Proliferation And Apoptosis Of T-lymphoma Cell Lines Jurkat And Multiple Myeloma U266Cell Line Treated With Decitabine

Objective： Decitabine (Decitabine, DAC) is a nucleoside analogue,decitabine phosphorylation involved in DNA synthesis, covalent binding andDNA methyltransferase (DNMTs), inhibiting its activity, resulting in lowDNA methylation, activation of tumor suppressor genes cause celldifferentiation or apoptosis. The FDA has officially approved decitabine forthe treatment of patients with myelodysplastic syndrome (MDS). Decitabinetreatment refractory acute myeloid leukemia, recent these reports also oftenfound.While decitabine treatment of tumors of the lymphatic system reportedless.Domestic and foreign research has confirmed abnormal hypermethylationexist in lymphoma patients and their cell lines, often occurs in the rich CpGislands of tumor suppression gene,(e.g. P15INK4B gene, SHP-1gene, P53gene) promoter region, and lead to the silencing of the gene. The basis ofstudies have shown that the DAC is capable of inducing lymphoma cell lineRaji in P15INK4B, gene demethylation and enhanced expression, therebyinhibiting tumor cell proliferation. Recent studies have shown that Gore andGarcia-Manero reported respectively with5-azacytidine and butyric acidbenzyl ester, decitabine and valproic acid (butyric acid benzyl ester, valproicacid group protein deacetylase inhibitor) jointmedication, leukemia and T-celllymphoma patients, the first and the second phase of clinical trials are showingobvious synergies. And studies have shown that in vivo, decitabine canincrease the sensitivity of the cells to biological therapy, such as retinoic acid,and to increase the expression of apoptotic factors.Multiple Myeloma (Multiple myeloma, MM)occurred in the malignantplasma cell disease in B lymphocytes, and still can not be cured. T-celllymphoma (T-cell lymphoma), T-cell NHL predilection in the Asian region, sources with B-cell NHL as invasive, is a derived from malignant clonalproliferation of T lymphocytes diseases, but the prognosis is more poor.Although the T-cell NHL achieved remission after conventional treatment insome patients, but a part remission in the short run, there may be a recurrenceor progression, currently with relapsed or refractory T-cell NHL, no standardsalvage therapy program. Need to find effective drugs and their combinationregimens to improve the efficacy of T-cell lymphoma patients improveprognosis. Epigenetic therapeutic effect of the tumor growing attention, whichmainly include the demethylation treatment.For further exploring mechanisms of proliferation and apoptosis withdecitabine in lymphoma cell, we choose multiple myeloma cell line U266,human T-cell lymphoma Jurkat cells in this experiment, to observe U266celllines, Jurkat cells inhibition of proliferation and induction of apoptosis andinfluence with decitabine.In addition, T lymphocytes involved in the immune response, their effecton tumor cell killing and inhibition of tumor growth plays an essential role. Tlymphocytes, respectively, expression of the two major subsets of CD4andCD8molecules, which are referred to as T Helper Lymphocytes (Th) and TCytotoxic T lymphocyte (Tc).Th1-type cytokines including INF-α, IL-2, andtumor necrosis factor-β (tumor necrosis factor-β, TNF-β)-mediatedcellular immune response, and the body’s anti-tumor, antiviral activity relatedTH2cytokines including, IL-4, IL-5, IL-6, IL-10, IL-13, may also includeIL-9, mediated by humoral immune responses, with graft tolerance, inhibitionof autoimmunity. The present study shows that lymphocytes can effectivelysuppress the growth of malignant tumor cells through the activation ofcytokines such as INF-α, TNF, Th and Tc cells, and most of the presence of atumor patient is often a very low immune function According to studiesreported in this mainly due to Since the tumor cells secreteimmunosuppressive factors, while inhibition of T lymphocyte function.Decitabine impact on the function of T lymphocytes, has not been reported yet.Jurkat human T lymphoma cell line can secrete IL4, IFN-gamma and IL-10, Jurkat cells as a template for in vitro study of T cell