体外和体内研究伊可替尼与培美曲塞联合应用对人肺癌细胞系的协同细胞毒性作用与序列依赖性。

Sequence-dependent synergistic cytotoxicity of icotinib and pemetrexed in human lung cancer cell lines in vitro and in vivo.

机构信息

Department of Oncology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 519000, China.

State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China.

出版信息

J Exp Clin Cancer Res. 2019 Apr 5;38(1):148. doi: 10.1186/s13046-019-1133-z.

BACKGROUND

Recent Clinical trials of administration of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in combination with standard first-line chemotherapy have failed to improve survival in patients with advanced NSCLC, However, the sequential treatment with EGFR-TKIs and chemotherapy is expected to improve survival of NSCLC. The aim of this study is to test the antiproliferative effect of pemetrexed combined with icotinib in different sequences on non-small cell lung cancer (NSCLC) cell lines to determine the optimal combination schedule, and subsequently elaborated the potential mechanisms.

METHODS

Six human lung cancer cell lines with wild-type or mutant EGFR gene were exposed to pemetrexed and icotinib combined in different sequences. Cell proliferation was examined by cell counting kit-8 (CCK-8) and colony formation assay; cell cycle and apoptosis were evaluated by flow cytometry; cell migration and invasion were measured by wound healing and transwell invasion assays respectively; protein expression was by detected by Western blot.

RESULTS

The growth inhibition effect of pemetrexed combined with icotinib on NSCLC cells were schedule-dependent in vitro and in vivo. Treatment with pemetrexed followed by icotinib (P-I) had significantly stronger anticancer ability than treatment with icotinib followed by pemetrexed (I-P) and concomitant treatment with pemetrexed and icotinib (P + I). Cell cycle analysis revealed that pemetrexed blocked cells in S phase, whereas icotinib arrested cells in G1 phase. We also found that icotinib markedly enhanced the pro-apoptotic activity of pemetrexed via cytochrome-C/Caspase/Bcl-2 signaling pathway. In addition, our results showed that pemetrexed alone increased the levels of p-EGFR, p-AKT and p-MAPK, which were inhibited by icotinib. Finally, we showed that the washout period of icotinib was no less than 96 h.

CONCLUSIONS

Sequential treatment of NSCLC cells with pemetrexed followed by icotinib had powerful antiproliferative effect, and it could become a novel effective combination therapy for NSCLC patients.

背景

最近表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）联合标准一线化疗治疗晚期 NSCLC 患者的临床试验未能提高生存率，但 EGFR-TKIs 序贯化疗有望改善 NSCLC 患者的生存率。本研究旨在测试培美曲塞联合厄洛替尼在不同序列对非小细胞肺癌（NSCLC）细胞系的增殖抑制作用，以确定最佳联合方案，并随后阐述潜在的机制。

方法

将 6 株具有野生型或突变型 EGFR 基因的人肺癌细胞系分别用培美曲塞和厄洛替尼联合不同序列处理。用细胞计数试剂盒-8（CCK-8）和集落形成实验检测细胞增殖；用流式细胞术检测细胞周期和凋亡；用划痕愈合和 Transwell 侵袭实验分别检测细胞迁移和侵袭；用 Western blot 检测蛋白表达。

结果

培美曲塞联合厄洛替尼对 NSCLC 细胞的抑制作用在体外和体内均具有时间依赖性。培美曲塞序贯厄洛替尼（P-I）组的抗癌能力明显强于厄洛替尼序贯培美曲塞（I-P）组和培美曲塞与厄洛替尼同时给药（P+I）组。细胞周期分析显示，培美曲塞将细胞阻滞在 S 期，而厄洛替尼将细胞阻滞在 G1 期。我们还发现，厄洛替尼通过细胞色素 C/Caspase/Bcl-2 信号通路显著增强了培美曲塞的促凋亡活性。此外，我们的结果表明，培美曲塞单独增加了 p-EGFR、p-AKT 和 p-MAPK 的水平，而厄洛替尼抑制了这些水平。最后，我们发现厄洛替尼的冲洗期不少于 96 小时。