健脾消癌方对人结肠癌HCT116细胞AKT、mTOR表达的影响

Effect of Jianpi Xiaoai prescription on the expression of protein kinase B and mammalian target of rapamycin in human colon cancer HCT116 cells

目的:探讨健脾消癌方(JPXA)含药血清对人结肠癌HCT116细胞丝氨酸/苏氨酸蛋白激酶(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)表达以及对细胞增殖、细胞周期及凋亡的影响。方法:取不同浓度的JPXA含药血清作用于HCT116细胞,筛选JPXA含药血清处理HCT116细胞的半抑制浓度(IC50),并将HCT116细胞分为实验组和对照组,予以相应处理后,采用CCK-8法检测细胞增殖能力,PI-FACS法检测细胞周期变化,Annexin V-APC单染法流式细胞仪检测凋亡水平,并采用Rt-PCR检测AKT mRNA、mTOR mRNA水平,采用Western Blot检测AKT、p-AKT、mTOR及磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)水平。结果:与对照组相比,实验组HCT116细胞增殖能力显著下降,能使细胞周期停留在G1期及S期,且细胞凋亡水平上升,差异均有统计学意义(P<0.05)。与对照组相比,实验组AKT mRNA、mTOR mRNA表达水平及AKT、p-AKT、mTOR、p-mTOR蛋白含量明显下降(P<0.05或P<0.01)。结论:JPXA能抑制AKT、mTOR表达,对结肠癌细胞的增殖、克隆能力有一定抑制作用,并可在一定程度上促进结肠癌细胞的凋亡。

Objective: To investigate the effect of serum containing Jianpi Xiaoai prescription(JPXA) on the expression of serine/threonine protein kinase B(AKT) and mammalian target of rapamycin(mTOR) in human colon cancer HCT116 cells and the proliferation, cell cycle, and apoptosis of HCT116 cells.Methods: HCT116 cells were treated with different concentrations of serum containing JPXA to screen out the half-maximal inhibitory concentration of serum containing JPXA in the treatment of HCT116 cells.HCT116 cells were divided into experimental group and control group;after corresponding treatment, CCK-8 assay was used to measure cell proliferative capacity, PI-FACS was used to measure the change in cell cycle, Annexin V-APC single-staining flow cytometry was used to measure the level of cell apoptosis, RT-PCR was used to measure the mRNA expression levels of AKT and mTOR,and Western blot was used to measure the protein expression levels of AKT,phosphorylated AKT(p-AKT),mTOR,and phosphorylated mTOR(p-mTOR).Results: Compared with the control group, the experimental group had a significant reduction in the proliferative capacity of HCT116 cells, with most cells arrested in G1/S phase, and a significant increase in the level of cell apoptosis(P<0.05).Compared with the control group, the experimental group had significant reductions in the mRNA expression levels of AKT and mTOR and the protein expression levels of AKT,p-AKT,mTOR,and p-mTOR(P<0.05 or P<0.01).Conclusion: JPXA can inhibit the expression of AKT and mTOR and the proliferation and clone abilities of colon cancer cells, and it can also promote the apoptosis of colon cancer cells to a certain extent.