Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 PARs, thrombin may also affect cell function via the activation of metalloproteinase-2 (MMP2) [12]. Apart from serine proteinases that can activate PARs to affect cancer cell behavior, MMPs have for some time been known to be involved in cancer metastasis and invasion [13-17]. Surprisingly, MMP1 has been observed, like thrombin, to regulate invasion and tumorigenesis of breast cancer-derived cells by a process involving PAR1 [18], providing an important link between tumor gener- ated metalloproteinases and PAR signaling. Additionally the existence of cross-talk between GPCR and EGFR signaling systems has been established in various cancer cells and has been found to promote cancer cell prolif- eration and migration through EGFR transactivation in colon cancer and renal cell carcinoma. MMPs are required by some GPCRs which suggest a possible role for MMPs in the PAR1 activation system as PAR1 is a subfamily of GPCR [19,20]. In prostate cancer-derived cells, PAR1 over-expression has also been documented and has been linked to PAR1-stimulated activation of NF- B, with an increase in NF- B-regulated inflamma- tory cytokines like IL-6 and IL-8 [21]. The exact role and mechanism of action of PAR1 in this process remains unclear. In our previous work, using an immunohistochemical approach with gastric carcinoma tissue, we found that the expression of PAR1, along with a metalloproteinase known to activate PAR1 (MMP1) was associated with poorer prognosis, compared with expression-negative tumors [22]. In this study, we hypothesized that PAR1 would play an important role in gastric carcinoma cells. To test this hypothesis, we evaluated the impact of PAR1 activation in gastric cancer-derived cells. Our data show that the signaling pathways stimulated by PAR1 in the gastric cancer-derived cells mediate proliferation and invasion, and Tenascin-C (TN-C) might play an important role in this signaling pathways stimulated by PAR1. Methods Reagents An antibody against PAR1 (clone WEDE15) was pur- chased from BECKMAN COULTER (Fullerton, CA, USA). Anti-TN-C was purchased from IBL (Gunma, Japan) and TN-C-neutralizing antibody (Clone BC24) [23] was from Sigma-Aldrich (St. Lois, MO, USA). Anti- Bcl-2, phospho-specific antibodies against EGFR (clone 20G3) and phosphotyrosyl-1173 EGFR (clone 9H2) were purchased from Upstate Biotech (Temecula, CA, USA). Anti-NF- B-p50 and -p52 were from Santa-Cruz Bio- technology (Santa-Cruz, CA, USA). Anti-cIAP1 was from R&D systems (Minneapolis, MN, USA). Anti- GAPDH was from IMGENEX (San Diego, CA, USA). Human a-thrombin was purchased from Sigma-Aldrich Page 2 of 14 (catalog #T1063). The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [24]. The NF- B inhibitor Caffeic acid phenethyl ester (CAPE) (IC50 = 25 μg) was purchased from Biomol (Plymouth Meeting, PA, USA) [25]. Cell Culture The human gastric cancer cell lines, MKN28, MKN45 MKN74, NUGC2, NUGC3, and KATOIII cells were obtained from the Riken Cell Bank (Tsukuba, Japan). TMK-1 was a gift from Dr. S Fushida (Kanazawa Uni- versity, Japan). Cells were cultured at 37°C in 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Cells were propagated by mechanical re-suspen- sion using a scraper, without the use of trypsin. Reverse transcription-PCR and quantitative RT-PCR analysis Total RNA was extracted from gastric cancer cells with ISOGEN reagent (NipponGene, Tokyo, Japan). Single- strand cDNA prepared from 3 μg total RNA using MMLV reverse transcriptase (GIBCO, Calabasas, CA, USA) with an oligo (dT)14 primer that was used as a tem- plate for reverse transcription-PCR (RT-PCR) or quanti- tative-PCR (qPCR). The following primer pairs were used: GAPDH/5′-GGGAGCCAAAAGGGTCATCATCT- 3′ and 5′-GACGCCTGCTTCACCACCTTCTTG-3′; and PAR1/5′-TGTGAACTGATCATGTTTATG -3′ and 5′- TTCGTAAGATAAGAGATATGT -3′. qPCR analysis was also done with a PCR mixture con- taining each primer and SYBR Green master mix (Qia- gen, Hilden, Germany). The PCR primer pairs for the NF- B target genes were custom made (Hokkaido Sys- tem Science, Hokkaido, Japan). Each sample was exam- ined in triplicate and the amounts of cDNAs were normalized with respect to those of a GAPDH internal control. Construction of PAR1 expression plasmid A human PAR1 cDNA sequence was isolated by PCR from a NUGC3. We amplified the PAR1 cDNA using a primer set as follows: PAR1-CX for the 5′ primer, GGGGATCCCGGCAGAGCCCGGGACAATG; and PAR1-DX for the 3′ primer, GGGAATTCTCCCAG- CAGTCCCTTTTCC. Both primers incorporated 5′- BamH1 and 3′-EcoR1 sites, respectively. We amplified the BamH1 and EcoR1 site-tagged full-length PAR1 fragments, and cloned them into a pcDNA3.1 (Invitro- gen, Carlsbad, CA, USA). Positive clones (pcDNA3.1- PAR1) were isolated and validated by DNA sequencing. The sequence agreed with the Genebank record: NM001992.