Metformin Combined with Hyperthermia Induces Apoptosis in U937 Cells and Its Mechanisms
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.
metforminhyperthermiaapoptosisP38 MAPK signal pathway
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