The Effects And Mechanisms Of Histone Deacetylase Inhibitor Combined With Arsenic Trioxide Acid On The Proliferation And Apoptosis Of HL-60 Cell

Objective: Acute myeloid leukemia(AML)is a class of hematological malignancies originating from hematopoietic stem cells.High-dose chemotherapy and hematopoietic stem cell transplantation after inducing combined chemotherapy are the main methods for the treatment of AML.But there are many problems still,such as primary drug resistance,relapse and refractory,large side effects of chemotherapy,transplantation donor limitation and GVHD(graft versus host disease),which may be eventually leading to treatment failure.In order to improve the effect of treatment,reduce the traditional side effects of chemotherapy,cross-drug resistance and other defects,the new therapeutic drugs and the role of the target needed to be explored.Telomerase is a reverse transcriptase,relying on its own RNA as a template to reverse the way to extend shortened telomeres gradually over cell division,which generates cell immortalization and cancer.Telomerase has high expression in acute myeloid leukemia cells and is confirmed to be a specific marker of the tumor.Studies have shown that active of telomerase,using the change of epigenetics to adjust the level of acetylation of histones,can regulate gene expression without changing the DNA sequence.In this way,can inhibit telomerase activity,and thus produce inhibition of tumor cell proliferation,prevent cell cycle,induce apoptosis and other anti-cancer effect.Histone deacetylases inhibitors(HDACi),for example,chidamide by inhibiting histone deacetylase to improve the level of cell protein acetylation,can selectively regulate a large number of gene transcriptions,and produce specific tumors killing effect.Arsenic trioxide can reduce the activity of telomerase in tumor cells,change the expression of cell cycle proteins and apoptosis-related proteins,inhibit tumor angiogenesis,inhibit tumor cellproliferation,regulate apoptosis gene and so on.Thus chidamide play a role in the treatment of acute myeloid leukemia.Therefore,by changing the level of histone acetylation,regulation of telomerase activity,chidamide provides a new way of thinking for the treatment of acute myeloid leukemia.Chidamide is a new chemical structure of our new research and listed as a national patented histone deacetylase inhibitor(HDACi).It is also a new type of anti-tumor drugs.Chidamide has been successfully applied to a variety of blood system tumors,such as cutaneous T cell lymphoma,peripheral T cell lymphoma,and vascular immune T cell lymphoma,and the drug is studied for a variety of clinical trials in treating malignant tumors.Arsenic trioxide is the main component of traditional Chinese medicine arsenic,and has definite effect on acute promyelocytic leukemia(APL),which can induce the differentiation and apoptosis of promyelocytic leukemia cells.US NCCN AML treatment guidelines suggest that when the primary disease does not relieve or relapse,it can be used as the preferred treatment of alternative treatment.At the same time,arsenic trioxide in combination with a variety of anti-tumor drugs has synergistic effect,in particularly,increasing the therapeutic effect,reducing the dose of single dose caused by excessive toxicity.In clinical applications,arsenic trioxide played a very good therapeutic effect.In recent years,histone deacetylase inhibitors as hot anti-tumor target in research field,has been used in a plenty of clinical and experimental researchs.For AML patients who have been failed or intolerate in standard chemotherapy regimens,even recurrence,seeking a relatively safe and effective treatment is an important issue.Therefore,this topic uses chidamide,arsenic trioxide and the combination of two drugs in the HL 60 cells(human acute myeloid leukemia cell line),to observe the effects of chidamide,arsenic trioxide and two drugs on the apoptosis of HL-60 cells.To investigate the effect of telamylase reverse transcriptase on the apoptosis of acute myeloid leukemia cells and the mechanism of telomerase activity in the treatment of acute myeloid leukemia cells,and to explore the effects of chidamide,arsenictrioxide and their combination on the selective induction of acute myeloid are the aims of the study.To explore the mechanism of leukemia cell apoptosis,in order to broaden the strategy of clinical treatment for acute myeloid leukemia patients,and improve the overall survival rate,providing more theoretical basis is important.Methods: AML HL-60 cells selected as the research object and randomly grouped into: blank control group,chidamide group(0.5?mol/L,2?mol/L,4?mol/L)and AS2O3 group(1.25?mol/L,2.5?mol/L,5?mol/L),chidamide combined with AS2O3 group(0.5?mol/L+1.25?mol/L,4?mol/L+5?mol/L).Evaluating proliferation and apoptosis of HL 60 cell in different concentration of drug intervention after different time;Evaluating h TERTm RNA telomerase activity and subunits(telomerase reverse transcriptase m RNA)expression level of HL 60 cells in different drug-concentrations interfereing with the different time;Discussing the apoptosis mechanism of HL 60 cell in different groups.Each experiment is repeated three times in each group.1.Using CCK8 method to explore the effect of different concentrations of drugs for 24 h,48h,72 h on the proliferation inhibition rate of HL-60 cells.2.Using flow cytometry(FCM)to study the effect of different concentrations of chidamide and arsenic trioxide for 24 h,48h,72 h on the apoptosis rate of HL-60 cells.3.Using RT-PCR method to detect the effect of different concentrations of drugs in HL-60 cells