甘草甜素对人急性髓系白血病细胞增殖、凋亡及AMPK/MTOR信号通路的影响

Effect of Glycyrrhizin on proliferation, apoptosis and AMPK/MTOR signaling pathway in human acute myeloid leukemia cells

目的 探讨甘草甜素对人急性髓系白血病HL-60细胞增殖和凋亡的影响,并基于腺苷单磷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(MTOR)信号通路分析其可能的作用机制。方法 将HL-60细胞分成对照组、甘草甜素低剂量组、甘草甜素中剂量组和甘草甜素高剂量组,甘草甜素低剂量组、甘草甜素中剂量组和甘草甜素高剂量组分别给予0.25μg/mL、0.5μg/mL、1μg/mL的甘草甜素处理细胞,对照组给予二甲基亚砜处理细胞。采用CCK-8法和细胞克隆法分别检测细胞的活力和有效克隆数情况;采用流式细胞术和Hoechst 33342染色法检测细胞凋亡;采用Western blot检测增殖细胞核抗原(PCNA)、细胞周期蛋白D1(cyclin D1)、活化型Caspase-3(cleaved Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、AMPK、磷酸化AMPK、MTOR和磷酸化MTOR的蛋白表达水平。结果 对照组、甘草甜素低剂量组、甘草甜素中剂量组、甘草甜素高剂量组的HL-60细胞存活率依次降低,有效克隆数依次减少,凋亡率依次升高,PCNA、cyclin D1、Bcl-2、磷酸化MTOR蛋白的表达水平依次降低,Bax、cleaved Caspase-3、磷酸化AMPK蛋白的表达水平依次升高(均P<0.05)。结论 甘草甜素可抑制HL-60细胞增殖,促进细胞凋亡,且具有一定的剂量依赖性,其作用机制可能与促进AMPK磷酸化水平、抑制MTOR磷酸化有关。

Objective To investigate the effect of Glycyrrhizin on proliferation and apoptosis of human acute myeloid leukemia HL-60 cells,and to analyze its possible mechanism based on adenosine monophosphate activated protein kinase(AMPK)/mammalian target of rapamycin(MTOR)signaling pathway.Methods HL-60 cells were divided into control group,Glycyrrhizin low-dose group,Glycyrrhizin medium-dose group or Glycyrrhizin high-dose group.The Glycyrrhizin low-,medium-,and high-dose groups were given 0.25μg/mL,0.5μg/mL and 1μg/mL Glycyrrhizin to process the cells,respectively,and dimethyl sulfoxide was given to the control group to process the cells.The cell viability and number of valid clones were detected by CCK-8 assay and cell cloning assay.The cell apoptosis was detected by flow cytometry and Hoechst 33342 staining.The protein expressions with respect to proliferating cell nuclear antigen(PCNA),cyclin D1,cleaved Caspase-3,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),AMPK,phosphorylated AMPK,MTOR,and phosphorylated MTOR were detected by the Western blot.Results The survival rate of HL-60 cells in the control group,and the Glycyrrhizin low-,medium-and high-dose groups decreased successively,the number of valid clones reduced successively,and the apoptosis rate increased successively.The protein expression levels of PCNA,cyclin D1,Bcl-2,and phosphorylated MTOR in the control group,and the Glycyrrhizin low-,medium-and high-dose groups decreased sequentially,while the protein expressions of Bax,cleaved Caspase-3,and phosphorylated AMPK increased sequentially(all P<0.05).Conclusion Glycyrrhizin can inhibit HL-60 cell proliferation and promote cell apoptosis in a dose-dependence manner to a certain extent.Its mechanism can possibly be related to promote AMPK phosphorylation level,and to inhibit MTOR phosphorylation.