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• W2116724134 abstract "Background & Aims: The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Methods: Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A2 [cPLA2]). Results: The inducible expression of Ha-RasV12 increased the production of prostaglandin (PG)F2α and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-RasV12 also up-regulated the mRNA and protein levels of cPLA2, COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA2. Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI2 alone also induced the expression of VEGF. Conclusions: Inducible Ha-RasV12 increases the production of PGI2 through the coordinate up-regulation of cPLA2, COX-2, and prostacyclin synthase (PGIS). The production of PGI2 leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis. Background & Aims: The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Methods: Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A2 [cPLA2]). Results: The inducible expression of Ha-RasV12 increased the production of prostaglandin (PG)F2α and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-RasV12 also up-regulated the mRNA and protein levels of cPLA2, COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA2. Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI2 alone also induced the expression of VEGF. Conclusions: Inducible Ha-RasV12 increases the production of PGI2 through the coordinate up-regulation of cPLA2, COX-2, and prostacyclin synthase (PGIS). The production of PGI2 leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis. Colorectal cancer is the second leading cause of cancer-related deaths in the United States and is a significant health concern in most industrialized countries. There will be over 107,000 estimated new cases diagnosed and over 48,000 estimated deaths caused by colorectal cancer this year alone in the United States.1Jemal A. Thomas A. Murray T. Thun M. Cancer statistics, 2002.CA Cancer J Clin. 2002; 52: 23-47Crossref PubMed Scopus (2930) Google Scholar The development of most colorectal cancers is thought to occur through a multistep process that involves the progression of normal colonic epithelium to an adenoma, then to an adenocarcinoma.2Leslie A. Carey F.A. Pratt N.R. Steele R.J. The colorectal adenoma-carcinoma sequence.Br J Surg. 2002; 89: 845-860Crossref PubMed Scopus (504) Google Scholar Several mutations in key regulatory genes have been shown to occur during this process.3Srivastava S. Verma M. Henson D.E. Biomarkers for early detection of colon cancer.Clin Cancer Res. 2001; 7: 1118-1126PubMed Google Scholar An early mutation occurs in the adenomatous polyposis coli tumor-suppressor gene in about 80% of cases involving chromosomal instability and this mutation is thought to play a crucial role in the process.4Lamlum H. Papadopoulou A. Ilyas M. Rowan A. Gillet C. Hanby A. Talbot I. Bodmer W. Tomlinson I. APC mutations are sufficient for the growth of early colorectal adenomas.Proc Natl Acad Sci U S A. 2000; 97: 2225-2228Crossref PubMed Scopus (152) Google Scholar This often is followed by mutations in Ras5Vogelstein B. Fearon E.R. Hamilton S.R. Kern S.E. Preisinger A.C. Leppert M. Nakamura Y. White R. Smits A.M. Bos J.L. Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Google Scholar and the increased expression of cyclooxygenase-2 (COX-2). Furthermore, the loss of p53 function is observed late in colorectal carcinoma development via the chromosomal instability pathway.6Vogelstein B. Fearon E.R. Hamilton S.R. Kern S.E. Preisinger A.C. Leppert M. Nakamura Y. White R. Smits A.M. Bos J.L. Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5910) Google Scholar The precise role of Ras during the progression to colorectal cancer still is under investigation, but clearly Ras signaling has dramatic effects on the regulation of cell proliferation and programmed cell death. Ras has been implicated to serve a role at the earliest stages in this process because mutations in this gene can occur in up to 92% of aberrant crypt foci in humans,7Takayama T. Katsuki S. Takahashi Y. Ohi M. Nojiri S. Sakamaki S. Kato J. Kogawa K. Miyake H. Niitsu Y. Aberrant crypt foci of the colon as precursors of adenoma and cancer.N Engl J Med. 1998; 339: 1277-1284Crossref PubMed Scopus (531) Google Scholar, 8Takayama T. Ohi M. Hayashi T. Miyanishi K. Nobuoka A. Nakajima T. Satoh T. Takimoto R. Kato J. Sakamaki S. Niitsu Y. Analysis of K-ras, APC, and beta-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 9Pretlow T.P. Brasitus T.A. Fulton N.C. Cheyer C. Kaplan E.L. K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (220) Google Scholar, 10Yamashita N. Minamoto T. Ochiai A. Onda M. Esumi H. Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon.Gastroenterology. 1995; 108: 434-440Abstract Full Text PDF PubMed Scopus (132) Google Scholar and at significant levels in aberrant crypt foci of carcinogen-treated rodents.11Vivona A.A. Shpitz B. Medline A. Bruce W.R. Hay K. Ward M.A. Stern H.S. Gallinger S. K-ras mutations in aberrant crypt foci, adenomas and adenocarcinomas during azoxymethane-induced colon carcinogenesis.Carcinogenesis. 1993; 14: 1777-1781Crossref PubMed Scopus (135) Google Scholar, 12Shivapurkar N. Tang Z. Ferreira A. Nasim S. Garett C. Alabaster O. Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet.Carcinogenesis. 1994; 15: 775-778Crossref PubMed Scopus (74) Google Scholar, 13Tachino N. Hayashi R. Liew C. Bailey G. Dashwood R. Evidence for ras gene mutation in 2-amino-3-methylimidazo[4,5-f]quinoline-induced colonic aberrant crypts in the rat.Mol Carcinog. 1995; 12: 187-192Crossref PubMed Scopus (53) Google Scholar One intriguing effect of Ras in epithelial cells is an increase in the production of prostaglandins (PGs).14Shao J. Sheng H. DuBois R.N. Peroxisome proliferator-activated receptors modulate K-ras-mediated transformation of intestinal epithelial cells.Cancer Res. 2002; 62: 3282-3288PubMed Google Scholar, 15Taylor M.T. Lawson K.R. Ignatenko N.A. Marek S.E. Stringer D.E. Skovan B.A. Gerner E.W. Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expression in human colon cancer cells.Cancer Res. 2000; 60: 6607-6610PubMed Google Scholar Recent studies have shown the importance of PGs in tumor growth and metastasis. For example, the treatment of prostate, breast, or colon cancer cells with PGE2 induces an increase in growth and migration.16Sheng H. Shao J. Washington M.K. DuBois R.N. Prostaglandin E2 increases growth and motility of colorectal carcinoma cells.J Biol Chem. 2001; 276: 18075-18081Crossref PubMed Scopus (578) Google Scholar, 17Tjandrawinata R.R. Dahiya R. Hughes-Fulford M. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells.Br J Cancer. 1997; 75: 1111-1118Crossref PubMed Scopus (213) Google Scholar, 18Bing R.J. Miyataka M. Rich K.A. Hanson N. Wang X. Slosser H.D. Shi S.R. Nitric oxide, prostanoids, cyclooxygenase, and angiogenesis in colon and breast cancer.Clin Cancer Res. 2001; 7: 3385-3392PubMed Google Scholar Furthermore, PGI2 can induce the activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta, which recently has been shown to increase the number and size of intestinal polyps in adenomatous polyposis coli(min) mice.19Gupta R.A. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Prostacyclin-mediated activation of peroxisome proliferator-activated receptor delta in colorectal cancer.Proc Natl Acad Sci U S A. 2000; 97: 13275-13280Crossref PubMed Scopus (358) Google Scholar, 20Gupta R.A. Wang D. Katkuri S. Wang H. Dey S.K. DuBois R.N. Activation of nuclear hormone receptor peroxisome proliferator-activated receptor-delta accelerates intestinal adenoma growth.Nat Med. 2004; 10: 245-247Crossref PubMed Scopus (254) Google Scholar PGI2 also can promote angiogenesis through the production of vascular endothelial growth factor (VEGF).21Pola R. Gaetani E. Flex A. Aprahamian T.R. Bosch-Marce M. Losordo D.W. Smith R.C. Pola P. Comparative analysis of the in vivo angiogenic properties of stable prostacyclin analogs a possible role for peroxisome proliferator-activated receptors.J Mol Cell Cardiol. 2004; 36: 363-370Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar The production of PGs occurs via the metabolism of arachidonic acid (Figure 1). Phospholipase A2 (cPLA2), the rate-limiting enzyme in prostaglandin production, generates arachidonic acid through the cleavage of a phosphodiester bond at the sn-2 position of cell membrane phospholipids. The resulting arachidonate, a 20-carbon-containing fatty acid, then is converted to PGH2 by prostaglandin endoperoxide synthase, also termed cyclooxygenase.22Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases structural, cellular, and molecular biology.Annu Rev Biochem. 2000; 69: 145-182Crossref PubMed Scopus (1546) Google Scholar, 23Vane J.R. Bakhle Y.S. Botting R.M. Cyclooxygenases 1 and 2.Annu Rev Pharmacol Toxicol. 