Interaction between head and neck squamous cell carcinoma cells and fibroblasts in the biosynthesis of PGE2
2012; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês
10.1194/jlr.m019695
ISSN1539-7262
AutoresSonia Alcolea, Rosa Antón, Mercedes Camacho, Marta Soler, Arántzazu Alfranca, Francesc Xavier Avilés‐Jurado, Juan Miguel Redondo, Miquel Quer, Xavier León, Luı́s Vila,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoProstaglandin (PG)E2 is relevant in tumor biology, and interactions between tumor and stroma cells dramatically influence tumor progression. We tested the hypothesis that cross-talk between head and neck squamous cell carcinoma (HNSCC) cells and fibroblasts could substantially enhance PGE2 biosynthesis. We observed an enhanced production of PGE2 in cocultures of HNSCC cell lines and fibroblasts, which was consistent with an upregulation of COX-2 and microsomal PGE-synthase-1 (mPGES-1) in fibroblasts. In cultured endothelial cells, medium from fibroblasts treated with tumor cell-conditioned medium induced in vitro angiogenesis, and in tumor cell induced migration and proliferation, these effects were sensitive to PGs inhibition. Proteomic analysis shows that tumor cells released IL-1, and tumor cell-induced COX-2 and mPGES-1 were suppressed by the IL-1-receptor antagonist. IL-1α levels were higher than those of IL-1β in the tumor cell-conditioning medium and in the secretion from samples obtained from 20 patients with HNSCC. Fractionation of tumor cell-conditioning media indicated that tumor cells secreted mature and unprocessed forms of IL-1. Our results support the concept that tumor-associated fibroblasts are a relevant source of PGE2 in the tumor mass. Because mPGES-1 seems to be essential for a substantial biosynthesis of PGE2, these findings also strengthen the concept that mPGES-1 may be \a target for therapeutic intervention in patients with HNSCC. Prostaglandin (PG)E2 is relevant in tumor biology, and interactions between tumor and stroma cells dramatically influence tumor progression. We tested the hypothesis that cross-talk between head and neck squamous cell carcinoma (HNSCC) cells and fibroblasts could substantially enhance PGE2 biosynthesis. We observed an enhanced production of PGE2 in cocultures of HNSCC cell lines and fibroblasts, which was consistent with an upregulation of COX-2 and microsomal PGE-synthase-1 (mPGES-1) in fibroblasts. In cultured endothelial cells, medium from fibroblasts treated with tumor cell-conditioned medium induced in vitro angiogenesis, and in tumor cell induced migration and proliferation, these effects were sensitive to PGs inhibition. Proteomic analysis shows that tumor cells released IL-1, and tumor cell-induced COX-2 and mPGES-1 were suppressed by the IL-1-receptor antagonist. IL-1α levels were higher than those of IL-1β in the tumor cell-conditioning medium and in the secretion from samples obtained from 20 patients with HNSCC. Fractionation of tumor cell-conditioning media indicated that tumor cells secreted mature and unprocessed forms of IL-1. Our results support the concept that tumor-associated fibroblasts are a relevant source of PGE2 in the tumor mass. Because mPGES-1 seems to be essential for a substantial biosynthesis of PGE2, these findings also strengthen the concept that mPGES-1 may be \a target for therapeutic intervention in patients with HNSCC. Eicosanoids derived from polyunsaturated fatty acids are soluble mediators that exert a key role in the physiopathology of many disorders, including inflammation, thrombosis, and cancer. Prostanoids derived from arachidonic acid (AAc) through the cyclooxygenase (COX) pathway are particularly relevant. The increasing interest in the role of prostanoids in the context of cancer originates in the large epidemiological trials that showed that COX-inhibiting nonsteroidal anti-inflammatory drugs could be beneficial against the development and growth of malignancies (1Marnett L.J. Aspirin and the potential role of prostaglandins in colon cancer.Cancer Res. 1992; 52: 5575-5589PubMed Google Scholar). Prostaglandin (PG)H2 is the common cyclic-peroxide intermediate in the biosynthesis of prostanoids derived from AAc. The other prostanoids are formed in reactions catalyzed by specific synthases acting on PGH2 (2Vila L. Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in atherosclerosis.Med. Res. Rev. 2004; 24: 399-424Crossref PubMed Scopus (93) Google Scholar). In contrast with the ubiquitous expression of COXs, expression of downstream synthases confers a cell-specific prostanoid profile. COX-2 receives the most attention because, unlike COX-1, which is widely expressed, its expression is restricted in nonpathologic settings to a few cell types and tissues, but it is over-expressed in a wide range of cell types in tumors and inflamed tissues. COX-2 is transiently and selectively induced by pro-inflammatory cytokines, tumor promoters, growth factors, and hormones (2Vila L. Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in atherosclerosis.Med. 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Unlike COX-2, COX-1 activity exhibits a cooperative dependence on the substrate concentration [reviewed in Refs. (3Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2449) Google Scholar) and (6Kulmacz R.J. Regulation of cyclooxygenase catalysis by hydroperoxides.Biochem. Biophys. Res. Commun. 2005; 338: 25-33Crossref PubMed Scopus (42) Google Scholar)]. This difference is relevant for the relative prominence of both isoenzymes in vivo; it may permit COX-2 to compete more effectively for the substrate when both the isoenzymes are expressed in the same cells. These properties, and the evidence accumulated from coexpression studies, have led to the concept that COX-2 is functionally coupled with downstream synthases such as microsomal PGE-synthase-1 (mPGES-1) (7Murakami M. Nakatani Y. Tanioka T. Kudo I. 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Prostaglandins in squamous cell carcinoma of the head and neck: a preliminary study.Laryngoscope. 1985; 95: 307-312Crossref PubMed Google Scholar–11Greenhough A. Smartt H.J.M. Moore A.E. Roberts H.R. Williams A.C. Paraskeva C. Kaidi A. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment.Carcinogenesis. 2009; 30: 377-386Crossref PubMed Scopus (981) Google Scholar, 12Wang D. DuBois R.N. Prostaglandins and cancer.Gut. 2006; 55: 115-122Crossref PubMed Scopus (713) Google Scholar), and because it has several biological activities that are compatible with tumor progression. PGE2 promotes cancer cell growth and survival by several mechanisms, including increased proliferation, inhibition of apoptosis, increased migration and invasiveness, angiogenesis, suppression of immune attack, and chronic inflammation [reviewed in Refs. (11Greenhough A. Smartt H.J.M. Moore A.E. Roberts H.R. Williams A.C. Paraskeva C. Kaidi A. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment.Carcinogenesis. 2009; 30: 377-386Crossref PubMed Scopus (981) Google Scholar) and (12Wang D. DuBois R.N. Prostaglandins and cancer.Gut. 2006; 55: 115-122Crossref PubMed Scopus (713) Google Scholar)]. Conversion of PGH2 to PGE2 is catalyzed by PGE-synthases (PGESs). Three PGES isoenzymes have been characterized: two microsomal isoforms (mPGES-1 and mPGES-2) and one cytosolic isoform (cPGES) (7). mPGES-1 is inducible by pro-inflammatory cytokines, and it seems to be the essential PGES isoenzyme involved in PGE2 biosynthesis under inflammatory conditions (13Soler M. Camacho M. Escudero J.R. Iñiguez M.A. Vila L. Human vascular smooth muscle cells but not endothelial cells express prostaglandin E synthase.Circ. Res. 2000; 87: 504-507Crossref PubMed Scopus (99) Google Scholar–14Camacho M. Gerbolés E. Escudero J.R. Anton R. García-Moll X. Vila L. 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Kudo I. Prostaglandin E synthase: a novel drug target for inflammation and cancer.Curr. Pharm. Des. 2006; 12: 943-954Crossref PubMed Scopus (125) Google Scholar). Accumulated evidence indicates that interactions between tumor cells and stromal cells may dramatically influence tumor progression (23Bhowmick N.A. Moses H.L. Tumor-stroma interactions.Curr. Opin. Genet. Dev. 2005; 15: 97-101Crossref PubMed Scopus (367) Google Scholar, 24Hu M. Polyak K. Microenvironmental regulation of cancer development.Curr. Opin. Genet. Dev. 2008; 18: 27-34Crossref PubMed Scopus (278) Google Scholar). Fibroblasts seem to be particularly relevant among stromal cells, and carcinoma-associated fibroblasts are frequently observed in the stroma of human carcinomas. Their presence in large numbers is often associated with the development of high-grade malignancies and poor prognoses. The molecular mechanisms underlying tumor-promoting capabilities of carcinoma-associated fibroblasts include the release of cytokines and growth factors, which promote tumor cells growth and migration, angiogenesis, and evasion of the immune response (reviewed in References 25Shimoda M. Mellody K.T. Orimo A. Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.Semin. Cell Dev. Biol. 2010; 21: 19-25Crossref PubMed Scopus (265) Google Scholar and 26Franco O.E. Shaw A.K. Strand D.W. Hayward S.W. Cancer associated fibroblasts in cancer pathogenesis.Semin. Cell Dev. Biol. 2010; 21: 33-39Crossref PubMed Scopus (293) Google Scholar). Tumor cell-fibroblast cross talk causes mutual influence, which results in a promotion of tumor progression. Tumor cells release soluble factors that could act on the neighboring stroma cells, inducing expression of genes, for example chemokines, which amplify inflammation (27Brú A. Souto J.C. Alcolea S. Antón R. Remacha A. Camacho M. Soler M. Brú I. Porres A. Vila L. Tumour cell lines HT-29 and FaDu produce proinflammatory cytokines and activate neutrophils in vitro. Possible applications for neutrophil-based anti-tumour treatment.Mediators Inflamm. 2009; 2009: 817498Crossref PubMed Scopus (14) Google Scholar, 28Souto J.C. Vila L. Brú A. Polymorphonuclear neutrophils and cancer. Intense and sustained neutrophilia as a treatment against solid tumors.Med. Res. Rev. 2011; 31: 311-363Crossref PubMed Scopus (71) Google Scholar). The present work was conducted to test the hypothesis that the cross-talk between HNSCC and fibroblast could substantially enhance PGE2 biosynthesis through COX-2/mPGES-1 induction in fibroblasts. FaDu and SSC-25, permanent pharynx, and tongue squamous cell carcinoma cell lines were obtained from American Type Culture Collection (ATCC HTB-43 and CRL-1628). Cells were grown in DMEM containing 10% FBS and supplemented with 2 mM/l L-glutamine, 1 mM/l sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Biological Industries, Kibbutz Beit Haemek, Israel). When the cells reached early confluence, the medium was replaced with fresh medium containing 1% FBS. Conditioned medium was collected from cultures 48 h later. The conditioned medium was centrifuged and stored at –80°C until fibroblast stimulation. Human dermal fibroblasts were isolated and cultured as described (29Godessart N. Camacho M. López-Belmonte J. Antón R. García M. de Moragas J.M. Vila L. Prostaglandin H-synthase-2 is the main enzyme involved in the biosynthesis of octadecanoids from linoleic acid in human dermal fibroblasts stimulated with IL-1β.J. Invest. Dermatol. 1996; 107: 726-732Abstract Full Text PDF PubMed Scopus (28) Google Scholar). Cells in the 3-4 passage were seeded in 6-well plates and cultured in DMEM containing 10% FBS. Confluent fibroblasts were stimulated by replacing the medium with DMEM containing 1% FBS (control) or conditioned medium from tumor cells. Fibroblasts were kept in the culture chamber for different periods of time before prostanoids and enzyme expression were analyzed. COX-2 and mPGES-1 protein expression induced by tumor cell-conditioned medium were also analyzed in a tumor-derived fibroblast cell line CCD-18Co. The human colon carcinoma-derived fibroblast cell line CCD-18Co was obtained from ATCC (ATCC-CRL-1459) and was cultured as indicated in the product information sheet from ATCC. This cell line was treated as described for dermal fibroblasts. Dermal fibroblasts were cultured in 12-well culture plates, and tumor cells were seeded in 0.4 μm pore cell culture inserts (Becton Dickinson Labware, Franklin Lakes, NJ). When cells reached confluence, inserts were placed in 12-well culture plates containing the fibroblasts, and plates were incubated for 24 h in the culture chamber. Thereafter, culture media and cells were recovered for analysis. Dermal fibroblasts were cultivated in 75 cm2 culture flasks. When the fibroblasts reached early confluence, the medium was replaced with fresh medium (DMEM) containing 1% FBS or FaDu-conditioned medium (FaDu-CM). Conditioned media were collected from cultures 48 h later. The conditioned media were then centrifuged and stored at –80°C until the angiogenesis, migration, and proliferation assays. To obtain conditioned media free of prostanoids, all procedures, including those to obtain FaDu-CM, were performed in the presence of 10 µM/l indomethacin. Cells were incubated with 25 μM/l of [14C]AAc, (55–58 mCi/mmol; GE Healthcare, Buckinghamshire, UK) for 10 min as described (30Camacho M. López-Belmonte J. Vila L. Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression and PGI-synthase activity.Circ. Res. 1998; 83: 353-365Crossref PubMed Scopus (123) Google Scholar). Prostanoids were analyzed by HPLC as previously described (29Godessart N. Camacho M. López-Belmonte J. Antón R. García M. de Moragas J.M. Vila L. Prostaglandin H-synthase-2 is the main enzyme involved in the biosynthesis of octadecanoids from linoleic acid in human dermal fibroblasts stimulated with IL-1β.J. Invest. Dermatol. 