Abberant Energy Metablism In Chemo-resistance In Patients With Myeloid Ieukemias

BackgroundThe drug resistance of leukemic cells remains a major obstacle to chemotherapy in patients with myeloid leukemia, which always lead to treatment failure consequently. The altered expression of p-glycoprotein(P-gp), multidrug resistance-associated protein (MRP), type II topoisomerase (Topo Ⅱ) and lung resistance related protein (LRP) mediates lung resistance related protein (MDR) in previous studies. However, the complex mechanism in chemoresistance causes a growing concern for looking for some new therapeutic targets and improve the sensitivity of chemotherapy.Metabonomics established an analysis method for make some qualitation and quantitation analysis qualitation and quantitation of metabolites of biology and cells, which revealed the relation between metabolites and physiological and pathological changes. Recently, researchers have have used metabolomics technology to explore the differences of metabolomics between imatinib (IM)-resistant and sensitive chronic myeloid leukemia (CML) cells, and thus to clarify the mechanism of IM resistance, provide new targets to the treatment of IM resistance. The data showed that increased glycolytic activity and phospholipid flip related with IM drug resistance, and the IM-resistant K562-r and LAMA84-r cells remained the high glycolytic metabolic phenotype of elevated glucose uptake and lactate production. As glycolytic rate directlly correlated with tumorigenesis and chemoresistance, it provides a new idea for cancer treatment. By inhibiting the activity of glycolytic enzymes, can block the aerobic glycolysis, so that the tumor cells died due to the lack of energy supply, but normal cells unaffected, which is expected to become a new targeted cancer therapy. Although the tumor cells demonstrated a over-reliance for glycolytic pathway, but studies show that just relying on the inhibition of aerobic glycolysis, is not sufficient to produce significant clinical significance anti-tumor effect. This may be due to depletion of ATP only reach a certain threshold to activate apoptosis or necrosis process, eventually leading to cell death, and because all of the tumor cells have a mitochondrial structure, when glycolysis was inhibited, also there may be generated ATP by oxidative phosphorylation.Some large-scale gene expression analysis recently showed that in hematopoietic malignancies the genes encoding component of glycolytic pathway selectively upregulated. These studies confirmed that there is also abnormal glycolysis in hematopoietic tumor cells.3-BrPA can effectively induce multidrug resistance in acute myeloid leukemia cell lines HL-60/AR apoptosis, suggesting that blocking the cell’s energy supply is likely to overcome the blood tumor cell multidrug resistance. The recent studies revealed that prednisolone-resistant significantly related with acute lymphoblastic leukemia (ALL) cells high glucose consumption, through the inhibition of glycolysis can make prednisolone-resistant ALL cell lines sensitive to glucocorticoid. Additional, the inhibiton of glycllysis for reversal of glucocorticoid resistance is not limited to cell lines, the same as primary ALL cells. This improved sensitization for chemotherapy may be mediated through the activate protein kinase (AMPK), an important cell energy sensor. Inhibition of glycolysis can activate AMPK, leading to inhibition of mTOR, and thus make the anti-apoptotic protein Mcl-1downregulation. Hitherto, none of glycolytic inhibitor combined with chemotherapeutic drugs for reversing chemoresistance were reported. So, the study according to the new discovery of metabolomics, use the methods of cellular and molecular biology to investigate the relations between the altered energy metabolism and chemoreisitance in myeloid leukemias. The project is expected to clarify the mechanism of energy metabolism in myeloid leukemia patients with chemoresi stance, thus it may provide a new targeted therapy of reversal of chemoresistance.Part1. The expression of glycolysis associated moleculars and chemoresistance in myeloid leukemiasObjective:1. To study the relationship between the expression of glycolysis activity and chemoresistance in myeloid leukemias.2. To study the relationship between the expression of glycolysis associated moleculars and chemoresistance in myeloid leukemias.3. To study the relationship between the expressions of human ATP