1998; 38: 97-120Crossref PubMed Scopus (2607) Google Scholar There are 2 known forms of cyclooxygenase: COX-1 and COX-2. COX-1 is expressed in many tissues constitutively but can be up-regulated in some cancers.24Gupta R.A. Tejada L.V. Tong B.J. Das S.K. Morrow J.D. Dey S.K. DuBois R.N. Cyclooxygenase-1 is overexpressed and promotes angiogenic growth factor production in ovarian cancer.Cancer Res. 2003; 63: 906-911PubMed Google Scholar However, the expression of COX-2 is inducible in many circumstances by a variety of mitogens and tumor promoters and levels of COX-2 are increased dramatically in many tumors.25Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases structural, cellular, and molecular biology.Annu Rev Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2464) Google Scholar, 26Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas.Gastroenterology. 1994; 107: 1183-1188Abstract PubMed Google Scholar, 27Hida T. Yatabe Y. Achiwa H. Muramatsu H. Kozaki K. Nakamura S. Ogawa M. Mitsudomi T. Sugiura T. Takahashi T. Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas.Cancer Res. 1998; 58: 3761-3764PubMed Google Scholar, 28Half E. Tang X.M. Gwyn K. Sahin A. Wathen K. Sinicrope F.A. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ.Cancer Res. 2002; 62: 1676-1681PubMed Google Scholar PGH2 is an extremely unstable compound and is converted quickly to PGE2, PGI2, PGD2, PGF2, or thromboxane A2 by their respective PG synthases. An increasing interest in the role of COX-2 in tumor growth and development has emerged over the past few years. The first observations indicating the potential role of prostaglandins in intestinal carcinogenesis arose from population studies showing up to a 50% decrease in the mortality rate of patients taking nonsteroidal anti-inflammatory drugs such as aspirin on a regular basis.29Greenberg E.R. Baron J.A. Freeman Jr, D.H. Mandel J.S. Haile R. Reduced risk of large-bowel adenomas among aspirin users. The Polyp Prevention Study Group.J Natl Cancer Inst. 1993; 85: 912-916Crossref PubMed Scopus (294) Google Scholar, 30Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath Jr, C.W. Aspirin use and risk of fatal cancer.Cancer Res. 1993; 53: 1322-1327PubMed Google Scholar, 31Giovannucci E. Rimm E.B. Stampfer M.J. Colditz G.A. Ascherio A. Willett W.C. Aspirin use and the risk for colorectal cancer and adenoma in male health professionals.Ann Intern Med. 1994; 121: 241-246Crossref PubMed Scopus (736) Google Scholar Since these initial findings, the use of nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors has been shown to inhibit tumor growth and metastasis in many cell culture and animal models,32Williams C.S. Sheng H. Brockman J.A. Armandla R. Shao J. Washington M.K. Elkahloun A.G. DuBois R.N. A cyclooxygenase-2 inhibitor (SC-58125) blocks growth of established human colon cancer xenografts.Neoplasia. 2001; 3: 428-436Crossref PubMed Scopus (35) Google Scholar, 33Sheng H. Shao J. Kirkland S.C. Isakson P. Coffey R.J. Morrow J. Beauchamp R.D. DuBois R.N. Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.J Clin Invest. 1997; 99: 2254-2259Crossref PubMed Scopus (699) Google Scholar, 34Liu X.H. Kirschenbaum A. Yao S. Lee R. Holland J.F. Levine A.C. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo.J Urol. 2000; 164: 820-825Abstract Full Text Full Text PDF PubMed Google Scholar, 35Lim J.T. Piazza G.A. Han E.K. Delohery T.M. Li H. Finn T.S. Buttyan R. Yamamoto H. Sperl G.J. Brendel K. Gross P.H. Pamukcu R. Weinstein I.B. Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines.Biochem Pharmacol. 1999; 58: 1097-1107Crossref PubMed Scopus (189) Google Scholar as well as to regulate angiogenesis.34Liu X.H. Kirschenbaum A. Yao S. Lee R. Holland J.F. Levine A.C. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo.J Urol. 2000; 164: 820-825Abstract Full Text Full Text PDF PubMed Google Scholar, 36Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cyclooxygenase regulates angiogenesis induced by colon cancer cells.Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar, 37Daniel T.O. Liu H. Morrow J.D. Crews B.C. Marnett L.J. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis.Cancer Res. 1999; 59: 4574-4577PubMed Google Scholar Although oncogenic Ras has been shown to increase the expression of cPLA2 and COX-2 in lung, fibroblast, and intestinal cell lines,38Heasley L.E. Thaler S. Nicks M. Price B. Skorecki K. Nemenoff R.A. Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer.J Biol Chem. 1997; 272: 14501-14504Crossref PubMed Scopus (163) Google Scholar, 39Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway.J Biol Chem. 1998; 273: 22120-22127Crossref PubMed Scopus (179) Google Scholar, 40Sheng H. Shao J. DuBois R.N. K-Ras-mediated increase in cyclooxygenase 2 mRNA stability involves activation of the protein kinase B1.Cancer Res. 2001; 61: 2670-2675PubMed Google Scholar the coordinate regulation of the enzymes involved in prostaglandin production have not been studied carefully. Therefore, we sought to determine if the expression of Ha-RasV12 could coordinately induce the expression and activation of cPLA2, COX-2, and PGI2 synthase (PGIS) in nontransformed intestinal epithelial cells (IECs). Furthermore, we also have examined what role COX-2 and PGI2 may serve in the induction of angiogenesis, a vital process in the early stages of tumor growth and metastasis. Antibodies to COX-2, cPLA2, PGIS, and VEGF were purchased from Santa Cruz (Santa Cruz, CA). Antibodies to Ras and β-actin were purchased from Oncogene (Boston, MA) and Sigma (St. Louis, MO), respectively. Celecoxib (SC-58635) was provided as a kind gift from G. D. Searle and Co. (St. Louis, MO). Scalaradial (SC) and methyl arachidonyl fluorophosphonate were obtained from Biomol (Plymouth Meeting, PA) and tranylcypromine was purchased from Sigma. The rat intestinal epithelial (RIE)-iHa-Ras(inducible) cell line was generated as described previously.41Sheng H. Shao J. Dixon D.A. Williams C.S. Prescott S.M. DuBois R.N. Beauchamp R.D. Transforming growth factor-beta1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of.J Biol Chem. 2000; 275: 6628-6635Crossref PubMed Scopus (189) Google Scholar Cells were maintained in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 400 μg/mL G418 (Life Technologies, Inc., Carlsbad, CA), and 150 μg/mL hygromycin B (Calbiochem, San Diego, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2 atmosphere. Isopropyl-1-thio-β-D-galactopyranoside (IPTG; Life Technologies, Inc.), at a concentration of 5 mmol/L, was used to induce the expression of Ha-RasV12. Prostaglandin profiles for the RIE-iHa-Ras cells were measured and quantified by using a gas chromatography/negative ion chemical ionization mass spectrometric assay as previously reported.42DuBois R.N. Awad J. Morrow J. Roberts L.J. Bishop P.R. Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester.J Clin Invest. 1994; 93: 493-498Crossref PubMed Scopus (378) Google Scholar Total cellular RNA was isolated by using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. RNA samples were separated on 1.0% formaldehyde agarose gels (20 μg/lane) and blotted onto nitrocellulose membranes. The blots were hybridized with complementary DNA probes labeled with [α32P]deoxycytidine triphosphate by random primer extension (Stratagene, La Jolla, CA). After hybridization and washing, the blots were subjected to autoradiography with BioMax film (Fisher, Pittsburgh, PA). 28s RNA was used as a control to confirm equal loading of samples and the quality of RNA. Cells were washed 1× in cold phosphate-buffered saline and lysed in phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mg/mL phenylmethyl sulfonyl fluoride, 10 mg/mL aprotinin, 10 mg/mL pepstatin, and 10 mg/mL leupeptin. The protein lysates (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% dry milk and primary antibody incubations were performed in Tris-buffered saline with 0.1% Tween 20 with 5% dry milk overnight at 4°C. After washing, the membranes were incubated with the appropriate secondary peroxidase-conjugated antibody for 1 hour in Tris-buffered saline with 0.1% Tween 20 with 5% dry milk. Immunoreactive proteins were visualized using the enhanced chemiluminescence system from Amersham Pharmacia (Arlington Heights, IL). Cells were collected by centrifugation (2000g for 10 min) and resuspended at 7.5 × 105 cells/mL in HEPES buffer (50 mmol/L HEPES, pH 7.4, with 1 mmol/L ethylenediaminetetraacetic acid). The cells were sonicated, centrifuged at 10,000g for 15 minutes at 4°C, and the supernatant was collected. The activity of cPLA2 was measured using the cPLA2 assay kit from Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s protocol. Four fragments of the cPLA2 promoter were