Sequence-dependent Effects and Mechanism of Pemetrexed and Gefitinib on Human Lung Adenocarcinoma Cells
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摘要: 目的 探讨培美曲塞与吉非替尼不同时序应用对肺腺癌细胞A549和PC-9生长及凋亡的影响， 并阐述其可能机制。方法 MTT法检测各组细胞的增殖抑制情况，流式细胞仪检测各组细胞凋亡及细胞周期分布，Western印迹法检测对EGFR下游信号通路及TS酶蛋白水平表达的影响。结果 培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼对PC-9和A549细胞增殖抑制率及凋亡率较单药组均提高（P<0.05），培美曲塞可以提高EGFR、AKT 、ERK磷酸化水平，而吉非替尼表现为抑制作用， 同时吉非替尼降低TS酶表达。培美曲塞序贯吉非替尼，培美曲塞同步联合吉非替尼抑制EGFR、AKT 、ERK磷酸化水平较单药更强。吉非替尼主要将PC-9、A549细胞阻滞在G0/G1期；培美曲塞主要将细胞阻滞在S期。培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼较其他组G2/M期细胞比例提高（P<0.05）。结论 培美曲赛序贯吉非替尼、培美曲赛同步联合吉非替尼在PC-9、A549细胞中均起到协同增效作用，且培美曲赛序贯吉非替尼协同作用更为显著，可能主要与培美曲赛诱导EGFR、AKT 、ERK磷酸化及吉非替尼降低TS酶作用有关。Abstract: Objective To investigate the anti-proliferative and apoptotic effect of different sequences of combined pemetrexed and gefitinib on human lung adenocarcinoma cells A549 and PC-9, and to probe the possible mechanisms. Methods MTT assay was used to measure cell proliferation. Flow cytometry using annexin V-FITC/PI staining was employed to measure cell apoptosis and cell cycle. The expression of phosphorylation of EGFR, AKT, ERK and TS protein were detected by Western blot. Results Sequential pemetrexed followed by gefitinib or cocurrent pemetrexed and gefitinib remarkably enhanced the antiproliferation effects (P<0.05). Sequential pemetrexed followed by gefitinib or cocurrent pemetrexed and gefitinib enhanced the apoptosis effect on PC-9 and A549 cells. Pemetrexed induced the phosphorylation of EGFR, AKT and ERK, while gefitinib significantly suppressed the phosphorylation of EGFR,AKT,ERK and TS expression. Gefitinib mainly blocked the cells in G0/G1 phase and pemetrexed blocked the cells in S phase. The percentage of cells in G2/M phase was increased in sequential pememtrexed followed by gefitinib or cocurrent pemetexed and gefitinib group (P<0.05). Sequential pemetrexed followed by gefitinib or concurrent pemetrexed and gefitinib had a more significant effect on decreasing the expression of p-EGFR，p-AKT and p-ERK. Conclusion Both concurrent pemetrexed and gefitinib or sequential pemetrexed followed by gefitinib have synergistic effects on the expression of p-EGFR,p-AKT and p-ERK, moreover sequential pemetrexed followed by gefitinib has more synergistic effects than concurrent pemetrexed and gefitinib. The phosphorylation of EGFR,AKT and ERK induced by pemetrexed and TS expression suppressed by gefitinib may contribute to the synergistic effects.
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Key words:
- Lung adenocarcinoma /
- Pemetrexed /
- Gefitinib /
- Cell cycle /
- Cell apoptosis
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