吉非替尼与培美曲塞对人肺腺癌细胞SPC-A1生长及细胞周期的作用

Effects of gefitinib and pemetrexed on the proliferation and cell cycle of human lung adenocarcinoma cell SPC-A1

目的:探索培美曲塞与表皮生长因子酪氨酸激酶抑制剂(EGFR-TKIs)吉非替尼联合应用对人肺腺癌细胞SPC-A1生长的影响,探索二者序贯用药是否比单药更有效,并从细胞周期角度分析其可能的细胞学机制。方法:RT-PCR法检测人肺腺癌细胞SPC-A1中EGFR mRNA表达,Western blot法检测SPC-A1细胞中EGFR蛋白表达,四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide,MTT]显色分光光度法检测细胞增殖,流式细胞术检测细胞周期。分别观察培美曲塞与吉非替尼单独、同时及间隔24小时序贯作用。结果:SPC-A1细胞中有EGFR mRNA和蛋白的表达,且其表达量为过表达。吉非替尼和培美曲塞分别在0.1pmol/L-1μmol/L和10-9-10-1g/L浓度范围内明显抑制SPC-A1细胞生长,呈浓度依赖性,IC50分别为:61.2nmol/L(吉非替尼)和16.2μg/ml(培美曲塞),二者在IC50下抑制作用呈时间依赖性,96h时达最大抑制。二者联合作用于细胞时对生长的影响与二者加药的次序有关,跟单药组相比,先用培美曲塞后用吉非替尼组对抑制细胞生长有明显增强作用(P<0.05),同时给药或先用吉非替尼后用培美曲塞组对抑制细胞生长无显著增强作用(P>0.05)。细胞周期研究显示:吉非替尼和培美曲塞作用于不同的细胞周期,分别将SPC-A1细胞阻滞于G1期(G0/G1)和S期,先用吉非替尼后用培美曲塞组G1(G0/G1)期细胞显著增多,G2期(G2/M)细胞显著减少(P<0.05)。同时用药组G1期(G0/G1)和S期细胞比例无显著变化,G2期(G2/M)细胞显著增多(P<0.05)。先用培美曲塞后用吉非替尼组G1期(G0/G1)细胞显著减少,S期细胞比例无显著变化,G2期(G2/M)细胞显著增多(P<0.05)。结论:吉非替尼和培美曲塞都能抑制SPC-A1细胞生长,二者联合作用对细胞生长的影响与给药次序有关,先用吉非替尼后用培美曲塞对抑制细胞生长无显著增强作用,而先用培美曲塞后用EGFR-TKIs则表现有明显的协同性,且这种关系可能与它们对细胞周期的影响相关。

Objective：To assess the sequential administration of pemetrexed and epidermal growth factor receptor tyrosine kinase inhibitors （EGFR -TKIs） geiftinib on the cell proliferation of lung adenocarcinoma cell SPC -A1 and to explore its mechanism by observing their effects on the cell cycle. Methods：The expression of EGFR mRNA and EGFR protein were examined by RT - PCR and Western blot respectively. MTF was used to measure the cell prolifer- ation of SPC - A1 ceils. Cell cycle was detected by flow cytometry. The cells were treated with the pemetrexed alone, gefitinib alone,or a combination of two given medicines together or sequentially 24 hours apart. Results：There were expressions of EGFR mRNA and EGFR protein in SPC - A1 cells, and both of them were overexpressed. Gefitinib and pemetrexed inhibited the cell proliferation of SPC - A1 cells in a dose - dependent manner from 0. Ipmol/L to lp, mol/L and 10-9g/L to 10 -lg/L respectively in vitro. The ICso was 61.2nmol/L for gefitinib and 16.21xg/ml for pemetrexed. These inhibitory effects were also time - dependent. Both maximal inhibitory effects were reached at 96 hours. The effects of pemetrexed in combination with gefitinib on cell proliferation depended on the sequence. Sequen- tial administration of gefitinib following pemetrexed enhanced the effect of pemetrexed on the cell proliferation. Other- wise, no significant additive effects on cell proliferation had been found when they were used simultaneously or ge- fitinib was added before pemetrexed. Cell cycle studies showed that pemetrexed and gefitinib induced S phase and Go/ G1 phase arrest respectively. The Go/G1 phase arrest was observed when pemetrexed and gefitinib was used simultane- ously or gefitinib was added before pemetrexed. In contrast, sequential administration of gefitinib following pemetrexed induced S phase arrest. Conclusion：Both pemetrexed and gefitinib inhibited the proliferation of SPC - A1 cells. The additive effects on cell proliferation were sequential - dependent. The concomitant and the sequential treatment of ge- fitinib followed by pemetrexed exerted no significant additive effects on cell proliferation and resulted in accumulation of cells in G0/G1 phase, which may decrease the effectiveness of pemetrexed in subsequent cycles. The additive effects on the cell proliferation were observed when gefitinib was sequentially administrated following pemetrexed, which re- suited in the G2/M phase arrest. Both of the above relationship could be partly explained by their effect on cell cycle.