generously provided by Dr. Rapheal A. Nemenoff (University of Colorado Health Science Center, Denver, CO). Cells were cotransfected with 0.5 μg of the cPLA2 promoter construct and 0.01 μg of the pRL-CMV plasmid that contains the Renilla luciferase gene (Promega, Madison, WI) with FuGene6 transfection reagent (Roche, Indianapolis, IN) per the manufacturer’s instructions. Transfected cells were cultured for 24 hours. The media was replaced with Dulbecco’s modified Eagle medium 10% fetal bovine serum containing 5 mmol/L IPTG. After incubation for 24 hours, the cells were harvested. Twenty micrograms of lysate were used for both the firefly and Renilla luciferase assay. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) and a monolight 3010 Luminometer (PharMingen, San Diego, CA). Firefly luciferase values were normalized relative to Renilla luciferase. The concentration of VEGF in cultured media was measured by enzyme-linked immunosorbent assay with the QuantikineM Mouse VEGF Immunoassay kit from R&D Systems (Minneapolis, MN) according to the manufacturer’s protocol. The induction of constitutively active Ras is known to transform a variety of cell types. As our laboratory has reported previously,41Sheng H. Shao J. Dixon D.A. Williams C.S. Prescott S.M. DuBois R.N. Beauchamp R.D. Transforming growth factor-beta1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of.J Biol Chem. 2000; 275: 6628-6635Crossref PubMed Scopus (189) Google Scholar the induction of Ha-RasV12 can transform IECs. Normal RIE cells were stably transfected with the LacSwitch eukaryotic expression system (Stratagene) containing Ha-RasV12. In this system, the expression of Ha-RasV12 occurs only in the presence of IPTG. Noninduced cells have the same morphologic appearance as parental RIE cells. However, a morphologic change of RIE-iHa-RasV12 cells was observed within 24–48 hours after IPTG treatment (Figure 2A). During this time, the cells transformed into a spindle shape with condensed nuclei and exhibited a loss in contact-dependent growth regulation. Increased levels of prostaglandins have been reported in a variety of carcinoma cell lines and solid malignancies.43Bing R.J. Miyataka M. Rich K.A. Hanson N. Wang X. Slosser H.D. Shi S.R. Nitric oxide, prostanoids, cyclooxygenase, and angiogenesis in colon and breast cancer.Clin Cancer Res. 2001; 7: 3385-3392PubMed Google Scholar, 44Shao J. Sheng H. Inoue H. Morrow J.D. DuBois R.N. Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells.J Biol Chem. 2000; 275: 33951-33956Crossref PubMed Scopus (248) Google Scholar Because mutant Ras and increased COX-2 levels are observed in many tumor tissues and cell lines, we determined if the induction of Ha-RasV12 could stimulate prostaglandin production in intestinal epithelial cells in a coordinate manner. The production of PGI2 requires the presence of not only the COX enzyme, but also prostacylin synthase and phospholipase. After IPTG treatment for 24 hours the levels of PGF2α and PGI2 were increased 2- and 13-fold, respectively (Figure 2B). Basal levels of PGF2α increased from 4.63 to 9.9 ng/mL with the induction of Ha-RasV12. The levels of PGI2 showed a marked increase from 0.8 to 10.8 ng/mL. Our laboratory also has observed that Ras can induce the production of PGI2 in IEC cells.14Shao J. Sheng H. DuBois R.N. Peroxisome proliferator-activated receptors modulate K-ras-mediated transformation of intestinal epithelial cells.Cancer Res. 2002; 62: 3282-3288PubMed Google Scholar IEC cells, similar to RIE cells, are also a normal intestinal epithelial cell line. Other prostaglandins (PGD2, PGE2, and thromboxane A2) were not produced in the presence or absence of Ras activation. As shown in Figure 2C and D, the media concentration of 6-k-PGF1α (a stable metabolite of PGI2) was maximal 48 hours after the induction of Ha-RasV12, and began to increase within 8 hours of IPTG treatment. The production of prostaglandins occurs via the presence of at least 3 enzymes. cPLA2 generates arachidonic acid through the cleavage of a phosphodiester bond at the sn-2 position of phospholipids. The liberated arachidonate then is converted to PGH2 by COX. PGH2 is an extremely unstable compound and then is converted to various prostaglandins by their respective PG synthases. Because Ha-RasV12 induces an increase in the production of PGI2 (Figure 2B), we sought to understand whether activation of Ha-RasV12 had any