The Expression Of Mitochondrial ATP Synthase β Subunit(ATPsyn-β) In Refractory/Relapsed Acute Myelogenous Leukemia And Its Effect On Drug Resistance And Prognosis

BackgroundLeukemia is a group of heterogeneous diseases characterized by either a block in differentiation, aberrant and accelerated growth, or a combination of both with considerable diversity in terms of clinical behavior and prognosis. Acute myelogenous leukemia (AML) is the most common type of leukemia in adults. The main therapy is still chemotherapy induction and consolidation. However, nearly70%of responding patients will eventually relapse and die of the disease. One of the major causes of failure in the treatment is drug resistance. But the mechanisms underlying the development of multidrug resistance in AML are not fully understood. Therefore, it is significant to explore novel molecular targets for drug-resistant reversal in AML patients.Recently, a rebirth of the interest in the energetic metabolism of cancer spurred, and mitochondria has become a central player. The mitochondrial ATP synthase is the enzyme complex that carries out the synthesis of ATP. The β subunit of mitochondrial ATP synthase (ATPsyn-β) is the rate-limiting component of mitochondria oxidative phosphorylation. Down-regulated expression of mitochondrial ATPsyn-β has been reported in many types of tumors,including colon、lung、breast、 esophagea、stomach and kidney.Our previous study shows that down-regulated expression of mitochondria ATPsyn-β is associated with the development of drug resistance in chronic myeloid leukemia (CML). Here, we studied the expression of mitochondrial ATPsyn-β and the activity of mitochondrial ATPase in AML patients.Chapter Ⅰ The expression of mitochondrial ATPsyn-β on bone marrow mononuclear cells and CD34+cells form AML patients and the correlation between ATPsyn-β level and sensitivity to adriamycin in vitro.Objective:To investigate the expression of mitochondrial ATPsyn-β in AML patients and the association with chemotherapy response.Methods:1. Bone marrow samples were collected from118non M3AML patients (42of relapsed/refractory,38of complete remission duration,38of newly diagnosis) and20patients with benign hematological diseases(for control)from Oct,2010to Jun,2012. Expression of mitochondrial ATPsyn-β mRNA and protein in bone marrow mononuclear cells (BMMCs) of AML patients were detected by RT-PCR, western blot and flow cytometry respectively.2. The purified CD34positive cells were acquired from BMMCs of20AML patients using the CD34positive selection immunomagnetic beads. Then the expression of ATPsyn-β mRNA and protein were analyzed by RT-PCR and western blot.3. Sensitivity of BMMCs to adriamycin in relapsed/refractory and remission duration AML patients was detected by MTT assay in vitro. Correlation between expression of ATPsyn-β and adriamycin sensitivity in the two groups was used linear correlation to analysis.Results:1. Expression of ATPsyn-β mRNA and protein in AML patients (n=118) was downregulated (P<0.01). 2. Different levels of mitochondrial ATPsyn-β mRNA and protein were detected in AML patients. Compared with remission duration patients(n=38), expression of ATPsyn-β in relapsed or refractory AML patients(n=42)was significantly downregulated both in BMMCs and CD34positive cells, P<0.05.3. The relative level of ATPsyn-β mRNA was higher in CD34+cells than in CD34-cells, P<0.05.4. IC50value of adriamycin to BMMCs from relapsed/refractory and remission duration AML patients was13.69μM and1.549μM respectively.5. The mRNA and protein expression of ATPsyn-β significantly inversely correlated with IC50of adriamycin to BMMCs in relapsed/refractory AML patients, P<0.05.Conclusion:The mRNA and protein expression of mitochondrial ATPsyn-β was significantly downregulated in non-M3AML patients. Compared with complete remission patients, the mRNA and protein level of ATPsyn-β in relapsed/refractory AML patients were lower in both bone marrow mononuclear cells and CD34positive cells. And this decrease correlated with resistance of primary leukemia cells to adriamycin in vitro, suggesting that down-regulation of ATPsyn-β in AML is associated with drug resistance. Chapter II The activity of mitochondrial ATP synthase in drug-resistant leukemia cell lines and primary cells of AML