Facilitating The Neogenesis Of Blood Vessels Of Cyclo-oxygenase-2 Inhibitor Celecoxib And Its Effection On Expression Of Neurosponglioma Angiogenin-2

IntroductionTumor is characteristic angiogenesis-dependent disease,which means that the growth and metastases of tumor depends on the formation of blood vessels.The formation of new blood vessels is tightly regulated by a large number of factors and it is the result of the imbalance between promoters and inhibitors of tumor angiogenesis.New blood vessels increase the blood flow perfusion and promote tumor growth,invasion and metastasis. Now it is one of the methods of tumor therapy that regarding inhibition of angiogenesis as treatment target.Cyclooxygenase(COX)is the key enzyme in conversion of arachidonic acid into prostaglandin and is associated with angiogenesis and growth of many kinds of tumors. Angiogenin-2 can affect on vascular endothelial cell specially,which play an important role in angiogenesis and keep the balance of blood vessels.Study results indicate that Cyclo-oxygenase-2 inhibitor can suppress tumor growth and increase tumor necrosis,but there is no report on its effect on expression of angiogenin-2 in tumor blood vessels.In the experiment,we cultivate human umbilical vein endothelial cells(HUVECs) and human neurosponglioma cells in vitro,and set up a stable angiogenesis culture system by Matrigel in order to discuss the influence of the role of celecoxib in facilitating human vascular endothelial cells and expression of neurosponglioma angiogenin-2 .Further discuss the relationship between celecoxib and neogenesis of tumor blood vessels and its mechanism. MethodsHUVECs are cultured by modified Jaffe method and induced to develop tubular structure by Matrigel. Thus set up a stable angiogenesis system and added the stimulating factors. Neurosonglioma cells are fractional cultivated in vitro, the conditional medium of tumor cells were prepared when cell approach to sub-confluence The concentration of celecoxib in our experiments is 20(?)m/l 40(?)m/l 0(?)m/l respectively using Dimethyl Sulfoxide (DMSO) as dissolvent.Experiment are divided into two parts: Group is, dynamic observing of the circumstances of HUVECs induced to form tubular structure by Matrigel , further divided into three group:one is control group (serum-free DMEM culture medium) and tumor cells conditional medium stimulating groups in varying concentrations ( the ratio of the upper clear liquid of tumor cells to DMEM culture medium is 1:2, 1:1 and 2:1, respectively) then acting 24h. Second Group is divided into celecoxib stimulating groups in varying concentrations(20(?)ml/(?) 40(?)m/l(?) 60(?)m/l) and control group added into DMEM.Third group is celecoxib stimulating groups in varying time (12h(?) 24h(?) 48h ) and control group added into DMEM.Groups two detect the expression of Ang-2 protein and divided into stimulating groups in varying concentrations and varying time,too. The various groups are observed and photography by inverted phase contrast microscope , then fixed by 95% ice alcohol.GFAP and Ang-2 are detected by immunocytochemical method.The experiment results are tested by calculating the number of tubules in microscopic obversation and computer image-analysis. Finally the data was analyzed by statistics.ResultsHUVECs can be induced to develop tubular structure by Matrigel in vitro. Aftertreatment with the conditional medium of tumor cells, the number of tubules increase ,and the contrary result are received when treated with celecoxib . In certain ranges results show a dose-dependent and time-dependent manner (P<0．01) ; with the number of tubules decreased, the expression of neurosonglioma angiogenin-2 and in certain ranges showing a dose-dependent and time-dependent manner (P<0．01) .DiscussionCox(?)2 is induced by cytokines,growth factors and tumor promoters and is over expressed in inflamed and malignant tissues.The enzyme is localized in neoplastic cells,endothelial cells, stromal tissues and contributes to tumor angiogenesis and tumor growth. So selective pharmacological inhibition of Cox-2 represents a promising therapeutic strategy for the treatment of malignant solid tumors.But the effection on glioma of Cyclo-oxygenase-2 inhibitor celecoxib has not been reported.In the present study we evaluated the possible mechanisms by which a highly selective Cox-2 inhibitor,celecoxib,affects angiogenesis and expression of neurosponglioma angiogenin-2．Tumor angiogenesis is controlled by both proangiogenic and antiangiogenic factors.In vivo tumor angiogenic factors are mainly secreted by tumor cells and stimulate vascular endothelial cells to proliferate and form new blood vessels. In this experiment we observedthat after the cultured HUVECs treated by the conditional medium of the tumor cells,the number of tubules of the cultured system increased and in certain ranges showing a dose-dependent effect.It can be inferred that tumor cells promoted tumor angiogenesis via secreting growth factors. In this experiment we also observed that after the cultured HUVECs treated by celecoxib with different concentration and different time the number of tubules of the cultured system decreased in celecoxib compared to in controls and in certain ranges showing a dose-dependent and time-dependent effectAng-2 is a member of angiogenin.In the condition of hypoxia tumor cells secreted more VEGF and Ang-2．Ang-2 promote the angiogenesis around necrosic tissue of tumor with synergism of VEGF. In this experiment we observed that the expression of Ang-2 obviously decreased in celecoxib compared to in Controls and in certain renges showing a dose-dependent and time-dependent effect.It suggested selective pharmacological inhibition of Cox-2, celecoxib,inhibit tumor angiogenesis by down regulation the expression of Ang-2 via pathway of Cox-2 dependent.Conclusions1．tumor cell can promote in vitro induced tubular structure by Matrigel and in certain ranges showing a dose-dependent fashion.2．celecoxib can inhibit promoting to form new blood vessels and in certain ranges showing a dose-dependent and time-dependent fashion.3．celecoxib can downregulate Ang-2 expression in human neurosonglioma cell expression and in certain ranges showing a dose-dependent and time-dependent fashion.