synthase and chemoresistance in myeloid leukemias.Materials and Methods:1. Cell cultureHL-60, HL-60/ADM, K562, K562-R; myeloid leukemia cell lines were cultured at37℃in a5%humidified atmosphere in RPMI1640plus10%fetas calf serum,100U/ml penicillin,100μg/mlstreptomycin. ADM resistant AML cell line HL-60/ADM and IM resistant CML cell line K562-R were generated from ADM-sensitive AML cell line HL-60and IM-sensitive CML cell line K562following the corresponding drug induced method, repectively.2. Patient samples The bone marrow samples obtained from with newly diagnosised (n=45) and relapsed (n=9) patients with AML (M3excluded) in our hospitals during October2010to November2011. The AML blasts in every sample was exceed to80percent. The total54AML cases with average age of41.6±17.8years were included in31male and23female patiens. The constituent ratio of age and sex in19control cases showed no significant difference with compared to those in AML patients by chi square test (p>0.05).3. Experimental method(1) The glycolytic rates of myeloid leukemia cell lines with different drug sensitivities were detected by glucose consumption assay.(2) The mRNA expression of glycolysis associated moleculars (e.g. HIF-la, FBP1,HK-Ⅱ,GLUT1) in myeloid leukemia primary cells and cell lines with different drug sensitivities were measured by real time polymerase chain reaction (PCR).(3) The serum LDH level in AML patients with different drug sensitivities was detected by enzymatic synthesis assay.(4) The expression of CD147on bone marrow cells of AML patients with different drug sensitivities was analyzed by flow cytometry.(5) The expression of β subunit of human mitochondrial F1F0-ATP synthase (ATP5B) in myeloid leukemia primary cells and cell lines with different drug sensitivities was measured by the method of western blot assay.4. Statistical analysisAll the statisticala nalyses were perfomed with the statistical software package SPSS16.0. Student’s t-test was used to determine the significance of the differences between the mean values. One-way ANOVA test was performed and post hoc multiple comparisons were made using LSD test under homogeneity of variance, while mean comparison of multi-group samples were analyzed by Dunnett’s T3method under homogeneity of variance. Results are presented as; p<0.05was regarded as statistically significant.Results1.Correlation between glycolysis rateand chemoresistance in myeloidleukemia cellThe drug-resistant HL-60/ADM and K562-R cell lines exhibited a significantly higher glycolysis rates than the corresponding wild type sensitive leukemic cells HL-60and K562, respectively.2. The mRNA expression of glycolysis associated genes in myeloid leukemia primary cells and cell lines with different drug sensitivities(1) The mRNA expression of glycolysis associated genes in myeloid leukemia cell lines with different drug sensitivitiesThe levels of HIF-1αand GLUT1mRNA expression of the two drug-resistant HL-60/ADM and K562-R cells were higher than that of drug-sensitive HL-60and K562cells (p<0.01、p<0.05). However, the mRNA expression of HK-Ⅱ, another key enzyme in glycolytic pathway, in ADM-resistant HL-60/ADM cells was lower than that of wild type HL-60cells (p<0.01), while it was elevated in IM-resistant K562-R cells with comparison to IM-sensitive K562cells(p<0.01). Additional, the mRNA expression of FBP1, an inhibitory enzyme of glycolysis, in ADM-resistant HL-60/ADM cells was higher than that of wild type HL-60cells (p<0.01), while it was down-regulated in IM-resistant K562-R cells with comparison to IM-sensitive K562cells (p<0.01)(2) The mRNA expression of HIF-la in blasts from AML patients with different chemotherapy responsivenessThe mRNA expression of HIF-1α in AML patients with NR were higher than that in groups of health control, CR and PR, while the expression level of HIF-1α mRNA exhibited an increasing tendency in groups of health control, CR and PR.(3) The mRNA expression of FBP1in blasts from AML patients with different chemotherapy responsivenessWe found considerable interindividual differences of the mRNA expression of FBP1in AML patients with CR, PR, and NR, respectively. There were no statistically significant difference, however, between these groups (p>0.05).(4) The mRNA expression of HK-Ⅱ in blasts from AML patients with different chemotherapy responsivenessThe mRNA expression of HK-Ⅱ in AML patients with NR were higher than that in groups of health control, CR and PR, while the expression level of HK-Ⅱ mRNA exhibited an increasing tendency in groups of health control, CR and PR.