Effect Of Protease-activated Receptor 2 On Proliferation, Invasion And Metastasis Of Esophageal Cancer EC109 Cells

Objective: Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled seven-trans-membrane-domain receptor that is activated by trypsin-like proteinases. Many studies reveal PAR-2 level is higher in digestive system tumors and tumor microenvironment than that in normal tissues. Moreover, cancers with higher level of PAR-2 have a poor prognosis and shorter disease-free survival. Esophageal tumor is still a major cause of tumor related deaths worldwide.Nowadays, some reports reveal that PAR-2 is expressed in esophageal tumor and special intestinal metaplasia tissues. Moreover, PAR-2 is expressed in the luminal part of the esophageal tissues. The localization of the PAR-2 receptor indicates that the receptor can be cleaved and activated by trypsin in duodenogastric esophageal refluxate and may take part in esophageal cancer progression. In this study ,we aimed to investigate the expression of protease activated receptor-2 (PAR-2) in human esophageal cell line EC109, and to evaluate the effect of PAR-2 on proliferation, invasion and metastasis of esophageal cancer EC109 cells.Methods: 1 Human esophageal cell line EC109 was cultured. The expression of PAR-2 on EC109 cells was determined by immunocytochemistry and reverse transcription PCR(RT-PCR). 1 MTT assay and cell proliferation cure were performed to examine whether endogenous PAR-2 acitivator trypsin and PAR-2 activating peptide SLIGKV could promote EC109 cell proliferation. 3 Cell cycle of EC109 treated with trypsin and PAR-2 activating peptide SLIGKV was estimated by flow cytometry. 4 Cell invasion and migration assay were performed to measure wheather trypsin and SLIGKV could promote cell invasion and migration in vitro. 5 The chang of MMP-2, MMP-9, PAR-2 mRNA expression in EC109 treated with trysin and SLIGKV were detected by RT-PCR. 6 The gelatinase activity of MMP-2 and MMP-9 in EC109 treated with trypsin and SLIGKV were detected by Zymographic analysis.Results:PAR-2 mRNA and protein expression in EC109 cells1 The human esophageal cell EC109 showed PAR-2 positive. The PAR-2 protein was predominantly localized to membrane and plasma.2 PAR-2 mRNA was expressed in EC109 cells. PAR-2 mRNA was upregulated in cells treated with trypsin or PAR-2 activating peptide SLIGKV(0.781±0.045)(0.653±0.029)vs (0.491±0.032)(P<0.05), but not with reverse PAR-2 activating peptide VKGILS(P>0.05).Effect of PAR-2 agonist on proliferation of esophageal cancer EC109 cells1 The proliferation rate of EC109 cells treated with trypsin or PAR-2 activating peptide SLIGKV was significantly enhanced. The proliferation rate of EC109 was promoted in a dose-and time-dependent fashion when cells were stimulated with trypsin or SLIGKV. But there was no difference of statistical significance between reverse sequence peptide VKGILS and control group.2 The cell cycle was accelerated, along with the up-regulation of the percentage of phase G0/G1 and the down-regulation of the percentage of phase and phase G2/M in EC109 cells.Effect of PAR-2 agonist on invasion and migration of esophageal cancer EC109 cells1 After treated with trypsin or SLIGKV, the number of EC109 cells that passed through the millicell inserts was significantly increased in the invasion assay(80.8±10.9)(62.6±8.9) vs (41.3±7.2)(45.8±8.5),P<0.05) and in the migration assay(72.5±9.2) (59.4±8.7) vs (31.6±6.6) (36.2±9.8),(P<0.05).2 Compared with control group and VKGILS group, the mRNA level of MMP-9 but not MMP-2 in trypsin and SLIGKV groups were significantly increased (0.719±0.034) (0.466±0.042) vs (0.341±0.032) (0.370±0.021),(P< 0.05). Moreover, the changes of MMPs gelatinolytic activities showed similar trends(75.6±6.1) (60.4±4.6) vs (44.9±4.2) (39.3±5.2),(P<0.05).Conclusion:1 PAR-2 mRNA and protein were expressed in EC109 cells. Moreover, the mRNA level of PAR-2 could be upregulated by PAR-2 agonist.2 PAR-2 agonist could increase the proliferation of EC109 cells in a dose-and time-dependent manner through accelerating the cell cycle progression.3 PAR-2 agonist played a role in the invasion and metastasis of human esophageal carcimoma cells by stimulating MMP-9 production, but not MMP-2.