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• W2023183189 abstract "Prostaglandin (PG) E2 (PGE2) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE2 is accomplished by conversion of the cyclooxygenase (COX) product PGH2 by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Δ12,14-PGJ2 and PGA2 downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor γ or PGD2 receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE2. Our data suggest that there is a feedback mechanism between PGE2 and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC. Prostaglandin (PG) E2 (PGE2) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE2 is accomplished by conversion of the cyclooxygenase (COX) product PGH2 by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Δ12,14-PGJ2 and PGA2 downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor γ or PGD2 receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE2. Our data suggest that there is a feedback mechanism between PGE2 and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC. Population-based studies have demonstrated that long-term use of nonsteroidal antiinflammatory drugs (NSAIDs) reduces the relative risk of developing colorectal cancer (CRC) by 40–50% (1Gwyn K. Sinicrope F.A. Chemoprevention of colorectal cancer.Am. J. Gastroenterol. 2002; 97: 13-21Crossref PubMed Google Scholar). In addition, increased levels of cyclooxygenase type 2 (COX-2) are found in colorectal adenomas and adenocarcinomas compared with normal mucosa (2Rigas B. Goldman I.S. Levine L. Altered eicosanoid levels in human colon cancer.J. Lab. Clin. Med. 1993; 122: 518-523PubMed Google Scholar, 3Eberhart 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 Full Text PDF PubMed Scopus (2338) Google Scholar). NSAIDs inhibit the enzymatic activity of both isoforms of COX (COX-1 and COX-2). Because prolonged use of NSAIDs is associated with considerable side effects, which are believed to arise from the inhibition of the constitutively expressed COX-1 (4Vane J.R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs.Nat. New Biol. 1971; 231: 232-235Crossref PubMed Scopus (7305) Google Scholar), selective COX-2 inhibitors were developed. Indeed, this new class of drugs retains the antiinflammatory activity and antitumoral effects of the NSAIDs while reducing gastrointestinal toxicity by up to 50% (5Warner T.D. Mitchell J.A. Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic.FASEB J. 2004; 18: 790-804Crossref PubMed Scopus (528) Google Scholar). Unfortunately, prolonged use of higher doses of selective COX-2 inhibitors was recently shown to be associated with an increase in adverse cardiovascular events, resulting in the withdrawal of rofecoxib and valdecoxib. Prostaglandins are formed from a common unstable endoperoxide intermediate, prostaglandin (PG) H2 (PGH2), which in turn is generated via enzymatic oxygenation of arachidonic acid (AA) catalyzed by COX-1 or COX-2. Among the various downstream metabolites of COX-2-derived PGH2, PGE2 and its receptors have been demonstrated to play a predominant role in the promotion of colorectal carcinogenesis. This prostaglandin, which is found at increased levels in human colorectal adenomas and cancer (2Rigas B. Goldman I.S. Levine L. Altered eicosanoid levels in human colon cancer.J. Lab. Clin. Med. 1993; 122: 518-523PubMed Google Scholar, 6Pugh S. Thomas G.A. Patients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E2.Gut. 1994; 35: 675-678Crossref PubMed Scopus (241) Google Scholar), promotes a multitude of biologic actions related to colorectal carcinogenesis. PGE2 facilitates tumor progression by stimulation of cellular proliferation and angiogenesis, inhibition of apoptosis, and modulation of immunosuppression (for review, see 7Turini M.E. DuBois R.N. Cyclooxygenase-2: a therapeutic target.Annu. Rev. Med. 2002; 53: 35-57Crossref PubMed Scopus (547) Google Scholar). Treatment of rodent models with PGE2 has demonstrated increased cell proliferation and enhanced survival of epithelial cells in the gastrointestinal tract (8Watanabe K. Kawamori T. Nakatsugi S. Ohta T. Ohuchida S. Yamamoto H. Maruyama T. Kondo K. Ushikubi F. Narumiya S. et al.Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis.Cancer Res. 1999; 59: 5093-5096PubMed Google Scholar, 9Mutoh M. Watanabe K. Kitamura T. Shoji Y. Takahashi M. Kawamori T. Tani K. Kobayashi M. Maruyama T. Kobayashi K. et al.Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis.Cancer Res. 2002; 62: 28-32PubMed Google Scholar, 10Kawamori T. Uchiya N. Sugimura T. Wakabayashi K. Enhancement of colon carcinogenesis by prostaglandin E2 administration.Carcinogenesis. 2002; 24: 985-990Crossref Scopus (153) Google Scholar). Together, these findings suggest that the selective pharmacological inhibition of PGE2 production downstream of COX-2 is the best treatment for inhibiting carcinogenesis and that this may result in fewer side effects. In addition to prostaglandin E2 receptors (EPs), one such target for pharmacological intervention comprises the group of terminal prostaglandin E synthases (PGES). To date, three PGES isoforms have been identified, two perinuclear membrane-bound enzymes termed microsomal prostaglandin E synthase-1 (mPGES-1) and mPGES-2 and a cytosolic isoform [cytosolic prostaglandin E synthase (cPGES)]. Both microsomal proteins have been found to be overexpressed in human colorectal adenoma and cancer (11Yoshimatsu K. Golijanin D. Paty P.B. Soslow R.A. Jakobsson P.J. DeLellis R.A. Subbaramaiah K. Dannenberg A.J. Inducible microsomal prostaglandin E synthase is overexpressed in colorectal adenomas and cancer.Clin. Cancer Res. 2001; 7: 3971-3976PubMed Google Scholar, 12Murakami M. Nakashima K. Kamei D. Masuda S. Ishikawa Y. Ishii T. Ohmiya Y. Watanabe K. Kudo I. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2.J. Biol. Chem. 2003; 278: 37937-37947Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), emphasizing their importance as drug targets. In contrast, there are no data suggesting that cPGES contributes to colorectal carcinogenesis. Unlike PGE2, which has been demonstrated to definitely contribute to the promotion and survival of CRC, cyclopentenone prostaglandins (CyPGs), especially 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), are emerging as potent antitumor agents. In a variety of malignancies including CRC, 15d-PGJ2 displays growth-inhibitory and proapoptotic actions (13Takashima T. Fujiwara Y. Higuchi K. Arakawa T. Yano Y. Hasuma T. Otani S. PPAR-gamma ligands inhibit growth of human esophageal adenocarcinoma cells through induction of apoptosis, cell cycle arrest and reduction of ornithine decarboxylase activity.Int. J. Oncol. 2001; 19: 465-471PubMed Google Scholar, 14Chen Z.Y. Tseng C.C. 15-Deoxy-Delta12,14 prostaglandin J2 up-regulates Kruppel-like factor 4 expression independently of peroxisome proliferator-activated receptor gamma by activating the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signal transduction pathway in HT-29 colon cancer cells.Mol. Pharmacol. 2005; 68: 1203-1213Crossref PubMed Scopus (47) Google Scholar). 15d-PGJ2 is a CyPG of the J2 series derived from PGD2. Interestingly, the dependence of PGJ2 synthesis on PGD2 production suggests that the formation of PGJ2 adducts is delayed relative to the synthesis of other prostaglandins and that 15d-PGJ2 may participate in the resolution of PGE2-mediated inflammation (15Gilroy D.W. Colville-Nash P.R. Willis D. Chivers J. Paul-Clark M.J. Willoughby D.A. Inducible cyclooxygenase may have anti-inflammatory properties.Nat. Med. 1999; 5: 698-701Crossref PubMed Scopus (1115) Google Scholar). In addition, 15d-PGJ2 itself has been shown to regulate COX-2 expression via both peroxisome proliferator-activated receptor γ (PPARγ)-dependent and -independent mechanisms (16Inoue H. Tanabe T. Umesono K. Feedback control of cyclooxygenase-2 expression through PPARgamma.J. Biol. Chem. 2000; 275: 28028-28032Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 17Tsubouchi Y. Kawahito Y. Kohno M. Inoue K. Hla T. Sano H. Feedback control of the arachidonate cascade in rheumatoid synoviocytes by 15-deoxy-Delta(12,14)-prostaglandin J2.Biochem. Biophys. Res. Commun. 