for 48 h on expression of h TERTm RNA.4.Using Western Blot method to detect the effect of different concentrations of drugs in HL-60 cells for 48 h on expression of h TERT protein.5.Using statistical methods to analyze the results.Results:1 Effects of chidamide and AS2O3 on the proliferation of HL-601.1 Different concentrations of chidamide,AS2O3 respectively act on the HL-60 alone for 24 h,48h,72 h,with the increase of drug concentration and the prolongation of time,the number of apoptotic cells in HL-60 increase,theinhibition of proliferation is obvious,and the inhibition rate of proliferation increase gradually.Chidamide on HL-60 cell,proliferation inhibition rate is(16.01 + 0.34)% to(97.09 + 0.91)%,The inhibition rate of AS2O3 on HL-60 cells is increased from(13.93±1.03)% to(84.40±1.50)%.The difference between the treatment groups is statistically significant(P=0.00).It is said that chidamide,AS2O3 single drug can inhibit the proliferation of HL-60 cells,and have dose-time dependence.(Fig.1,Fig.2,Table 1,Table 2)1.2 Different concentrations of chidamide combined with AS2O3 act on HL-60 cells 24h,48 h,72h,With the increase of drug concentration and the extension of time,the inhibition rate increase from(70.34±1.59)% to(98.22±0.77)%,and the inhibition rate of HL-60 cells in the combination of the two drugs is significantly higher than that of the single drug group,The difference between the treatment groups is statistically significant(P=0.00).That chidamide joint AS2O3 has a synergistic effect on HL-60 cell proliferation inhibition.(Fig.3,Table 3)2 Effects of chidamide and AS2O3 on apoptosis of HL-60 cells2.1 Different concentrations of chidamide,AS2O3 respectively act on the HL-60 alone for 24 h,48h,72 h,,with the increase of drug concentration and the prolongation of time,the apoptosis rate increase gradually.The HL-60cells' apoptosis rate of Chidamide is increased by(7.30 + 0.47)% to(25.62 +1.53)%,the HL-60 cells' apoptosis rate of AS2O3 is increased by(12.16 +1.08)% to(40.97 + 1.73)%.The difference between the treatment groups is statistically significant(P=0.00).It is said that chidamide,AS2O3 alone can induce apoptosis of HL-60 cells,and has dose-time dependence.(Fig.4,Table4,Table 5)2.2 Different concentrations of chidamide combined with AS2O3 act on HL-60 cells 24h,48 h,72h,with the increase of drug concentration and the prolongation of time,the apoptosis rate is increased from(19.56±0.77)% to(78.72±2.47)%,and the apoptosis rate of each treatment group is significantly higher than that of the single drug group.There is statistical difference between the treatment groups and the control groups(P=0.00).It is said thatchidamide joint AS2O3 has a synergistic effect on the induction of apoptosis in HL-60 cells.(Fig.4,Table 6)3 Effects of chidamide and AS2O3 on the expression of h TERTm RNA in HL-60 cells3.1 Different concentrations of chidamide,AS2O3 respectively act on the HL-60 alone for 48 h,with the increase of drug concentration,the expression level of h TERTm RNA decreases gradually.The expression of chidamide on the amount of h TERTm RNA from 0.95±0.02 reduces to 0.72±0.05,the amount of h TERTm RNA on the expression of AS2O3 reduces from0.91±0.01 to 0.66±0.02.There is statistical difference between the treatment groups and the control groups(P=0.00).It is said that chidamide,AS2O3 alone can reduce the expression level of h TERTm RNA in HL-60 cells,in a dose-dependent manner.(Fig.5,Fig.6,Table 7,Table 8)3.2 After 48 h of different concentrations of chidamide combined with AS2O3 act on HL-60 cells,with the increase of drug concentration,the expression of h TERTm RNA decreases from 0.55±0.02 to0.15±0.02.There is statistical difference between the treatment groups and the control groups(P =0.00).It is said that chidamide joint AS2O3 can reduce the expression level of h TERTm RNA in HL-60 cells.(Fig.5,Fig.6,Table 9)4 Effects of chidamide and AS2O3 on the expression of h TERT protein in HL-60 cells4.1 Different concentrations of chidamide,AS2O3 respectively act on the HL-60 alone for 48 h,the expression of h TERT protein decreases with the increase of drug concentration,chidamide in HL-60 cells after 48 h,the expression of h TERT protein decreases to 0.39±0.03 from 0.88±0.09,AS2O3 in HL-60 cells after 48 hours,the expression of h TERT protein declines from0.88±0.02 to 0.40±0.03.There is statistical difference between the treatment groups and the control groups(P=0.00).It is said that single drug chidamide,AS2O3 could reduce the expression level of h TERT protein in a dose-dependent manner.(Fig.7,Fig.8,Table 10,Table 11)4.2 Different concentrations of chidamide combined with AS2O3 act on HL-60 cells after 48 h,with the increase of drug concentration,the expression of h TERT protein decreases gradually from 0.63±0.05 to 0.17± 0.02.Compared with the control group,the expression of h TERT protein is significantly decreased in the combined group.The difference between the groups is statistically significant(P=0.00).That chidamide joint AS2O3 can synergistically reduce the expression level of HL-60 protein in h TERT cells.(Fig.9,Table 12)Conclusions:1 Chidamide and arsenic trioxide alone can cause apoptosis and inhibit proliferation of HL-60 cells in both dose and time-dependent manner.2 Chidamide and arsenic trioxide has a synergistic effect,can enhance the effect of inhibition in HL-60 cells.3 Chidamide,alone or in combination with arsenic trioxide can inhibit the proliferation of HL-60 cells,induce apoptosis.The antitumor mechanism maybe relative to inhibit the expression of h TERTm RNA and h TERT protein and down-regulate the activity of telomerase.