| Wogonin is a kind of natural flavonoids as one of active components from barbata. Its molecular formula is C16H12O5. Wogonin has been shown to have a wide range of effects, such as antioxidant, antibacterial, anti-inflammatory, sedative and anti-tumor activity. However, studies of anti-tumor activity of wogonin were mostly concentrated on tumor cytotoxicity ex vivo. Whether wogonin antagonizes gastric cancer growth in vivo, and how wogonin affects function of dendritic cells (DCs) and T/NK lymphocytes is waiting for further study.This thesis firstly observed whether VEGF expression in human gastric cancer and serum VEGF concentrations correlated with infiltration and activity of dendritic cells respectively based on clinical data of gastric cancer. Secondly whether wogonin inhibited growth of gastric cancer in vivo was observed in a transplanted tumor model. Effects and mechanisms of wogonin on DCs activities under normal state or gastric cancerous microenvironment were analyzed. Finally direct effects of wogonin on functions of T and NK cells, and whether wogonin synergized with DSS to induce colitis in mice were both observed. Thus mechanisms of anti-tumor activity of wogonin in vivo would be elucidated deeply.Our study was divided into four parts.Part I. Correlation analysis of VEGF expression in gastric cancer, serum VEGF concentration with density of DCs in tumor tissues and DC activitiesObjective:To analyze whether VEGF expression level in gastric cancer tissue, serum VEGF levels correlates with density of DCs infiltration and DCs activities or not, and to clarify effects of VEGF on CD86expression in DCs.Methods:VEGF expression on tumor cells and CDllc+cells was detected by immunofluorescence. Serum VEGF level was measured by an enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) from both gastric cancer patients and healthy controls were isolated and induced to immature DCs with stimulations of GM-CSF and IL-4. Expression level of HLA-DR and CD86was compared between gastric cancer patients and healthy controls. At last variation of CD86expression on DCs with VEGF stimulation was observed.Results:The level of VEGF expression was significantly higher in gastric cancer tissues than in adjacent tissues, VEGF expression level and density of DC infiltration was inversely correlated. VEGF expression level was positively correlated with size, depth of invasion, lymph node metastasis and TNM stage of gastric cancer. The density of CD11c+DC infiltration was negatively correlated with lymph node metastasis, TNM stage. The frequency of CD11c+VEGF+cells was higher in tumor tissues than in adjacent tissues. The density of CD11c+VEGF+cells in tumor tissues was positively correlated with TNM stage and lymph node metastasis. Serum VEGF concentration in patients with gastric cancer was significantly higher than in healthy individuals. Compared with healthy individuals, PBMC-derived DCs from patients with gastric cancer decreased their expression of HLA-DR and CD86. Recombinant VEGF reduced surface expression of CD86of DCs in gastric cancer patients.Conclusions:Intensity of VEGF expression in gastric cancer cells was negatively correlated with density of DC infiltration in tumor tissue. Enhanced level of serum VEGF was related with decreased expression of HLA-DR and CD86on DCs derived from PBMC. The frequency of VEGF+DC in tumor tissues was positively with the development of gastric cancer. It could be inferred that methods that involved targeted inhibition of VEGF secretion of tumor cells and enhancement of DC function can achieve good anti-tumor effects.Part Ⅱ. Activities and preliminary mechanisms of bidirectional regulation on suppression of gastric cancer and immune-enhancing function mediated by wogoninObjective:To explore effects of wogonin on apoptosis, secretion of VEGF, expression of MHC class Ⅰ chain-related protein A (MICA) and B7H1on gastric tumor cells. To observe whether apoptotic bodies induced by wogonin are susceptible to be engulfed by DCs. To verify anti-tumor activities mediated by wogonin in vivo based on a mouse xenograft tumor model.Methods:Firstly serial concentrations of wogoin were added into SGC7901and MFC cell culture system for12hours, apoptosis was detected by flow cytometry. In addition, VEGF, HMGB1concentration in culture supernatants were detected by ELISA. CRT, MICA, B7H1expression on tumor cells was measured by flow cytometry. Next PBMC of gastric cancer patients was isolated and induced to immature DCs. Serial concentrations of wogoin were co-cultured with immature DCs for48hours, frequency of CD11C+CFSE+cells was detected by flow cytometry. MFC cells (1x106/ml) were inoculated to armpits of615mice in order to form solid tumors. Then serial doses of wogonin were intraperitoneally injected every day for3weeks Tumor growth and mouse survival conditions were documented. At last Frequencies of CD4+T、 CD8+T、NK cells in spleens of tumor-bearing mice were detected, and expression of VEGF, RAE-1, B7H1on transplanted-tumor tissues, and densities of CD4+、CD8+T、NK cells in tumor tissues were measured by immunofluorenscence. Results:Wogonin induced apoptosis of gastric cancer cells (SGC-7901and MFC) in a dose-dependent manner, and down-regulated SGC-7901and MFC cells to produce VEGF and to express B7H1and RAE-1(MICA analogs in mouse). In addition, apoptotic bodies from SGC-7901cells induced by wogonin were susceptible to be engulfed by immature DCs. The growth of transplanted gastric tumor was suppressed with the therapy of wogonin, particularly in the dose of50mg/kg. Wogoin significantly prolonged survival of tumor-bearing615mice. Frequencies of CD4+T、CD8+T、NK cells in spleens