Role of Protease-Activated Receptor-2 Activation on The Expression of VEGF mRNA and Its Protein in MKN28 Gastric Cancer Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 ZHANGCheng , GAOGuang-rong , LIDa , LÜChi , SHANYong-qi ,  ZHANGXue-feng

Department of General Surgery, General Hospital of Shenyang Military Area Command, Shenyang 110840, Liaoning Province, China;

Corresponding author：

ZHANGXue-feng, Email: cz1791@126.com

Keywords：

Protease-activated receptor-2; Vascular endothelial cell growth factor; Gastric cancer

DOI：

10.7507/1007-9424.20150173

Video：

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Objective To investigate the role of protease-activated receptor-2 (PAR-2) activation on the expression of vascular endothelial growth factor (VEGF) in MKN28 gastric cancer cells. Methods ①MKN28 cells were treated with increased concentrations of trypsin (0, 0.1, 1.0, 10.0, and 100.0 nmol/L respectively) for 6 hours, or treated with 10.0 nmol/L trypsin for 3, 6, 12, and 24 hours (blank control group was treated with PBS) respectively, then the expression levels of VEGF mRNA and its protein in MKN28 cells were detected by real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western bolt method, with the concentration of VEGF protein in broth was detected by enzyme linked immunosorbent assay (ELISA) method.②MKN28 cells were divided into blank control group (treated with PBS), trypsin group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group, then the expression levels of VEGF mRNA and its protein in MKN28 cells were detected by qRT-PCR method and Western bolt method respectively. Results ①The effect of different concentration of trypsin. Compared with blank control group, the expression levels of VEGF mRNA and its protein in 0.1, 1.0, 10.0, and 100.0 nmol/L group were higher (P < 0.05); compared with 0.1 nmol/L group, the expression levels of VEGF mRNA and its protein in 1.0, 10.0, and 100.0 nmol/L group were higher (P < 0.05); compared with 1.0 nmol/L group, the expression levels of VEGF mRNA and its protein in 10.0 and 100.0 nmol/L group were higher (P < 0.05); but there was no significant difference between 10.0 nmol/L and 100.0 nmol/L group (P > 0.05). The broth concentration of VEGF protein in blank control group, 0.1, 1.0, 10.0, and 100.0 nmol/L group crept upward (P < 0.05).②The effect of different treated time of 10.0 nmol/L trypsin. The expression levels of VEGF mRNA in blank control group, 3, 6, 12, and 24 hours group crept upward, and there was significant difference between any 2 groups (P < 0.05). But the expression of VEGF protein was not similar with VEGF mRNA. Compared with blank control group, the expression levels of VEGF protein in 3, 6, 12, and 24 hours group were higher (P < 0.05); compared with 3 hours group, the expression levels of VEGF protein in 6, 12, and 24 hours group were higher (P < 0.05); but there was no significant difference among 6, 12, and 24 hours group (P > 0.05). The broth concentration of VEGF protein in blank control group, 3, 6, 12, and 24 hours group crept upward, and there was significant difference between any 2 groups (P < 0.05).③The effect of extracellular regulated protein kinase (ERK) inhibitor (PD98059) and p38 inhibitor (SB203580). The expression levels of VEGF mRNA and its protein in trypsin group were all higher than corresponding indexes of blank control group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group (P < 0.01), but there was no significant difference among blank control group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group (P > 0.05). Conclusion Activation of PAR-2 can induce the expressions of VEGF mRNA and its protein in MKN28 gastric cancer cells, that is mediated by ERK1/2-and p38-dependent pathway.

Citation： ZHANGCheng, GAOGuang-rong, LIDa, LÜChi, SHANYong-qi, ZHANGXue-feng. Role of Protease-Activated Receptor-2 Activation on The Expression of VEGF mRNA and Its Protein in MKN28 Gastric Cancer Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2015, 22(6): 665-670. doi: 10.7507/1007-9424.20150173 Copy

