Effect Of Metformin On Proliferation Capacity,Apoptosis And Glycolysis In K562 Cells

Background:AML is a malignant disease of the hematopoietic system,clonal expansion of undifferentiated myeloid progenitors/precursors,resulting in impaired normal hematopoiesis and bone marrow failure.For the last four decades,the standard of care of AML has been a 3+7 chemotherapy regimen,induction therapy of cytarabine and anthracycline drugs is very effective in killing leukemic cells.Although the complete remission rate is high,the overall survival rate is very low due to recurrence.Metabolic reprogramming,such as the Warbug effect(a key metabolic change consisting in the capacity of cancer cells to consume more glucose than non-proliferating cells,but to preferentially ferment glucose to lactate regardless of oxygen availability)is a common phenotypic feature of cancer cells,allowing their rapid adaptation in response to the high energetic requests necessary to support their high rate of proliferation.Therefore,the treatment of leukemia from the perspective of energy metabolism may become an opportunity to improve the prognosis of leukemia and reduce recurrence.Metformin,a widely used anti-diabetic molecule,has attracted a strong interest in the last 10 years as a possible new anti-cancer molecule.Numerous investigations worldwide rapidly demonstrated direct anti-cancer eff ects of metformin on various models.In vitro,metformin exhibits a strong anti-proliferative action on cancer cell lines derived from breast,colon,ovaries,pancreas,lung,and prostate.Previous studies have shown that metformin can induce apoptosis of k562 cells and block the cell cycle,which together inhibit the proliferation of k562 cells.However,there are few studies on the mechanism ofmetformin affecting the proliferation and apoptosis of acute leukemia through glycolysis energy metabolism at home and abroad.Objective:To investigate the effect of metformin on the proliferation and energy metabolism of k562 cells and the possible mechanism.Methods:Different doses(0,5,10,20 and 30 mmol/L)of metformin was added on the k562 cells,cultivated 24 h,48 h and 72 h,used the inverted optical microscope to observe the cell growth,CCK 8 was to detect the cell vitality,selected the appropriate metformin doses(0,10,20 and 30 mmol/L)and the best time(48 h)for subsequent experiments.Annexin V-FITC /PI flow cytometer was used to detect apoptosis,glucose detection kit and lactate detection kit were used to detect glucose consumption and lactate production,fluorescence quantitative PCR was used to detect glycolysis related gene expression,and Western blot was used to detect protein expression.Results:Metformin inhibited the proliferation of k562 cells in a dose-dependent manner(r=0.92),and the relative survival in the 30mmol/L group was as low as 19.84% at72 h.When treated with metformin for 48 h,the apoptosis rates of 0,10,20 and30mmol/L groups were 5.14%,12.19%,26.29% and 35.5%.Compared with the control group,the glucose consumption and lactate secretion of k562 cells treated with metformin were significantly reduced(P<0.05),and showed a dose-dependent effect(r=0.94,r=0.93,respectively).Metformin inhibited the relative genes expression of GLUT1,LDHA,ALDOA,PDK1,and PGK1 of k562 cells(P<0.05),and the gene expression level decreases as the concentration of metformin increases.Metformin inhibited the relative proteins expression of P-Akt,P-S6,GLUT1,LDHA(P<0.05)among k562 cells,and the expression of metformin decreases as the concentration of metformin increases.Conclusion:Metformin could inhibit the growth and proliferation of k562 cells and promote the apoptosis of k562 cells by inhibiting glycolysis energy metabolism.PI3K/Akt/mTOR signaling pathway may be one of the molecular mechanisms of metformin on k562 cells.