Clinical significance of vascular endothelial growth factor expression and neovascularization in colorectal carcinoma.

AIM: To clarify the association of vascular endothelial growth factor (VEGF) and microvascular density (MVD) expression with the angiogenesis and prognosis of colorectal cancer. METHODS: A total of 97 cases of colorectal carcinomas were examined by immunohistochemical staining (SP method), using anti-VEGF and anti-factor CD34(+) monoclonal antibodies. RESULTS: VEGF positive staining was obtained in 68 out of 97 cases (70.1 %), and observed mainly in the cytoplasm of tumor cells, and also frequently in stromal cells. VEGF expression was more intense in poorly differentiated adenocarcinoma in comparison with others, but there was no significant correlation between VEGF expression and age, sex and stage. A significant correlation was found between the MVD and grades, and there was no significant relationship between the MVD and age, sex, and stage. The MVD in the VEGF positive group (68 cases) was higher than that in the negative group. Upon multivariate analysis, the significant variables were stage, tumor grade and MVD; VEGF expression was not an independent prognostic factor. CONCLUSION: The expression of VEGF has a significant correlation with MVD; MVD expression has prognostic value but VEGF has not in colon cancer.

[Subcutaneous transplantation of microencapsulated Chinese hamster ovary (CHO) cells/pcDNA 3.1/mIL-12 and subcutaneous transplantation of microencapsulated CHO/pcDNA3.1/mIL-12 combined with 5-fluoro-uracil in treatment of tumor-burdened mice].

OBJECTIVE: To investigate the effect of subcutaneous transplantation of microencapsulated Chinese hamster ovary cells (CHO)/pcDNA3.1/mIL-12 and subcutaneous transplantation of microencapsulated CHO/pcDNA3.1/mIL-12 combined with 5-fluoro-uracilum (5-FU) in treatment of tumor-burdened mice. METHODS: CHO/pcDNA3.1/mIL-12 and CHO/pcDNA3.1 were suspended in solution of sodium alginate and made into microcapsules. Sixty mice were divided into 6 groups of 10 mice: (1) IL-12 group, microencapsulated CHO/pcDNA3.1/mIL-12 was injected subcutaneously, (2) combined treatment group: CHO/pcDN with 5-FU, (3) 5-FU group: 5-FU was injected intraperitoneally, (4) blank vector group: CHO/pcDNA3.1 was injected subcutaneously, (5) tumor-burdened group: without any treatment, and (6) blank control group: normal mice without any treatment. Except the mice of the blank control group, all mice were injected subcutaneously into the inner side of right thigh with mice colonic adenoma cells. The volume of tumor was measured every third day. 20 days after the treatment, 5 mice in each group were killed to examine the serum Th1 type cytokines: interferon (IFN)-gamma, IL-12, and Th2 type cytokins: IL-4, IL-10 by double antibody sandwich ELISA. The spleens were made into suspension of lymphocytes to examine the activity of natural killer cell (NK) and cytolytic T lymphocyte (CTL). The survival period of the remaining 5 mice in each group was observed till the 60th day. RESULTS: The activity of NK and CTL were significantly much more in the IL-12 group than in other groups. The activity of NK was significantly much more in combined treatment group than in the tumor-burdened group. The levels of Th1 type cytokines were the lowest in the tumor-burdened group. There was no difference in the levels of Th1 type cytokine between the tumor-burdened group and 5-FU group. However, the levels of Th1 type cytokine were significantly higher in the IL-12 group than in other groups. The levels of Th2 type cytokines were rather high in the tumor-burdened group. There was no difference in the levels of Th2 type cytokine between the tumor-burdened group and 5-FU group. However, the levels of Th2 type cytokine were the lowest in the IL-12 group than in other groups. There was no significant difference in volume of tumor among the tumor-burdened group, blank vector group, and 5-FU group. The mean diameters of tumor in the IL-12 group and combined treatment group were significantly smaller than in the 5-FU, tumor-burdened, and blank vector groups (P < 0.05), however, without a difference between the IL-12 group and combined treatment group. The survival periods of the IL-12 group and combined treatment group were significantly longer than those in the blank vector, tumor-burdened and 5-FU groups (P < 0.05). CONCLUSION: Microencapsulated CHO/pcDNA3.1/mIL-12 transplanted subcutaneously significantly improves the immune function of tumor-burdened mice and partially overcomes immune suppression caused by chemotherapy, and is effective in slowing the growth of tumor and lengthening the survival period of tumor-burdened ice.

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy.

AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene. METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated. RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-gamma, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice. CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy.

Inducible nitric oxide synthase expression is related to angiogenesis, bcl-2 and cell proliferation in hepatocellular carcinoma.

In this study, we examined the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistochemical staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC. Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissue close to the tumor edge was stronger than that of HCC tissue, and the strongest was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84.8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P = 0.000) between iNOS and VEGF expression. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but without p53 expression. DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proliferating Index) and SPF (S-phase fraction) in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group. The present findings suggested that iNOS expression was significantly associated with angiogenesis, bcl-2 and cell proliferation of HCC.