Autophagy Inhibition Overcomes the Antagonistic Effect Between Gefitinib and Cisplatin in Epidermal Growth Factor Receptor Mutant Non-Small-Cell Lung Cancer Cells
Autophagy Inhibition Overcomes the Antagonistic Effect Between Gefitinib and Cisplatin in Epidermal Growth Factor Receptor Mutant Non-Small-Cell Lung Cancer Cells
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自噬抑制克服了吉非替尼和顺铂在表皮生长因子受体突变非小细胞肺癌细胞中的拮抗作用
DOI：
10.1016/j.cllc.2015.03.006
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发表时间：
2015-09-01
影响因子：
3.6
通讯作者：
Sun, Guo-Ping
中科院分区：
文献类型：
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作者：
Liu, Jia-Tao;Li, Wen-Cheng;Sun, Guo-Ping
临床试验表明，表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）与化疗药物同时给药并不能改善非小细胞肺癌患者的总生存期。自噬可能在 EGFR-TKI 和/或化疗药物的耐药性中发挥重要作用。在本研究中，我们发现吉非替尼和顺铂联合使用对EGFR-TKI敏感细胞产生拮抗作用，而先前用氯喹抑制自噬可以通过增加细胞凋亡产生协同作用。 背景：四项大型临床试验表明，同时给予表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI），例如 吉非替尼或厄洛替尼等化疗药物并不能改善未经选择的晚期非小细胞肺癌 (NSCLC) 患者的总生存期 (OS)。在本研究中，研究了吉非替尼和顺铂联用对EGFR-TKI敏感的人肺癌细胞系中自噬的作用。此外，还探讨了同时自噬抑制治疗是否会增加抗肿瘤活性。材料和方法：将 PC9 细胞单独或一起暴露于吉非替尼或顺铂。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物测定法测量细胞活力。使用药物相互作用系数（CDI）分析确定吉非替尼和顺铂之间的细胞毒性相互作用。使用流式细胞术测量细胞周期分布和凋亡。使用蛋白质印迹法测量自噬和凋亡信号通路的变化。结果：吉非替尼和顺铂联合给药对肿瘤细胞增殖产生拮抗活性。然而，CDI 分析和膜联蛋白 V-FITC/碘化丙啶 (PI) 测定表明，添加自噬抑制剂氯喹 (CQ) 克服了拮抗作用。此外，吉非替尼给药导致细胞G1期停滞，这可能导致吉非替尼和顺铂之间的拮抗活性。然而，CQ 的添加并没有解除细胞周期停滞的调节，这表明可能还涉及其他机制。 Annexin V-FITC/PI检测结果显示，CQ的添加显着增加了凋亡细胞的比例。此外，免疫印迹检测显示 Bax 增加和 Bcl-2 减少，表明 CQ 抑制自噬可以增加细胞凋亡，从而克服拮抗作用。结论：吉非替尼联合顺铂对EGFR-TKI敏感细胞产生拮抗作用。然而，抑制自噬会产生协同作用，这表明吉非替尼和顺铂与自噬抑制剂（尤其是 CQ）联合可能是克服 EGFR-TKI 与化疗药物之间拮抗作用的有益策略。 (C) 2015 Elsevier Inc. 保留所有权利。
Clinical trials have demonstrated that concurrent administration of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) with chemotherapy agents does not improve overall survival in patients with non-small-cell lung cancer. Autophagy might play an important role in the resistance to EGFR-TKIs and/or chemotherapy agents. In the present study, we found that a combination of gefitinib and cisplatin generated antagonistic effects in EGFR-TKI-sensitive cells and that previously inhibiting autophagy by chloroquine could produce synergistic effect by increasing apoptosis.Background: Four large clinical trials have shown that concurrent administration of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), such as gefitinib or erlotinib, with chemotherapy agents does not improve overall survival (OS) in unselected patients with advanced non-small-cell lung cancer (NSCLC). In the present study, the role of autophagy in the combination of gefitinib and cisplatin on EGFR-TKI-sensitive human lung cancer cell line was investigated. Moreover, whether simultaneous autophagy inhibition treatment increases the antitumor activity was explored. Materials and Methods: The PC9 cell was exposed to either gefitinib or cisplatin alone or together. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cytotoxic interaction between gefitinib and cisplatin was determined using coefficient of drug interaction (CDI) analysis. The cell cycle distribution and apoptosis were measured using flow cytometry. Alterations in the autophagy and apoptosis signaling pathway were measured using Western blot assays. Results: Coadministration of gefitinib and cisplatin resulted in antagonistic activity to tumor cell proliferation. However, the addition of chloroquine (CQ), an autophagy inhibitor, overcame the antagonistic effects, as demonstrated by CDI analysis and Annexin V-FITC/propidium iodide (PI) assays. In addition, gefitinib administration led to cell G1 phase arrest, which might have contributed to the antagonistic activity between gefitinib and cisplatin. However, the addition of CQ did not deregulate cell cycle arrest, indicating that other mechanisms might be involved. The Annexin V-FITC/PI assays showed that the addition of CQ significantly the increased the ratio of apoptosis cells. Also, immunoblotting assays exhibited increased Bax and decreased Bcl-2, suggesting that autophagy inhibition by CQ could increase cell apoptosis and thus overcome the antagonistic effects. Conclusion: The combination of gefitinib with cisplatin generates antagonistic effects on EGFR-TKI-sensitive cells. However, inhibiting autophagy produces a synergistic effect, suggesting that gefitinib and cisplatin combined with an autophagy inhibitor (especially CQ) might be a beneficial strategy to overcome the antagonistic effects between EGFR-TKIs and chemotherapeutic agents. (C) 2015 Elsevier Inc. All rights reserved.