| Objective：T cell lymphoma is a kind of malignant cloning hyperplasticdisease that comes from T lymphocytes.At present the main treatment is thestandard chemotherapy, poor effect and easy relapse.So we need to look forthe effective drugs and their combination treatments to improve the curativeeffect and the prognosis of T cell lymphoma patients.Epigenetics is ofparticular interest to the tumor therapy effect, including methylation treatmentand histone acetylation treatment.The domestic and foreign research has confirmed that lymphoma patientsand its cell lines exist in abnormal hypermethylation. Hypermethylationusually happens in the CpG abound island of tumor suppressor genes (such asP15INK4B gene, SHP-l gene, P53genes) promoter area, and lead to the genesilence. At the same time histone excessive acetylation also exists inlymphoma patients and its cell lines.Decitabine(DAC) is a nucleosideanalogue.Phosphorylated decitabine participates in DNA synthetic, and thencovalent bond to DNA phenol-o-methyl shift enzyme (DNMTs), restrain theactivity, cause low DNA methylation, activate the tumor-suppressor genes andcause cell differentiation and apoptosis. The FDA has approved decitabine tobe used in the treatment of myelodysplastic syndromes (MDS).The basicresearch shows that decitabine has the ability to induce P15INK4B genesdemethylation and to express enhancement in lymphoma cell lines Raji cell,thus inhibit tumor cell proliferation.Valproic acid sodium (VPA) is used totreat epilepsy since1970.Recent research shows that, VPA can restrainhistone acetylation enzyme, adjust the cell cycle, induce the apoptosis, andinhibit angiogenesis of lymphoma,and also have antitumor function tolymphoma. So DAC and VPA both have probable to combinate with eachother. To further confirm the mechanism of proliferation and apoptosis induced byDAC in lymphoma cells, this experiment choose human T cell lymphomaJurkat cell lines, to observe proliferation suppression and apoptosis-orientedfunction by DAC, and to observe the effect by the combination of the DACand VPA,and to detect the expression changes of the methylation enzymeDNMT3AmRNA.To provide new treatment theory basis for T cell lymphoma.Methods:1The conventional method to cultivate a human T cell lymphoma Jurkat celllines.The cells were half passed every24hours.Logarithmic growth cells wereused in the experiment.2MTT method to detect different concentrations DAC, VPA and two singledrug combination to the proliferation influence in Jurkat lymphoma cell: theconcentration of the DAC respectively10,50100μ mol/L,the concentrationof the VPA1,2,3mmol/L, DAC (10μ mol/L) respectively combinatedVPA1,2,3mmol/L three concentration.Observing Jurkat cells inhibition rateafter single drug groups were roled for24h,48h,72h, combined treatmentgroups were roled for48h.And JinZhengJun formula calculate combination issynergy or antagonist role, computation formula is: Q=EAB/(EA+EB-EAEB). EA and EB is respectively inhibition rate of each drug, EAB is theshare inhibition rate of the two drugs.Q quartile is more than0.85and lessthan1.15for adding role, Q quartile1.15for synergy, or0.85for antagonistrole.3AnnexinV/PI double staining to detect different concentrations of DAC,VPA single medicine and the joint use in Jurkat lymphoma cell apoptosisinfluence, the drug concentration was the same as MTT experiment.Observingthe apoptosis rate after single-agent24h,48h,72h,and the combination ofthe24h;4Half quantitative RT-PCR method to test the expression of DNMTs mRNAby DAC,VPA and two single drug combination in Jurkat cells: the drugconcentration was the same as MTT experiment, Observing DNMTs mRNAquantity after single-agent24h,48h,72h,and the combination of the48h. 5Statistical analysis:The software of SPSS13.0was used.Mean±standarddeviation expresses grouped data.The comparisons betwen two groups use ttest,One-way analysis of variance was used for comparing means in groupsmore than two.P<0.05was indicated statistical significance.Results:1Effect of DAC alone on the proliferation of Jurkat cells：Differentconcentration