Liu Jia-Tao, Li Wen-Cheng, Gao Shuang, Wang Fang, Li Xiao-Qiu, Yu Han-Qing, Fan Lu-Lu, Wei Wei, Wang Hua, Sun Guo-Ping
Department of Oncology, First Affiliated Hospital of Anhui Medical University, Hefei, China.
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, China.
Clin Lung Cancer. 2015 Sep;16(5):e55-66. doi: 10.1016/j.cllc.2015.03.006. Epub 2015 Apr 2.
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.
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.
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.
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.
四项大型临床试验表明，对于未筛选的晚期非小细胞肺癌（NSCLC）患者，同时给予表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI），如吉非替尼或厄洛替尼，与化疗药物联合使用并不能提高总生存期（OS）。在本研究中，探讨了自噬在吉非替尼和顺铂联合作用于EGFR-TKI敏感的人肺癌细胞系中的作用。此外，还研究了同时进行自噬抑制治疗是否会增加抗肿瘤活性。
将PC9细胞单独或联合暴露于吉非替尼或顺铂。使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐法测定细胞活力。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。使用蛋白质免疫印迹法检测自噬和凋亡信号通路的变化。
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，如CDI分析和膜联蛋白V-异硫氰酸荧光素/碘化丙啶（PI）检测所示，添加自噬抑制剂氯喹（CQ）可克服这种拮抗作用。此外，给予吉非替尼导致细胞G1期阻滞，这可能是吉非替尼和顺铂之间拮抗活性的原因之一。然而，添加CQ并未解除细胞周期阻滞，表明可能涉及其他机制。膜联蛋白V-异硫氰酸荧光素/PI检测显示，添加CQ显著增加了凋亡细胞的比例。此外，免疫印迹检测显示Bax增加而Bcl-2减少，表明CQ抑制自噬可增加细胞凋亡，从而克服拮抗作用。
吉非替尼与顺铂联合对EGFR-TKI敏感细胞产生拮抗作用。然而，抑制自噬可产生协同效应，这表明吉非替尼和顺铂与自噬抑制剂（尤其是CQ）联合使用可能是克服EGFR-TKI与化疗药物之间拮抗作用的有益策略。
