Action And Mechanism Of Integrin And PAR1 Particapating In Thrombin Promoting Tumor Metastasis

Invasion and metastasis are the essential charateristics of malignancy. Prevention and treatment of tumor invasion or metastasis is one of the important measures to decrease tumor mortalities.The relation of thrombosis to cancer was observed by Professor Armand Trousseau in 1865. Abnormalities of blood coagulation have been recognized in many cancer patients with solid tumor, especially for those with a huge tumor mass or lung metastasis. Experimental evidence shows that the activation of systemic or local blood coagulation may accelerate tumor metastasis. It has been revealed that many coagulation factors are involved in the development of tumor, among which thrombin,as a key component of the coagulation cascade, plays an important role in tumor metastasis.Previous studies suggested that thrombin promotes tumor metastasis mainly through the protease-activated receptors (PARs). PARs is a family of G-protein-coupled receptors. There are four members of PARs that have been found recently,and PAR1 is the major receptor to mediate the function of thrombin in tumor metastasis. The interaction of thrombin with PAR1 involves two subdomains in thrombin, exosite domain and catalysis domain. The exosite domain binds to hirudin-like sequence of PAR1, subsequently the specific sites of extracellular compartment of PAR1 is cleaved by catalysis domain of thrombin to reveal a new N-terminal sequence that binds to the receptor and initiates transmembrane signaling, that is,the activation of PAR1 depends on the activation of the catalysis domain of thrombin. Previous studies indicated that PAR1, expressed in vascular endothelial cells, stimulates tumor metastasis by inducing angiogenesis. Recently, it has been reported that PAR1 is also expressed on the surfaces of many tumor cells. Hence, thrombin can promote the proliferation, migration and adhesion of tumor cells.Integrin is a membrane receptor, which participates in the promotion of tumor metastasis,but the mechanism of which is not very clear .The ligands of integrin such as fibronectin,laminin and collagen contain RGD sequence, a crucial structural domain interacting with integrin. It is reported that thrombin also has RGD sequence, but this sequence is buried in the 220-loop, therefore,which could not establish a functional conformation. When immobilized or exposed to high salt solution, the conformation of thrombin would be changed and the RGD sequence would be exposed.So, we presume that thrombin may interact with integrin and this interaction may be mediated by other molecules.PAR1 should be considered as one of the molecules.Thrombin binds to PAR1,then would be fixed to PAR1 by it's exosite domain.The catalysis domain of conformation has been changed and the RGD sequence was exposed to activate thrombin. Andrew JL, et al. reported that in platelet, the exosite domain of thrombin binds to PAR1, and the catalysis domain of thrombin also can bind to PAR4.The purpose of our work is to clarify the role of PAR1 and integrin played in tumor metastasis. And a fusion protein containing hirudin and RGD sequence is constructed to inhibit tumor metastasis.Methods and results:①Anti-PAR1 antibody, anti-αv antibody, GRGDS and HV are added to test the cell potential of migration and adhesion. We discovered that both antagonists of PAR1 and integrinαv inhibit the tumor metastasis induced by thrombin, hence we concluded that both PAR1 and integrinαv participate in the thrombin-induded tumor metastas.②The interaction between PAR1 and integrinαv was initially demonstrated by immunoprecipition.③The detection of ERK phosphorylation in the thrombin-induced tumor metastasis indicates that integrin may play its role in regulating tumor cell activity through ERK signaling pathway.④By construction,cloning expression of recombinant pPIC9K-RGD-TH,and the fermentation,separation and purification of expressed product, the fusion protein RGD-TH were obtained.⑤The results showed that thrombin at the concentration of 5U/ml stimulates the proliferation of the lung cancer cell,while this effect could be inhibited by RGD-TH , whereas it was also revealed that RGD-TH inhibited the adheresion of tumor cell simulated by thrombin , nevertherless there was no distinguished impact of RGD-TH on tumor cell migration .Conclusion: In our study, we found that the actions of thrombin in inducing tumor metastasis and adhesion are inhibited by GRGDS and anti-αv antigen,which explained that integrin is involved in the process of thrombin promoting lung cancer metastasis and adhesion. The interaction between PAR1 and integrinαv is initially demonstrated by immunoprecipition test. Integrin may regulate tumor cell activity by ERK signal pathway. RGD-TH can inhibit tumor cell proliferation and adhesion, but cannot affect tumor cell migration.