2001; 283: 750-755Crossref PubMed Scopus (82) Google Scholar). Finally, in vitro studies have demonstrated that 15d-PGJ2 can bind to mPGES-1, resulting in decreased PGES activity (18Quraishi O. Mancini J.A. Riendeau D. Inhibition of inducible prostaglandin E(2) synthase by 15-deoxy-Delta(12,14)-prostaglandin J(2) and polyunsaturated fatty acids.Biochem. Pharmacol. 2002; 63: 1183-1189Crossref PubMed Scopus (59) Google Scholar). Both PGE2 and 15d-PGJ2 are derived from the same precursor by the action of COX, but they have opposing effects on inflammatory processes and tumorigenesis, so some additional means to regulate the production of these prostaglandins relative to each other must exist. Primary candidates are the PGES. It can be hypothesized that new insights into the regulation of these enzymes may result in novel approaches for the treatment and perhaps also the prevention of cancer. In this study, therefore, we aimed to elucidate the regulatory mechanisms of 15d-PGJ2 on terminal PGES, in particular mPGES-1 and mPGES-2, in CRC. 15d-PGJ2 was purchased from Alexis Co. (Carlsbad, CA). All other prostaglandins and MCC555 were obtained from Cayman Chemical Co. (Ann Arbor, MI). All other reagents were from Sigma (Deisenhofen, Germany) and were of the highest analytical grade. Cell culture media and supplements were purchased from Gibco BRL (Lofer, Austria). Oligonucleotides were from Biospring (Frankfurt, Germany). RT-PCR reagents were obtained from Applied Biosystems (Branchburg, NJ). Secondary horseradish peroxidase-conjugated antibodies were from Vector Laboratories (Burlingame, CA), and the chemiluminescence reagent (ECL) and Hyperfilm-MP were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Human colon cancer cell lines (Caco-2 and HCT 116) were obtained from the European Animal Cell Culture Collection. PPARγ dominant-negative mutant Caco-2 cells were kindly provided by V. K. Chatterjee (Department of Medicine, University of Cambridge, Cambridge, UK). Construction of plasmids as well as transfection of cells were performed as described previously (19Ulrich S. Waächtershaäuser A. Loitsch S. von Knethen A. Bruäne B. Stein J. Activation of PPARgamma is not involved in butyrate-induced epithelial cell differentiation.Exp. Cell Res. 2005; 310: 196-204Crossref PubMed Scopus (13) Google Scholar). Before experiments, diminished transcriptional activity of the mutant was routinely checked by comparing the transactivation of wild-type and transfected cells to increasing doses of the PPARγ ligand BRL49653. All cell lines were maintained in DMEM containing 4.5 g/l glucose and 25 mM HEPES supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% nonessential amino acids, and 1% pyruvate. The medium was changed every second day. Cells were checked for Mycoplasma infection at monthly intervals. To investigate the effect of prostaglandins and MCC555 on mPGES-2 expression in colon cancer cells, 2.5 × 103 cells/100 μl were seeded and incubated at 37°C under 6% CO2 and 94% air until the cells were ∼50% confluent. The new medium containing the respective ligand or solvent vehicle was then added, and cells were incubated for the indicated periods of time. Cellular proliferation/survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) colorimetric method (Roche, Mannheim, Germany) according to the manufacturer’s instructions. In brief, after defined periods of culture, cells grown in DMEM were harvested using a 10% trypsin-PBS solution and resuspended in DMEM. Cell suspensions (2.5 × 103 cells/100 μl) were plated onto 96-well