Potential Role of Microsomal Prostaglandin E Synthase-1 in Tumorigenesis
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m213290200
ISSN1083-351X
AutoresDaisuke Kamei, Makoto Murakami, Yoshihito Nakatani, Yukio Ishikawa, Toshiharu Ishii, Ichiro Kudo,
Tópico(s)NF-κB Signaling Pathways
ResumoMicrosomal prostaglandin E2 synthase-1 (mPGES-1) is a stimulus-inducible enzyme that functions downstream of cyclooxygenase (COX)-2 in the PGE2-biosynthetic pathway. Given the accumulating evidence that COX-2-derived PGE2 participates in the development of various tumors, including colorectal cancer, we herein examined the potential involvement of mPGES-1 in tumorigenesis. Immunohistochemical analyses demonstrated the expression of both COX-2 and mPGES-1 in human colon cancer tissues. HCA-7, a human colorectal adenocarcinoma cell line that displays COX-2- and PGE2-dependent proliferation, expressed both COX-2 and mPGES-1 constitutively. Treatment of HCA-7 cells with an mPGES-1 inhibitor or antisense oligonucleotide attenuated, whereas overexpression of mPGES-1 accelerated, PGE2 production and cell proliferation. Moreover, cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when implanted subcutaneously into nude mice. cDNA array analyses revealed that this mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to proliferation, morphology, adhesion, and the cell cycle. These results collectively suggest that aberrant expression of mPGES-1 in combination with COX-2 can contribute to tumorigenesis. Microsomal prostaglandin E2 synthase-1 (mPGES-1) is a stimulus-inducible enzyme that functions downstream of cyclooxygenase (COX)-2 in the PGE2-biosynthetic pathway. Given the accumulating evidence that COX-2-derived PGE2 participates in the development of various tumors, including colorectal cancer, we herein examined the potential involvement of mPGES-1 in tumorigenesis. Immunohistochemical analyses demonstrated the expression of both COX-2 and mPGES-1 in human colon cancer tissues. HCA-7, a human colorectal adenocarcinoma cell line that displays COX-2- and PGE2-dependent proliferation, expressed both COX-2 and mPGES-1 constitutively. Treatment of HCA-7 cells with an mPGES-1 inhibitor or antisense oligonucleotide attenuated, whereas overexpression of mPGES-1 accelerated, PGE2 production and cell proliferation. Moreover, cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when implanted subcutaneously into nude mice. cDNA array analyses revealed that this mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to proliferation, morphology, adhesion, and the cell cycle. These results collectively suggest that aberrant expression of mPGES-1 in combination with COX-2 can contribute to tumorigenesis. Clinical, genetic, and biochemical evidence has suggested that prostaglandin (PG) 1The abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; PLA2, phospholipase A2; mPGES-1, microsomal prostaglandin E2 synthase-1; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; TBS, Tris-buffered saline; IL, interleukin; HEK, human embryonic kidney. E2 produced via the cyclooxygenase (COX)-2-dependent pathway plays a crucial role in the development of colorectal cancer and possibly other cancers (1Williams C.S. Mann M. DuBois R.N. Oncogene. 1999; 18: 7908-8791Crossref PubMed Scopus (1293) Google Scholar). Non-steroidal anti-inflammatory drugs, which inhibit COX-2, reduce the incidence of colorectal cancer (2Marnett L.J. Cancer Res. 1992; 52: 5575-5589PubMed Google Scholar, 3Rao C.V. Rivenson A. Simi B. Zang E. Kelloff G. Steele V. Reddy B.S. Cancer Res. 1995; 55: 1464-1472PubMed Google Scholar, 4Sheng H. Shao J. Kirkland S.C. Isakson P. Coffey R.J. Morrow J. Beauchamp R.D. DuBois R.N. J. Clin. Invest. 1997; 99: 2254-2259Crossref PubMed Scopus (699) Google Scholar). The major prostanoid produced by several types of cancer is PGE2, which is produced by three biosynthetic reactions involving phospholipase A2 (PLA2), COX, and terminal PGE2 synthase (PGES). PGE2 promotes survival and motility of colon cancer cells in vitro and promotes tumorigenesis and angiogenesis in vivo (5Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 6Levy G.N. FASEB J. 1997; 11: 234-247Crossref PubMed Scopus (284) Google Scholar, 7Reinhart W.H. Muller O. Halter F. Gastroenterology. 