| Objective:Despite advances in understanding the etiology and pathogenesis of myeloid leukemia,the treatment of refractory or relapsed myeloid leukemia remains a daunting clinical challenge.Approximately 20%of acute myeloid leukemia(AML)patients do not respond to induction chemotherapy,and 40-60%of patients relapse.Therefore,investigation of strategies aiming to reverse resistance to drugs in AML is critical.Many cancer cells have elevated rates of glycolysis and lactate production even in the presence of oxygen.A few metabolism-associated molecules,including glucose transporter 1(GLUT1),hexokinase ?(HK?)and lactate dehydrogenase A(LDHA),which are over expressed in certain cancer types,have been proven to be potential therapeutic targets.In addition,drug resistance in AML cells was associated with increased glycolytic activity and low efficiency of oxidative phosphorylation,which was observed by the elevated expression of glycolysis-associated molecules,such as hypoxia inducible factor-1(HIF-1),HK-?,GLUT1 and LDHA.Autophagy is a vesicular trafficking pathway that targets intracellular substrates,from entire organelles to protein aggregates and specific proteins,for lysosomal degradation and recycling.Autophagy-related E1 ligase 7(ATG7)protein,a key molecule in autophagy vesicle elongation,is involved in two essential ubiquitin-like reactions.There has been no definitive research documenting the relation of metabolic changes and autophagy in AML.It has been reported that ATG7-mediated autophagy inhibition in chronic myeloid leukemia(CML)cells leads to a decrease in glycolysis,and using the nonspecific autophagy inhibitor hydroxychloroquine.On the other hand,multiple studies have suggested that autophagy limits glycolytic metabolism in AML.Therefore,it is unclear whether autophagy is increased glycolysis or not because of a lack of knowledge.Wnt pathway is highly conserved in evolution and plays an important physiological role in human body.There are three downstream transduction ways of Wnt signaling,and the canonical Wnt/?-catenin pathway has the closely relationship with tumors.A lot of studies indicate that Wnt/?-catenin pathway involves in the pathogenesis of a range of disease including many kinds of carcinomas.The abnormal activation of Wnt/?-catenin signaling pathway in myeloid leukemia cells is involved in the regulation of the development and progression of acute leukemia.In recent years,it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain,which would represent a new role for Wnt signaling in cell metabolism.In the present study,we illuminate the relationship between glycolytic activity and autophagy in myeloid leukemia.Then we investigated whether autophagy promotes glycolysis in myeloid leukemia cells by activating the Wnt/?-catenin signaling pathway.Materials and methods:1.Object of studyBone marrow(BM)of primary acute myeloid leukemia(AML)specimens and healthy controls were collected from patients during routine diagnostic assessments.RT-PCR and Western blot were applied to detect the expression of Autophagy-related E1 ligase 7(ATG7),glucose transporter 1(GLUT1),hexokinase ?(HK?)and lactate dehydrogenase A(LDHA).2.Glycolytic inhibitionFollowing 24 h of treatment of the HL-60 cells and K562 cells with 2-deoxy-D-glucose(2-DG),Glucose and lactate were detected by Beckman Coulter AU5800 biochemical analyzer.CCK8 was performed to detect the proliferation change of cells.Annexin V-FITC/PI apoptosis detection Kit was applied to detect the apoptosis change of cells after treated with cytarabine(Ara-C).3.Sh ATG7-mediated autophagy inhibition(1)The infection efficiencies were determined by GFP fluorescence and flow cytometer.The expression of ATG7 was detected by RT-PCR and Western blot.(2)The levels of LC3B and P62 were detected by Western blot.(3)CCK8 was performed to detect the proliferation change of cells.Annexin V-FITC/PI apoptosis detection Kit was applied to detect the apoptosis change of cells after treated with Ara-c.(4)Glucose and lactate were detected by Beckman Coulter AU5800 biochemical analyzer.(5)GLUT1,HK-? and LDHA was detected by RT-PCR and Western blot4.Autophagy activation induced by rapamycin(1)HL-60 cells and K562 cells were treated with rapamycin(2)The levels of LC3B and p62 were detected by Western blot after treated by rapamycin.(3)CCK8 was performed to detect the proliferation change of cells.Annexin V-FITC/PI apoptosis detection Kit was applied to detect the apoptosis change of cells after treated with Ara-c.(4)Glucose and lactate were detected by Beckman Coulter AU5800 biochemical analyzer.(5)GLUT1,HK-II and LDHA were detected by RT-PCR and Western blot.5.The relationship between autophagy and Wnt/?-catenin signaling pathway?-catenin was detected by RT-PCR and Western blot after the changes of autophagy.6.Si?-catenin mediated Wnt/?-catenin signaling pathway downregulation The infection efficiencies were determined by GFP fluorescence and flow cytometer.The expression of ?-catenin was detected by RT-PCR and Western blot.7.The influence of ?-catenin downregulation to autophagy-induced glycolysis(1)The levels of LC3B and P62 were detected by Western blot after treated by rapamycin.(2)Glucose and lactate were detected by Beckman Coulter AU5800 biochemical analyzer.(3)GLUT1,HK-? and LDHA were detected by RT-PCR and Western blot.8.Statistical analysisAll statistical analyses were performed using the statistical software package SPSS 21.0.Results:1.Comparative analysis of ATG7 expression was conducted on AML samples and control through real-time quantitative PCR and Western blot.ATG7 mRNA was found to be differentially overexpressed in AML samples,whereas it was weakly expressed in healthy controls and western blotting yielded similar results.The expressions of ATG7 in NR group were obviously higher than in CR group.2.The expressions of HK-?,GLUT1 and LDHA in AML group were obviously higher than in control group.3.Glycolytic inhibitor reduced proliferation and increases apoptosis of HL-60 cells and K562 cells.4.ATG7-mediated autophagy inhibition sensitized HL-60 cells and K562 cells to Ara-c-induced cell death.Glucose consumption and lactate production were reduced after ATG7 knocked down.The levels of HK-? GLUT1 and LDHA were lower in ATG7 knockdown cells.5.After autophagy activation induced by rapamycin,the sensitivity to Ara-c of HL-60 cells and K562 cells decreased.Glucose consumption and lactate production were increased.The levels of HK-?,GLUT1 and LDHA were higher after using rapamycin.6.Western blotting and RT-PCR showed increased ?-catenin expression in the rapamycin-treated cells as compared with the control,while ?-catenin expression was decreased following ATG7-mediated autophagy inhibition.7.The level of ?-catenin was lower in p-catenin knockdown cells.8.?-catenin downregulation inhibited autophagy-induced glycolysis and the levels of HK-?,GLUT1 and LDHA.Conclusions:Our findings suggest that the over expression of ATG7 is associated with the prognosis of AML.We demonstrate that apoptosis of HL-60 and K562 cells distinctly promotes while glycolysis is inhibited after treatment with 2-DG.Furthermore,apoptosis is promoted in ATG7 knockdown cells in comparison to those transfected with negative control sh RNA.The decrease in the level of GLUT1,HKII and LDHA is associated with the decrease of ATG7,and the glycolysis of ATG7 knockdown cells is reduced.Activation of autophagy can promote metastasis and glycolysis in AML cells and autophagy promote glycolysis expression by up-regulation GLUT1,HK ?and LDHA via Wnt/?-catenin signaling pathway.Our study describes the connection between autophagy and glucose metabolism in myeloid leukemia cells and may provide a potential therapeutic target for myeloid leukemia treatment. |
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