and tumor tissues were enhanced in wogonin treatment groups. Meanwhile, VEGF, RAE-1, and B7H1expression on tumor tissues were significantly decreased with the wogonin treatment.Conclusions:Wogonin not only directly induced apoptosis of gastric cancer cells, but also improved local tumor immunosuppressive microenvironment. In addition, Wogonin could induce tumor cell immunogenic death and promote DCs to phagocyte apoptotic bodies, thus tumor-specific immune response was effectively generated. Wogonin could be a candidate in natural drugs which would have both anti-tumor and immune-enhancing activities for tumor therapy.Part III. Effects of wogonin on activities of dendritic cellsObjective:To observe effects of wogoin on phenotype and function of DCs ex vivo, and to verify whether the anti-tumor activity mediated by wogonin could depend on DC function in mice transplanted with gastric cancer cells.Methods:Expression of HLA-DR, CD86, B7H1, and MICA on human or mouse DCs, and productions of IL-1β, IL-12, and IL-10of DCs with the wogonin treatment were detected by flow cytometry. Effect of wogonin on CD86expression on DCs were analyzed when DCs were co-stimulated with LPS/VEGF and wogonin. Frequencies of CD4+CD69+, CD4+IFN-y+, CD4+CD25+Foxp3+cells were detected by flow cytometry after mouse splenocytes were incubated with DCs pre-treated with wogoin for48hours. Finally frequencies of CD11c+, CDllc+CD86+, CDllc+B7H1+cells in spleens and tumor tissues from tumor-bearing mice were observed, and density of DCs infiltration in tumor tissues were measured by immunofluorenscence.Results:Wogonin down-regulated expressions of HLA-DR, CD86, B7H1and MICA on DCs in dose-dependent manner from both gastric cancer patients and healthy controls. Similarly wogonin down-regulated CD86and B7H1expressions of mouse DCs in vitro, but had no significant influences on CD86and B7H1expressions of mouse in vivo. Wogonin antagonized up-regulation of CD86expression induced by LPS, and synergized with VEGF to down-regulate CD86expression in DCs. IL-1β, IL-12and IL-10productions of DCs were inhibited with the wogonin treatment. DCs pre-treated with wogonin promoted CD4+T cells to express CD69and secret IFN-y, and inhibited survival of Treg cells in a dose-dependent manner. Frequencies of DCs in spleens and tumor tissues of tumor-bearing mice with the wogonin treatment were enhanced.Conclusions:Although wogonin down-regulated expressions of some important membrane-molecules and secretions of cytokines of DCs, DCs pre-treated with wogonin promoted functions of CD4+T cells. The wogonin treatment also enhanced DC frequencies in spleens and tumor tissues. Effects of wogonin on DCs may be one of mechanisms mediated by wogonin to suppress tumor growth.Part IV. Effects of wogonin on functions of T and NK cellsObjective:To analyze direct effects of wogonin on T and NK cell function, and to clarify whether this effect synergizes with DSS to induce colitis in mice.Methods:Expressions of activating receptors on spleen CD4+T, CD8+T and NK cells were detected with wogonin stimulation both in vitro and in vivo, respectively. Cytotoxicity of CD8+T and NK cells against target cells, and IFN-y production of CD4+T, CD8+T and NK cells was analyzed with the wogonin treatment. Different doses of (50,100mg/kg) wogonin were intrapritoneally injected at the same time with DSS drinking. Weight changes were documented and onset of enteritis was checked. Infiltration of CD4+T, CD8+T, NK cells into inflamed enteric tissues were measured by immunofluorescence. Frequencies of CD4+T, CD8+T, NK cells in inflamed intestine and activating receptors on these lymphocytes were detected by flow cytometry.Results:Splenic CD4+T, CD8+T cells and NK cells were activated with wogonin stimulations ex vivo. Meanwhile, the generation of regulatory T cells was suppressed. With injection of wogonin in vivo, activation of CD4+T cells and NK cells were not affected, and CD8+T cells were weakly activated. Cytotoxicty of CD8+T cells against MFC cells and cytotoxicity of NK cells were both enhanced with the wogonin stimulation, and IFN-y production of CD4+T, CD8+T cells and NK cells were enhanced. Wogonin synergized DSS to induce colitis, and degree of weight loss was more intensed, and intestinal necrosis was also more evident. Frequencies of CD4+T, CD8+T cells and NK cell infiltration were significantly enhanced in inflamed enteric tissues with the wogonin treatment. With co-stimulations of wogonin and DSS, frequencies of splenic CD4+CD69+T, CD8+CD69+T, and NK1.1+CD69+cells were higher than the DSS treatment alone. In addition, frequencies of CD4+T, CD8+T, and NK1.1+cells in intestines were significantly enhanced, and most lymphocytes were effector cells.Conclusions:Wogonin directly stimulated T and NK cell activation, and synergized with DSS to induce colitis in mice. These results demonstrated that wogonin had activities to up-regulate cellular immune function.In summary, this paper explored effects of wogonin on growth of gastric cancer cells and effects of wogoin on local tumor immunosuppressive microenvironment in depth. With confirmation of anti-tumor activities of wogonin in vivo, the mechanisms of woginin against gastric cancer could be involved with induction of tumor cell immunogenic death, promotion of crosstalk between DCs and CD4+T cells, direct stimulation of T and NK cell activation. Our study not only preliminarily clarified cellular and molecular mechanisms of wogonin against gastric cancer, but also provided important experimental data for clinical therapy with combination of common chemical drugs and wogonin. |
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