1983; 85: 1003-1010Abstract Full Text PDF PubMed Scopus (83) Google Scholar). High levels of constitutive expression of COX-2 have been found in various cancer cells and tissues (8Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar, 9Kargman S.L. O'Neill G.P. Vickers P.J. Evans J.F. Mancini J.A. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar), and studies employing overexpression, antisense suppression, and specific inhibitors of COX-2 have demonstrated that COX-2 contributes to the progression of several types of cancer (10Liu C.H. Chang S.H. Narko K. Trifan O.C. Wu M.T. Smith E. Haudenschild C. Lane T.F. Hla T. J. Biol. Chem. 2001; 276: 18563-18569Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 11Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2228) Google Scholar, 12Tsujii M. DuBois R.N. Cell. 1995; 83: 493-4501Abstract Full Text PDF PubMed Scopus (2150) Google Scholar). More direct evidence for the role of COX-2 and its product PGE2 in colorectal tumorigenesis has been provided by gene targeting studies. Gene disruption of either COX-2 (13Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2296) Google Scholar) or the PGE receptor EP2 (14Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat Med. 2001; 7: 1048-1051Crossref PubMed Scopus (533) Google Scholar) results in reduction of the number and size of intestinal polyps in Apc mutant mice, a model for human familial adenomatous polyposis. In another model, disruption of the genes for the PGE receptors EP1 (15Watanabe K. Kawamori T. Nakatsugi S. Ohta T. Ohuchida S. Yamamoto H. Maruyama T. Kondo K. Ushikubi F. Narumiya S. Sugimura T. Wakabayashi K. Cancer Res. 1999; 59: 5093-5096PubMed Google Scholar) or EP4 (16Mutoh M. Watanabe K. Kitamura T. Shoji Y. Takahashi M. Kawamori T. Tani K. Kobayashi M. Maruyama T. Kobayashi K. Ohuchida S. Sugimoto Y. Narumiya S. Sugimura T. Wakabayashi K. Cancer Res. 2002; 62: 28-32PubMed Google Scholar) suppresses the development of colorectal cancer induced by carcinogen. Moreover, gene knockout of cytosolic PLA2α (cPLA2α), which supplies the substrate arachidonic acid to COX-2, also leads to reduced polyposis in Apc mutant mice (17Takaku K. Sonoshita M. Sasaki N. Uozumi N. Doi Y. Shimizu T. Taketo M.M. J. Biol. Chem. 2000; 275: 34013-34016Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Hong K.H. Bonventre J.C. O'Leary E. Bonventre J.V. Lander E.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3935-3939Crossref PubMed Scopus (125) Google Scholar). PGES catalyzes the conversion of PGH2, which is produced from arachidonic acid by COX-1 or COX-2, to PGE2. Recent advances in this field have led to identification of at least three PGES enzymes, including cytosolic PGES (cPGES) (19Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar), microsomal PGES (mPGES) -1 (20Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 22Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), and mPGES-2 (23Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar). Among them, microsomal PGES-1 (mPGES-1) has received much attention, as this enzyme is induced by proinflammatory stimuli, down-regulated by anti-inflammatory glucocorticoids, and functionally coupled with COX-2 in marked preference to COX-1 (20Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 22Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). In comparison, cPGES (the heat shock protein-associated protein p23) is constitutively and ubiquitously expressed and is selectively coupled with COX-1 (19Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). mPGES-2 does not show homology with mPGES-1 and has a unique N-terminal hydrophobic domain and a glutaredoxin-like domain (23Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar), although its cellular function has not yet been addressed. mPGES-1 is a member of the MAPEG (for membrane-associated proteins involved in eicosanoid and glutathione metabolism) superfamily, to which other proteins involved in arachidonic acid metabolism, such as 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase, also belong (20Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 22Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Induced expression of mPGES-1 has been postulated to be associated with various pathophysiological events in which COX-2-derived PGE2 has been implicated, such as rheumatoid arthritis (24Stichtenoth D.O. Thoren S. Bian H. Peters-Golden M. Jakobsson P.J. Crofford L.J. J. Immunol. 