Effect Of Combination Of Metformin And 2-deoxyglucose On P-gp-mediated Multidrug Resistant And Its Mechanism

Part 1 Targeting P-glycoprotein expression and cancer cell energy metabolism:combination of metformin and 2-deoxyglucose reverses the multidrug resistance of K562/Dox cells to doxorubicinPurpose:To elucidate the possible mechanisms for whether combination of metformin and 2-deoxyglucose reverses the MDR of K562/Dox cells and tried to elucidate the possible mechanisms.Methods:Cell viability was determined by MTT assay.The relation between metformin and P-gp was comfirmed using the bidirectional transport assay.The changes in P-gp expression were determined at the mRNA,protein and functional level.Western blot analysis was performed to study its molecular mechanisms of action.The changes in metabolism were determined by glucose uptake,lactate production,adenosine triphosphate(ATP)depletion and autophagy.Apoptosis were determined by Annexin V-FITC/PI assay and the cleaved-caspase3.Results:The combination of metformin and 2-deoxyglucose selectively enhanced the cytotoxicity of doxorubicin against MCF-7/Dox cells.Metformin was not a substrate of P-gp but suppressed the elevated level of P-gp in K562/Dox cells.The down-regulation of P-gp may be partly attributed to the inhibition of extracellular signal-regulated kinase pathway.The addition of 2-deoxyglucose to metformin inhibited glucose uptake and lactate production in K562 and K562/Dox cells leading to a severe depletion in ATP.P-gp substrate selectively aggravated this ATP depletion effect and increased cell apoptosis in K562/Dox cells.Conclusions:Metformin decreases P-gp expression in K562/Dox cells via blocking phosphorylation of extracellular signal-regulated kinase.P-gp substrate increases K562/Dox cell apoptosis via aggravating ATP depletion induced by combination of metformin and 2-deoxyglucose.Part 2 Targeting p53,P-glycoprotein and energy metabolism:combination of metformin and 2-deoxyglucose reverses the multidrug resistance ofmcf-7/Dox cells to doxorubicinPurpose:To clarify the possible mechanisms for whether combination of metformin and 2-deoxyglucose reverses the MDR of MCF-7/Dox cells and tried to elucidate the possible mechanisms.Methods:Cell viability was determined by MTT assay.The changes in metabolism were determined at glucose,lactate,fatty acid adenosine and triphosphate(ATP)levels.The changes in P-gp were determined at the mRNA,P-gp ATP enzyme and functional level.Cell cycle and caspase3 activity were determined to investigate the effect of drugs on cell cycle and apoptotic activity.Western blot analysis was performed to clarify the molecular mechanisms of the actions mentioned above.Results:The combination of metformin(0.5mM)and 2-deoxyglucose(0.5mM)had a stronger antiproliferative action in MCF-7/Dox cells compared with MCF-7 cells and selectively enhanced the cytotoxicity of doxorubicin against MCF-7/Dox cells.The combination of two drugs arrested cells cycle in G2/M and increased caspase3 activity in both MCF-7 and MCF-7/Dox cells.In MCF-7/Dox cells,metformin and 2-deoxyglucose increased p53 expression via inhibiting the overexpression of MDM2 and MDMX.The increased p53 significantly enhanced the cytotoxicity of doxorubicin in MCF-7/Dox cells.The combination of two drugs had no effect on P-gp mRNA expression and P-gp ATP enzyme activity but increased doxorubicin accumulation in MCF-7/Dox cells.The increased doxorubicin accumulation maybe associated with metabolic stress induced by metformin and 2-deoxyglucose.The combination of metformin and 2-deoxyglucose initiated a strong metabolic stress in both cell lines via inhibiting glucose uptake、lactate、fatty acid、ATP production and AKT/mTOR pathway.Doxorubicin selectively aggravated this metabolic stress effect and increased cell apoptosis in MCF-7/Dox cells.Conclusions:1.The combination of metformin and 2-deoxyglucose could increased p53 expression via inhibiting the overexpression of MDM2 and MDMX to enhance the cytotoxicity of doxorubicin against MCF-7/Dox cells.2.Doxorubicin selectively increases MCF-7/Dox cell apoptosis via aggravating metabolic stress induced by combination of metformin and 2-deoxyglucose.