| Background:Acute lymphoblastic leukemia(ALL)is the most common malignant tumor in children.and the immunophenotype of B cell type accounts for about 80%.With the standardization of treatment,the 5-year event-free survival rate of childhood ALL in China has exceeded 80%.Because of the adverse effects of chemotherapy on long-term growth of children.Targeted therapy has become the therapeutic goal of childhood ALL.Tumor cells rely on metabolic reprogramming to support their malignant proliferation under nutrient-deficient conditions,which has been proved to be widespread in solid tumors.In recent years,metabolic reprogramming has also been found in hematological diseases research,among which the metabolic reprogramming of activating glycolysis,improving pentose phosphate pathway and increasing mitochondrial respiration is the most prominent.However,there are few studies on the metabolic characteristics of BALL in children and the relationship with the progression of leukemia.1,5-anhydroglucitol(1,5-AG)is a monosaccharide with six-carbon structure,which is an analog of glucose.It is widely present in the diet and is rapidly absorbed in the intestine.Currently,functional studies on 1,5-AG have mainly focused on the cardiovascular and endocrine systems.1,5-AG can be combined with hemoglobin A1c to reflect the very short-term glycemic levels.At the same time,low levels of 1,5-AG correlate with the severity of coronary artery disease.However,in the field of tumor research,1,5-AG is rarely reported.In recent years,high levels of 1,5-AG have been found to be associated with male cancer mortality.However,few reports on the role of 1,5-AG have been reported in hematologic malignancies.In order to reveal the metabolic characteristics of B-ALL in children.the peripheral blood(PB)samples of children with B-ALL and healthy donors were collected for untargeted metabolomic analysis.The high abundance of 1,5-AG,a product of glycometabolism,was found in children with initial diagnosis B-ALL.To further explore the role of 1,5-AG in hematological tumors,we set out from glucose metabolism.The role of 1.5-AG in the activation of glycolysis and interference with redox homeostasis was found.The mechanism of 1,5-AG in the progression of B-ALL was revealed and verified by RNA-seq and animal models.This study suggests a possible therapeutic direction for the clinical treatment of B-ALL children with higher 1,5-AG.Objective:1.To explore the characteristics of metabolites in the peripheral plasma of children with initial diagnosis B-ALL;2.To study the effect of metabolite 1,5-AG on the biological function of B-ALL cell lines.3.To explore the metabolic mechanism of 1,5-AG promoting B-ALL.4.To investigate the role of antioxidants in the treatment of B-ALL children with high 1,5-AG.Methods:Part 1.Detection of peripheral blood plasma metabolites in children with primary acute B-lymphoblastic leukemia and the effect of 1,5-anhydroglucitol on the biological function of B-ALL cell lines.1.PB samples of 27 children with B-ALL at the time of initial diagnosis prior to chemotherapy and healthy donors undergoing physical examination were collected.Samples were centrifuged and untargeted metabolomic analysis.Blood cells were separated from lymphocyte separation solution,and PB-MNCs were frozen after flow cytometry identification.2.Establishment of B-ALL mice model.Peripheral blood,spleen cells,and bone marrow cells were obtained after modeling.The percentage of CD19+in B-ALL model were detected by flow cytometry.PB plasma was collected for the targeted detection of 1,5-AG.3.The effects of 1,5-AG on proliferation,apoptosis and cell cycle of B-ALL cell lines Reh and HAL-01 were detected by using CCK8 assay,Annexin-V FITC/PI double staining method,and flow cytometry respectively.4.GraphPad Prism 8.0.1(GraphPad Software.Inc.,La Jolla.CA.USA)was used for statistical analyses.For the analysis of non-targeted metabolomics,orthogonal partial least squares discriminant analysis(OPLS-DA)was subsequently applied.All data were tested for normality using the Kolmogorov-Smirnov test.The Bonferroni test was performed between groups.Two-tailed