Expression Of Cyclooxygenase-2 (COX-2) And Vascular Endothelial Growth Factor-C (VEGF-C) In Breast Cancer And The Correlation With Prognosis

IntroductionBreast cancer is a severse disease to the female.Lymphatic metastasis is a major metasis path in breast cancer,which is also correlated with prognosis in breast cancer patients.However,it is needed some new the mechanism of lymphogenesis is uncertain. Lymph vessel is the structural foundationg of lymph node metastasis.Recently,it was reported that vascular endothelial growth factor-C(VEGF-C)are able to promote lymphogenesis significantly.In some abroad studies,there are obvious relationgship between VEGF-C and the lymph node metastasis of patients.In the lung cancer cells,COX-2 can upregulate the expression of VEGF-C through Prostaglandin E2(PGE2),which may participate in lymph node metastasis.COX-2 is the key enzyme involved in prostaglandin production,which is induced by mitogen,cytokines,growth factor or hormone,at the time of inflammation and cancer.Cyclooxygenase-2(COX-2) is highly expressed in many tumours.Many studies suggest that there are significant correlations between COX-2 and the stages of tumour,lymphatic metastasis, prognosis.So,COX-2 has a important role in tumourigenesis.It is significant to research the relationship between COX-2 and VEGF-C in lymph node metastasis mechanism of breast cancer.To research COX-2 and VEGF-C in breast cancer,we study at levels of the tissues and cells.At the tissues level,and we analyse the relationship between COX-2,VEGF-C,and prognosis in the tissue microarray of breast cancer using SP method;A pNLR-COX-2 vector was transient transfected in MCF-7 cell,then we observe the changes of the two proteins'expressions and their correlations,with or without the treatment of celecoxib. Methods1.Patients and samples Breast cancer samples(n=117)were assembled in a tissue microarray,using immunohistochemical SP methods,which came from The First Hospital of China Medical University and underwent modified radical mastectomy or concerving surgery.64 patients had lymph node metastasis.The average ages of these patients is 51.4,none of them underwent chemotherapeutics or actintherapy before operation.There are 88 cases with invasion ductal carcinoma,29cases with other type;80 cases with stageⅠandⅡ,37cases with stageⅢ;We have the whole follow-up data.And the follow-up time ranged from 4 to 70 months.The cut-off time of follow-up was 1,February,2008.2.Reagent:the antibodies against COX-2 was purchased from Wuhan Boster Biotechnology,the antibodies against VEGF-C was purchased from R&D Systems,the second antibodies was purchased from Beijing Zhongshan Goldenbrige Biotechnology.Celecoxib is from Pharmacia Corporation,the pNLR-COX-2 vector is a gift from Professor Min-Liang Kuo(Institute of Toxicology,College of Medicine, National Taiwan University)3.Immunohistochemistry:Make the breast cancer tissue microarray.And using the immunohistochemical SP method to detect the expressions of COX-2 and VEGF-C, according the procedure of the SP KIT.4.Cell culture and transient transfection:Breast cancer cell MCF-7 was obtained from the oncology lab of The First Hospital of China Medical University,and it was maintained in routine conditions.Cells were seeded in 5 cm dishes,every dish was seeded about 2×10~5 cells.After 24 hours,using transfection reagent Fugene-6 as the procedure.5.Western Blot:After induced transfected 24 hours,cells was treated with or not with different concentrations of celecoxib(0,10,20uM)for 24 hours.Then prepared the cellular lysates.Equal amounts of protein were resolved on 10%SDS-PAGE and transferred to PVDF membrane.After blocking,blots were incubated with specific COX-2 and VEGF-C antibodies.After TTBS washing and incubating with secondary. antibodies,immunoreactive proteins were visualized by the enhanced chemiluminescence detection system.6.Immunohistochemical Result Evaluation:outcome definition of COX-2 was stained palm yellow particle in cytosol,positive cells＞10%as positive,≤10%as negative.The VEGF-C immunohistochemical staining was defined as below:-,negative;+,focal expression＜5%of cancer tissues;++,focal expression in 5-20%of cancer tissues;and +++,diffuse expression＞20%of cancer tissues.The tissue with ++ and +++ staining of VEGF-C was classified as high expression group and tissue with negative and positive staining was assigned as low expression group.7.Statistical Analysis:statistical methods,X~2 test,Spearman rank correlation test, Disease free survival(DFS)curves were obtained using the Kaplan-Meier method and compared using the log-rank test.Ps of＜0.05 were considered to be statistically significant.These data performed by SPSS soft ware version 13.0.Results1.Expression of COX-2 and VEGF-C in breast cancer tissue,and the relationship between them and patients' characters.COX-2 and VEGF-C are highly expressed in the 117 breast cancer tissue,the positive expression of them was 55.8%and 58.1% respectively.And the correlation between them was positive,(r=0.356, P=0.000).Expression of COX-2 was positive correlated with clinical stage(r=0.193, P=0.033),ER(r=0.224,P=0.015),Her-2(r=0.254,P=0.006),recurrence(r=0.312, P=0.001),lymph node metastasis(r=0.430,P=0.000).Expression of VEGF-C was positive correlation with clinical stage(r=0.317,P=0.001),lymph node metastasis (r=0.202,P=0.029),recurrence(r=0.205,P=0.027).2.The relationship between COX-2,VEGF-C and the prognosis of breast cancer patients.The DFS of COX-2 positive expressed patients are much shorter(log-rank test,P=0.004),and so are the DFS of VEGF-C highly expressed patients(log-rank test,P=0.016).And the DFS of patients that COX-2 positive expressed and VEGF-C highly expressed,COX-2 positive expressed or VEGF-C highly expressed were shorter than the DFSof patients that COX-2 negative expressed and VEGF-C low expressed p (log-rank test,P=0.002,P=0.022,respectively).3.The relationship between COX-2 and VEGF-C in breast cancer cell.After transfected the p-NLR-COX-2 vector about 24 hours,COX-2 protein was increased,as well as VEGF-C protein was upregulated.4.In breast cancer cells,the influence of celecoxib on the expression of VEGF-C.After transfected the p-NLR-COX-2 vector about 24 hours,treated with the celecoxib of final concerntration 20 uM,COX-2 protein was not downregulated,but VEGF-C protein was downregulated.Conclusion1.COX-2 and VEGF-C are high expressed in the breast cancer tissue,and the correlation of them was positive,COX-2 and VEGF-C are both the negative prognostic indicators in the breast cancer,which are correlation with the disease free survival(DFS) of breast cancer patients.2.The combined detection of COX-2 and VEGF-C are more useful in clinical for the prognosis of breast cancer,DFS are much shorter in patients with COX-2 positve and VEGF-C high expressed.3.In breast cancer cell MCF-7,COX-2 protein can regulate the expression of VEGF-C protein.It was also suggest that the low concerntrations of celecoxib can downregulate the expression of VEGF-C protein,which may not affect the expression of COX-2 protein.