Experimental Application Of IL-24 In Basic Study On Myelogenous Leukemia Biotherapy

Part Ⅰ The effect on biological functions of CIK cells with supplement of IL-24 to the culture medium systemObjective:To explore the effect of IL-24 on biological functions of CIK cells and find an effective method to enhance the cytotoxic activity of CIK cells.Methods: CIK cells were induced by culturing human peripheral blood mononuclear cells(PBMC) in the culture medium with or without IL-24. The cells obtained were named CIK-IL-24 or CIK. The proliferation of CIK cells was evaluated by cell number counting method. The phenotypes of CIK cells were examined by flow cytometric analysis, the concentration of IFN-γ and TNF-α in supernatant of CIK was determined by ELISA. The ability of CIK to produce perforin, Granzyme B and IFN-γ was measured by FCM and homolysis method. MTT and FCM were used to determine the cytotoxicity of CIK cells to the leukemia cells.Results: CIK cells were successfully prepared. The proliferation rate of CIK cells without IL-24 treatment was higher than that with IL-24 treatment. Although the expression rate of CD3+、CD4+and CD3+CD56+ cells had no significant change in CIK cells, supplement of IL-24 to CIK culture medium significantly augmented the number of CD3+CD8+, CD3+CD107a+ and CD8+TCM cells. The expression of Granzyme B and IFN-γ in CIK cells was significantly increased, and the expression level of adhesion molecule CD54 on CD3+ cells was also increased. The rate of CD4+CD25+CD127- cells was significantly decreased compared with that of the control group. CIK-IL-24 cells produced markedly higher levels of IFN-γ and TNF-α as compared with the CIK cells. CIK cells could efficiently induce apoptosis of various leukemia cells. In comparison with CIK cells without IL-24 treatment, CIK cells with IL-24 treatment had a significantly higher lytic activity against leukemia cells.Conclusion: CIK cells were successfully prepared by in vitro differentiation of human PBMC in culture medium containing the cytokines, and they had normal biological functions. IL-24 could enhance the functions of antitumor immunity of CIK cells, the mechanism of which might be associated with the increased number of CD3+CD8+ and CD8+TCM cells, high level secretion of IFN-γ and TNF-α, up-regulation expression of CD107 a, Granzyme B and adhesion molecules, reduction of the rate of regulatory T cells in CIK cells. Part Ⅱ Enhanced cytotoxicity of cytokine-induced killer cells co-cultured with IL-24 gene-modified dendritic cells to leukemia cellsObjective:To study the antitumor effect and mechanism of CIK cells co-cultured with autologous IL-24 gene modified DC on leukemia cells.Methods: DC and CIK cells were prepared routinely from PBMC. Recombinant adenovirus AdVGFP/IL-24(Ad-IL-24) expressing IL-24 was constructed. IL-24 gene was transduced into DC via Ad-IL-24, which was already loaded with tumor antigen. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in DC-IL-24. After 72 hours of transfection with Ad-IL-24, IL-10 was added in the culture medium. The phenotype changes of DC were examined by flow cytometric analysis, the concentration of IL-12 and TNF-α in supernatant of DC was determined by ELISA. FCM was used to determine the cytotoxicity of CIK cells co-cultured with autologous IL-24 gene modified DC to the leukemia cells.Results: Dendritic cells and CIK cells were successfully prepared by in vitro differentiation of PBMC in medium containing corresponding cytokines. The high titre IL-24 recombinant adenovirus(Ad-IL-24) was obtained. IL-24 gene was transferred into DC with high efficiency via adenovirus-mediated infection. The percentage of GFP+ DC detected by fluorescence microscopy was markedly high. The expression rates of CD80, CD83, HLA-DR, CD40 and CXCR4 on DC-IL-24 were significantly increased compared with those of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with those of DC. IL-10 had an obviously negative effect on the maturation of the monocyte-derived immature DC, and could drive immature DC to differentiate into the granulocytes or macrophages. However, IL-10 had not negative effect on DC-IL-24. In comparison with CIK cells co-cultured with non-transfected DC, CIK cells co-cultured with transfected DC had a significant higher lytic activity against leukemia cells. Moreover, this effect could not be suppressed by IL-10 in the culture medium.Conclusion: IL-24 gene-modified DC co-cultured with CIK cells elicited a prominent augmentation of antitumor immunity against myelogenous leukemia cells, which may be closely