2001; 167: 469-474Crossref PubMed Scopus (254) Google Scholar), febrile response (25Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar), reproduction (26Filion F. Bouchard N. Goff A.K. Lussier J.G. Sirois J. J. Biol. Chem. 2001; 276: 34323-34330Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 27Lazarus M. Munday C.J. Eguchi N. Matsumoto S. Killian G.J. Kubata B.K. Urade Y. Endocrinology. 2002; 143: 2410-2419Crossref PubMed Scopus (66) Google Scholar), bone metabolism (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar), and Alzheimer's disease (28Satoh K. Nagano Y. Shimomura C. Suzuki N. Saeki Y. Yokota H. Neurosci. Lett. 2000; 283: 221-223Crossref PubMed Scopus (57) Google Scholar). A recent gene targeting study of mPGES-1 has shown that PGE2 production by lipopolysaccharide-stimulated peritoneal macrophages depends almost entirely on this enzyme (29Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-6816Crossref PubMed Scopus (276) Google Scholar). Induced expression of mPGES-1 is regulated by the NF-IL-6 pathway (29Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-6816Crossref PubMed Scopus (276) Google Scholar) or the mitogen-activated protein kinase pathway (30Han R. Tsui S. Smith T.J. J. Biol. Chem. 2002; 277: 16355-16364Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), the latter of which may switch on the inducible transcription factor Egr-1 that in turn binds to the proximal GC box in the mPGES-1 gene promoter, leading to mPGES-1 transcription (31Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-Ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). A possible linkage of mPGES-1 with tumorigenesis has been provided by a recent observation that mPGES-1 is constitutively expressed in several cancers, most of which also express COX-2 constitutively (32Yoshimatsu K. Altorki N.K. Golijanin D. Zhang F. Jakobsson P.J. Dannenberg A.J. Subbaramaiah K. Clin. Cancer Res. 2001; 7: 2669-2674PubMed Google Scholar–34). In this study, we have used colon cancer cell lines and mPGES-1-transfected cells to examine the expression of mPGES-1 in colorectal cancer tissues and cells and evaluate its potential role in tumorigenesis. Materials—Human embryonic kidney (HEK) 293 cells (Human Science Research Resources Bank) and human colon adenocarcinoma HCA-7 cells (a generous gift from Dr. M. Tsujii (Osaka University) and Dr. R. DuBois (Vanderbilt University Medical Center and VA Medical Center) were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co.) containing 10% (v/v) fetal calf serum (Bioserum). The cDNAs for human mPGES-1 and its mutant R110S (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar), human COX-1 and COX-2 (35Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and human cPGES (19Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar) were described previously. cDNA probes for human rhoA and human c-myc were donated by Dr. M. Shibanuma (Showa University). HEK293 cells stably expressing COX-2 and/or mPGES-1 were described previously (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). The enzyme immunoassay kits for PGE2 and PGF2α and the COX-2 inhibitor NS-398 were purchased from Cayman Chemicals. The MAPEG inhibitor MK-886 was from Toronto Research Chemicals. The rabbit anti-human cPLA2α, goat anti-human COX-1 and goat anti-human COX-2, goat anti-human RhoA and goat anti-human ezrin antibodies were purchased from Santa Cruz. Mouse monoclonal antibody against human ErbB3 and mouse monoclonal anti-FLAG antibodies were from Sigma. Rabbit