二甲双胍联合热疗诱导U937细胞凋亡及其机制研究

Metformin Combined with Hyperthermia Induces Apoptosis in U937 Cells and Its Mechanisms

探讨二甲双胍(Met)联合热疗(HT)诱导人白血病U937细胞凋亡及其可能的作用机制.MTT法检测二甲双胍联合温热(HT+Met)处理对U937细胞增殖的影响;Hoechst 33342染色观察U937细胞形态变化;流式细胞术检测U937细胞凋亡、周期、活性氧以及线粒体膜电位的变化情况;Western Blot检测U937细胞中凋亡相关蛋白的表达情况.通过细胞计数及活力实验确定Met 10 mmol/L和HT 44℃、15 min为化疗联合热疗的实验条件.Hoechst 33342荧光染色及Annexin V-FITC/PI流式细胞术分析表明,与Met或HT单独处理相比,HT+Met诱导U937细胞凋亡作用增强、活性氧(ROS)产生增加、线粒体膜电位(MMP)降低及G2/M期阻滞.Western Blot结果表明,与Met或HT单独处理相比,HT+Met组Bax/Bcl-2蛋白表达上调,Caspase-3和Caspase-8蛋白表达下调,周期蛋白依赖性激酶CDK1和周期蛋白Cyclin B1表达下调,p-P38/P38水平显著升高,加入NAC(一种ROS清除剂)后可显著恢复HT+Met组诱导的p-P38蛋白和Caspase-3蛋白表达.二甲双胍联合热疗能够诱导U937细胞凋亡,其可能的作用机制为通过死亡受体(外源性)途径、ROS介导调节线粒体(内源性)途径以及P38 MAPK信号通路诱导人白血病U937细胞凋亡.

To investigate the induction of apoptosis in human leukemia U937 cells by metformin(Met)combined with hyperthermia(HT)and its possible mechanism of action.MTT assay was used to detect the effect of metformin combined with warm(HT+Met)treatment on the proliferation of U937 cells.Hoechst 33342 staining was used to observe the morphological changes of U937 cells.Flow cytometry was used to detect the changes of apoptosis,cycle,reactive oxygen species,and mitochondrial membrane potential in U937 cells,and Western Blot assay was used to detect the expression of apoptosis-related proteins in U937 cells.The expression of apoptosis-related proteins in U937 cells was detected by Western Blot.The cell counting and viability assays determined that Met 10 mmol/L and HT 44℃for 15 min were the experimental conditions for the combination of chemotherapy and hyperthermia.Hoechst 33342 fluorescence staining and Annexin V-FITC/PI flow cytometry analysis showed that HT+Met induced enhanced apoptosis,increased reactive oxygen species(ROS)production,and mitochondrial membrane potential in U937 cells,as compared with Met or HT treatment alone.ROS production was increased,mitochondrial membrane potential(MMP)was decreased,and G2/M phase was blocked.Western Blot showed that Bax/Bcl-2 protein expression was up-regulated,Caspase-3 and Caspase-8 protein expression was down-regulated,CDK1 and Cyclin B1 expression was down-regulated in HT+Met compared with Met or HT alone,p-P38/P38 flow cytometry analysis showed that HT+Met induced apoptosis in U937 cells.The expression of Caspase-3 and Caspase-8,CDK1 and Cyclin B1 was down-regulated.The level of p-P38/P38 was significantly elevated,and the addition of NAC,a ROS scavenger,significantly restored the HT+Met-induced expression of p-P38 and Caspase-3 proteins.Metformin combined with hyperthermia induced apoptosis in U937 cells,and its possible mechanism of action is to induce cell death in human leukemia U937 cells through the death receptor(exogenous)pathway,ROS-mediated regulation of the mitochondrial(endogenous)pathway,and the P38 MAPK signaling pathway.