Expression And Significance Of VEGF-D And COX-2 In Breast Cancer

INTRODUCTIONThe breast cancer has turned in the most common maligant tumor, Lymphatic metastasis is the common pathway of cancer dissemination, the metastasis and reoccur are leading causes of cancer - related mortality. Angiongensis and lym-phayenesis play a important role in the growth, spread and metastatic of tumor. but the mechanisms of this phenomenon remain unclear. VEGF - D is a new member of VEGF family, and it is alymphatic endothelial growth factor. VEGF — D can abind to the receptor flk — 1 and flt - 4 and activate them. VEGF - D promote the mitotic in the endothelial cell. Cox - 2 was none or a low expression in the normal tissue. but it can express induced by cell growth factor and imflama-tion factor and so on. In the present, the study on the role of COX - 2 in cell groth and apoptosis have obtain great progress. but the research in the correlation between the COX - 2 and VEGF has been little. In our study , we detected the VEGF - D COX - 2 expression and the correlation between these factors and clinicopathological parameters and prognosis. we assessed the their roles and importance in the metastasis and prognosis of breast cancer.MATERIALS AND METHODS1. Tissue samplesA total of sixty cases of breast cancer tissue samples were obtained from patients underwent surgical resection in the Anshan Tumor Hospital between 1995 and 1999, fixed in 4% paraformaldehyde, embedded in paraffin2. ReagentsThe primary antibody anti - rabbir VEGF - D, VEGFR - 3and COX - 2 mouse monoclonal antibody was provided by American Santa Cruz. The anti -mouse CD34 rabbit molyclonal antibody, the Ultro - sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company.3. Methods3.1 Immunohistochemistry for VEGF - D and COX -2Immunohistochemical staining was performed using a Ultro - sensitive S - P kit according to the instructions provided by the manufacturer. Color was developed using DAB/H2O solution. After the areas of tumor tissue had been chosen by random under low power, one hundred cells within tumors were counted in each of 10 fields at 200 magnification. Cases defined as positive were regarded by both staining intensity and the percentage of immunoreactive tumor cells. Mi-crovessel was determined by immunohistochemistry for CD^. After the areas of highest vascularization had been chosen under low power, vessel count within tumors was carried . The mumber of VEGFR - 3 - positive endothelial cells was determined in the same manner.3. 2 in situ hybridization of VEGF - Dand COX -2Paraffin - embedded specimens (4u,m) were deparaffinized and rehydra-ted. The steps of hybridization were performed using a kit according to the recommendations of the manufacturer.4. Statistical analysisStatistical Product and Services Solutions (SPSS) statistical software ( version 10.00) was applied for data analysis. Chi - square and Student's t test were used to compare the clinicopathological characteristics with the enzyme expression. Postoperative survival periods were computed by the Kaplan - Meier method and compared by the Log rank test. A P - value less than 0.05 was considered significant. The Cox stepwise regression analysis of which the level of significance was set at P < 0.1 was used to evaluate the statistical strength of independent association between selected covariates and patient survival.RESULTS1. Expression of VEGF - D and COX - 2 in breast cancer.The result of the Immunohistochemical: VEGF - D antigen were observed in the cytoplasmic and no staining was observed in the normal tissue. COX - 2 antigen were observed in the cytoplasmic. The result of the in situ hybridization, the positive rate of VEGF - DmRNA and COX -2Mrna is 80% and 73. 33% respectively. , which followed closely that of protein - expression.2. Correlation between the expression of VEGF - D and COX -2 and MVD or LVD.The MVD and LVD of VEGF - D - positive tumors was significantly greater than that of VEGF - D - negative tumors. The MVD of COX - 2 - positive tumors was significantly greater than that of COX -2 - negative tumors.3. The correlation between VEGF - D expression and COX -2 expression A significant positive correlation was found between VEGF - D expressionand COX - 2 expression in the breast cancer.4. Correlation between clinicopathological factors and VEGF - D and COX -2.There was a significant correlation between the expression of VEGF - D protein and the size of tumor,lymphatic metastasis. However, it was not correlated with the age, ER and PR. There was a significant correlation between the expression of COX -2 protein and the the size of tumor,lymphatic metastasis. However, it was not correlated with the age, ER and PR.5. Correlation between the expression of VEGF - D and COX -2 and prognostic in the breast cancer.Log rank analysis showed that the postoperative survival period was significantly shorter in VEGD - D positive expression group comparing with that in negative one. In multivariate analysis, only VEGF - D, VEGFR - 3 and LVD can be considered as prognostic factors.