Experimental Study Of The Correlation Of Survivin,COX-2 With Angiogenesis In Cholangiocarcinoma And Effects On Expression Of Angiogenic Related Factors Through COX-2 In Human Cholangiocarcinoma Cells

Cholangiocarcinoma (CCC) is one of the most common malignancies in the biliary tract.Despite overall advances in the ability to diagnose and treat patients with CCC,the prognosis for this disease still remains poor.One of the most important factors influencing the dismal prognosis of CCC is based on late diagnosis at an advanced stage of disease,when tumor cell invasion into blood and lymphatic vessels has resulted in metastatic spread,and curative resection can no longer be achieved.Angiogenesis is suggested as an important role in progression, metastasis, and prognosis in various types of tumor,so anti-angiogenesis represents a novel therapeutic approach in cancer treatment.CCC is relatively hypovascular in contrast to hepatocellular carcinoma (HCC),but high angiogenesis correlates with progression,metastasis,and prognosis of CCC along with the penetrating study to the carcinogenesis mechanism of CCC.Survivin is a recently described inhibitor member of apoptosis protein (IAP) family,which is abundantly expressed in fetal tissues and in a variety of human tumors,but it is characterized by a unique structure.Some researches have found that Survivin played an important role in the gene regulation of cell apoptosis and cell cycle progression , and it has some intimate correlation with regulation of tumor angiogenesis . Therefore , it could become a new target of gene therapy of tumor.Cyclooxygenase-2(COX-2) is one of the rate-limiting enzymes which catalyzes the convertion of arachidonic acid to prostaglandins.Many experimental studies have also indicated its relevance to tumor invasion,metastasis,cell apoptosis,cell cycle,and angiogenesis, and that its up-regulation regarded as rate-limiting step of tumor angiogenesis.Few reports on the role of COX-2 and Survivin in tumor angiogenesis are available.Our previous experimental studies have also indicated that COX-2 and Survivin had some correlation with cell apoptosis and cell proliferation in CCC.The aim of this study was to illustrate the relationship between angiogenesis and COX-2 , Survivin and potential mechanism of angiogenesis in CCC.We hope that we could provide a few certain proofs for clinic therapy of anti-angiogenesis in CCC.Objective: To illustrate the relationship between angiogenesis and COX-2 , Survivin and potential mechanism of angiogenesis in CCC.Methods : Survivin , COX-2 , VEGF and CD34 were detected with immunohistochemistry(IHC) technique in specimens of CCC. The QBC939 cells were cultured in vitro and seeded into culture plates.At the state of culturing, observe the morphology of cells in culture flask by invert microscope every day.Following exposure to a selective COX-2 inhibitor nimesulide at different concentrations in different times, then QBC939 cells were harvested and investigated.The effect of nimesulide on the secretion of VEGF in QBC939 cells were measured by enzyme linked immunosorbant assay (ELISA) .RT-PCR technique was used to investigate the transcriptional changes of gene COX-2/VEGF/Survivin . Furthermore, immunocytochemistry was used to examine the changes of their protein expression.Results: (1) MVD and positive expression rates of COX-2,VEGF and Survivin in CCC tissues were significantly higher than those in normal bile duct tissues by means of Immunocytochemistry.(2) There were positive relation between the MVD,expression of VEGF and expression of COX-2 in CCC tissues by means of Immunocytochemistry.(3) There were positive relation between the MVD,expression of VEGF and expression of Survivin in CCC tissues by means of Immunocytochemistry.(4) The secretion of VEGF in QBC939 cells increased in a dose dependent manner detected by means of ELISA. (5) Nimesulide induced down regulation of COX-2,VEGF and Survivin by means of Immunocytochemistry in QBC939 cells.(6) Nimesulide induced down regulation of COX-2,VEGF and Survivin by means of ELISA in QBC939 cells.Conclusions: (1) The high expression of Survivin and COX-2 have some close correlation with angiogenesis in CCC possibly.(2) The selective COX-2 inhibitor , nimesulide, can significantly inhibit the expression of related agents of angiogenesis in a dose-dependent and time-dependent manner in QBC939 cells in vitro,which were associated with the downregulation of COX-2 expression.