| Objective: Acute myeloid leukemia(AML)is a kind of hematopoietic stem cell malignant clonal disease,which is a serious threat to human health.In recent years,after the standard“3+7” regimen,the complete remission rate of adult patients with AML Can be as high as 70-80%.However,due to the existence of minimal residual disease after remission,the recurrence of the disease still inevitable.Only approximately 20% to 30%of the patients enjoy Iong term disease—free survival.Currently only allogeneic hematopoietic stem cell transplantation is one of the most effective treatments for AML.But due to the patient’s physical condition,source of donor,economy and other reasons,the number of patients receiving transplantation is limited.Therefore,the search for new targeted drugs to improve the effectiveness of chemotherapeutic drugs is a field that we continue to explore.Recent studies showed that the majority of acute myeloid leukemia(AML)patients have more than 2 aberrant methylation.Because DNA methylation is a versible process of genetic modification,which can used by cancer or precancerous lesions,so we can achieve the purpose of cancer prevention by the way of restoreing the gene expression.Thus,DNA methylation have been provided a new idea in cancer treatment.Decitabine(decitabine,DAC),also known as 5-aza-2 ’-deoxycytidine,a cytosine nucleoside analogue for demethylation specific DNA transferase inhibitor,which have been studied as a demethylation can reverse CpG island methylation.In the purpose of treatment of cancer,it may inhibit tumor cell growth,because methylation or loss ascrible to many tumor suppressor gene expression。Arsenic trioxide(AS2O3)is the effective components of arsenic in Traditional Chinese Medicine and one of the classical drug for the treatment of acute promyelocytic leukemia.The drug play its role in two ways--inducing apoptosis and differentiation of leukemia cells.With the development of the drug treatment,have been used in other types of malignant hematological diseases at present.Nowadays,it has not only been applied to the patients with myelodysplastic syndrome,but also to the elderly patients with AML,especially relapsed and refractory AML.Therefore,it is an important task to study new target drugs to improve drug efficacy and prolong disease-free survival.The experimental study of DAC and AS2O3 on proliferation and apoptosis of HL-60 cells(human acute myeloid leukemia cell line),suggest that has an influence of changes in the level of gene and protein expression of the telomerase activity of HL-60 cells.So as to provide a theoretical basis for future clinical using,exploring the mechanism of demethylation of AML drugs is necessary.Methods:Selection the HL-60 cell lineas of the object of study,and randomly divided into groups: control group,DAC group(1μmol/L,10μmol/L,50μmol/L)and AS2O3 group(1.25μmol/L,2.5μmol/L,5μmol/L),DAC combined with AS2O3 group(10μmol/L+2.5μmol/L,10μmol/L+5μmol/L,50μmol/L+2.5μmol/L,50μmol/L+5μmol/L),DAC(50μmol/L)pretreatment+ AS2O3 group(1.25μmol/L,2.5μmol/L,5μmol/L).The effects of different concentrations of drugs on the proliferation and apoptosis of HL-60 cells and the expression of telomerase gene and protein were detected.Each experiment was repeated 3 times.1 The inhibition rates of 24 h,48h and 72 h on the proliferation of HL-60 cells were detected after different concentrations of drugs,CCK8 method was used to screen the rates of proliferation;2 Detect the apoptosis rate of HL-60 cells after different concentrations of 24 h and 48 h by flow cytometry(FCM);3 RT-PCR method was used to detect the effect of different concentrations of drugs in HL-60 cells for 48 h on expression of h TERT mRNA;4 Western Blot method was used to detect the effect of different concentrations of drugs in HL-60 cells for 48 h on protein expression of hTERT;5 All the data were analyzed with the SPSS 21.0 software.All the data were expressed by mean value of 3 independent experiments.The results of the experiment were all expressed by mean + standard deviation(X + S).The results of the two groups were compared with t test,and the difference between the two groups was analyzed by P<0.05.Results: 1 the proliferation of HL-60 cells by DAC and AS2O3 1.1 Different concentration of DAC,AS2O3 alone on the HL-60 cells after 24 h,48h,72 h,with the increase of drug concentration and the extension of time,the inhibition rate increased gradually.After cells were treated with single drug DAC,the inhibition rate was increased from(10.85±2.04)% to(56.01±2.04)%,AS2O3 inhibited the proliferation of HL-60 cells increased from(14.55±1.68)% to(85.09±3.67)%.There was significant difference between the treatment groups(P=0.00).These results indicated that DAC and AS2O3 could inhibit the proliferation of HL-60 cells in a time-dose-dependent manner.