| Part one Expression of CD40 and growth-inhibitory activity of CD40 ligand in colon cancer in vitroObjective To investigate the expression of CD40 molecule on colon cancer and evaluated the growth-inhibitory effect of recombinant soluble human CD40L (rshCD40L) on colon cell lines.Methods The expression of CD40 was examined by immunohistichemistry in colon cancer tissues (65 cases) and adjacent normal tissues (10 cases). The correlations of CD40 expression with clinicopathologic features in colon cancer were analyzed by usingχ~2 test. The expression of CD40 on colon cell lines (HCT116, Caco-2, SW48, and COLO-320) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, its change by interferon-γ(IFN-γ) was examined by flow cytometry. The growth-inhibitory activity of rshCD40L on colon cancer cell lines was examined by MTT assay and the proportion of apoptotic tumor cells was examined in the TUNEL assay. In addition, the growth-inhibitory activity of combined rshCD40L and IFN-γwas examined.Results CD40 positive staining was observed both on membrane surfaces and in the cytoplasm of tumor cells. The high positive expression rate of CD40 was 41.5% in colon tumor tissues. There was a statistically significant correlation between tumor-node-metastasis (TNM) stage and CD40 high expression levels (P < 0.05). High expression of CD40 of colon cancer was more associated with lymph node metastasis (P < 0.05) than low expression.The three examined colon cell lines (HCT116, Caco-2, and, SW48) displayed variable but positive CD40 expression levels by RT-PCR and flow cytometry. CD40 expression could be induced by IFN-γ. RshCD40L significantly inhibited the proliferation of the CD40~+ colon cancer cell lines (growth-inhibitory activity: 33.5±5.5% of HCT116, 21.5±3.6% of Caco-2, and 30.1±4.2% of SW48). CD40~- colon cell line, COLO-320 was not inhibited by rshCD40L (P < 0.05). RshCD40L induced apoptosis of the CD40~+ colon cancer cell lines (HCT116: 25.6±4.52 % of apoptotic cells, Caco-2: 15.71±2.27 % of apoptotic cells, SW48: 18.0±3.7 % of apoptotic cells). However, rshCD40L did not induce apoptosis of the CD40~- colon cell lines, COLO-320 (P < 0.05). In addition, the inhibition and apoptosis could also be enhanced by IFN-γ.Conclusion CD40 molecule might play an important role in the carcinogenesis of colon cancer. Combined rshCD40L and IFN-γcould significantly induce apoptosis and inhibit the proliferation of the CD40~+ colon cancer cell lines, had potential anti-tumor effect.Part two CD40L treated DCs-based Vaccine anti-colon cancer cells efficiencyObjective This study was designed to investigate mobilization of dendritic cells (DCs) into the circulation by adiministration of RANTES, furthermore, explore the antitumor efficiency of the special cytotoxic T lymphocytes (CTLs) activated by DCs loaded with colon cancer antigens and CD40L ex vivo.Methods (1) C57BL/6 (B6, WT) mice were injected, via the tail vein, with RANTES. Peripheral blood was obtained after 24 h. Peripheral blood mononuclear cells (PBMNCs) were prepared from peripheral blood. F4/80~-B220~-CD11c~+ cells were sorted from these PBMNCs by FACS. Freshly isolated B220~-CD11c~+ cells and B220~-CD11c~+ cells cultured with cytokines mGM-CSF, IL-4, and mTNF-αwere analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction (MLR). (2) Colon cancer antigens were extracted by thawing and freezing. After treated in vitro with tumor lysates and CD40L, DCs were used to induce T lymphocytes into cytotoxic T lymphocytes for colon cancer cells. The cytotoxicity of CTLs to colon cancer cells was assayed by MTT. IFNγproduction was determined with ELISA kit.Results (1) Freshly isolated B220~-CD11c~+ cells did not show the character of mature DC. These cells cultured with cytokines mGM-CSF, IL-4, and mTNF-αbear typical morphologic and phenotypic characteristics of dendritic cells, high expressed MHCⅡ(69.70±2.35%), DEC-205 (61.60±1.02%), CD80 (49.65±0.73%), CD86 (53.42±1.52%), CD40 (42.30±1.35%), and no expressed F4/80 (0.45±0.09%), CD8α(0.26±0.04%), gained the powerful capacity to stimulate allogeneic T cells. (2) CTLs stimulated with DCs loaded colon cancer antigen and CD40L showed the specific kill effect on colon cancer cells, produced high levels of IFNγ, compared with DCs loaded tumor antigen, there was a significant difference (P < 0.05). In addition, CTLs stimulated with DCs unloaded colon cancer antigen did not show the specific kill effect on colon cancer cells.Conclusion The tumor antigen-treated DCs induced by RANTES from peripheral blood produced eficient and specific anti-tumor immunity, CTLs derived from cultures containing DCs treated with CD40L show the strongest cytolytic activities. |
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