plates in DMEM with 15d-PGJ2 (10 μM), PGE2 (1 nM to 1 mM), or combinations of 15-PGJ2 (10 μM) and various concentrations of PGE2 as indicated. The plates were incubated for 12, 24, 48, or 72 h at 37°C before the addition of MTT solution, which was followed by spectrophotometric measurement of absorbance at 570 nm. Changes in cell number were deduced from the absorbance data using the linear part of standard absorbance curves produced with predetermined cell numbers. Cytotoxicity was excluded using a lactate dehydrogenase release assay (LDH kit; Roche). Cultured cells were washed with PBS and trypsinated in 1× trypsin/EDTA for 10 min at 37°C. Thereafter, culture medium was added and cells were centrifuged at 500 g for 10 min, followed by two additional washing steps in PBS. The cell pellets were then resuspended in 1 ml of homogenization buffer consisting of potassium phosphate buffer (0.1 M, pH 7.4), 1× Complete™ protease inhibitor cocktail, and sucrose (0.25 M). The samples were then sonicated for 3 × 20 s at 100 W with a ultrasonic cell disruptor (Microson™; SPI Supplies, West Chester, PA) and subjected to differential centrifugation at 1,000 g for 10 min, 10,000 g for 15 min, and 100,000 g for 1.5 h at 4°C. After the last centrifugation step, microsomal fractions were resuspended in 100 μl of homogenization buffer, and total protein concentration in cytosolic and microsomal fractions was determined by the Coomassie protein assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). RNA isolation was conducted with RNAzol (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s protocol. Briefly, total RNA (1 μg) in water was heated (65°C, 12 min), cooled, and reverse-transcribed (20 min, 42°C) in PCR buffer [5 mM MgCl2, 1 mM deoxynucleoside triphosphates, 1 U/μl Moloney murine leukemia virus (MuLV) reverse transcriptase, 5 μM oligo d(T)16, and 0.5 U/μl RNase inhibitor]. After denaturation (1.5 min, 95°C), samples were amplified in PCR buffer (1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, 0.2 μM primer, and 0.05 U/μl Taq polymerase) with the primers and conditions described in Table 1using a Perkin-Elmer/GeneAmp PCR system 2400 (Applied Biosystems, Branchburg, NJ). Aliquots of the PCR mixtures (10 μl) were analyzed by electrophoresis using a 1% agarose gel containing 0.5 μg/ml ethidium bromide. For semiquantitative analysis of amplified PCR products, the fluorescent dye Pico Green® (dsDNA Quantitation Kit; Molecular Probes/Invitrogen, Karlsruhe, Germany) was used according to the manufacturer’s instructions (20Singer V.L. Jones L.J. Yue S.T. Haugland R.P. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.Anal. Biochem. 1997; 249: 228-238Crossref PubMed Scopus (641) Google Scholar).TABLE 1Sequences of oligonucleotides and PCR conditionsGenePrimer SequenceAnnealing TemperatureNo. of CyclesGAPDHForward: 5′-GCACCGTCAAGGCTGAGAAC-3′45°C24Reverse: 5′-CCACCACCCTGTTGCTGTAG-3′Microsomal prostaglandin E synthase-1Forward: 5′-GCACGCTGCTGGTCATCAAGATGTA-3′49.5°C38Reverse: 5′-CCGCTTCCCAGAGGATCTGCAGA-3′Microsomal prostaglandin E synthase-2Forward: 5′-CCTGCAGCTGACCCTGTACCAGTA-3′51°C31Reverse: 5′-CCCACTTGTCAGCAGCCTCATAGA-3′Cytosolic prostaglandin E synthaseForward: 5′-GCAAAGTGGTACGATCGAAGGGACTAT-3′48°C33Reverse: 5′-CCCAGTCTTTCCAATTATTGAAGTCGA-3′Cyclooxygenase-1Forward: 5′-GTGGGCTCCCAGGAGTACAGCTAC-3′48°C37Reverse: 5′-GCAATCTGGCGAGAGAAGGCATC-3′Cyclooxygenase-2Forward: 5′-CCCTTCTGCCTGACACCTTTCAAATT-3′48°C35Reverse: 5′-GCTCTGGATCTGGAACACTGAATGAAGT-3′ Open table in a new tab Aliquots (15 μg of protein) of samples in loading buffer were separated by SDS-PAGE on a 12% Tris-glycine, precasted, linear-gradient polyacrylamide gel and electroblotted onto nitrocellulose membranes. Transfer efficiency was visualized using Ponceau S stain (Sigma-Aldrich, St. Louis, MO). Membranes were then blocked overnight using Tris-HCl, pH 7.5, containing 100 mM NaCl, 0.1% Tween-20 (TBS-T), and 