(Fig.1,Fig.2,Table 1,Table 2)1.2 DAC combined with AS2O3 on HL-60 cells 24 h,48h,72 h,with the increase of drug concentration and action time,the proliferation inhibition rate Increased from(35.68±1.98)% to(94.89±1.02)%,the treatment group with the single drug group compared the proliferation inhibition rate increased significantly,and the difference has statistical significance(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(50μmol/L)pretreatment,different concentrations of AS2O3 effect on HL-60 cells after 24 h,48h,72 h,increased with the drug concentration and action time,the proliferation inhibition rate increased from(26.98±0.67)% to(93.10±0.80)%.Compared with AS2O3 monotherapy,proliferation inhibition rate was significantly enhanced,and the difference was statistically significant(P=0.00).that 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 the different concentrations of DAC and AS2O3 on the apoptosis of HL-60 cells 2.1 Different concentration of DAC,AS2O3 in HL-60 cells in the role of 24 h,48h,72 h later,with the extension of drug concentration and action time,the apoptosis rate increased gradually.In DAC group the HL-60 cell apoptosis rate increased from(7.61±1.02)% to(36.33±0.51)%,In AS2O3 group the HL-60 cell apoptosis rate increased from(9.80±0.26)% to(40.56±0.90)%.There was significant difference between the treatment group and the control group(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 with AS2O3 on HL-60 cells 24 h,48h,72 h later with the increase of drug concentration and action time,the rate of apoptosis increased from(18.00±1.37)% to(57.76±0.61)%,The apoptosis rate of treatment group compared with single drug group was significantly enhanced,and the difference was statistically significant(P=0.00).The results showed that DAC combined with AS2O3 could induce apoptosis of HL-60 cells.(Fig.5,Table7)2.3 After the DAC(50 μmol/L)pretreatment with different concentrations of AS2O3 on HL-60 cells 24 h,48h and 72 h,with the increase of drug concentration and action time,the rate of apoptosis increased from(17.93±0.55)% to(60.96±0.70)%.Comprared with the AS2O3 monotherapy group,the difference was statistically significant(P=0.00).These results indicated that DAC pretreatment could enhance the effect of AS2O3 on apoptosis of HL-60 cells.(Fig.5,Table 8)3 Effects of DAC and AS2O3 on the expression of hTERT mRNA of the HL-60 cells 3.1 Different concentrations of DAC,AS2O3 monotherapy on the HL-60 cells after 48 h,with the increase of drug concentration,the expression level of hTERT mRNA decreased gradually.The expression of DAC(0μmol/L、1μmol/L、10μmol/L、50μmol/L)on the amount of hTERT mRNA decreased from 0.993±0.007 to 0.578±0.004,while the expression level of AS2O3(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L)on hTERT mRNA decreased from 0.993±0.007 to 0.657±0.034.The difference and control group for between treatment group hTERT mRNA levels were statistically significant(P=0.00).The results indicated that DAC and AS2O3 single agent could decrease the expression of hTERT mRNA gene in HL-60 cells in a dose-dependent manner(Fig.6,Table9,Table10)3.2 Different DAC combined with AS2O3 treated on the HL-60 cells after 48 h,with the increase of drug concentration,the expression level of hTERT mRNA decreased from 0.528±0.022 to 0.213±0.023.The treatment group compared with single drug group and the control group,the expression level of hTERT mRNA gene was significantly decreased,and the differences were statistically significant(P=0.00).The results showed that DAC combined with AS2O3 could decrease the expression level of hTERT mRNA gene in HL-60 cells,and they had synergistic effect.(Fig.6,Table 11)3.3 After the DAC(50μmol/L)pretreatment,different concentrations of AS2O3 treated on the HL-60 cells after 48 h,with the increase of drug concentration,the expression of hTERT mRNA decreased from 0.161±0.022 to 0.065±0.008.Compared with the AS2O3 single drugs group,the expression level of hTERT mRNAgenes was significantly decreased,and the difference was statistically significant(P=0.00).The results indicated that DAC pretreatment could increase the inhibitory effect of AS2O3 on telomerase hTERT mRNA of HL-60 cells.(Fig.6,Table 12)4 Effects of DAC and AS2O3 on the expression of hTERT protein of the HL-60 cells 4.1 The effect of DAC、 AS2O3 on HL-60 cells in different concentrations of 48 h,the expression of hTERT protein decreased with the increase of drug concentration.The relative hTERT protein expression level of DAC(0μmol/L、1μmol/L、10μmol/L、50μmol/L)group was decreased from 1.046±0.073 to0.576±0.063,and which in AS2O3(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L)group decreased from 1.046±0.073 to 0.426±0.014.There was significant difference between the treatment group and control group(P=0.00).The results indicated that DAC and AS2O3 could decrease the expression level of telomerase in HL-60 cells in a dose-dependent manner.(Fig.7,Table 13,Table 14)4.2 The effect of DAC combined with AS2O3 on HL-60 cells after 48 h treatment,with the decrease of drug concentration,the relative expression of hTERT protein decreased from 0.674 ± 0.016 to 0.111 ± 0.015,and the difference was statistically significant(P=0.00).The results showed that DAC combined with AS2O3 could increase the expression level of telomerase gene in HL-60 cells,and they had synergistic effect。(Fig.8,Table 15)4.3 The DAC(50 μmol/L)after pretreatment with different concentration of AS2O3 on HL-60 cells after 48 h,with the increase of drug concentration,the relative expression of telomerase and hTERT protein decreased from 0.311±0.015 to 0.055 ± 0.011,compared with AS2O3 monotherapy group the differences had statistically significant(P=0.00).The results indicated that DAC pretreatment could enhance the effect of AS2O3 on hTERT protein expression in HL-60 cells.(Fig.9,Table 16)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 manner.3 After decitabine pretreatment can increase the sensitivity of arsenic trioxide on HL-60 cells,the mechanism may be related with the decreased expression of telomerase hTERT and telomerase hTERT may be one of the regulatory gene of DAC induced apoptosis in HL-60 cells.. |
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