Effect Of Decitabine On Multidrug Resistance Gene MDR1 And P170 In K562 Cells, K562/ADR Cells And Primary Drug-resistant AML Primary Cells

Objective: Acute myeloid leukemia(AML)is a type of clonal malignant disease characterized by abnormalities of hematopoietic stem cells.Chemotherapy and hematopoietic stem cell transplantation are the main treatment strategies.However,chemotherapy is prone to multidrug resistance(MDR)which leading to drug resistance and relapse of AML.The main mechanism of multidrug resistance is the over-expression of multidrug resistance gene MDR1 and its product P glycoprotein(P170)which caused by DNA methylation,and the drug is pumped out of the cells to reduce intracellular chemotherapy drug concentration,resulting in multidrug resistance.According to incomplete statistics,the incidence of drug resistance in patients with leukemia is about 30%.With the closed study of AML found that most of the AML patients with abnormal gene methylation.Decitabine(DAC),also known as DNA methyltransferases(DNMTs)inhibitors is a drug directed against the cell nuclear,DNMT is irreversibly inhibited by covalently binding to DNMT.Cause of DNA hypomethylation,activating silent tumor suppressor genes,and returning cells to normal terminal differentiation,senescence,and apoptosis.Decitabine has excellent clinical effects on myelodysplastic syndrome(MDS)and AML which significantly improved survival and overall response rates compared with those not treated by decitabine.This in vitro experiment suggests that decitabine can promote apoptosis and inhibit cells proliferation in resistant leukemic cells by inhibiting MDR1 gene expression and P170 protein activity.Methods: The experiment randomly divided into: the drug-free control group,decitabine group,cytosine arabinoside and aclacinomycin(AA)group,decitabine and AA group treated in K562 cells,K562 / ADR cells and primarydrug-resistant AML primary cells.Detect the three kinds of cells’ proliferation,apoptosis and the expression of MDR1 and P170 protein in different time.Each group was repeated 3 times.Results:1.The effect of different concentration of decitabine plus or not of AA on K562 cells,K562 / ADR cells,primary drug-resistant AML primary cells detected by CCK-8.With the increase of drug concentration and prolonged time,the proliferation inhibition rate gradually increased,and the difference was statistically significant(P<0.05).2.Assessment of apoptosis and P170 protein expression with FCMDifferent concentration of decitabine plus or not of AA can induce three kinds of cells apoptosis rate increased.The difference was statistically significant compared with the control group(P<0.05).The P170 protein expression levels which were decreased with increasing drug concentration.3.Test the mRNA expression levels of MDR1 with Realtime-PCRThree kinds of cells were treated with decitabine after 48 hours,with the increasing drug concentration,MDR1 mRNA expression decreased.The difference was statistically significant compared with the control group(P<0.05,P<0.01).Conclusions:1.Decitabine can inhibit the proliferation and induce the apoptosis of K562 cells,K562 / ADR cells,primary drug-resistant AML primary cells,with time and concentration dependency.Decitabine combined with AA in the treatment of primary drug-resistant AML primary cells is more effective than AA group.2.Under a certain concentration scope,decitabine can down-regulateMDR1 gene and P170 protein expression levels of the three kinds of cells which was the reason that inhibits cell proliferation and induces apoptosis.