5-甲氧基色醇对急性髓细胞性白血病U937细胞增殖和迁移的影响及其机制研究

Effect of 5-methoxychromol on proliferation and migration of acute myeloid leukemia cell line U937 and its mechanism

目的研究不同浓度5-甲氧基色醇对急性髓细胞性白血病U937细胞增殖和迁移的影响并初步探讨其作用机制。方法采用人胆囊收缩素/缩胆囊素八肽(CCK-8)法,检测不同浓度(8、16、32、64μmol/L)的5-甲氧基色醇对急性髓细胞性白血病U937细胞的增殖抑制率;流式细胞术检测U937细胞各处理组的凋亡比例;Transwell实验检测不同浓度5-甲氧基色醇对细胞U937迁移能力的影响;基于5-甲氧基色醇的药效团,应用生物信息学技术预测5-甲氧基色醇作用的蛋白靶点,并对蛋白靶点做通路富集分析;蛋白质印迹法检测不同浓度5-甲氧基色醇对显著富集通路中的蛋白靶点磷脂酰肌醇3激酶(PI3K)和Akt激酶(AKT)的蛋白表达水平影响。结果CCK-8结果显示,8、16、32、64μmol/L 5-甲氧基色醇以时间和浓度依赖方式抑制U937细胞的增殖[增殖抑制率24 h:(17.4±1.2)%、(31.6±2.6)%、(43.5±3.2)%、(58.28±4.5)%,72 h:(21.5±1.8)%、(43.4±2.4)%、(67.4±6.1)%、(85.6±3.9)%;均P<0.05];流式细胞术实验结果显示,与对照组凋亡率(3.93±0.19)%相比,5-甲氧基色醇明显促进细胞U937的凋亡[(7.5±0.33)%、(8.99±0.20)%、(13.71±0.39)%];Transwell实验结果显示,与对照组每视野下迁移细胞数(205±14)个相比,8、16、32、64μmol/L 5-甲氧基色醇明显抑制细胞U937的迁移能力[(152±18)个、(124±9)个、(78±22)个](P<0.05);生物信息学及通路富集结果显示,5-甲氧基色醇存在100个作用的蛋白靶点,这些蛋白靶点显著富集在包括PI3K-AKT信号通路等;对PI3K-AKT信号通路相关蛋白进行蛋白质印迹法检测其表达,结果显示,5-甲氧基色醇处理可明显减少U937细胞中PI3K和AKT蛋白的磷酸化水平(均P<0.05)。结论5-甲氧基色醇抑制PI3K-AKT信号通路的蛋白表达水平,进而抑制急性髓细胞性白血病U937细胞的增殖和迁移,促进U937的凋亡。

Objective To investigate the effects of different concentrations of 5-methoxytryptophol on the proliferation and migration of acute myeloid leukemia cell line U937 and its mechanism.Methods Human cholecystokinin octapeptide(CCK-8)ELISA was used to detect the proliferation inhibition rate of 5-methoxytryptophol at different concentrations(8,16,32 and 64μmol/L)on U937 cells of acute myeloid leukemia.The apoptotic ratios of U937 cells in each treatment group were determined by flow cytometry.Transwell assay was performed to determine the effects of different concentrations of 5-methoxychromol on the migration of U937 cells.Based on the pharmacophore of 5-methoxytryptophol,the protein targets of 5-methoxytryptophol were predicted by bioinformatics,and the protein targetsreceived pathway enrichment analysis.Western blotting was used to detect the effects of different concentrations of 5-methoxychromol on the expression levels of protein targets in the significantly enriched pathway.Results CCK-8 results showed that 8,16,32 and64μmol/L 5-methoxytryptophol inhibited the proliferation of U937 cells in a time-dependent and concentration-dependent manner[proliferation inhibition rate 24 h:(17.4±1.2)%,(31.6±2.6)%,(43.5±3.2)%,(58.28±4.5)%,72 h:[(21.5±1.8)%,(43:4±2.4)%.(67.4±6.1)%,(85.6±3.9)%;all P<0.05].Flow cytometry results showed that compared with the apoptotic rate in the control group[(3.93±0.19)%],t 5-methoxytryptophol significantly promoted the apoptosis of acute myeloid leukemia cell line U937[(7.5±0.33)%,(8.99±0.20)%,(13.71±0.39)%].Transwell assay results showed,compared with the number of migrated cells per field of view[(205±14)cells]in the control group,8,16,32,64μmol/L 5-methoxytryptophol significantly inhibited the migration of acute myeloid leukemia cell line U937(P<0.05)[(152±18)cells,(124±9)cells,(78±22)cells].Bioinformatics and pathway enrichment results showed that there were100 functional protein targets of 5-methoxytryptophol,which were significantly enriched in the phosphatidylinositol 3-kinase and AKT kinase(PI3 K-AKT)signaling pathway.The expression of proteins related to the PI3 K-AKT signaling pathway was detected by Western blotting,and the results showed that 5-methox)tryptophol treatment significantly reduced the phosphorylation levels of PI3 K and AKT proteins in U937 cells(both P<0.05).Conclusion 5-methoxytryptophol inhibits the protein expression level of the PI3 K-AKT signaling pathway,thereby inhibiting the proliferation and migration of U937 cells in acute myeloid leukemia and promoting U937 apoptosis.