舒尼替尼联合多西他赛对A549细胞增殖、凋亡、细胞周期及c-met、mek、erk mRNA表达的影响

摘要

目的:探讨舒尼替尼单药以及联合多西他赛不同顺序给药对A549细胞增殖、凋亡、细胞周期及c-met、mek、erk mRNA表达的影响.方法:取对数生长期的A549细胞进行以下实验.实验Ⅰ:对照组加入RPMI 1640培养基;多西他赛单药组给予多西他赛(终质量浓度16 mg/L,下同);舒尼替尼单药组给予舒尼替尼(终浓度7.5 μmol/L,下同);舒尼替尼+多西他赛组,加入同时含两药的培养液.实验Ⅱ:多西他赛→舒尼替尼组(D→S组)先加入100 μL含多西他赛的培养液培养24 h,洗脱,再加入100 μL含舒尼替尼的培养液继续培养24 h;舒尼替尼→多西他赛组(S→D组),同上反向处理.采用MTT法检测以上各组细胞增殖抑制率,流式细胞术检测细胞周期分布及细胞凋亡,RT-PCR检测A549细胞c-met、mek、erk mRNA的表达水平.结果:Ⅰ:联合用药组较单独用药组细胞增殖抑制率、晚期凋亡率和总凋亡率升高,S期细胞减少,erk表达上调(P＜0.05).Ⅱ:D→S组较S→D组细胞增殖抑制率和凋亡率升高,G0/G1期细胞减少,S期、G2/M期细胞增多,c-met、mek mRNA表达下调,erk mRNA表达上调(P＜0.05).结论:舒尼替尼和多西他赛均能抑制A549细胞的生长,联合用药优于单独用药;多西他赛后序贯舒尼替尼能产生明显的协同效应,效果优于舒尼替尼后序贯多西他赛.%Aim:To investigate the effects of sunitinib combined docetaxel under different administration schedules on the proliferation,apoptosis and cell cycle of A549 cells as well as the expressions of c-met, mek, and erk mRNA in A549 cells.Methods:Ⅰ:Control group was added RPMI 1640 medium only, docetaxel only (final concentration of 16 mg/L, the same below) , sunitinib only (final concentration of 7.5 μmol/L, the same below) , and the sunitinib + docetaxel group was added RPMI 1640 medium containing 2 drugs at the same time.Ⅱ:Docetaxel sequential sunitinib group(D→S) incubated with 100 μL RPMI 1640 medium containing docetaxel for 24 h, followed by replacing the docetaxel with 100 μL RPMI 1640 medium containing sunitinib for another 24 h.Sunitinib sequential docetaxel group (S→D) , reversed the above process.MTT assay was used to measure proliferation inhibition of each group.Flow cytometry was used to analyze the alteration of cell apoptosis and cycle in A549 cells.RT-PCR was employed to measure the expressions of c-met, mek, and erk mRNA in the cell line.Results: I:Com-pared with single drug treatment, in combination treatment group, cell proliferation inhibition rate, late apoptosis rate, and total apoptosis rate increased; S phase cells decreased; the mRNA expression level of erk increased (P <0.05).Ⅱ:Compared with S→ D group, in D→S group, cell proliferation inhibition rate and apoptosis rate increased; G0/G1 phase cells decreased, and S and G2/M phase cells increased; the mRNA expression levels of c-met and mek decreased, but that of erk increased(P <0.05).Conclusion: Sunitinib and docetaxel could inhibit the growth of A549 cell.Combination treatment is better than single drug treatment.The way using docetaxel before sunitinib shows obvious synergistic effect.