Effects Of The SIRT2 On Multidrug Resistance In Acute Myelogenous Leukemia Cells

BackgroundAcute myeloid leukemia (AML) is one of the most common hematologic malignancies, and there are many methods to treat it. However, there are many patients can not get a complete remission after treatment and a large part of the patients will relapse after complete remission. The primary and recurrent drug resistance of leukemic cells is one of the main reasons for the failure of leukemia treatment. One of the key issues for cure leukemia is the further study of molecular mechanism for multidrug resistance and improve the sensitivity of leukemic cells to drug treatment. SIRT2 is a member of nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family protein, which can participate in cell differentiation, proliferation, migration, intracellular signal transduction and so on by deacetylase in vivo and in vitro. It has been found that SIRT2 can involve in the proliferation, differentiation and survival of acute myeloid leukemia (AML) cells. However, its effect on drug reststance on chemoresistant AML cells is unclear.ObjectiveThe present study is aimed to detect the expression level of SIRT2 in newly diagnosed and relapsed AML bone marrow samples, AML cell lines HL60 and multidrug reststance HL60 cells HL60/A and to investigate the biological effects of SIRT2 on the multidrug reststance of AML and the related mechanisms.Methods1. Bone marrow samples were obtained from 25 children acute lymphoblastic leukemia (children-ALL) patients,30 acute lymphoblastic leukemia (ALL) patients,10 essential thrombocythemia (ET) patients,11 chronic myeloid leukemia (CML) patients,8 polycythemia vera (PV) patients,11 myeloproliferative neoplasm (MPN) patients,15 acute myeloid leukemia (AML) patients in our hospital from April 2013 to January 2014. Bone marrow samples of 6 healthy volunteers were used as control. Bone marrow mononuclear cells were prepared by Ficoll—Hypaque density gradient centrifugation. The expression level of SIRT2 in primary bone marrow cells was assayed by real-time quantitative PCR.2. Bone marrow samples were obtained from 15 newly diagnosed acute myeloid leukemia (AML) patients and 10 relapse AML patients in our hospital from April 2013 to January 2014. Bone marrow mononuclear cells were prepared by Ficoll—Hypaque density gradient centrifugation. The expression level of SIRT2 in primary bone marrow cells was assayed by real-time quantitative PCR.3. The expression level of SIRT2 in AML cell lines HL60 and multidrug reststance HL60 cells HL60/A was detected by real-time quantitative PCR and Western Blot.4. shRNA expressing plasmids specifically targeting SIRT2 (termed as SIRT2-shRNAl and 2) were constructed, transfected into HL60/A cells. Using RT-PCR, immunofluorescence assay and Western Blot detected the expression level of SIRT2. immunofluorescence assay and flow cytometry was used to measure the accumulation of intracellular doxorubicin. MTT assay was used to detected cell viability after the cells were treated with daunorubicin/arabinocytidine. Hoechst33258 staining, Annexin V-FITC/PI and Western Blot apoptosis analysis were used to detect apoptosis induced by daunorubicin/arabinocytidine in HL60/A cells.5. SIRT2-CD512B-1 plasmid was constructed according to molecular cloning and then stably transfected into HL60 cells, which made HL60 cells overexpress SIRT2. Using RT-PCR, immunofluorescence assay and Western Blot detected the expression level of SIRT2. immunofluorescence assay and flow cytometry was used to measure the accumulation of intracellular doxorubicin. MTT assay was used to detected cell viability after the cells were treated with daunorubicin/arabinocytidine. Hoechst33258 staining, Annexin V-FITC/PI and Western Blot apoptosis analysis were used to detect apoptosis induced by daunorubicin/arabinocytidine in HL60 cells.6. The Western Blot technique was used to detect MRP1, ERK1/2, P-ERK1/2, P53 and BCL2 proteins expression.ERK1/2 inhibitor PD98059 was used to study the effects of ERK1/2 signaling pathway in the progress of SIRT2 regulation the AML multidrug reststance.Results1. The mRNA expression level of SIRT2 was statistically higher in different hematologic malignancies than normal control and higher in relapse AML patients than preliminarily diagnosed (P<0.05).2. The mRNA expression level and protein expression level of SIRT2 was lower in HL60 cells compared with HL60/A cells (P<0.05).3. SIRT2 knock-down increased the accumulation of intracellular doxorubicin in HL60/A cells. SIRT2 knock-down in HL60/A cells enhanced the sensitivity to daunorubicin/arabinocytidine.4. SIRT2 overexpression reduced the accumulation of intracellular doxorubicin in HL60/A cells. SIRT2 overexpression in HL60/A cells attenuated the sensitivity to daunorubicin/arabinocytidine.5. In HL60/A cells, SIRT2 knock-down repressed expressions of MRP1, ERK1/2, P-ERK1/2 and BCL2 while up-regulated the expression of P53; By contrast, SIRT2 overexpression up-regulated the expressions of MRP1, ERK1/2, P-ERK1/2 and BCL2 meanwhile decreased the expression of P53 in HL60 cells. ERK1/2 inhibitor PD98059 could block ERK1/2 activity in SIRT2 knock-down HL60/A cells and sensitized these cells to daunorubicin/arabinocytidine. Furthermore, in SIRT2 overexpression HL60 cells, ERK1/2 inhibitor PD98059 could enhance the reststance to daunorubicin/arabinocytidine.ConclusionsSIRT2 was highly expressed in hematologic malignancies like:ALL, CML, ET, PV, MPN and so on. The expression of SIRT2 was higher in relapse AML than preliminarily diagnosed and in HL60 cells than multidrug reststance cells HL60/A. SIRT2 weakened the multidrug reststance of AML cells probably partially via regulating the ERK1/2 signaling pathway.