differentiation, analogdecitabine T lymphocyte function. Detection DAC T cells secrete cytokinesIL-4, IL-10and INF-α level of impact, and thus provide a theoretical basisfor the immunotherapy of malignant lymphoma patients.Methods:1The conventional method cultured U266cell line, human T-celllymphoma Jurkat cells.The cells were half passaged once per24hours.Logarithmic growth cells were used in the experiment.2CCK-8method to detect different concentrations DAC to U266cells,Jurkat lymphoma cell proliferation: the DAC concentration, respectively, for50、100、250μg/ml; Observing U266cells and Jurkat cells inhibitionrate after drug groups were roled for24h、48h、72h3AnnexinV/PI double staining to detect different concentrations ofDAC use in U266cells and Jurkat lymphoma cell apoptosis influence, thedrug concentration was the same as CCK-8experiment.Observing theapoptosis rate after24h、48h、72h4Enzyme-linked immunosorbent assays (ELISA) experiments, theconcentration of drug action with CCK-8experiment, to observe change ofexpression of the cytokines in Jurkat lymphoma cells by the role of decitabinefor48h5Statistical analysis:The software of SPSS13.0wasused.Mean±standard deviation expresses grouped data.The comparisonsbetwen two groups use t test,One-way analysis of variance was used forcomparing means in groups more than two.P<0.05was indicated statisticalsignificance.Results:1The affect of decitabine on U266cell proliferation and apoptosisDecitabine on the proliferation of U266cells with different concentrationsof DAC role in U266cells24h,48h,72h, proliferation inhibition rate of23.6±0.12%with the increase of the concentration of the drug action andprolonged duration of action, increased to65.06±0.03%, the statistical analysis between the treatment groups, and between the treatment and control groups,the difference was statistically significant (P <0.05).Decitabine on the apoptosis of U266cells by flow cytometry analysisshowed that, DAC act on U266cells, extended over time and the increase ofdrug concentration, the apoptosis rate from9.7±0.74%to69.4±0.24%Statistical analysis showed that between each treatment group, the treatmentgroup and the control group were statistically significant (P <0.05) DACpromote apoptosis in Jurkat cells was dose and time dependent.2The affect of decitabine on Jurkat cell proliferation and apoptosisDecitabine on the proliferation of Jurkat cells with different concentrationsof DAC role in Jurkat cells24h,48h,72h, proliferation inhibition rate of17.53±0.02%with the increase of the concentration of the drug action andprolonged duration of action, increased to83.63±0.57%, the statistical analysisbetween the treatment groups, and between the treatment and control groups,the difference was statistically significant (P <0.05).Decitabine on the apoptosis of Jurkat cells by flow cytometry analysisshowed that, DAC act on Jurkat cells, extended over time and the increase ofdrug concentration, the apoptosis rate from29.9±0.17%to82.4±0.03%Statistical analysis showed that between each treatment group, the treatmentgroup and the control group were statistically significant (P <0.05) DACpromote apoptosis in Jurkat cells was dose and time dependent.3Decitabine affect Jurkat cells functionDecitabine affect Jurkat cells function by enzyme-linked immunosorbentassay analysis showed that decitabine can promote Jurkat cells secretecytokines,Low concentration group decitabine can significantly promote thesecretion of IL-4, decline the ratio of Th1/Th2, enhanced immune tolerance;,medium and high concentrations of decitabine can significantly promote thesecretion of INF-α, the ratio of Th1/Th2obviously increased, enhancedanti-tumor function.Statistical analysis showed significant difference betweenthe blank control group and the treatment group (P <0.05)Conclusions: 1DAC inhibits U266cell proliferation, and promotes Jurkat cellapoptosis, in time and dose dependent.2DAC inhibits Jurkat lymphoma cell proliferation, and promotes U266cell apoptosis, in time and dose dependent.3Decitabine can promote T lymphocyte secretion of IL-4, IL-10andINF-α cytokines,enhanced T helper lymphocyte function.