(5) The mRNA expressions of GLUT1in blasts from AML patients with different chemotherapy responsivenessThe mRNA expression of GLUT1in AML patients with NR were higher than that in groups of health control, CR and PR, while the expression level of GLUT1mRNA exhibited an increasing tendency in groups of health control, CR and PR.3. The serum LDH level in AML patients with different drug sensitivitiesThe serum level of LDH in AML patients with NR was higher than that in groups from health control, CR and PR, while the level of LDH exhibited an increasing tendency in groups of health control, CR and PR.4. The expression of CD147on bone marrow cells of AML patients with different drug sensitivitiesThe expression of CD147on bone marrow blasts from AML patients with NR was higher than that in groups of CR and PR.5. The expression of β subunit of human mitochondrial F1F0-ATP synthase ATP5B in myeloid leukemia primary cells and cell lines with different drug sensitivities The expression of ATP5B protein in drug-resistant HL-60/ADM and K562-R cell lines were lower than that in the corresponding wild type sensitive leukemic cells HL-60and K562, respectively. Likewise, the level of ATP5B protein in AML patients with NR was lower than that in CR group(p<0.05).Conclusion1.The drug-resistant HL-60/ADM and K562-R cells showed elevated glycolytic activities than the two drug-sensitive myeloid leukemia cell lines.2. The level of HIF-1α and GLUT1mRNA expression in drug-resistant HL-60/ADM and K562-R cells were higher than those in the two drug-sensitive myeloid leukemia cell lines.3. The level of HIF-1α, HK-][and GLUT1mRNA expression in drug-resistant AML patients were higher than that in drug-sensitive group.4. There were no statistically significant difference in FBP1mRNA expression between drug-resistant and sensitive AML patients.5. The serum level of LDH in drug-resistant AML patients was higher than that in drug-sensitive group.6. The expression of CD147on bone marrow blasts from drug-resistant AML patients was higher than that in drug-sensitive group.7. The expression of mitochondrial synthase ATP5B protein were decreased in the two drug-resistant HL-60/ADM and K562-R cells, and the level of ATP5B protein in drug-resistant group was lower than that in drug-sensitive group.Part2. Inhibition of glycolysis modulates chemoresistancein myeloid leukemic cellsObjective1.To study the effect of inhibition of glycolysis on chemoresistance in myeloid leukemia cells. 2. To study the mechanism of inhibition of glycolysis modulates chemotherapy sensitivity in myeloid leukemic cells.Materials and Methods:1. Cell cultureHL-60, HL-60/ADM, K562, K562-R; myeloid leukemia cell lines were cultured at37℃in a5%humidified atmosphere in RPMI1640plus10%fetas calf serum,100U/ml penicillin,100μg/mlstreptomycin. ADM resistant AML cell line HL-60/ADM and IM resistant CML cell line K562-R were generated from ADM-sensitive AML cell line HL-60and IM-sensitive CML cell line K562following the corresponding drug induced method, repectively.2. Experimental method(1) The effect of glycolysis inhibition on cellular proliferation of myeloid leukemic cells were measured by the method of trypan blue exclude assayResponsiveness of myeloid leukemia cells (e.g. HL-60, HL-60/ADM, K562, K562-R) to glycolysis inhibitors, cytotoxitic drugs, and glycolysis inhibitor combination cytotoxitic drug was determined by the3-day in vitro trypan blue exclude assay. A possible synergistic effect was tested according to Weeb coefficient formula.(2) The effect of glycolysis inhibition on cellular proliferation of myeloid leukemia cells were measured by the method of MTT assayResponsiveness of myeloid leukemia cells (e.g. HL-60, HL-60/ADM, K562, K562-R) to glycolysis inhibitors, cytotoxitic drugs, and glycolysis inhibitor combination cytotoxitic drug was determined by the3-day in vitro MTT drug resistance assay. A possible synergistic effect was tested according to Weeb coefficient formula.(3) The glycolytic rates of myeloid leukemia cells were detected by glucose consumption assayGlucose consumption was measured as the conversion of glucose to6-phosphogluconate and NADH with the Glucose (HK) Assay Kit, as described by the manufacturer (Sigma-Aldrich). Briefly,106myeloid leukemia cells (e.g. HL-60, HL-60/ADM, K562, K562-R) after different treatments were grown in RPMI containing2g/L glucose. After4days, the medium was collected by centrifugation to remove the cells, and incubated for2hours with glucose assay buffer. The conversion of NAD to NADH can be measured by the increase in absorbance at340nm, which is directly proportional to the glucose concentration.