unpaired Student’s t tests were used for all comparisons between two groups.Oneway ANOVA with Dunnett’s test was used to compare groups,and results with P values less than 0.05 were considered significant.All data are presented as means ± standard deviations,and each experiment was biological repeated three times.Part 2.1,5-AG participates in glucose metabolism in B-ALL.1.The rate of glycolysis by B-ALL cells 24 h after intervention with 1,5-AG was detected by seahorse.The intracellular lactate levels were determined by the Lactate Colorimetric Assay Kit after intervention with different concentrations of 1,5-AG.2.The differentially expressed genes in B-ALL cells after the addition of 1,5-AG were detected by RNA-seq.3.The expression of PDK4 in B-ALL was down-regulated by siRNA,and the knockdown efficiency was verified by real-time quantitative PCR(RT-qPCR)and Western blot.4.The effects of PDK4 on B-ALL cells were detected by Western blot,CCK8,and OCR,respectively.5.GraphPad Prism 8.0.1 was used for statistical analyses.All data are presented as means ± standard deviations.Oneway ANOVA with Dunnett’s test was used to compare different 1,5-AG concentrations.Two-way ANOVA with Tukey’s test was used for analysis of different interventions in the presence of 1,5-AG,and results with P values less than 0.05 were considered significant.Each experiment was biological repeated three times.Part 3.In vivo and in vitro mechanism of 1,5-AG regulating glucose metabolism in B-ALL.1.NADP+/NADPH assay kit was used to detect the intracellular total NAPDH level after 1.5-AG intervention.Flow cytometiy was used to detect the level of intracellular reactive oxygen species(ROS)after different concentrations of 1,5-AG intervention.2.Resuscitated PB-MNCs of children with B-ALL were cultured in 37℃incubator.The viability and intracellular ROS level of leukemic cells added with different concentrations of 1,5-AG were detected by CCK8 and flow cytometry.3.The mechanism of 1,5-AG promoting the proliferation of B-ALL cells was verified by adding ROS scavenger NAC or vitamin C.4.Effect of 1,5-AG on leukemia progression in B-ALL model mice:construction of mCherry-Reh cells by lentivirus transfection.Leukemia burden in mice of different treatment groups was detected by the In Vivo Imaging System(IVIS).The relative quantification of 1,5-AG in PB plasma was determined by liquid chromatography-mass spectrometry-based metabolomic analysis.The percentage of CD19+PB,bone marrow(BM),and spleen samples in B-ALL model was detected by flow cytometry.5.Western blot was used to detect the effect of 1,5-AG on the protein levels of MAPK/ERK signaling pathway in B-ALL cell lines.6.HE staining was used to analyze the pathological differences of spleen and bone marrow sections in each group.7.The expression of p-ERK protein in spleen and bone marrow was analyzed by immunohistochemistry.8.GraphPad Prism 8.0.1 was used for statistical analyses.All data are presented as means ± standard deviations.Oneway ANOVA with Dunnett’s test was used to compare different 1,5-AG concentrations.Two-way ANOVA with Tukey’s test was used for analysis of different interventions in the presence of 1,5-AG,and results with P values less than 0.05 were considered significant.Each experiment was biological repeated three times.Results:Part 1.Detection of peripheral blood plasma metabolites in children with primary acute B-lymphoblastic leukemia and the effect of 1,5-anhydroglucitol on the biological function of B-ALL cells.1.The results showed that the metabolites in the peripheral blood plasma of patients with B-ALL were different from healthy children.The abundance of 37 metabolites was up-regulated and 23 metabolites was down-regulated in B-ALL group compared with healthy children.B-ALL significantly alters the metabolic profile of the PB in initial diagnosis children.2.Compared with the healthy children,1,5-AG was significantly higher in the blood plasma of patients with B-ALL(p=0.00055).At the same time,after successfully constructing the B-ALL model,the abundance of 1,5-AG was also higher in the model group than the blank group(0.4892±0.1464 vs.0.1538±0.03592;p<0.01).3.The