Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 10 of 14 Figure 11 Effect of NF- B inhibitor CAPE and a PAR1 antagonist SCH79797 on trans-criptional activation by thrombin stimulation of NF- B target genes. NF- B inhibitor or PAR1 antagonist was added at 25 μg/ml or 70 nM, respectively, to the culture medium with 15 nM a- thrombin. Six hour later, the treated cells were harvested for preparing total RNA, which was subjected to quantification for mRNA expression of four NF- B target genes, such as, TN-C, Bcl-2, cIAP1 and EGFR by real time RT-qPCR. Each mRNA expression level after 6 hr thrombin stimulation is 100%, the suppressive effect of inhibitor or antagonist was expressed as % inhibition. (Date were expressed as mean values ± SD from triplicate experiments.) increase in EGFR protein was also observed 6 hr after the addition of a-thrombin, and increased tyrosine- phosphorylation of the EGFR was clearly detected fol- lowing 12 hr of stimulation by a-thrombin in MKN45/ PAR1 (Figure 13). We hypothesized that the increased activation/phos- phorylation of the EGFR might be due to the autocrine action of the increased TN-C or of other high molecu- lar-weight constituents produced by the cells. To test this hypothesis, two separate cultures of MKN45/PAR1 cells were exposed to a-thrombin, one for a period of 3 hr and the other for a period of 12 hr, and high molecu- lar weight proteins were isolated and concentrated from each of the resultant conditioned mediums. Subsequent cultures of MKN45/PAR1 were incubated for 6 hr with one of each of the resultant concentrates and activation/ phosphorylation of the EGFR was quantified and com- pared. The lysates from the MKN45/PAR1, which were incubated with concentrate (deficient in proteins smaller than 200 kDa) derived from the 12 hr a-thrombin sti- mulated conditioned medium presented an increase in phospho-EGFR, but the lysates from the MKN45/PAR1 which were cultured with the concentrate derived from the 3 hr a-thrombin stimulated conditioned medium presented no detectable bands (Figure 14 left). The level of TN-C was minimal in the concentrate derived from the 3 hr a-thrombin stimulated conditioned medium, but abundant in the concentrate derived from the 12 hr a-thrombin stimulated conditioned medium. These results clearly indicate that a-thrombin-stimulated MKN45/PAR1 produced high-molecular-weight consti- tuents (possibly, TN-C), which can activate EGFR in an autocrine manner. We suspected that TN-C would be present along with constituents with sizes greater than 200 kDa. To deter- mine if TN-C might be responsible for the ability of the concentrated medium to activate EGFR, we repeated the experiment described above in the presence of a TN-C- neutralizing antibody. The antibody markedly reduced the ability of the high-molecular-weight cutoff medium to stimulate the phosphorylation of the EGFR (Figure 14 right). And we showed by means of histograms that phosphor-EGFR signal quantitatively diminished in the presence of TN-C-neutralizing antibody (Figure 15).