| Background:Acute myeloid leukemia(AML)is a myeloid,heterogeneous,clonal disease that is the most common type of leukemia in adult acute leukemia.At present,AML treatment mainly includes chemotherapy,hematopoietic stem cell transplantation,targeted therapy and immune cell therapy.multi-drug combined chemotherapy and hematopoietic stem cell transplantation have significantly improved the prognosis of AML.However,due to the aging of the incidence of AML,primary or recurrent drug resistance,serious side effects associated with chemotherapeutic drugs,lack of suitable donor for transplantation and risk associated with transplantation,and so on.Only a few patients have long-term survival due to their inability to carry out routine chemotherapy.Therefore,it is of great significance to explore new treatment methods to improve the long-term survival and quality of life of AML patients.In recent years,with the deepening of epigenetics research,it has been found that it also plays an important role in the occurrence and development of AML.The most important factors in the regulation of AML epigenetic abnormalities are abnormal methylation of DNA and deacetylation of histone.Therefore,the apparent genome of AML has become a new target for the treatment of demethylation drugs combined with histone deacetylase inhibitors.Objective:In this study,we investigate the proliferation and apoptosis of DNA methyltransferase inhibitor decitabine(DAC)alone or comibined with histone deacetylase inhibitors chidamide(CS055)by different concentrations,and the regulation of DNA methyltransferase 1(DNMT1)expression and histone H3 acetylation(Ac-H3)level.The aim of this study is to explore the possible mechanism of the combination of the two drugs and to provide theoretical basis for clinical application.The aim of this study is to explore possible mechanisms of action between the two drugs,and to provide theoretical andexperimental evidence for clinical application.Methods:1.The proliferation inhibition rate of chidamide alone or comibined with decitabine by different concentrations on U937 cells for 24 h,48h and 72 h was assessed by MTT assay.2.The apoptosis of chidamide alone or comibined with decitabine on U937 cells for72 h was detected by flow cytometry(FCM).3.The expression of DNMT1 m RNA was detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)in U937 cells treated with chidamide alone or comibined with decitabine for 72 hours.4.The expression of acetyl histone H3 and DNMT1 were analyzed by Western blot in U937 cells treated with chidamide alone or comibined with decitabine for 72 hours.Results:1.The results of MTT assay showed that the growth of U937 cells was significantly inhibited after treatment of U937 cells with different concentrations of CS055 and DAC for24 h,48h and 72 h.With the increase of CS055 and DAC drug concentration and the prolongation of intervention time,compared with the blank control group,the inhibition of U937 cells by CS055 and DAC groups was more and more obvious,and the difference was statistically significant.It indicated that CS055 and DAC showed a significant dose-time inhibition rate of U937 cell proliferation,and the IC50 values of CS055,DAC at 72 h were0.42μmol/L and 5.33 μ mol/L,respectively.The inhibition of the combination of the two drugs in the same period of time was stronger than that of the single drug.The analysis of variance analysis showed that there was a significant synergistic anti-leukemia effect between the two drugs.Through statistical factorial design analysis,there was interaction between the two drugs(F values for 24 h,48h and 72 h were 9.230,7.077 and 22.250.P <0.01,respectively),which indicated that there was obvious synergistic antileukemia effect between the two drugs.2.The results of FCM assay showed that the apoptosis rate of the 72 h time piont control group was(0.67±0.12)%,while the observation group 0.25μmol/LCS055,2.5μmol/LDAC apoptosis rates were(23.43±0.50)%,(8.47±0.32)%.The apoptosis rate of the combined drug group was(32.73 ±0.42)%,which was significantly higher than that of the single drug group.There are statistically significant differences between group(P<0.01).3.The results of RT-PCR showed that the expression of DNMT1 m RNA in U937 cells treated with 0.25 μmol/L CS055,2.5 μmol/L DAC for 72 h was significantly lower than that of the control group.The combination of the two drugs was more obvious than the single drug group,and there was statistical difference(P<0.01).4.Western blot analysis showed that the expression of DNMT1 protein decreased significantly and the level of histone H3 acetylation increased significantly in U937 cells treated with 0.25 μmol/L CS055,2.5μmol/L DAC for 72 h compared with the control group(all P<0.01).In the combination of the two drugs,the decrease of DNMT1 protein and the increase of histone H3 acetylation were significantly higher than those in the single drug group,and there was statistical difference(P<0.01).Conclusions:1.CS055,DAC could inhibit the growth of leukemia cell line U937 in a time-dose-dependent manner.2.After treatment with CS055,DAC,the expression of DNMT1 decreased and the level of Ac-H3 increased in U937 cells.3.CS055 combined with DAC has synergistic effect on proliferation inhibition and apoptosis of U937 cells.The mechanism is that histone deacetylase inhibitors have demethylation effects and demethylation drugs also have effect of histone deacetylase inhibitors.The combination of both enhances demethylation and enhances acetylated histone action. |
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