1.	Ammendola M, Leporini C, Marech I, et al. Targeting mast cells tryptase in tumor microenvironment:a potential antiangiogenetic strategy. Biomed Res Int, 2014, 2014:154702.
2.	Chang LH, Pan SL, Lai CY, et al. Activated PAR-2 regulates pancreatic cancer progression through ILK/HIF-α-induced TGF-αexpression and MEK/VEGF-A-mediated angiogenesis. Am J Pathol, 2013, 183(2):566-575.
3.	Carneiro-Lobo TC, Lima MT, Mariano-Oliveira A, et al. Expression of tissue factor signaling pathway elements correlates with the production of vascular endothelial growth factor and interleukin-8 in human astrocytoma patients. Oncol Rep, 2014, 31(2):679-686.
4.	Wang Y, Lin M, Weng H, et al. ENMD-1068, a protease-activated receptor 2 antagonist, inhibits the development of endometriosis in a mouse model. Am J Obstet Gynecol, 2014, 210(6):531.e1-531.e8.
5.	Rasmussen JG, Riis SE, Frøbert O, et al. Activation of protease-activated receptor 2 induces VEGF independently of HIF-1. PLoS One, 2012, 7(9):e46087.
6.	Sedda S, Marafini I, Caruso R, et al. Proteinase activated-receptorsassociated signaling in the control of gastric cancer. World J Gastroenterol, 2014, 20(34):11977-11984.
7.	Wang GJ, Wang YB, Li DN, et al. Expression of protease-activated receptor-2 in human gastric stromal tumor and its clinicopathological significance. Hepatogastroenterology, 2013, 60(128):2125-2128.
8.	Ossovskaya VS, Bunnett NW. Protease-activated receptors:contribution to physiology and disease. Physiol Rev, 2004, 84(2):579-621.
9.	Iablokov V, Hirota CL, Peplowski MA, et al. Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells. J Biol Chem, 2014, 289(49):34366-34377.
10.	Maeda S, Ohno K, Uchida K, et al. Intestinal protease-activated receptor-2 and fecal serine protease activity are increased in canine inflammatory bowel disease and may contribute to intestinal cytokine expression. J Vet Med Sci, 2014, 76(8):1119-1127.
11.	Fujimoto D, Hirono Y, Goi T, et al. Expression of protease activated receptor-2 (PAR-2) in gastric cancer. J Surg Oncol, 2006, 93(2):139-144.
12.	Jahan I, Fujimoto J, Alam SM, et al. Role of protease activated receptor-2 in tumor advancement of ovarian cancers. Ann Oncol, 2007, 18(9):1506-1512.
13.	Caruso R, Pallone F, Fina D, et al. Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation. Am J Pathol, 2006, 169(1):268-278.
14.	Darmoul D, Gratio V, Devaud H, et al. Protease-activated receptor 2 in colon cancer:trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation. J Biol Chem, 2004, 279(20):20927-20934.
15.	Hirota CL, Moreau F, Iablokov V, et al. Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2012, 303(1):G111-G119.
16.	van der Merwe JQ, Hollenberg MD, MacNaughton WK. EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2008, 294(2):G441-G451.
17.	Michel N, Heuzé-Vourc'h N, Lavergne E, et al. Growth and survival of lung cancer cells:regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor. Biol Chem, 2014, 395(9):1015-1025.
18.	Shimamoto R, Sawada T, Uchima Y, et al. A role for protease-activated receptor-2 in pancreatic cancer cell proliferation. Int J Oncol, 2004, 24(6):1401-1406.
19.	Syeda F, Grosjean J, Houliston RA, et al. Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways. J Biol Chem, 2006, 281(17):11792-11804.
20.	Kawaguchi M, Kanemaru A, Sawaguchi A, et al. Hepatocyte growth factor activator inhibitor type 1 maintains the assembly of keratin into desmosomes in keratinocytes by regulating protease-activated receptor 2-dependent p38 signaling. Am J Pathol, 2015, [Epub ahead of print].
21.	Defea KA, Zalevsky J, Thoma MS, et al. Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol, 2000, 148(6):1267-1281.
22.	Xiao YQ, Malcolm K, Worthen GS, et al. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-beta. J Biol Chem, 2002, 277(17):14884-14893.
23.	Fyfe M, Bergström M, Aspengren S, et al. PAR-2 activation in intestinal epithelial cells potentiates interleukin-1beta-induced chemokine secretion via MAP kinase signaling pathways. Cytokine, 2005, 31(5):358-367.

