| Objective: Acute myeloid leukemia(AML) is a kind of malignant clone disease that originated in hematopoietic stem cells, and its incidence has a tendency to rise year by year.In the past few decades, the treatment of AML has made considerable progress, current treatment methods mainly include chemotherapy and hematopoietic stem cell transplantation on the basis of chemotherapy.Side effects and drug resistance of recurrence of traditional chemotherapy has a serious impact on the overall survival rate of the patients, while bone marrow transplantation is the radical method, but due to human leukocyte antigen mismatched transplantation, and the risk of GVHD after transplantation, thus limits its clinical widely carried out.Therefore, we need to find new drugs to optimize treatment, reduce the side effects of drugs and cross resistance, in order to improve patients’ overall survival rate.At present, the study found leukemia cell gene promoter region significantly increased the incidence of DNA methylation, especially those which has the function of tumor suppressor gene promoter region of high methylation.Unlike the gene mutation, DNA methylation is a reversible process of genetic modification,so in the tumor or precancerous lesions can go through the demethylation treatment restored gene expression, so as to achieve the purpose of prevention and treatment of tumor.Therefore, DNA demethylation will provide new ideas for the treatment of hematological malignancies.Decitabine(DAC), also known as 5-aza-2’-deoxycytidine, as a kind of DNA methyltransferase inhibitor on AML patients including resistance, relapse patients have shown the preliminary curative effect.At present, DAC has been successfully applied to a variety of cancers of the blood, but research on combined application are less.Arsenic trioxide(AS2O3) in China is excavated from the medicine in the treatment of acute promyelocytic leukemia(APL) effect of certain drugs, has been the AML guidelines from the American NCCN as a recurrence of the disease does not relieve or relapse salvage treatment drug of choice, is a classic example of successful treatment of integrated traditional Chinese and western medicine.In recent years, demethylation drugs being used in clinic, especially for not suitable for the application of standard chemotherapy to induce remission in patients with AML.however, how to improve drug efficacy and reduce the side effects is currently an important topic.The experimental study of DAC, AS2O3 on HL-60 cells(human acute myeloid leukemia cell line) proliferation, apoptosis and the role of death associated protein kinase(DAPK) on gene expression, and to explore the mechanism of demethylation drugs in the treatment of AML, aim to provide more theoretical basis for the future clinical application.Methods:Selection of AML cell line HL-60 as the object of study, and randomly divided into group: control group, DAC group(20μmol/L, 40μmol/L, 80μmol/L) and AS2O3 group(1μmol/L, 2.5μmol/L, 5μmol/L), DAC combined with AS2O3 group(40μmol/L+2.5μmol/L, 80μmol/L+2.5μmol/L, 40μmol/L +5μmol/L, 80μmol/L+5μmol/L), DAC(80μmol/L) pretreatment+AS2O3 group(1μmol/L, 2.5μmol/L, 5μmol/L).Effect on the proliferation, apoptosis of HL-60 cells was evaluated for different concentrations at different time after drug intervention and the influence on the expression of DAPK gene.Each experiment was repeated 3 times.1 Sieving concentration and detect the effect of different concentrations of drugs for 24 h, 48 h, 72 h on the proliferation inhibition rate of HL-60 cells by CCK8method;2 Detecting the effect of different concentrations of decitabine and Arsenic trioxide for 24 h, 48 h, 72 h on the apoptosis rate of HL-60 cells by flow cytometry(FCM);3 RT-PCR method was used to detect the effect of different concentrations of drugs in HL-60 cells for 72 h on expression of DAPKm RNA;4 All the data were analyzed with the SPSS 13.0 software.Results: 1 Effects of DAC and AS2O3 on the proliferation of HL-60 cells 1.1 Different concentration of DAC, AS2O3 effect on the HL-60 cells after 24 h, 48 h, 72 h, along with the prolonging of the drug concentration and the reaction time, the inhibition rates of proliferation increased gradually. DAC inhibited the proliferation of HL-60 cells increased from(10.97±1.09)% to(61.95±1.24)%, AS2O3 inhibited the proliferation of HL-60 cells increased from(13.81±3.09)% to(84.18±2.94)%. The differences of treatment groups were statistically significant(P=0.00). It suggested that DAC and AS2O3 alone could inhibit the proliferation of HL-60 cells, and showed time and dose dependence.