Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 12 of 14 Figure 14 Detection of phosholyrated EGFR by TN-C. The conditioned medium of PAR1/MKN45 culture pre-stimulation by 15 nM a- thrombin for 3 and 12 hr was collected and partially purified by repeated concentration using ultra-filtration spin columns able to cutoff macromolecules below 200 kDa. MKN45/PAR1 were incubated either with the concentrated conditioned medium for 6 hr, which were pre- stimulated for 3 hr or 12 hr by 15 nM a-thrombin. These whole lysates of MKN45/PAR1 treated with the conditioned medium pre-stimulated for 12 hr by 15 nM a-thrombin was detected remarkably enhanced tyrosine phospholyration of the EGFR, but these whole lysates of MKN45/PAR1 with the conditioned medium pre-stimulated for 3 hr by 15 nM a-thrombin was not detected the tyrosine phospholyration of the EGFR (left). The whole lysates of MKN45/PAR1 treated with the concentrated conditioned medium with TN-C-neutralizing antibody (25 μg/ml), which was pre-stimulated for 12 hr by 15 nM a-thrombin, show no-increased in phosphor-EGFR protein quantity (right). Our qPCR analysis of the spectrum of NF- B target genes up-regulated by PAR1 activation revealed quite a number of proteins, for example TN-C, Bcl-2 and cIAP1, for which up-regulation has been previously associated with a tumorigenic phenotype (Figure 9&10). The prolonged time frame over which the mRNAs levels are elevated after a-thrombin stimulation (up to 12 hr) Figure 15 Phospho-EGFR signal quantitatively diminished in the presence of TN-C-neutralizing antibody by histograms. The phospho-EGFR signal quantitatively diminished in the presence of TN-C-neutralizing antibody by histograms. The bar height indicates the calculated fold activation values. It was based on the condition with the concentrated conditioned medium, which was obtained after at 3 hr 15 nM a-thrombin stimulation with or without TN-C- neutralizing antibody. Data are expressed as intensity units obtained by densitometric analysis. (Date were expressed as mean values ± SD from triplicate experiments.) implies that signals in addition to the one triggered by PAR1 may be involved. For instance, the direct activa- tion of PAR1 by a-thrombin or PAR1-activating peptide would be expected to be down-regulated over a rela- tively short time frame, as can be observed for the sti- mulation of intracellular calcium transients [29,30] or for the activation of Mitogen-activated protein kinase (MAPK) (often maximal at 5 minutes, declining to base- line within 1 hr). Yet, in contrast with a PAR1-activating peptide, a-thrombin as an agonist is known to cause a prolonged activation of MAPK and enhanced mitogen- esis [31]. It has been hypothesized that these long-term actions of a-thrombin, in contrast with the effects of PAR-activating peptides may be mediated by receptors and mechanisms other than those encompassing PAR1 [32]. Thus, although we also have shown that NF- B and EGFR activation initially were triggered by activa- tion of PAR1 in the early phase (Figures 8 &13) [19,33] the sustained responses very likely are mediated by ‘feed-forward’ mechanisms, possibly involving the pro- duction of autocrine stimulatory factors like the one(s) detected in the concentrated cell supernatants and/or the sequential and synergistic cooperation of several transcription factors in addition to NF- B. This sequence of events set in motion by PAR1 activation may reflect a generalized ‘oncogenic signal matrix’ that may be initiated by a variety of mitogenic agents like thrombin. Apart from cell-derived proteinases as potential auto- crine/paracrine factors, our work points to the possible autocrine importance of secreted TN-C that was observed to be a PAR1-induced gene and that could be recovered in concentrate of conditioned medium of cul- tures of a-thrombin-treated MKN45/PAR1 (RT-PCR data; ELISA assay and identification by mass spectro- scopy). PAR1 activation accounted for a-thrombin-