3% nonfat dry milk. After washing the membranes with TBS-T, polyclonal antiserum against mPGES-2 was added at a 1:5,000 dilution in TBS-T and incubated for 2.5 h. After three washing steps, the membranes were incubated for 2 h at 25°C with a horseradish peroxidase-linked goat anti-rabbit IgG antibody (1:5,000 dilution) in TBS-T. The washing steps were repeated, and subsequently, enhanced chemiluminescence detection was performed. SDS-PAGE immunoblots were quantitated with scanning densitometry using a Desaga CabUVIS scanner and Desaga ProViDoc software (Wiesloch, Germany). PGES enzyme activity was determined according to Thoreén and Jakobsson (21Thoreén S. Jakobsson P.J. Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4.Eur. J. Biochem. 2000; 267: 6428-6434Crossref PubMed Scopus (190) Google Scholar). Microsomal or cytosolic fraction samples were diluted in potassium phosphate buffer (0.1 M, pH 6.5) containing 0.5 mM DTT. PGH2 (4 μl) dissolved in acetone (0.28 mM) was kept in separate vials at −80°C. Before incubation, both the substrate and samples were equilibrated at 4°C for 2 min. The reaction was started by the addition of the sample (100 μl) to the tubes containing PGH2 (final concentration, 10 μM) and then terminated by the addition of 400 μl of stop solution (25 mM FeCl2, 50 mM citric acid), decreasing the pH to 3, giving a total concentration of 20 mM FeCl2 and 40 mM citric acid. The reaction mixture was then diluted and assayed for PGE2 using an enzyme immunoassay kit (Cayman Chemical Co.). If not stated otherwise, data are expressed as means ± SD of three independent experiments performed in duplicate. Data were analyzed by one-way ANOVA and Student’s t-test. P < 0.05 was considered statistically significant. To investigate the effect of 15d-PGJ2 on mRNA and protein expression of cPGES, mPGES-1, mPGES-2, COX-1, and COX-2 in CRC cell lines HCT 116 and Caco-2 were treated with increasing concentrations of 15d-PGJ2. Whereas mRNA expression of cPGES, mPGES-1, COX-1 (expressed in HCT 116), and COX-2 (expressed in Caco-2) remained unchanged after treatment with this CyPG (data not shown), 15d-PGJ2 downregulated mPGES-2 mRNA in both cell lines compared with unstimulated or vehicle-treated cells. As can be seen in Fig. 1A, B, the suppressive effect of 15d-PGJ2 was time- and dose-dependent. Maximal reduction in mPGES-2 mRNA expression was observed after 4 h of treatment, which gradually leveled off to baseline levels thereafter. At 4 h, a 50% suppression of mPGES-2 mRNA was observed at ∼10 μM 15d-PGJ2 in Caco-2 cells and at ∼5 μM 15d-PGJ2 in HCT 116 cells. The downregulation of mPGES-2 mRNA expression was followed by a subsequent transient reduction of this enzyme at the protein level. Thus, at a concentration of 1 μM 15d-PGJ2, the suppressive effect on protein expression in HCT 116 cells was observed after an incubation period of 12 h, whereas in the Caco-2 cell line a similar response was found after 36 h of stimulation (Fig. 2). When challenged with 10 μM 15d-PGJ2, inhibition of mPGES-2 protein expression reached its maximum at 6 h (HCT 116) and 24 h (Caco-2), followed by a return to baseline levels thereafter (Fig. 2). HCT 116 and Caco-2 cells were also challenged with 10 μM 15d-PGJ2 and PGE2 as well as 16,16-dimethyl-PGE2, a more stable analog of PGE2, at concentrations ranging from 10−9 to 10−6 M for 4 h. However, the inhibitory effect of 15d-PGJ2 on mPGES-2 mRNA and protein expression persisted in the presence of PGE2 or 16,16-dimethyl-PGE2 at any concentration used, thus excluding a potential counteractive effect of the product of PGES activity, PGE2, on the downregulation of mPGES-2 mRNA and protein expression by 15d-PGJ2 (data not shown).Fig. 1Inhibition of microsomal prostaglandin E synthase-2 (mPGES-2) mRNA expression by 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2) in the colorectal cancer (CRC) cell lines Caco-2 and HCT 116. A: