Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 R E S E A R C H A R T I C L E Open Access The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion Daisuke Fujimoto1*, Yasuo Hirono1, Takanori Goi1, Kanji Katayama1, Shigeru Matsukawa2, Akio Yamaguchi1 Abstract Background: In addition to regulating platelet function, the G protein-coupled sub-family member Proteinase- activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer. Methods: We established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (a-thrombin and TFLLR-NH2). We also quantified NF- B activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of a-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR). Result: PAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF- B and EGFR phosphorylation initially were triggered by the activation of PAR1 with a-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF- B target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C- neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed. Conclusion: Our data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF- B, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer. Background A dysregulation of the coagulation cascade in the setting of human tumors has been recognized for over a cen- tury [1]. In particular, active thrombin has been found to play an important role in terms of tumor behavior, affecting a variety of cancer-related processes including * Correspondence: rui5218@yahoo.co.jp 1First Department of Surgery, Faculty of Medicine and Division of Bioresearch Laboratories, University of Fukui, Fukui, 910-1193, Japan Full list of author information is available at the end of the article invasion, metastasis and tumor cell growth [2,3]. In large part, thrombin initiates cellular effects by cleaving and thus activating a novel set of Proteinase-activated receptors (PARs 1 and 4; but not PAR2), that are mem- bers of the G-protein-coupled receptor (GPCR) super- family [4-8]. Although able to activate PARs 1 and 4, thrombin is not able to activate PAR2, which is a target for trypsin [9]. PAR1 has been found to be instrumental in cell growth and invasion of tumor-derived cells [10,11]. In addition to regulating cell function by the © 2010 Fujimoto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.