antibody against human Egr-1 was provided by Dr. H. Naraba (National Cardio-vascular Center Research Institute, Japan). Fluorescein isothiocyanate-conjugated anti-goat IgG, Cy3-conjugated anti-rabbit IgG, and horseradish peroxidase-conjugated anti-goat, -mouse, and -rabbit IgGs were purchased from Zymed Laboratories Inc.. Rabbit antiserum for human cPGES was prepared as described previously (19Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Human interleukin (IL)-1β was purchased from Genzyme. LipofectAMINE 2000, oligofectamine, Opti-MEM medium, TRIzol reagent, geneticin, hygromycin, zeocin, and the pcDNA3.1 series of mammalian expression vectors were obtained from Invitrogen. Preparation of Antibody against Human mPGES-1—Human mPGES-1 cDNA was subcloned into the bacterial expression vector pET21c (Novagen) and transformed into the competent cell BL21-D3 (Stratagene). After culture with 0.5 mm isopropyl-β-d-(-)-thiogalacto-pyranoside, cells were spun down, freeze-thawed, and suspended in phosphate-buffered saline containing 1 mm phenylmethylsulfonyl fluoride, 3 μg/ml leupeptin, 3 μg/ml antipain, 1 mm dithiothreitol, and 1% (v/v) sodium N-dodecanoylsalcosilate. After sonication and centrifugation for 10 min at 10,000 × g, the resultant supernatant was dialyzed against 20 mm Tris-HCl (pH 7.4) containing 150 mm NaCl (TBS), 1 mm EDTA, and 0.5% (v/v) Triton X-100 overnight. Then the dialyzed sample was applied to a nickel-nitrilotriacetic acid-agarose column (Novagen), and the bound proteins were eluted with 40–80 mm imidazole at a flow rate of 10 ml/h. Fractions containing pure His6-tagged mPGES-1 protein were collected and dialyzed against phosphate-buffered saline. New Zealand White rabbits (male, 1 kg; Saitama Animal Center) were immunized subcutaneously with the purified mPGES-1 protein (0.3 mg/head) mixed with Freund's complete adjuvant (Difco). After several booster immunizations with Freund's incomplete adjuvant (Difco) at 2-week intervals, the blood was collected and the serum titer was assayed by enzyme-linked immunosorbent assay and Western blotting with recombinant mPGES-1 protein. The antiserum obtained was further purified on an immunoaffinity Hi-Trap NHS-activated column (Amersham Biosciences) that had been conjugated with mPGES-1 protein. The purified antibody was used in subsequent studies. Transfection Studies—Transfection of cDNAs into HEK293 cells was performed by lipofection as described previously (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 35Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Briefly, 1 μg of plasmid (mPGES-1 in pCDNA3.1/hyg and COX-1 or -2 in pCDNA3.1/neo) was mixed with 2 μl of LipofectAMINE 2000 in 100 μl of Opti-MEM for 30 min and then added to cells that had attained 40–60% confluence in 12-well plates (Iwaki Glass) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium and culture was continued at 37 °C in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium containing appropriate antibiotics (10 μg/ml hygromycin or 1 mg/ml G418). After culture for 3–4 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by RNA blotting. The established clones were expanded and used for the experiments described below. The C-terminal FLAG-tagged mPGES-1 cDNA was transfected into HCA-7 cells by the ViraPower lentiviral expression system (Invitrogen) according to the manufacturer's instructions. Briefly, the FLAG-tagged mPGES-1 cDNA insert was amplified by polymerase chain reaction with the Advantage cDNA polymerase mixture (Clontech) and was subcloned into the pLenyi6/V4-D-TOPO vector (Invitrogen). The resulting plasmid was transfected into 293FT cells (Invitrogen) with LipofectAMINE 2000, and an aliquot of the supernatant harvested 3 days after transfection was then added to HCA-7 cells. The cells were cultured in the presence of 40 μg/ml blastcidine (Invitrogen), and the antibiotic-resistant cells were used in subsequent studies. Antisense Experiments—HCA-7 cells (6 × 104 cells) were seeded into 6-well plates and cultured for 2 days. Then the mPGES-1 antisense S-oligonucleotide (0.2 