1. Ammendola M, Leporini C, Marech I, et al. Targeting mast cells tryptase in tumor microenvironment:a potential antiangiogenetic strategy. Biomed Res Int, 2014, 2014:154702.
2. Chang LH, Pan SL, Lai CY, et al. Activated PAR-2 regulates pancreatic cancer progression through ILK/HIF-α-induced TGF-αexpression and MEK/VEGF-A-mediated angiogenesis. Am J Pathol, 2013, 183(2):566-575.
3. Carneiro-Lobo TC, Lima MT, Mariano-Oliveira A, et al. Expression of tissue factor signaling pathway elements correlates with the production of vascular endothelial growth factor and interleukin-8 in human astrocytoma patients. Oncol Rep, 2014, 31(2):679-686.
4. Wang Y, Lin M, Weng H, et al. ENMD-1068, a protease-activated receptor 2 antagonist, inhibits the development of endometriosis in a mouse model. Am J Obstet Gynecol, 2014, 210(6):531.e1-531.e8.
5. Rasmussen JG, Riis SE, Frøbert O, et al. Activation of protease-activated receptor 2 induces VEGF independently of HIF-1. PLoS One, 2012, 7(9):e46087.
6. Sedda S, Marafini I, Caruso R, et al. Proteinase activated-receptorsassociated signaling in the control of gastric cancer. World J Gastroenterol, 2014, 20(34):11977-11984.
7. Wang GJ, Wang YB, Li DN, et al. Expression of protease-activated receptor-2 in human gastric stromal tumor and its clinicopathological significance. Hepatogastroenterology, 2013, 60(128):2125-2128.
8. Ossovskaya VS, Bunnett NW. Protease-activated receptors:contribution to physiology and disease. Physiol Rev, 2004, 84(2):579-621.
9. Iablokov V, Hirota CL, Peplowski MA, et al. Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells. J Biol Chem, 2014, 289(49):34366-34377.
10. Maeda S, Ohno K, Uchida K, et al. Intestinal protease-activated receptor-2 and fecal serine protease activity are increased in canine inflammatory bowel disease and may contribute to intestinal cytokine expression. J Vet Med Sci, 2014, 76(8):1119-1127.
11. Fujimoto D, Hirono Y, Goi T, et al. Expression of protease activated receptor-2 (PAR-2) in gastric cancer. J Surg Oncol, 2006, 93(2):139-144.
12. Jahan I, Fujimoto J, Alam SM, et al. Role of protease activated receptor-2 in tumor advancement of ovarian cancers. Ann Oncol, 2007, 18(9):1506-1512.
13. Caruso R, Pallone F, Fina D, et al. Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation. Am J Pathol, 2006, 169(1):268-278.
14. Darmoul D, Gratio V, Devaud H, et al. Protease-activated receptor 2 in colon cancer:trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation. J Biol Chem, 2004, 279(20):20927-20934.
15. Hirota CL, Moreau F, Iablokov V, et al. Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2012, 303(1):G111-G119.
16. van der Merwe JQ, Hollenberg MD, MacNaughton WK. EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2008, 294(2):G441-G451.
17. Michel N, Heuzé-Vourc'h N, Lavergne E, et al. Growth and survival of lung cancer cells:regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor. Biol Chem, 2014, 395(9):1015-1025.
18. Shimamoto R, Sawada T, Uchima Y, et al. A role for protease-activated receptor-2 in pancreatic cancer cell proliferation. Int J Oncol, 2004, 24(6):1401-1406.
19. Syeda F, Grosjean J, Houliston RA, et al. Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways. J Biol Chem, 2006, 281(17):11792-11804.
20. Kawaguchi M, Kanemaru A, Sawaguchi A, et al. Hepatocyte growth factor activator inhibitor type 1 maintains the assembly of keratin into desmosomes in keratinocytes by regulating protease-activated receptor 2-dependent p38 signaling. Am J Pathol, 2015, [Epub ahead of print].
21. Defea KA, Zalevsky J, Thoma MS, et al. Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol, 2000, 148(6):1267-1281.
22. Xiao YQ, Malcolm K, Worthen GS, et al. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-beta. J Biol Chem, 2002, 277(17):14884-14893.
23. Fyfe M, Bergström M, Aspengren S, et al. PAR-2 activation in intestinal epithelial cells potentiates interleukin-1beta-induced chemokine secretion via MAP kinase signaling pathways. Cytokine, 2005, 31(5):358-367.

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Role of Protease-Activated Receptor-2 Activation on The Expression of VEGF mRNA and Its Protein in MKN28 Gastric Cancer Cells

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