(4) The apoptosis of myeloid leukemic cells was analyzed by flow cytometryThe effect of glycolysis inhibition on apoptosis of myeloid leukemia cells (e.g. HL-60, HL-60/ADM, K562, K562-R) was analyzed by flow cytometry after different treatments for24h.。(5) RNA interference with shRNA sequences targeting GLUT1or HIF1α was used to detected the changes of ADM-induced seneitivities.First, we designed several pairs of shRNA sequence targeting GLUT1or HIFla, and transfected it into HL-60/ADM cells by the method of electroporation, and screened the optimal shRNA sequence by the efficiency of RNA interference which was monitored at RNA level by real-time quantitative PCR and at protein level by western blot.Next, the shRNA sequence was constructed into lentiviral vector through recombinant DNA technology. Then, Lenti-X293T cells were transfected with lentiviral shRNA vector for viral packaging, and the virus titers were tested through infecting HT-1080cells with recombinant virus. Meanwhile, the efficiency of RNA interference in HL-60/ADM cells was monitored after infected with lentiviral shRNA plasmid. After that, HL-60/ADM cell line silenced stably GLUT1or HIF1α gene expression was generated through screening with puromycin for three times. The effect of various concentrations of ADM on these newly constructed HL-60/ADM cells interfered for GLUT1or HIF1α was measured by MTT assay.3. Statistical analysisAll the statisticala nalyses were perfomed with the statistical software package SPSS16.0. Student’s t-test was used to determine the significance of the differences between the mean values. One-way ANOVA test was performed and post hoc multiple comparisons were made using Dunnett-t test. Results are presented as; p<0.05was regarded as statistically significant.Results1. The effect of glycolysis inhibition on cellular proliferation of myeloid leukemia cells2-DG or3BrPA, the glycolysis inhibitors, acts in synergy with ADM in ADM-resistant HL-60/ADM leukemic cells, while it didn’t synergize with ADM in wild type HL-60cells. Likewize,2-DG or3BrPA acts in synergy with IIM-resistant K562-R leukemic cells, while it didn’t synergize with IM in wild type K562cells.2. The effect of glycolysis inhibition on glycolytic rates of myeloid leukemia cellsTreatment of the drug-resistant or sensitive cell lines HL-60, HL-60/ADM, K562, and K562-R with2-DG or3BrPA, either alone or in comination with ADM or IM, resulted in a considerable reduction of glucose consumption compared with non-treated cells. This decrease in glucose consumption was not seen when cells incubated with ADM or IM alone.3. The effect of glycolysis inhibition on cellular apopotosis of myeloid leukemia cellsAfter24h treatment of the drug-resistant or sensitive cell lines HL-60, HL-60/ADM, K562, and K562-R with2-DG or3BrPA, either alone or in comination with ADM or IM, yet the cellular apoptosis rate in these different cell lines showed no significant difference (p>0.05). However, the glycolysis inhibitors in comination with ADM or IM increased markedly the necrosis in these cell lines.4. The changes of ADM-induced seneitivities in HL-60/ADM cells silenced stably GLUT1or HIF1α gene expression with shRNA sequencesWe constructed two HL-60/ADM cell lines infected with lentiviral shRNA plasmids for stably silencing GLUT1or HIFla gene expression. RNA interference with shRNA sequences targeting HIFla did not affect ADM sensitivity in HL-60/ADM cells. However, the silence of GLUT1gene expression with shRNA increased ADM sensitivity in HL-60/ADM cells.Conclusion1. The glycolytic inhibitors2-DG or3BrPA increased AMD-induced cytotoxity in ADM-resistant HL-60/ADM cells by decreasing glucose consumption. Also,2-DG or3BrPA exerted a synergism effect on cellular proliferation of K562-R cells with IM.2. The enhancements of chemotherapy-induced cytotoxity by2-DG or3BrPA may be due to the activiation of nonapoptotic, programmed cell death.3. RNA interference with shRNA sequences targeting GLUT1resulted in a reduction in gene expression, and increased ADM sensitivity of HL-60/ADM cells. However, the silence of HIF-1αgene with shRNA didn’t obviously change the ADM sensitivity in HL-60/ADM cells.