title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion.
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 (alpha-thrombin and TFLLR-NH2). We also quantified NF-kappaB 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 alpha-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).
  
  
  RESULTS
  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-kappaB and EGFR phosphorylation initially were triggered by the activation of PAR1 with alpha-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-kappaB 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.
  
  
  CONCLUSIONS
  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-kappaB, 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.
authors: First Department of Surgery, Faculty of Medicine and Division of Bioresearch Laboratories, University of Fukui, Fukui 910-1193, Japan. rui5218@yahoo.co.jp
authors: Daisuke
authors: D
authors: Fujimoto
authors: Yasuo
authors: Y
authors: Hirono
authors: Takanori
authors: T
authors: Goi
authors: Kanji
authors: K
authors: Katayama
authors: Shigeru
authors: S
authors: Matsukawa
authors: Akio
authors: A
authors: Yamaguchi
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D017077:Culture Media, Conditioned / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D044463:Receptor, PAR-1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*; D019063:Tenascin / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: D017077:Culture Media, Conditioned; D016328:NF-kappa B; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D019063:Tenascin; C512478:EGFR protein, human; D066246:ErbB Receptors; D013917:Thrombin