associated with the up-regulation expression of co-stimulatory molecules on DC, increased Th1-type cytokine production, promoted activation and maturation of DC, and subsequent activation of CIK cells leading to the generation of specific immunologic effector cells. Furthermore, this effect could not be inhibited by IL-10 in the medium.Part Ⅲ Antitumor effect and underlying mechanism of RGD-modified adenovirus mediated IL-24 expression on myeloid leukemia cellsObjective: To construct an RGD-engineered recombinant adenoviral vector, Ad.RGD-IL-24, and assess its effects on human myeloid leukemia cells.Methods: RGD-engineered recombinant adenoviral vector Ad.RGD-IL-24 was constructed. IL-24 gene was transduced into human myeloid leukemia cells via Ad.RGD-IL-24. Cellular morphologic changes associated with differentiation were detected with Wright-Giemsa staining. The expression of CD11 b, CD14, CD36 and CD41 was estimated by FCM. The effect of ectopic expression of IL-24 on the growth of the K562, K562/A02, THP-1 and MEG-01 cells was determined by MTT assay. Cell-cycle distribution and apoptosis of the leukemia cells were determined by FCM. Quantitative Real-Time PCR and western blot analysis were used to detect the expression levels of apoptosis-associated genes GRP78/Bip, GADD34, GADD153, GADD45α, Bax, Bcl-2 and Mcl-1. The activation of Caspase-3 was detected by the spectrophotometric method.Results: We constructed an RGD-engineered recombinant adenoviral vector Ad.RGD-IL-24. It mediates gene transfer into myeloid leukemia cells with high efficiency. Ectopic over-expression of IL-24 has significant growth inhibition and differentiation inducement effects on these cells. Treatment with Ad.RGD-IL-24 could potentially induce leukemia cells’ cell-cycle arrest. In addition, IL-24 expression could significantly induce apoptosis of the THP-1 cells, but no difference in the apoptotic rate was observed in the cell line of K562 or K562/A02. Ad.RGD-IL-24 had a potent effect on the up-regulation of the expression of GRP78/Bip, GADD34, GADD153, GADD45α and Bax, down-regulation of the expression of Bcl-2 and Mcl-1, and induction of the activation of Caspases-3, which may be responsible for its apoptosis-inducing effect on THP-1 cells.Conclusion: RGD-engineered recombinant adenoviral vector Ad.RGD-IL-24 could mediate gene transfer into myeloid leukemia cells with high efficiency. Intracellular IL-24 over-expression had growth inhibition and differentiation inducement effects on myelogenous leukemia cells and IL-24 expression could induce apoptosis of the THP-1 cells significantly. The potent effect on the regulation of the expression of apoptosis-related genes might be responsible for the apoptosis-inducing effect of IL-24 on THP-1 cells.Part Ⅳ Immunogenicity modulation effect of ectopic expression of IL-24 on myelogenous leukemia cellsObjective:To investigate the role of IL-24 gene expression in immunogenicity modulation of the myelogenous leukemia cells.Methods:The phenotypes changes of leukemia cells with or without IL-24 treatment were examined by flow cytometric analysis. The concentration of VEGF, TNF-α, IL-8 and IL-6 in culture supernatant was determined by ELISA. Cytotoxicity assay was performed to evaluate cytotoxic sensitivity changes of transfected leukemia cells to CIK cells. The tumor suppression effect of Ad.RGD-IL-24 on human leukemia cell was further observed using transplanted tumor in an athymic nude mouse model. The expression levels of VEGF, CD34, collagen IV, CD31, CD147, MT1-MMP, MMP-2 and MMP-9 in transplanted tumor were determined using immunohistochemistry analysis.Results: Myelogenous leukemia cells express low level of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominately regulate leukemia cells to produce immunize-associated cytokines, and improve cytotoxic sensitivity of these cells to immunologic effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. IL-24 had marked effects on down-regulating the expression of angiogenesis related proteins VEGF, CD31, CD34 and collagen IV, and metastasis-related factors CD147, MT1-MMP, MMP-2 and MMP-9 in transplanted tumors.Conclusion: IL-24 could enhance myelogenous leukemia cells’ immunogenicity, improve cytotoxic sensitivity of leukemia cells to immunologic effector cells. Intracellular IL-24 over-expression could retard leukemia cell transplanted tumor growth in vivo in an athymic nude mouse model. The antitumor activity elicited by IL-24 was closely associated with the effect of inhibition of tumor angiogenesis and reduction of tumor invasiveness.