell lung cancer cell line with high expression of Aurora-A

摘要

摘要:
背景 Aurora-A丝/苏氨酸激酶作为维持染色体稳定的中心体调节关键激酶，在许多肿瘤中表达，并与预后不良和放射抗拒相关。放疗联合表皮生长因子受体(epidermal growth factor receptor，EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitor，TKI)在Aurora-A高表达的非小细胞肺癌(non-small cell lung cancer，NSCLC)中的作用尚不明确。
目的探讨辐照联合EGFR-TKI对Aurora-A高表达的NSCLC细胞系的抑制作用。
方法在A549和GLC82细胞系中筛选Aurora-A相对高表达的NSCLC细胞系，从吉非替尼(Gefitinib)和埃克替尼(Icotinib)中选择针对EGFR作用更明显的靶向药物。比较辐照联合靶向药物作用后Aurora-A及EGFR下游信号通路相关蛋白(Akt和Stat1)表达、细胞周期分布与辐照剂量之间的关系；通过流式细胞仪检测Aurora-A siRNA干预前后辐照联合靶向药物对细胞凋亡的影响；通过细胞克隆形成实验，观察辐照联合靶向药物对细胞凋亡率的影响。
结果筛选发现GLC82细胞系是Aurora-A相对高表达的NSCLC细胞系，Icotinib对Aurora-A高表达GLC82细胞系的EGFR及其磷酸化蛋白水平抑制作用更明显。在GLC82细胞系中，与单纯Icotinib相比，Icotinib联合辐照2 Gy组的p-EGFR/EGFR、p-Aurora-A/Aurora-A、p-Akt/Akt和p-Stat1/Stat1蛋白表达水平均无统计学差异(P＞0.05)；Icotinib联合辐照10 Gy组，p-Aurora-A/Aurora-A和p-Akt/Akt表达明显下降(P＜0.05)。流式细胞术检测显示，Icotinib联合辐照后GLC82细胞周期分布呈线性改变，细胞周期阻滞在G₂/M期。常规辐照(2 Gy)未显著增加GLC82细胞凋亡(P＞0.05)，Aurora-A siRNA干预后明显增加Icotinib组、辐照(2 Gy，10 Gy)组和Icotinib联合辐照10 Gy组的敏感度，促进GLC82细胞凋亡(P＜0.05)。细胞克隆实验表明，与单纯辐照相比，辐照联合Icotinib可以明显促进GLC82细胞凋亡。
结论辐照联合Icotinib可以逆转Aurora-A引起的放射抗拒，促进GLC82细胞凋亡。

Abstract:
Background Aurora-A plays a key role in regulating centrosome kinase that maintains chromosomal stability, and its abnormal expression is observed in many tumors and associated with poor prognosis and resistant to radiation. However, it remains unclear whether radiation combined with epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) works in non-small cell lung cancer (NSCLC) cell lines with high Aurora-A expression.
Objective To investigate the effectiveness of radiation in combination with EGFR-TKI in NSCLC cell lines with relatively high expression of Aurora-A.
Methods NSCLC cell lines with relatively high expression of Aurora-A were screened in A549 and GLC82 cell lines, and targeted drugs with more obvious effect on EGFR were selected from Gefitinib and Icotinib. The relationship between the expression of Aurora-A and EGFR downstream signal pathway related proteins (Akt and Stat1), cell cycle distribution and irradiation dose after irradiation plus targeted drugs was compared. The effect of irradiation combined with targeted drugs on cell apoptosis before and after Aurora-A siRNA intervention was detected by flow cytometry, and its effect on the rate of apoptosis was observed by cell clone formation assay.
Results GLC82 cell line was a NSCLC cell line with relatively high expression of Aurora-A, and Icotinib had a more obvious inhibitory effect on the levels of EGFR and its phosphorylated protein in GlC82 cell line with high expression of Aurora-A. No significant alterations in expression of p-EGFR/EGFR, p-Aurora-A/Aurora-A, p-Akt/Akt and p-Stat1/Stat1 were observed in GLC82 cells with combination of Icotinib and radiation of 2 Gy compared to Icotinib alone. Icotinib combined with radiation of 10 Gy resulted in a significant decrease in expression of p-Aurora-A/Aurora-A and p-Akt/Akt (P＜0.05). Flow cytometry showed a linear change in GLC82 cell cycle distribution after Icotinib combined with radiation, and the tumor cells were significantly blocked in the G₂/M phase. The conventional radiation of 2 Gy did not significantly increase the apoptosis of GLC82 cells (P＞0.05), however, the GLC82 cell apoptosis were significantly promoted by increasing the sensitivity of Icotinib, radiation (2, 10 Gy) and Icotinib combined with radiation of 10 Gy after Aurora-A siRNA intervention (P ＜ 0.05). The cell clone formation assay showed that radiation combined with Icotinib significantly promoted GLC82 cell apoptosis compared with radiation alone.
Conclusion Radiation combined with Icotinib can reverse the Aurora-A-induced resistance to radiation and promote apoptosis of GLC82 cells.

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