Caco-2 (closed circles) and HCT 116 (open circles) cells were incubated for 4 h in the absence or presence of 15d-PGJ2 at concentrations of 0.1, 1, 5, 10, and 20 μM. B: Time course of mPGES-2 mRNA expression in Caco-2 cells (black bars) treated with 10 μM 15d-PGJ2 and in HCT 116 cells (white bars) stimulated with 5 μM 15d-PGJ2 for the indicated incubation periods. Total RNA was isolated as described in Materials and Methods and subjected to semiquantitative RT-PCR with the fluorescent dye PicoGreen®. All values for mRNA were normalized to the corresponding mRNA amount for the housekeeping gene GAPDH and represent means ± SD. The statistical significance of changes relative to vehicle-treated controls is expressed as follows: * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2Downregulation of mPGES-2 protein expression by 15d-PGJ2 in Caco-2 and HCT 116 cells. Western blot analysis of mPGES-2 protein expression in Caco-2 and HCT 116 cells incubated in the absence and presence of 1 or 10 μM 15d-PGJ2 for the times indicated. In all lanes, 15 μg of protein from the microsomal fraction of cells was analyzed. The results shown are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In accordance with the downregulation of mPGES-2 at the mRNA and protein levels, a substantial decrease in enzyme activity was observed after treatment with 10 μM 15d-PGJ2. As demonstrated in Fig. 3, enzymatic activity was found to be reduced only in the microsomal fraction, whereas the cytosolic PGES activity remained unchanged. The notable discrepancy between the two cell lines regarding the reduction in mPGES-2 activity can be explained, most likely, by a different sensitivity of HCT 116 and Caco-2 cells to 15d-PGJ2, which is reflected in the mRNA and protein expression studies. Direct interaction of 15d-PGJ2 with PPARγ to regulate gene transcription has been demonstrated to be one mode of action of this CyPG. To elucidate the signaling mechanism responsible for 15d-PGJ2-mediated regulation of mPGES-2, Caco-2 cells, transfected with a mutant receptor to inhibit wild-type PPARγ action, were subjected to 15d-PGJ2 treatment. As shown in Fig. 4, mPGES-2 mRNA and protein expression were reduced to a similar extent compared with nontransfected Caco-2 cells or the HCT 116 cell line. In addition, PGES activity in PPARγ dominant-negative Caco-2 cells was found to be decreased in the same manner as in nontransfected Caco-2 or HCT 116 cells (data not shown). This finding suggested that the effect of 15d-PGJ2 on mPGES-2 expression might be independent of PPARγ. To further corroborate this hypothesis, Caco-2 and HCT 116 cells were additionally stimulated with the thiazolidinedione homolog MCC555, a synthetic PPARγ agonist, at a concentration of 50 μM and cultured for various incubation periods (0–24 h). No changes in mRNA expression of mPGES-2 or any of the other enzymes examined (COX-1, COX-2, mPGES-1, and cPGES) were observed upon treatment with MCC555 (data not shown).Fig. 4Inhibition of mPGES-2 mRNA and protein expression by 15d-PGJ2 in peroxisome proliferator-activated receptor γ (PPARγ) dominant-negative mutant Caco-2 cells. A: PPARγ dominant-negative mutant Caco-2 cells were treated for 4 h in the absence or presence of the indicated concentrations of 15d-PGJ2. RT-PCR was performed on total RNA for cyclooxygenase-2 (COX-2), mPGES-1, mPGES-2, cytosolic prostaglandin E synthase (cPGES), and GAPDH during the linear phase of amplification. All values for mRNA are normalized to the corresponding mRNA amount for GAPDH. B: Immunoblot analysis of mPGES-2 protein expression in PPARγ dominant-negative mutant Caco-2 cells incubated in the absence or presence of 10 μM 15d-PGJ2 for the times indicated. The top panel shows a series of immunoreactive bands corresponding to mPGES-2 and β-actin (serving as an internal control). The bottom panel depicts a histogram obtained by densitometric analysis of