of DAC acted on Jurkat cells in the role of24h,48h,72h,found that the DAC inhibited Jurkat cell proliferation, and with the time, dosedependent. The proliferation inhibition rate increased from14.67%to80.6%.Satistical analysis between the treatment groups, and between the treatmentgroups and control group, the difference was statistically significant (P<0.05).2Effect of VPA alone on the proliferation of Jurkat cells： Differentconcentration of VPA acted on Jurkat cells in the role of24h,48h,72h,found that the DAC inhibited Jurkat cell proliferation, and with the time, dosedependent. The proliferation inhibition rate increased from14.47%to77.51%.Satistical analysis between the treatment groups, and between the treatmentgroups and control group, the difference was statistically significant (P <0.05).3Effect of DAC on the apoptosis of Jurkat cells: The results was analyzed byflow cytometry, with time extension, and drug concentration increased,cellapoptosis rate increased from18.9%to47.2%.Satistical analysis between thetreatment groups, and between the treatment groups and control group, thedifference was statistically significant (P <0.05).Jurkat cell apoptosis rate waspromoting with the DAC time dose dependent.4Effect of VPA on the apoptosis of Jurkat cells:Cell apoptosis rate increasedfrom10.2%to43.2%in the role of the influence of VPA.Satistical analysisbetween the treatment groups, and between the treatment groups and controlgroup, the difference was statistically significant (P <0.05).Jurkat cellapoptosis rate was promoting with the VPA time dose dependent.5Effect of DAC in combination with VPA on the proliferation and apoptosisof Jurkat cells:Cell growth inhibition rate after DAC (10μmol/L) and VPA1,2, 3mmol/L joint role in Jurkat cells for48h,compared with single drug,thedifference was statistically significant (P﹤0.05).Q value was ranged between0.85and1.15, and show the resust that VPA and DAC combinate on Jurkatcell,the effec twas Simple addition together. DAC and VPA combinate witheach other on Jurkat cells, that can enhance Jurkat cell growth inhibition effect.AnnexinV/PI double stainning measured24h apoptosis rate by threecombination compared with single drug,which was statistically significant (P﹤0.05), the joint action can increase cell apoptosis.6DAC and VPA on the influence of the DNMT3A mRNA expression:RT-PCR results showed that10,50,100μ mol/L three different concentrationof DAC in Jurkat cells24h~72h compared with the control group,DNMT3A mRNA expression level gradually reduced with time and drugconcentration, express quantity by0.67to0.30, each group was statisticallysignificant (P <0.05).1,2,3mmol/L three different density VPA acted onJurkat cells24h,48h,72h,compared with control group, DNMT3A mRNAexpression level had no change as VPA drugs concentration increased and theextension of time (P value>0.05). VPA acted on Jurkat cells, each groupcell methylation shift enzyme DNMT3A mRNA expression level wascompared no statistical difference (P﹥0.05).7VPA enhance the demethylation of DAC in Jurkat cells: RT-PCR resultsshowed that three different density VPA and10μ mol/L DAC two drugs in thejoint action on Jurkat cells after48h, Jurkat cells methylation shift enzymeDNMT3A mRNA was more reduced than a single drug, the largest amount ofDNMT3A mRNA expression level can drop to0.26;compared with singledrug effect0.33, statistical differences (P﹤0.05).Conclusions:1DAC inhibits Jurkat lymphoma cell proliferation, and promotes Jurkat cellapoptosis, in time and dose dependent.2VPA also has a time dose dependent for antiproliferaiton and proapopticeffects on Jurkat cells.3DAC can reduce the amount of DNMT3AmRNA expression, and with the time dose dependent.VPA has no significant effect on Jurkat's DNMT3AmRNA expression.4VPA can promote inhibition of proliferation and apoptosis-oriented effectinduced by DAC, increase to down the DNMT3AmRNA of Jurkat lymphomacells induced by DAC, both joint can enhance the sensitivity of Jurkat cells toDAC. |
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