patientsObjective:To investigate the activity of mitochondrial ATP synthase in AML patients and the clinic significance. Methods:After the isolation of mitochondia, the activity of ATPase in adriamycin resistant cell lines(K562/A02、HL-60/ADM) and BMMCs of AML patients was measured in the direction of ATP hydrolysis using a kit.Results:1. The mitochondrial ATPase activity of K562/A02(36.70±7.47U) and HL-60/ADM cells(38.22±2.00U) was lower than K562(65.43±1.31U) and HL-60cells(132.06±14.65U), P<0.05.2. The mitochondrial ATPase activity in relapsed or refractory samples (n=26) was obviously reduced when compared with those of complete remission samples (n=16). The mean value was20.33U vs.31.17U, P<0.05.3. Mitochondrial ATPsyn-β mRNA expression showed a positive correlation with ATPase activity (rs=0.294, P=0.031).Conclusion:The activity of mitochondrial ATP synthase was down-regulated in adriamycin resistant cell lines and relapsed/refractory AML patients. Mitochondrial ATPsyn-β mRNA expression had a positive correlation with ATPase activity. Chapter Ⅲ Expression of mitochondrial ATPsyn-β in adriamycin resistance AML cell lines and its effect on growth inhibition and apoptosis inductionObjective:To explore the influence of ATPsyn-β on proliferation-inhibiting capability and apoptosis-inducing effect of adriamycin in leukemia cells. To analyse whether the change of ATPsyn-β level could lead to or reverse drug resistance. Methods:1. Expression of mitochondrial ATPsyn-β in HL-60and HL-60/ADM cell lines was detected by RT-PCR, western blot and flow cytometry.2. Afer inhibition of ATPsyn-β expression by RNA interference or increase it by gene transfection, HL-60and HL-60/ADM cells were treated with different concentrations of adriamycin, then (1)cell viability was analyzed with MTT colorimetric assay test;(2) cellular uptake of adriamycin was detected by flow cytometry;(3) morphologic character of apoptosis was evaluated by Wrights-Giemsa staining and Hoechst33258staining;(4)the percentage of earlier apoptosis cell was analyzed by flow cytometry;(5)expression of MRP mRNA relative level was detected by RT-PCR.Results:1.The expression of mitochondrial ATPsyn-P in HL-60/ADM cells was lower than HL-60cells, and MRP mRNA expression was higher.2.Overexpression of ATPsyn-β in HL-60/ADM cell line restored cells’ sensitivity to adriamycin, promoted earlier cell apoptosis and decreased the expression levels of MRP. In contrast, suppression of ATPsyn-β expression weakened cell’sensitivity to adriamycin, blocked cell apoptosis and increased the expression of MRP.Conclusion:The expression of mitochondrial ATPsyn-p in HL-60/ADM cells was downregulated. Inhibition of mitochondrial ATP synthase β subunit led to increased chemo-resistance and apoptotic resistance in HL-60cells and up regulation of mitochondrial ATPsyn-β restored chemo-sensitivity and promoted earlier cell apoptosis in HL-60/ADM cells. Chapter Ⅳ Expression of ATPsyn-P in AML patients with prognostic analysisObjective:To analyze the impact of established prognostic factors on the clinical outcome in patients with AML. To determine the prognostic value of ATPsyn-β in AML patients.Methods:We analyzed3variables for their impact on complete remission (CR) duration and overall survival (OS) in118AML patients, including age, performance status and WBC counts. We also determined the role of ATPsyn-P as a poor prognostic factor in AML patients.Results:1. Multivariate analysis determined old age (older than45years), poor PS (score3-4), leukocytosis (WBC>30×109) as poor prognostic factors for OS.2. Much of the relation between ATPsyn-β and therapeutic resistance was observed. The median CR duration in newly diagnosis AML patients with low ATPsyn-β mRNA expression was4months, shorter than that in high ATPsyn-β expression patients (eight months). The overall survival of relapsed/refractoty AML patients with low ATPsyn-β mRNA expression was10.68±7.87months, shorter than that with high lower ATPsyn-P mRNA expression(21.28±1.26months), P<0.05。Conclusion:1. Age, performance status, WBC counts onset were independent prognostic factors for clinical outcome in AML.2. As a prognostic factor for survival, low level of mitochondrial ATPsyn-P was associated with an increased relapse risk, shorter CR duration and overall survival.