immunoblots from three independent experiments normalized to protein expression of β-actin. All values shown represent means ± SD. The statistical significance of changes relative to vehicle-treated controls is expressed as follows: ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One mechanism for 15d-PGJ2 action may involve the activation of prostaglandin D2 receptors (DPs). Previous studies indicate that 15d-PGJ2 has a weak agonist activity on DP1 receptors in certain cell types of hematopoietic origin (22Wright D.H. Metters K.M. Abramovitz M. Ford-Hutchinson A.W. Characterization of the recombinant human prostanoid DP receptor and identification of L-644,698, a novel selective DP agonist.Br. J. Pharmacol. 1998; 123: 1317-1324Crossref PubMed Scopus (61) Google Scholar). Recently, a second high-affinity receptor (DP2), also designated CRTH2, was identified, thus far exclusively expressed in Th2 cells, T cytotoxic cells, eosinophiles, and basophiles (23Hata A.N. Zent R. Breyer M.D. Breyer R.M. Expression and molecular pharmacology of the mouse CRTH2 receptor.J. Pharmacol. Exp. Ther. 2003; 306: 463-470Crossref PubMed Scopus (63) Google Scholar). From a recent study it was known that HCT 116 does not express either DP receptor (24Hawcroft G. Gardner S.H. Hull M.A. Expression of prostaglandin D2 receptors DP1 and DP2 by human colorectal cancer cells.Cancer Lett. 2004; 210: 81-84Crossref PubMed Scopus (13) Google Scholar), and our own preliminary results indicated that Caco-2 is equipped with DP1 mRNA but not DP2 mRNA (data not shown). Given that activation of DP1 leads to an increase in intracellular cAMP levels, we investigated the effect of increasing intracellular cAMP levels on mPGES-2 protein expression. Caco-2 cells were treated with forskolin, which directly stimulates adenylyl cyclase and thus increases intracellular cAMP levels. However, mPGES-2 protein expression was not affected after treatment with various concentrations of forskolin (data not shown), thus excluding the participation of DP1 in the regulation of mPGES-2 in Caco-2 cells. Biologic actions of 15d-PGJ2 have also been linked to changes in the intracellular thiol-disulfide redox status. To investigate whether oxidative stress may be involved in 15d-PGJ2-induced suppression of mPGES-2, Caco-2 and HCT 116 cells were preincubated with or without the antioxidant DTT for 2 h before stimulation with 15d-PGJ2 for 12 h. As shown in Fig. 5A, the inhibitory effect of 15d-PGJ2 on mPGES-2 protein expression was completely reversed by DTT at concentrations of 2 mM in both cell lines. The possible role of changes in the redox potential in mediating the downregulation of mPGES-2 by 15d-PGJ2 was further investigated by treating HCT 116 cells with arsenite, which among other molecular events has been demonstrated to attack critical thiols (25Valko M. Morris H. Cronin M.T. Metals, toxicity and oxidative stress.Curr. Med. Chem. 2005; 12: 1161-1208Crossref PubMed Scopus (3583) Google Scholar). Arsenite dose-dependently reduced mPGES-2 protein expression in HCT 116 cells, which was statistically significant at concentrations of 50 μM, thereby mimicking the effect of 15d-PGJ2. Again, attenuation of mPGES-2 protein expression could be abolished by preincubation of cells with 2 mM DTT for 2 h before stimulation of cells with 50 μM arsenite (Fig. 5B). Fin" @default.
• W2023183189 created "2016-06-24" @default.
• W2023183189 creator A5023947945 @default.
• W2023183189 creator A5040510726 @default.
• W2023183189 creator A5066637534 @default.
• W2023183189 creator A5078231949 @default.
• W2023183189 creator A5082042696 @default.
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• W2023183189 date "2006-05-01" @default.
• W2023183189 modified "2023-10-06" @default.
• W2023183189 title "15-deoxy-Δ12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells" @default.
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