塞来昔布联合培美曲塞对人肺腺癌A549细胞株增殖、凋亡及PI3K/AKT信号通路 相关蛋白的影响

Effect of Celecoxib combined with Pemetrexed on proliferation, apoptosis and PI3K/AKT signaling pathway-related proteins in human lung adenocarcinoma A549 cells

摘要:

目的 探讨塞来昔布（Cele）联合培美曲塞（Pem）对人肺腺癌A549细胞株增殖、凋亡的影响及其可能的机制。方法 分别采用不同浓度的Cele和Pem作用于A549细胞，48?h条件下，CCK-8法检测光密度（OD）值，计算半抑制浓度（IC50）作为后续实验的药物浓度；实验分组：对照组（加入培养液）和实验组（分别加入IC50浓度的Cele、IC50浓度的Pem、IC50浓度的Cele+Pem）；Cele、Pem单独用药或者联合用药在不同时间条件下（24、36和48?h）作用于A549细胞，CCK-8法检测细胞增殖，择最佳作用时间作为后续实验的干预时间，金氏法评估两药之间的相互关系，流氏细胞仪检测细胞凋亡及周期分布，Western blotting检测AKT、p-AKT蛋白表达情况。结果 与对照组比较，Cele和Pem组呈浓度依赖性抑制A549细胞的增殖，但低浓度Cele组（5?μmol/L） 与对照组比较，差异无统计学意义（P?>0.05）。Cele IC50值为30.51?μmol/L，Pem IC50值为1.33?μmol/L；与对照组比较，Cele和Pem组呈时间依赖性抑制A549细胞增殖，且Cele+Pem组效果最佳。最佳干预时间为48?h，Cele与Pem联合抑制A549细胞增殖；Cele+Pem组与对照组、Cele组及Pem组比较，Cele+Pem组细胞凋亡率和G0/G1期细胞比例升高，p-AKT/GAPDH值下调（P?<0.05），而AKT/GAPDH值各组间差异无统计学意义（P?>0.05）。结论 Cele与Pem联合可抑制A549细胞增殖，诱导细胞凋亡，其机制可能与诱导细胞G0/G1期阻滞及抑制PI3K/AKT信号通路相关蛋白AKT活性相关。

Abstract:

Objective To investigate the therapeutic effect of Celecoxib (Cele) and Pemetrexed (Pem) on human lung adenocarcinoma A549 cell line and possible mechanism. Methods A549 cells were treated with different concentrations of Celecoxib) or Pemetrexed for 48 h. Half concentration (IC50) was determined. A549 cells were treated with Celecoxib or Pemetrexed for 24?h, 36?h and 48?h to determine optimal concentration to inhibit the growth of cancer cells. Further cancer cells were treated with Celecoxib or Pemetrexed under optimal concentration and time of duration. Cell proliferation were identified by CCK-8 assay. Cell apoptosis and cycle distribution were determined by Flow Cytometry. Western blot was performed to detect the expression of AKT and p-AKT protein. Kim's method was applied for determination of crosstalk between the two drugs. Results Compared with the control group, proliferation of A549 cells was inhibited by Celecoxib or Pemetrexed in a concentration-dependent manner (P?