The Effects And Mechanisms Of Holothurian Glycosaminoglycan On Cogulation Process Mediated By Malignant Melanoma Cells

Background and Purpose:There is a close relationship between tumor malignant process and coagulation system.Trousseau syndrome is a kind of disease that characterizes the link between tumor and thrombosis,it illustrates that occurrence and development of tumor is always accompanied by the hypercoagulable state,and the hypercoagulable environment also facilitates tumor to metastasize,they have formed a vicious circle,so inhibiting the activation of coagulation system during the process of tumor metastasis preventing the procoagulant trait of tumor in the hypercoagulable state has,become a new way in recent anti-tumor therapy,meanwhile,investigating the procoagulant trait of tumor is helpful for the hypercoagulability.Holothurian glycosaminoglycan(hGAG)that constituted by galactosamine,glucuronic acid,fucose and sulfate is the main ingredient of sea cucumber.Recent studies have shown that hGAG has potential antitumor effect,but its mechanism remains to be elucidated.This study is to investigate the effects of hGAG in tumor and coagulation system and to observe the effects of hGAG on coagulation system mediated by B16F10 tumor cells,and also to illustrate its mechanism of action,which can provide a new visual angle for antitumor and antithrombosis therapy.Experimental Approach:In vitro,coagulation analyzer was used to measure the effect of B16F10 cells treated with hGAG on plasma recalcification time,symbolic factors of fibrinolytic system of B16F10 cells treated with hGAG were examined by ELISA,Furthermore,the levels of FXa were measured with luminescent substrate S-2222.The changes of Ca2+ and phosphatidylserine exposure were examined by Fluo-4AM fluorescent probe and Annexin V/PI double staining in B16F10 cells,respectively.The mRNA and protein levels of TF were measured by real-time PCR and western blot,respectively.Besides,MTS was used to examine the proliferation ability of B16F10 cells treated with hGAG.Would healling,transwell chamber experiment and dipstick of gold were used to investigate the migration ability of B16F10 cells treated with hGAG.In vivo,the experimental metastatic models were replicated with B16F10 cells treated with hGAG.Prothrombin Time(PT),Activated Partial Thromboplastin Time(APTT),ThrombinTime(TT)and Fibrinogen(FIB)of the blood taken from mice were measured on day 7,14 and 23.The metastasis was observed with HE staining in lung tissues on day 23 and the expression of TF in lung tissue by immunehistochemistry(IHC).In terms of mechanism,vectors of TF promoter containing different transcriptional factors binding sites were constructed and tranfected into the B16F10 cells,the effects of hGAG on this were examined by Luciferase Reporter Gene and TF upstream signaling pathways treated with hGAG by western blot.Key Results:hGAG can extend the coagulation time induced by B16F10 tumor cells in a dose dependent manner in vitro but it has no obvious effects on the symbolic factors of fibrinolytic system including uPA and PAI-1.hGAG can also decrease the FXa generation in a dose dependent manner,and FXa generation requires three independent factors:Ca2+,phosphatidylserine exposure and TF.hGAG has no obvious effects on the former two factors,but it can downregulate mRNA and protein expression of TF in a dose dependent manner.In malignant biological behavior,hGAG at the concentrations had no obvious effects both on proliferation and migration abilities of B16F10 cells.In vivo,hGAG can also extend the TT,PT and APTT on the day 7,14 and 23 of establishing experimental metastasis models,and can reduce the number of metastaic nodules on day 23.We also confirmed that hGAG can downregulate the expression of TF in lung tissue in a dose dependent manner.In order to investigate the mechanisms of hGAG downregulating TF expression,we constructed five vectors of TF promoter containing different transcriptional factors binding sites and found that hGAG can decrease the fluorescence values of two vectors that have NF-?B binding site,the effects of hGAG disappeared after mutated the NF-?B binding site of full length vector,which further confirmed hGAG can prevent NF-?B entering into nucleus and binding onto the TF promoter,and inhibiting the transcriptional process of TF.hGAG exerted the effects through inhibiting the phosphorylation of MAPK signaling pathway rather than affecting AKT,NF-?B and other signaling pathways.Conclusions and Implications:The outcomes of this study are as following:? hGAG had no obvious effects on proliferation and migration abilities of B16F10 cells at the concentration between 0.01 u M and 1?M.?hGAG could act at the tumor cells to extend the coagulation time in a dose dependent manner(0.01 ? M-1 ? M),which was induced by reducing fibrin generation rather than increasing fibrinolysis;?hGAG could inhibit FXa generation by downregulating TF expression rather than affecting the Ca2+ changes and phosphatidylserine exposure in the B16F10 cells;?hGAG could also extend TT,PT,APTT and reduce the metastatic nodules in the lung tissue in a dose dependent manner in vivo;?hGAG downregulated TF expression by preventing NF-? B entering into nucleus and binding onto the binding site of TF promoter,which is achieved by inhibiting the phosphorylation of MAPK signaling pathway.This project helped us illustrate the acted mechanisms of hGAG and provided a new way and visual angle for us to research tumor metastasis and thrombosis.