effects on the expression of the other key enzymes required for the biosynthesis of prostacyclin. As shown in Figure 3A, Northern analysis shows that treatment with IPTG induced an increase in messenger RNA (mRNA) encoding cPLA2, COX-2, and PGIS. The increase in the level of cPLA2 and PGIS mRNAs was first observed at 8 hours and was maximal at 48 hours. The level of COX-2 mRNA was increased by 24 hours and was maximal at 48 hours as well. An increase in the protein level of cPLA2 was seen as early as 8 hours and was highly expressed through 72 hours of IPTG treatment (Figure 3B). Similarly, the protein levels of COX-2 and PGIS were observed to increase within 24 hours and remained increased until 72 hours after IPTG treatment. An increase in Ha-Ras protein was seen as early as 8 hours after IPTG treatment and continued through 72 hours. Furthermore, we previously showed that Ras can induce the expression of COX-2 in IEC cells as well.40Sheng H. Shao J. DuBois R.N. K-Ras-mediated increase in cyclooxygenase 2 mRNA stability involves activation of the protein kinase B1.Cancer Res. 2001; 61: 2670-2675PubMed Google Scholar Because cPLA2 is normally thought to be the rate-limiting step in the biosynthesis of prostaglandins, the activity and expression of this enzyme is an important regulator of PGI2 synthesis. Oncogenic Ras has been shown to increase the activity of the cPLA2 promoter as well as the expression and enzyme activity of cPLA2 in human non-small-cell lung cancer cells.38Heasley L.E. Thaler S. Nicks M. Price B. Skorecki K. Nemenoff R.A. Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer.J Biol Chem. 1997; 272: 14501-14504Crossref PubMed Scopus (163) Google Scholar, 45Blaine S.A. Wick M. Dessev C. Nemenoff R.A. Induction of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun.J Biol Chem. 2001; 276: 42737-42743Crossref PubMed Scopus (72) Google Scholar, 46van P.V. Refaat Z. Dessev C. Blaine S. Wick M. Butterfield L. Han S.Y. Heasley L.E. Nemenoff R.A. Induction of cytosolic phospholipase A2 by oncogenic Ras is mediated through the JNK and ERK pathways in rat epithelial cells.J Biol Chem. 2001; 276: 1226-1232Crossref PubMed Scopus (61) Google Scholar Therefore, we determined if Ha-RasV12 affected cPLA2 enzyme activity and promoter transactivation in IECs. The induction of Ha-RasV12 stimulated an increase in the enzymatic activity of cPLA2 within 24 hours of IPTG treatment (Figure 4A). At 48 hours there was a 3-fold increase in the level of cPLA2 activity from 7.47 × 10−4 to 24.17 × 10−4 μmol/min/mL. Ha-RasV12 also induced the promoter activity of cPLA2 (Figure 4B). We observed an increase in promoter activity in the 2.4-kb, 2.0-kb, and 1.0-kb truncations of the cPLA2 promoter after treatment with IPTG for 24 hours. The 0.2-kb promoter construct did not induce an increase in luciferase activity. Ha-RasV12 induced the highest level of luciferase activity in the 1.0-kb fragment of the 5′-flanking region of the cPLA2 promoter. The involvement of COX is a critical step in the RasV12-induced synthesis of PGI2. Two isozymes of COX have been identified and are termed COX-1 and COX-2. COX-1 is expressed in almost all tissues, whereas COX-2 is more limited in its expression pattern. Because Ha-RasV12 induced the expression of COX-2 (Figure 3B) and COX-2 has been shown to be highly expressed in a variety of tumors, we determined the effect of selective inhibition of COX-2 on PGI2 production after Ras activation. As shown in Figure 5, Ha-RasV12 induced an increase in the production of 6-k-PGF1α from 7.95 to 24.3 ng/mL within 24 hours. Cotreatment of cells with celecoxib (a COX-2-specific inhibitor) and IPTG almost completely inhibited the accumulation of PGI2. Furthermore, treatment of cells under similar conditions with the metabolic product of cPLA2, arachidonate, had no effect on the inhibition of PGI2 production. The expression of oncogenic Ras has been shown to induce the production of VEGF in a variety of cell types.47Arbiser" @default.
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• W2116724134 date "2004-11-01" @default.
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• W2116724134 title "Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor" @default.
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• W2116724134 doi "https://doi.org/10.1053/j.gastro.2004.07.025" @default.
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