Effect Of Protease-activated Receptor 2 On Proliferation, Invasion And Metastasis Of Human Pancreatic Cancer SW1990 Cells

Objective: Proteinase-activated receptor 2 (PAR-2) is a kind of G protein-coupled seven-trans-membrane-domain receptor that is widely distributed in each systems and tissues. Trypsin, fibrinolytic enzyme, coagulation factor VIIa and Xa are endogenous activated peptides of PAR-2. And PAR-2 can also be activated by the synthetic selective agonist SLIGKV - NH2. A lot of studies reveal PAR-2 level is higher in digestive system tumors and tumor microenvironment than that in normal tissues. PAR-2 can also promote tumor cells on the proliferation, invasion and metastasis. Pancreatic cancer is a kind of malignant tumor of digestive system with poor prognosis. Due to its high invasion and migration rate, most patients hardly received radical therapy. Some researches showed that PAR-2 mRNA and protein were highly expressed in pancreatic cancer tissues, and PAR-2 can promote the proliferation of varied pancreatic cancer cells. It can also accelerate the growth and metastasis of subcutaneous transplanted pancreatic tumor in nude mouse. Both normal pancreatic acinus and pancreatic cancer cells can secrete trypsinogen, which transforms into trypsin after activation. And trypsin is the most effective agonists of PAR-2. These results indicated that PAR-2 is closely related to pancreatic cancer. So far, there is rare report around the world about PAR-2 promoting the invasion and metastasis of pancreatic cancer in vitro. And the studies on PAR-2 accelerate pancreatic cancer angiogenesis and lymphatic metastases are still in vivo. In this study ,we aimed to investigate the expression of protease activated receptor-2 (PAR-2) in human pancreatic cancer cell SW1990 line, and to evaluate the effect of PAR-2 on proliferation, invasion and metastasis of e pancreatic cancer.Methods: The expression of PAR-2 on SW1990 cells was determined by reverse transcription PCR(RT-PCR) and immunocytochemistry. MTT assay and cell proliferation cure were performed to examine whether endogenous PAR-2 acitivator trypsin and PAR-2 activating peptide SLIGKV could promote SW1990 cell proliferation.Cell cycle of SW1990 treated with trypsin and PAR-2 activating peptide SLIGKV was estimated by flow cytometry.Cell invasion and migration assay were performed to measure wheather trypsin and SLIGKV could promote cell invasion and migration in vitro.The chang of MMP-2, MMP-9, VEGF-A,VEGF-C,IL-8mRNA expression in SW1990 treated with trysin and SLIGKV were detected by RT-PCR.Gelatinase activity of MMP-2 and MMP-9 in SW1990 treated with trypsin and SLIGKV were detected by Zymographic analysis.The chang of VEGF-A,VEGF-C,IL-8 protein expression in cells supernatant fluid of SW1990 after treated 8h,16h,24h,32h with trysin and SLIGKV were detected by ELISA.Results:PAR-2 mRNA and protein expression in SW1990 cells1 The human Pancreatic cancer SW1990 cells showed PAR-2 positive. The PAR-2 protein was predominantly localized to membrane and plasma.2 PAR-2 mRNA was expressed in SW1990 cells. PAR-2 mRNA was upregulated in cells treated with trypsin or PAR-2 activating peptide SLIGKV,(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 SW1990 cells1 The proliferation rate of SW1990 cells treated with trypsin or PAR-2 activating peptide SLIGKV was significantly enhanced. The proliferation rate of SW1990 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 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 SW1990 cells which treated with trypsin or SLIGKV.Effect of PAR-2 agonist on invasion and migration of SW1990 cells1 After treated with trypsin or SLIGKV, the number of SW1990 cells that passed through the millicell inserts was significantly increased in the invasion assay the migration assay,(P<0.05).2 The mRNA level of MMP-2 but not MMP-9 in trypsin and SLIGKV groups were significantly increased,(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).3 ompared with control group and VKGILS group, the mRNA level of VEGF-A,VEGF-C,IL-8 in trypsin and SLIGKV groups were significantly increased, (P< 0.05).4 Compared with control group and VKGILS group, the protein level of VEGF-A,VEGF-C,IL-8 in trypsin and SLIGKV groups were showed similar trends,(P< 0.05).Conclusion:1 PAR-2 mRNA and protein are expressed in SW1990cells. Moreover, the mRNA level of PAR-2 can be upregulated by PAR-2 agonist(trypsin and SLIGKV).2 PAR-2 activated can accelerate cell cycle process, promote pancreatic cancer cells proliferation, in certain concentration and time, its promoting proliferation function in a dose-and time-dependent manner.3 PAR - 2 agonists may play a role in formation of blood vessels of human pancreatic carcinoma by stimulating VEGF-A, VEGF-C and IL-8 production.4 PAR - 2 agonists may play a role in promoting lymph node metastases and the formation of lymphatic vessel of human pancreatic carcinoma stimulating VEGF-A and VEGF-C production.5 PAR-2 agonist play a role in the invasion and metastasis of human pancreatic cancer cell by stimulating MMP-2,VEGF-A,VEGF-C,IL-8 production, but not MMP-9.