The neutralizing EGFR antibody didn’t interfere with the power of PAR-2 peptide to market GC cell growth (Figure 4D). sufferers with EGFR-positive GC possess a worse prognosis than people that have EGFR-negative GC, and blocking the EGFR signaling pathway continues to be utilized to inhibit the development of individual GC xenografts successfully.2C4 The precise molecular systems that trigger activation of EGFR inside the GC microenvironment aren’t fully understood. EGFR could be straight activated by members of the EGF family, including EGF, transforming growth factor (TGF)-, and amphiregulin, all produced in excess in GC tissue.3 Studies in EG01377 TFA other systems have also revealed that, during neoplastic transformation and/or progression, EGFR can be transactivated by various extracellular stimuli, unrelated to EGFR ligands, such as cytokines, and agonists of the G protein-coupled receptor, such as proteases-activated receptors (PARs).5C7 EG01377 TFA PARs are seven transmembrane-spanning domain G protein-coupled receptors, comprising four receptors: PAR-1, PAR-2, PAR-3, and PAR-4. Activation of PARs is an irreversible phenomenon in which the protease binds to and cleaves the amino-terminal exodomain of the receptor. The cleavage generates a new amino-terminal sequence that binds to the core receptor and serves as a tethered ligand.8 Whereas PAR-1, -3, and -4 are activated by thrombin, PAR-2 is activated by multiple trypsin-like enzymes, such as trypsin itself and mast cell tryptase.9,10 Evidence has been accumulated to show Rabbit polyclonal to ANXA8L2 that trypsin is produced in excess in many cancers of the digestive tract, including GC, and it is supposed to contribute to the growth and diffusion of cancer cells.11 In line with this, overexpression of exogenous trypsinogen cDNA in human gastric cancer cells has been reported to increase their tumorigenicity in nude mice.12 Whether the ability of trypsin to enhance GC tumorigenesis relies on PAR-2 activation remains unknown, however. These observations together with the demonstration that PAR-2 has been involved in the growth of epithelial cancer13 prompted us to explore the role of PAR-2 in human GC. To this end, we first used AGS and MKN28 gastric cancer cell lines as a model of GC to examine whether PAR-2 activation results in enhanced EGFR signaling and cell growth. Second, we dissected the molecular mechanism by which PAR-2 regulates EGFR activation. Finally, the expression of PAR-2 in human gastric cancer specimens was evaluated. Materials and Methods Human Samples GC specimens were taken from 15 patients undergoing subtotal gastrectomy. No patient had received preoperative chemotherapy. Seven GCs were of intestinal type, whereas the EG01377 TFA remaining were signet-ring cell carcinomas (diffuse), according to the Lauren classification. Additionally gastric biopsies were taken from eight patients with Hp-related gastritis and 12 Hp-negative patients EG01377 TFA (controls). All specimens were taken from the antrum. Cell Culture and Proliferation The gastric cancer cell lines AGS and MKN28 (kindly provided by Prof. Marco Romano, Dipartimento di Internistica Clinica e Sperimentale-Gastroenterologia, II University of Naples, Italy) were cultured in 25-cm2 plastic flasks and maintained at 37C in a humidified atmosphere of 5% CO2 in Dulbeccos modified Eagles and RPMI 1640 media (both from Sigma-Aldrich, Milan, Italy), respectively, supplemented with 10% inactivated fetal bovine serum (FBS, Sigma-Aldrich). To assess cell proliferation, AGS and MKN28 cells were starved in serum-free medium for 24 hours, then 3000 to 5000 cells/well were seeded in 96-well culture dishes in medium supplemented with 0.1% of bovine serum albumin (Sigma-Aldrich), allowed to adhere for 4 hours, and then stimulated with the PAR-2-activating peptide (SLIGKV-NH2) or -inactivating peptide (VKGILS- NH2, both used at a final concentration of 20 mol/L; Sigma-Aldrich) for 48 hours. In parallel, cells were preincubated with the EGFR tyrosine kinase inhibitor, AG1478 (20 mol/L) or the Src tyrosine kinases inhibitor, PP1 (20 mol/L; both from Inalco, Milan, Italy) or dimethylsulfoxide (DMSO, vehicle) for 60 minutes before adding the PAR-2-activating peptide. The optimal concentration of both AG1478.
The neutralizing EGFR antibody didn’t interfere with the power of PAR-2 peptide to market GC cell growth (Figure 4D)