Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 11 of 14 Figure 12 TN-C concentration determined by ELISA in the conditioned medium of a-thrombin stimulated MKN45/PAR1. Six cm diameter dishes with 50% confluency of MKN45/mock, MKN45/PAR1 were used for preparing the conditioned medium. a- thrombin was added at 15 nM to 6 dishes with MKN45/PAR1, and the conditioned medium was collected at indicated time (3, 6, and 12 hr) and cleared supernatant by centrifugation was subjected to ELISA assay which can detect only high molecular TN-C. The concentration of high molecular TN-C in the conditioned medium was expressed as μg/ml. (Date were expressed as mean values ± SD from triplicate experiments.) The data indicates that TN-C can contribute in an auto- crine manner to stimulate phosphorylation of EGFR. Discussion The main finding of our study is that activation of PAR1 triggers activation of NF- B and EGFR for a long per- iod, and TN-C, which is overexpressed by PAR1 activa- tion, may be associated with EGFR activations. Our data now show not only that the histological presence of PAR1 is correlated with the pathological findings asso- ciated with invasion and metastasis in gastric cancer [22], but also that this receptor and its activating protei- nases, including thrombin and other serine proteinases [8] can be seen as mechanistically important factors driving the process of gastric cancer cell proliferation and invasion. Because our work used a PAR1 cell as the host for PAR1 expression, our data clearly demonstrate the oncogenic potential of PAR1 itself in a gastric cancer cell background, apart from factors other than PAR1 that can confer the oncogenic phenotype. Both the absence of a-thrombin effects in the PAR1 cells and the ability of the PAR1-selective antagonist, SCH79797, to block the actions of a-thrombin in MKN45/PAR1 indicate that the actions of a-thrombin were due to PAR1 activation and not to other a-throm- bin targets, like PAR4 or triggering by metalloprotei- nases [12]. Figure 13 Increases in EGFR protein expression and phosphorylation of EGFR at Tyr1173 in MKN45/PAR1 stimulated with a-thrombin. MKN45/PAR1 were stimulated with 15 nM a-thrombin for 1, 3, 6, and 12 hr and harvested for preparing whole cell lysates. The clear extracts containing 100 μg protein was developed on SDS-PAGE. We detected by western blotting experiment using an antibody raised against total EGFR (dilution of 1: 500) was increased 3 hr after addition of 15 nM a-thrombin to MKN45/PAR1. But we detected using an antibody raised against phosphotyrosine (Tyr1173) that tyrosine phosphorylation of EGFR (dilution of 1:500) was slightly increased 1 hr after addition of 15 nM a- thrombin, and following 3 hr, phospho-EGFR was dramatically increased.