Study Of The Effects Of Decitabine Combined With Valproic Acid On Proliferation And Apoptosis Of U937Cell Lines

Objective:At present, refractory and relapsed leukemia are still themain reasons to block the long survival, especially for elderly patients andgeneral situation poor patients who are not possible candidate for intensivechemotherapy. Epigenetic drugs bring a new way to treat these patients.Nowcommonly used drugs are DAN methyltransferase inhibitors and histonedeacetylase inhibitors.Decitabine (DAC) is DNA methyltransferase inhibitor, it can restrainDNA-methyltransferase activity and reverse tumor-suppressor genes abnormalmethylation to reactive silent tumor-suppressor gene and make the leukemiacell back to normal differentiation and apoptosis. Compared with intensivechemotherapy, DAC is effective,safe, treatment-related mortality is low, andbecomes a new choice for leukemia.Current research shows that histone acetylation drugs can interfere withthe cell cycle, induce cell differentiation and apoptosis,and discover themhave synergistic anti-leukemia with methyltransferase inhibitors.Valproic acidsodium (Valproic acid, VPA) is a traditional antiepileptic drug, in recent yearshas found it can inhibit histone deacetylase activity and has the ability toinduce leukemia cell apoptosis.In this study, we try to explore the effect of DAC combined with VPA onproliferation and apoptosis of U937leukemia cells line, research theinfluence of on DNA methyltransferase and tumor-suppressor genes P15atmRNA level, and discusse the mechanism of the drug.Method：1Conventionally cultivate people leukemia cell lines U973and take cells ofthe logarithm growing period to experience;2proliferation inhibition effect of DAC and VPA on U937was measured by MTT:U937cells were treated by DAC at the concentration of0.1,0.8,1.6mmol/L; VPA at1.0,3.0,5.0mmol/L; DAC (0.8mmol/1) combined VPA(1.0,3.0,5.0mmol/L)for24,48,72hours, cell proliferation inhibition ratewere detected with the MTT3The percentage of apoptosis cells was measured by flow cytometry andstained by Annexin V-FITC/PI,0.8,1.6mmol/L; VPA at1.0,3.0,5.0mmol/L;DAC (0.8mmol/1) combined VPA (1.0,3.0,5.0mmol/L) for72hours,PIstained, flow cytometric detect cell apoptosis4cell cycle distribution was evaluated by flow cytometry with PIstaining:U937cells were treated by DAC at the concentration of0.1,0.8,1.6mmol/L for72hours,PI stained and flow cytometric detect cell cycledistribution5The mRNA expression level of DNA methyltransferase DNMT1\DNMT3A\DNMT3B and cyclin-dependent kinase inhibitor p15wasmeasured with RT-PCR: testing the mRNA expression level of cells beforeand after treated with drug6SPSS16.0software analysis, grouped data are experienced with mean±standard deviation, comparison differences between two groups with Ttest,comparison differences among many groups with the single factoranalysis of variance, P <0.05means differences have statistical significanceResults:1The proliferation effect of DAC\VPA alone or combination of the twodrugs on the U937cell:the results show that with the increasing ofconcentration and the extension of time of drug, U937cell proliferationinhibition rate of DAC increased from10.1%to67.0%,the statistical analysisshow P <0.05meaning differences have statistically significance; cellproliferation inhibition rates of VPA increased from17.6%to61.8%, thecomparison show that VPA at5.0mmol/L has no statistical differences of cellproliferation inhibition rate between48hours and24or72hours,P>0.05, at1.0,3.0mmol/L of VPA, differences between each group P <0.05;The biggestcell proliferation inhibition of combination is89.2%,the differences among groups have statistical significance,P <0.05. U937cell proliferation inhibitionrate increase along with concentration and time gradually. Through thedomestic guinness formula calculation, the two drugs has synergy.2the effect of apoptosis of DAC, VPA alone and combination of the twodrugs on U937cell:The results show that the apoptosis rate of U937treatedwith DAC for72h increased from13.17%to30.53%, showing that the cellapoptosis rate increased with the drug concentration, obviously higher thanthose in the control group (P <0.05); treated with VPA, cell apoptosis rateincreased from28.61%to47.30%, meaning the cell apoptosis rate are dosedependent, the comparison among groups P <0.05;treated with DACcombined VPA, cell apoptosis rates were significantly higher than the singlemedicine groups, the cell apoptosis rate can be as high as68.40%, thecomparison among groups P <0.05, the results showed that the two drugs hassynergy to induce apoptosis.3The changes of cell cycle with DAC for72hoursThe results showed that DAC act in S phase cells, along with the increasing ofconcentration of DAC, the ratio of G0/G1phase cells increased from50.85%to68.9%, S phase ratio gradually reduce from47.78%to28.11%. Thedifferences among groups have statistical significance respectively (P <0.05)4RT-PCR detect DNMT1, DNMT3A, DNMT3B and P15mRNA expressionlevel:the results show that DNA methyltransferases DNMT1, DNMT3A,DNMT3B in U937cells can express but P15mRNA has no expression no useof drug.the expression level of DNMT1andDNMT3A reduce treated withDAC for72hours.the expression level of DNMT1reduce from6.012to0.521,the expression level of the DNMT3A reduce from5.039to0.628(P <0.05),P15mRNA appears after treated with DAC and the quantity increased with theconcentration. P15mRNA increased from no to2.078, and VPA can enhancethe action of DAC to induce P15mRNA expression, two drug combination canmake P15mRNA increase to3.911(P <0.05), DNMT3BmRNA expressionhas no changes (P>0.05). Conclusions：1The DAC and VPA can inhibit the proliferation of U937cells significantly,and the two medicine are synergistic.2DAC and VPA can induce U937to apoptosis and two drugs aresynergistic;3DAC can reduce S phase U937cells and increase G0/G1phase cells;4The mechanisms of action of DAC are associated with suppressing DNMT1mRNA and DNMT3AmRNA expression and activating P15mRNAexpression and VPA can enhance DAC to induce P15mRNA expression.