nmol) 5′-GAGGAAGACCAGGAAGTGCAT-3′ was transfected into HCA-7 cells with oligofectamine reagent. After 48 h, cell numbers and the PGE2 released into the supernatants during culture were quantified. The cell lysates were subjected to Western blotting to verify mPGES-1 expression. Measurement of PGES Activity—PGES activity was measured by assessment of conversion of PGH2 to PGE2 as previously reported (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Briefly, cells were harvested from culture dishes with a cell scraper and disrupted by sonication with a Branson sonifier (10 s, 3 times, 50% duty) in 10 mm Tris-HCl (pH 8.0) containing 150 mm NaCl. After centrifugation of the sonicates at 100,000 × g for 1 h at 4 °C, the membrane fractions were used as an enzyme source. An aliquot (10 μg of protein equivalents) was incubated with 0.5 μg of PGH2 for 30 s at 24 °C in 0.1 ml of 0.1 m Tris-HCl (pH 8.0) containing 1 mm glutathione and 5 μg of indomethacin. After stopping the reaction by the addition of 100 mm FeCl2, PGE2 contents in the reactions were quantified by use of the enzyme immunoassay kit. Protein concentrations were determined by the bicinchoninic acid protein assay kit (Pierce) with bovine serum albumin as a standard. RNA Blotting—Approximately equal amounts (∼5 μg) of total RNA obtained from the cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Biosciences) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (35Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). SDS-PAGE/Immunoblotting—Cell lysates (2 × 105 cell equivalents) were subjected to SDS-PAGE using 7.5–12.5% gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) with a semi-dry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in TBS containing 0.05% (v/v) Tween 20 (TBS-Tween), the membranes were probed with the respective antibodies (1:5,000 dilution for mPGES-1, cPGES, cPLA2α, and COX-2, and 1:20,000 dilution for COX-1 and FLAG epitope in TBS-Tween) for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (for mPGES-1 and cPGES), anti-goat (for COXs), or anti-mouse (for FLAG) IgG (1:5,000 dilution in TBS-Tween) for 2 h, and were visualized with the ECL Western blot system (PerkinElmer Life Sciences) (35Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Semisoft Agar Assay—Cells (104 cells/ml) were suspended in cell culture medium containing 1% (w/v) low-melt agarose and plated on 60-mm culture dishes. After culture for 10 days at 37 °C in a CO2 incubator, colony numbers in each plate were counted. Relative colony size was determined by measuring 10 random colonies in each plate, and the mean for each treatment set was calculated and compared with that of controls. Experiments with Nude Mice—Cells (5 × 106 cells) were suspended in 100 μl of phosphate-buffered saline and injected subcutaneously into BALB/c-nu/nu mice (6-week-old males) (Crea Japan). After 3 months, solid tumors were removed surgically and fixed in 10% (v/v) formalin. After embedding in paraffin, thin sections (4∼6 μm thickness) of tumor tissues were prepared on glass slides. Immunohistochemistry—The tissue sections were incubated with Target Retrieval Solution (DAKO) as required, incubated for 10 min with 3% (v/v) H2O2, washed 3 times with TBS for 5 min each, incubated with 5% (v/v) skim milk for 30 min, washed 3 times with TBS-Tween for 5 min each, and incubated for 2 h with the first antibodies in TBS (1:50 and 1:200 dilutions for anti-mPGES-1 and anti-COX-2 antibodies, respectively). Then the sections were treated with the LSAB2 staining kit (for COX-2; DAKO) or the CSA system staining kit (for mPGES-1; DAKO). cDNA Array Analysis—mRNAs isolated from COX-2-expressing and COX-2/mPGES-1-coexpressing HEK293 cells (107 cells for each) were reverse-transcribed into cDNA and 32P-labeled with Atlas cDNA Expression Arrays kit (Clontech). Hybridization was performed on the Atlas Human 1.2 Array (Clontech). After exposure to an image plate (BAS III; Fuji Photo Film), signals were analyzed by AtlasImage 1.0 Software (Clontech). Other Procedures—Confocal laser microscopy was performed as described previously (21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Data were analyzed by Student's t test. Results are expressed as the mean ± S.E., with p = 0.05 as the limit of significance. mPGES-1 Is Expressed in Human Colorectal Cancer Tissues—Surgically resected human colorectal adenocarcinoma (Fig. 1, A and B) and adenoma (Fig. 1, C and D) tissues were fixed with formaldehyde, embedded in paraffin, and serial sections were immunostained with antibodies against mPGES-1 (Fig. 1, A, C, and E) and COX-2 (Fig. 1, B, D, and F). In the sections shown in Fig. 1, virtually all adenocarcinoma cells (panels A and B) and adenoma cells (panels C and D) were positively stained with anti-mPGES-1 and anti-COX-2 antibodies. Signal for mPGES-1 was distributed throughout the cytoplasm with a punctate pattern, whereas that of COX-2 was enriched in the luminal side of the nuclei. Considering that mPGES-1 is an integral membrane protein, it is likely that the observed mPGES-1 immunostaining reflects its distribution in the endoplasmic reticulum membrane. Immunoreactive signals for mPGES-1 and COX-2 were barely seen in normal colon sections (Fig. 1, E and F). Of several colon cancer tissue sections examined, 9 of 9 adenocarcinoma tissues and 5 of 7 adenoma tissues were positive for mPGES-1 immunoreactivity. Furthermore, 5 of 9 mPGES-1-positive adenocarcinoma tissues and 4 of 5 mPGES-1-positive adenoma tissues were also COX-2-positive. mPGES-1 Is Involved in the Growth of Human Colon Adenocarcinoma Cell Line HCA-7—Fig. 2A depicts the expression of the PGE2-biosynthetic enzymes cPLA2α, COX-1, and -2, cPGES and mPGES-1 in 3 human colon cancer cell lines (HCA-7, WiDr, and HCT116) as well as in HEK293 cells. Of these cell lines, only HCA-7, a colon adenocarcinoma cell line that has been reported to exhibit COX-2- and PGE2-dependent growth (4Sheng H. Shao J. Kirkland S.C. Isakson P. Coffey R.J. Morrow J. Beauchamp R.D. DuBois R.N. J. Clin. Invest. 1997; 99: 2254-2259Crossref PubMed Scopus (699) Google Scholar, 36Coffey R.J. Hawkey C.J. Damstrup L. Graves-Deal R. Daniel V.C. Dempsey P.J. Chinery R. Kirkland S.C. DuBois R.N. Jetton T.L. Morrow J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 657-662Crossref PubMed Scopus (296) Google Scholar, 37Sheng H. Shao J. Morrow J.D. Beauchamp R.D. DuBois R.N. Cancer Res. 1998; 58: 362-366PubMed Google Scholar), expressed COX-2 and mPGES-1 constitutively. mPGES-1 expression was also detected in WiDr and HCT116 cells, although rather more weakly than in HCA-7 cells. Indirect immunofluorescent cytostaining analysis by confocal microscopy revealed the colocalization of COX-2 and mPGES-1 in the perinuclear area (Fig. 2B). All these cell lines expressed cPLA2α and cPGES constitutively, whereas COX-1 expression was restricted to HCA-7 and WiDr cells (Fig. 2A). Stimulation of HCA-7 cells with IL-1β, a proinflammatory cytokine that increases the expression of COX-2 and mPGES-1 in various cell types (20Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 21Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 22Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 24Stichtenoth D.O. Thoren S. Bian H. Peters-Golden M. Jakobsson P.J. Crofford L.J. J. Immunol. 2001; 167: 469-474Crossref PubMed Scopus (254) Google Scholar, 25Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar), did not alter the expression of COX-2 (not shown) and mPGES-1 (Fig. 2C). We therefore chose HCA-7 cells as a model for further study. To examine the role of PGE2 produced by the COX-2/mPGES-1 pathway in the growth of HCA-7 cells, we first examined the effects of NS-398, a well known COX-2 inhibitor (38Futaki N. Takahashi S. Yokoyama M. Arai I. Higuchi S. Otomo S. Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (804) Google Scholar), and MK-886, an mPGES-1 inhibitor that also inhibits other MAPEG proteins such as FLAP and LTC4 synthase (22Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), on cell growth and PGE2 production. Treatment of HCA-7 cells with NS-398 almost completely abolished PGE2 pro
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