The Effect Of HDAC Inhibitor VPA On Inducing Leukemia Cells To Differentiate Into Leukemia-DCs And Approach To Its Key Mechanism

BackgroundThe immunotherapy based on dendritic cells (DCs) plays an important role in prolonging disease-free survival and eliminating minimal residual disease (MRD) after chemotherapy in patients with leukemia. The DCs are specialized antigen presenting cells with a unique ability to induce primary T cell response. Loaded with leukemia antigens, DCs were re-injected into the patients through different ways and then DCs can migrate to the T-cell areas of lymphoid organs to activate T cells and induce specific anti-leukemia immune response.The function of DCs in patients with leukemia is deficient and insufficient, such as the lack of MHC and co-stimulatory molecules on the surface of DCs, can lead to the deficiency in activating of T-cells to start effective anti-leukemia immune responses; on the other hand, the leukemia cells in growth can secrete many immunosuppressive factors such as interleukin-10 (IL-10), transforming growth factor-β(TGF-β) and vascular endothelial growth factor (VEGF), which can lead to functional defect of DCs; moreover, the immature DCs in patients with leukemia might induce regulatory or suppressive T cells impairing the quality of anti-leukemia immune responseand, and so on. Therefore, how to improve the function of DCs in patients with leukemia is the focus of current attention. As for a source of DCs, excepting for the CD34~+ hemopoietic stem cells and the CD14~+ monocyte, some Scholars tried to inducing leukemia blasts to differentiate into DC, and have been successful. Leukemia derived DCs not only act as antigen presenting cells, but also carry leukemia antigens, which solved the problem that most of the leukemia cells lack the specific antigen and it become an important source of DC vaccine. However, it was found that the traditional induing agents such as cytokines (CK) and calcium ionophore (CI) so far can not induce all subtypes of leukemia cells to differentiate into DCs. Otherwise, the leukemia-derived DCs have many defects, such as a long-induced cycle, low numbers and maturity is not high, which was easy to induce immune tolerance and restricted the clinical application of leukemia-derived DCs. Therefore some laboratories focus on exploring some more efficient inducing profile to induce leukemia DCs.Histone deacetylase inhibitors (HDACi) is one kind of compounds for regulating gene expression at the transcriptional level, it plays an important role in anti-tumor treatment, the mechanism include inhibiting cell proliferation, promoting apoptosis, blocking the cell cycle and inducing cell differentiation, and so on. It has been demonstrated that the key mechanism of leukemia was related to the abnormal recruitment of HDAC on some terget genes, which blocked the differentiation of leukemia. According to the above-mentioned theories and experiment results, we concluded that HDACi should play an important role not only the differentiation treatment of leukemia, but also inducing leukemia cells to differentiate into DCs as a new inducing drug.ObjectiveBased on the background above, we selected the combination of valproate acid (VPA) which a representative drugs of HDACi, CK and A23187 as the breakthrough point, observed the ability of the combination to inducing leukemia cells differentiation into DC, and hope to establish a more maturate and effective inducing system, and offer a new choose for expansion of DC in vitro. MethodsLeukemia cells (K562 cells and THP-1 cells) were treated with VPA, CK combination (GM-CSF, IL-4) and A23187 and induced to differentiate into DC, which choosing CK combination and A23187 as the positive control groups, taking VPA with CK combination and (or) A23187 as the experiment groups, CK combination group acted on 10 days, on the 8th day of culture, TNF-αwas added to the medium for the next 2 days of culture, other groups acted on 4 days, half medium was changed every other day and added drugs to initial concentration. The change of cell morphology was observed by inverted microscope during the whole process. The expressing of cell surface markers of DCs were detected by flow cytometry after induction finished, RT-PCR analyzed the changes in gene levels, mixed lymphocyte reaction (MLR) detected the ability to stimulating lymphocyte proliferation. Dates are expressed as mean and standard deviations. Comparisons between the treatments and control were performed using the t-test and one-way anova. A P-value<0.05 was considered to be statistically significant.ResultsWhile treating K562 and THP-1 cells with VPA alone, we can not observe typical dendritic-like morphology under Inverted microscope; but the expression of DCs marks (CD1a, CD83, HLA-DR, CD86) on K562 cells surface are significantly increased compared with the negative control group, and THP-1 cells can not test the same change; and the ability to stimulating lymphocyte proliferation have not been enhanced by VPA alone.While treating with VPA combined with CK and A23187, the morphology of K562 cells still have not significant change, but the DCs marks of cell surface were significantly increased compare with alone, parts of the marks have significant statistical difference; and the ability to stimulating lymphocyte proliferation also significantly increased.ConclusionsVPA can not induce leukemia to differentiate into DC by itself, but while combined with CK and A23187, it can obviously increase the ability of CK and A23187 to inducing leukemia cells to differentiate into DC, and shorten the differentiation cycle by CK, increase the purity and muture of leukemia derived DC, and improve the ability to stimulating lymphocyte proliferation. So that the combined scheme is a more effective way in inducing leukemia cells to differentiate into DCs.