(Fig.1, Fig.2, Table 1, Table 2) 1.2 Different concentrations of DAC combined with AS2O3 effect on the HL-60 cells after 24 h, 48 h, 72 h, with the extension of drug concentration and action time, the proliferation inhibition rate increased from(43.22±4.78)% to(93.12±3.71)%, the treatment group compared with single drug group was significantly enhanced proliferation inhibition rate, and the difference was statistically significant(P=0.00).It suggested that DAC combined with AS2O3 had a synergistic effect on HL-60 cell proliferation inhibition.(Fig.3, Table 3) 1.3 After the DAC(80μmol/L) pretreatment, different concentrations of AS2O3 effect on HL-60 cells after 24 h, 48 h, 72 h, with the extension of drug concentration and action time, the proliferation inhibition rate increased from(31.8±1.32)% to(91.44±0.78)%.Compared with AS2O3 monotherapy group, proliferation inhibition rate was significantly enhanced, and the difference was statistically significant(P=0.00).It suggested that DAC pretreatment could enhance the effects of AS2O3 on the proliferation inhibition of HL-60 cells.(Fig. 4, Table 4) 2 Effects of DAC and AS2O3 on the apoptosis of HL-60 cells 2.1 Different concentration of DAC, AS2O3 monotherapy in HL-60 cells in the role of 24 h, 48 h, 72 h later, with the extension of drug concentration and action time, the apoptosis rate increased gradually.DAC for the HL-60 cell apoptosis rate increased from(8.0±1.05)% to(36.6±1.35)%, AS2O3 for the HL-60 cell apoptosis rate increased from(10.2±1.35)% to(45.3±2.35)%.The difference of treatment group and between treatment group and control group were statistically significant(P=0.00).It suggested that DAC and AS2O3 alone could induce the apoptosis of HL-60 cells and showed time and dose depend- ence.(Fig.5,Table5,Table6) 2.2 Different concentrations of DAC combined with AS2O3 effect on the HL-60 cells after 24 h, 48 h, 72 h, with the extension of drug concentration and action time, the apoptosis rate increased from(19.6±0.56)% to(62.1±0.71)%, The apoptosis rate of treatment group compared with single drug group was significantly enhanced, and the difference was statistically significant(P=0.00).It suggested that DAC combined with AS2O3 had a synergistic effect on the induction of apoptosis of HL-60 cells.(Fig.5,Table7) 2.3 After the DAC(80μmol/L) pretreatment, different concentrations of AS2O3 effect on the HL-60 cells after 24 h, 48 h, 72 h, with the extension of drug concentration and action time, the apoptosis rate increased from(18.8±1.32)% to(71.4±0.79)%.Comprared with the AS2O3 monotherapy group,the apoptosis rate was significantly enhanced, and the difference was statistically significant(P=0.00).It suggested that DAC pretreatment could enhance the apoptosis- inducing effects of AS2O3 on HL-60 cells.(Fig. 5, Table 8) 3 Effects of DAC and AS2O3 on the expression of DAPKm RNA in HL-60 cells 3.1 Different concentrations of DAC, AS2O3 monotherapy on the role HL-60 cells after 72 h, with the increase of drug concentration, the expression level of DAPKm RNA increased gradually. The expression of DAC on the amount of DAPKm RNA increased from 1.5±0.34 to 3.2±0.56, while the expression level of AS2O3 on DAPKm RNA increased from 1.2±0.50 to 3.0±0.17.The difference of treatment group and between treatment group and control group were statistically significant(P=0.00).It suggested that DAC, AS2O3 alone could increase the expression level of DAPKm RNA in HL-60 cells with dose dependence.(Fig.6,Fig.7,Table9,Table10) 3.2 Different concentrations of DAC combined with AS2O3 on the role of HL-60 cells after 72 h, with the increase of drug concentration, the expression level of DAPKm RNA increased from 3.6±0.47 to 5.2±0.25.The treatment group compared with single drug group and the control group, the expression level of DAPKm RNA was significantly increased, and the differences were statistically significant(P=0.00).It suggested that DAC combined with AS2O3 could increase the expression level of DAPKm RNA in HL-60 cells, and they had synergistic effect.(Table 11) 3.3 After the DAC(80μmol/L) pretreatment, different concentrations of AS2O3 on the role of HL-60 cells after 72 h, with the increase of drug concentration, the expression of DAPKm RNA increased from 2.0±0.35 to 4.5±0.18. Compared with the AS2O3 single drugs group, the expression level of genes was significantly increased, and the difference was statistically significant(P=0.00).It suggested that DAC pretreatment could promote the expression level of AS2O3 on DAPKm RNA in HL-60 cells.(Table 12)Conclusions:1 Decitabine and arsenic trioxide alone or combined could inhibit the proliferation and induce the apoptosis of HL-60 cell lines and both had a synergistic effect.2 Decitabine and arsenic trioxide played a role with time and dose dependence.3 After decitabine pretreatment,could increase the sensitivity of arsenic trioxide on HL-60 cells, and its mechanism might be related to restore or increase the expression of DAPK gene, DAPK gene might be one of the regulating gene of DAC induced HL-60 cells’ apoptosis. |
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