results of CCK8 showed that the addition of 1,5-AG 24 h significantly promoted cell viability in Reh in a 0.2-0.8 mM and HAL-01 in a 0.8-2.0 mM(p<0.01;p<0.001).Compared with the control group,the 1,5-AG concentration of 0.4 mM and 0.8 mM cells were increased at S phase(p<0.01).Apoptosis assay showed that high concentration of 1,5-AG caused the death of B-ALL cells(p<0.05;p<0.001).Part 2.1,5-AG participates in glucose metabolism in B-ALL.1.The addition of 1.5-AG significantly improved both basal and compensatory glycolytic rates and increased lactate levels in both Reh and HAL-01 cells(p<0.05).2.The results of RNA-seq suggested that the differentially expressed genes were significantly enriched in carbohydrate metabolism processes.Among them,PDK4,a regulatory kinase in glycoly sis,was highly expressed(p<0.05).3.We knocked down PDK4 with siRNAs in B-ALL.Two siRNAs targeting PDK4 were used,and protein levels in B-ALL were determined by RT-qPCR and western blot.The siPDK4 infection rate was about 50%.4.The results shown that knockdown of PDK4 reduced cell viability and lactate levels of B-ALL compared with siNC-transfected cells to which 1,5-AG was added.Moreover,the addition of 1,5-AG significantly decreased cellular mitochondrial maximum respiration.Furthermore,the knockdown of PDK4 reversed this phenomenon(p<0.05).Part 3.In vivo and in vitro mechanism of 1,5-AG regulating glucose metabolism in B-ALL.1.The results indicated that the amount of total NADPH produced by 1.5-AG(0.2 mM-0.8 mM)was significantly higher than that produced by the control cells(p<0.05).At the same time.1.5-AG(0.2-0.8 mM)significantly increased cellular ROS levels in both the Reh and HAL-01 cell lines(p<0.05).2.In the three patient-derived B-ALL PB-MNCs samples,the addition of 1,5-AG also increased cell viability and intracellular ROS levels(p<0.05).3.Both NAC and VC were able to reverse the 1,5-AG-induced promoting effect(including an increase in cell viability,cell cycle progression,and induction of cell apoptosis)(p<0.05).4.Animal experiments showed that the levels of 1,5-AG were gradually upregulated(three to four times higher)in the AG-treated groups than those in the model group for 7 days(13.12±3.806 vs.1.940±0.5312;p<0.01).The effect of the ROS scavenger was also demonstrated at the NAC treatment mouse plasma level(13.12±3.806 vs.7.590±2.860;p<0.05).Compared with the control,the groups treated with 1,5-AG significantly promoted leukemogenesis on day 7(p<0.01),and NAC to a certain extent abolished this effect(p=0.0760).In addition,the spleen weight of the AG group was not significantly different from that of the model group.Flow cytometry results showed that 1,5-AG improved the proportion of leukemic cells in the PB(p=0.0642).BM and increased leukemia cell infiltration into the spleen(p<0.05).These findings also suggest that NAC slowdown 1,5-AG induces leukemic progression(p<0.05).5.1,5-AG(0.2-0.8 mM)markedly improved the activity of MAPK/ERK signaling in B-ALL cells,characterized by increased phosphorylation of ERK1/2(p<0.05).The inhibitory effect of 1,5-AG on the MAPK/ERK pathway was reversed by NAC and Vitamin C treatment in both Reh and HAL-01 cells(p<0.05).6.HE staining was performed in the spleen and BM of mice,and the pathological results showed no significant differences among all groups.7.Immunohistochemical results showed that 1,5-AG upregulated p-ERK1/2 positive cells in spleen and BM of mice and was also reversed by NAC(p<0.05).Conclusion:1.High level of 1.5-AG was generally present in peripheral blood plasma of children with B-ALL.2.1,5-AG may accelerate B-ALL proliferation and cell cycle by promoting anaerobic glycolysis through upregulation of PDK4,thereby providing pre-B cells with a competitive advantage.3.Elevated NADPH levels in B-ALL cells optimize ROS-driven cell proliferation by increasing their own antioxidant status.Eventually,the ROS-dependent MAPK/ERK pathway was activated,leading to the progression of B-ALL.4.Targeted knockout of PDK4 or antioxidant treatment(NAC/Vitamin C)can reverse the B-ALL children with higher 1,5-AG. |
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