1996; 107: 726-732Abstract Full Text PDF PubMed Scopus (28) Google Scholar). PGE2 and 6-oxo-PGF1α (stable hydrolysis product of PGI2) were analyzed by specific enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) following the manufacturer's instructions. Total RNA was extracted by chloroform isopropanol precipitation using Ultraspec (Biotecx Laboratories, Inc., Houston, TX) according to the manufacturer's instructions. Reverse transcription was performed with 0.5 μg of RNA per 10 μl reaction mixture, and enzyme mRNA levels were studied by real-time PCR as previously described (14Camacho M. Gerbolés E. Escudero J.R. Anton R. García-Moll X. Vila L. Microsomal-PGE synthase-1, which is not coupled to a particular COX-isoenzyme, is essential for PGE2 biosynthesis in vascular smooth muscle cells.J. Thromb. Haemost. 2007; 5: 1411-1419Crossref PubMed Scopus (49) Google Scholar). Gene expression data were normalized to β-actin as endogenous control, and RNA of untreated cells was used as a calibrator sample. Fibroblast protein extracts were analyzed by Western blot as described (14Camacho M. Gerbolés E. Escudero J.R. Anton R. García-Moll X. Vila L. Microsomal-PGE synthase-1, which is not coupled to a particular COX-isoenzyme, is essential for PGE2 biosynthesis in vascular smooth muscle cells.J. Thromb. Haemost. 2007; 5: 1411-1419Crossref PubMed Scopus (49) Google Scholar). Briefly, cell cultures were washed twice with PBS and lysed with lysis buffer 20 mM/l Tris-HCl (pH 7.4) containing protease inhibitor cocktail (Roche Diagnostics GmbH), 1 mM/l EDTA, and 0.1% Triton X-100. Protein concentration was determined by the Bradford method. Total protein equivalents were resolved by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore Millipore Ibérica, Spain). Membranes were incubated with antibodies against the human enzymes (Cayman Chemical). Bound antibody was detected using the appropriate horseradish peroxidase-conjugated antibody (Dako, Glostrup, Denmark) and a chemiluminescent detection system (Amersham ECL Plus Western Blotting Detection Reagents, GE Healthcare). Results were normalized by β-actin (Sigma) used as a loading control. For the cell cycle experiments, after the required treatment, cells were fixed in ice-cold 70% ethanol and stored at −20°C for at least 18 h. Cells were stained with 0.5 ml propidium iodide/Rnase staining buffer (BD Biosciences, San Diego, CA) for 15 min at room temperature and analyzed by flow cytometry (FACS Calibur; Beckton Dickinson) and Cell Quest Pro software. Apoptosis was determined by washing harvested cells with cold PBS and staining them with an annexin V-FITC apoptosis detection kit (Bender MedSystems GmbH, Viena, Austria) according to the manufacturer's instructions. Samples were then subjected to flow cytometry analysis. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described (31Camacho M. Godessart N. Antón R. García M. Vila L. Interleukin-1 enhances the ability of cultured umbilical vein endothelial cells to oxidize linoleic acid.J. Biol. Chem. 1995; 270: 17279-17286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In vitro angiogenesis assays were performed as described (32Alfranca A. López-Oliva J.M. Genís L. López-Maderuelo D. Mirones I. Salvado D. Quesada A.J. Arroyo A.G. Redondo J.M. PGE2 induces angiogenesis via MT1-MMP-mediated activation of the TGFbeta/Alk5 signaling pathway.Blood. 2008; 112: 1120-1128Crossref PubMed Scopus (59) Google Scholar). Briefly, 12.5 to 15 × 103 HUVECs were seeded onto growth factor-reduced Matrigel (Becton Dickinson Matrigel Basement Membrane Matrix) in 96-well plates and exposed to treatments as required. After 4.5 h of treatment, photographs were taken with a Nikon Digital Sight DS-2MBW camera mounted on a Nikon Eclipse TS100 microscope using a 10×/0.40 objective, and the number of closed polygons in the endothelial cell mesh was determined. The Radius™ 96-Well Cell Migration Assay Kit (Cell Biolabs, Inc., San Diego, CA) was used following the manufacturer's instructions. FaDu cells were seeded in the assay plate and subjected to the required treatment for 6.5 h. Afterward, photographs were taken with a Olympus C5050 digital camera attached to an Olympus BX50 microscope. Standardized size photographs were printed in a high-quality photograph paper, and the cell-free area was outlined and cut out. The percentage of initial cell-free area invaded was determined by gravimetry. For immunostaining studies, dermal fibroblasts or tumor-derived fibroblast cell line CCD-18Co were grown in 12 mm cover glasses (Marlenfeld GmbH and Co. KG, Lauda-Königshofen, Germany), treated as required, and fixed with methanol:acetone 1:1 at −20°C. Cells were stained using a Monoclonal Anti-Vimentin-Cy3 conjugate (C9080 Sigma-Aldrich Madrid, Spain). Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) 1 μg/ml diluted in the antibody solution. Fluorescent images were recorded in a contrast (bright-field) microscopy and fluorescence microscope (Axiovert-200M Zeiss, Jena, Germany). Dermal fibroblasts were incubated with conditioned medium of tumor cells for 48 h in the absence and in the presence of the indicated concentrations of GF109203× (a general inhibitor of protein kinase C), U0126 or PD98059 (inhibitors of mitogen-activated protein kinase kinase 1/2), SB203580 (a p38 mitogen-activated protein kinase [p38-MAPK] inhibitor), LY294002 (phosphoinoside 3-kinase inhibitor), rapamycin (mammalian target of rapamycin inhibitor) (all from Sigma) and Gö6976 (a Ca2+-dependent protein kinase C inhibitor), and Akt-inhibitor (1L6-Hydroxymethyl-chiro-inosito-2(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate; protein kinase B inhibitor), both from Calbiochem, Darmstad, Germany. Cells were incubated for 30 min with the inhibitors before the addition of the tumor cell-conditioned medium. Thereafter, COX-2 and mPGES-1 protein expression was evaluated as described above. Phosphorylation of p38-MAPK was determined by immunoblotting using a p38-MAPK polyclonal antibody and ant phosphor-p38-MAPK (Tyr180/Tyr182) mouse monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA) as described above. Tumor cell-conditioned media were heated for 30 min at 50, 75, and 100°C. Media were then stored at −80°C until their use to stimulate fibroblasts. Another set of experiments was performed using tumor cell-conditioned media filtered through exclusion molecular weight (MW) membranes of 10, 30, and 50 kDa (Amicon Ultra; Millipore, Carrigtwohill, Co. Cork, Ireland) following the manufacturer's instructions. The protein array test was performed using the Human Cytokines Antibody Array 3 (RayBiotech, Inc., Norcross, GA), and samples were processed following the manufacturer's protocol. Chemiluminiscent detection was performed with Amersham ECL Plus Western Blotting Detection Reagents. The density of the blots was measured in a GelDoc 2000 using Quantity One software (Bio-Rad Laboratories, Hercules, CA). Two-D nano-liquid chromatography mass spectrometry (MS) and database searching analysis of tumor cell conditioned media was performed in a liquid chromatograph (Ultimate 3000; LC Packings, Amsterdam, The Netherlands) coupled to an nESI-LTQ mass spectrometer (Thermo Electron Corp., Bremen, Germany). A detailed description of the method is supplied as supplementary material. Quantitative analysis of IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra) in the culture media was performed by specific ELISA following the manufacturer's instructions (IL-1α and IL-1ra were from R&D Systems, Minneapolis, MN; IL-1β was from eBioscience, San Diego, CA). To explore the possible involvement of IL-1 on tumor cell-stimulation of dermal fibroblasts, cells were exposed to human recombinant IL-1β or tumor cell-conditioned medium for the indicated period of time in the presence of recombinant human IL-1 receptor antagonist (IL-1ra) (PeproTech, London, UK). The present study was approved by the HSCSP Ethics Committee, and informed consent was obtained from each subject. A tissue sample of tumor and nontumoral mucosa (contralateral to the site of the primary tumor) was obtained at the pretreatment period from 20 patients with a HNSCC diagnosed and treated in our center (see Supplementary Table I for characteristics of patients). Immediately after extraction, tumor and nontumoral mucosa pieces of HNSCC were cut into small fragments with a surgical blade (≈1 mm) and used for experiments without further manipulation. Fragments of 100–200 mg were placed in 1 ml DMEM supplemented with 2 mM/l L-glutamine, 1 mM/l sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin and incubated for 48 h in the culture chamber. The culture medium was later recovered and kept at −80°C until IL-1α, IL-1β, and IL-1ra were analyzed. Sigma-Plot 11 software was used for statistical analysis. Statistical significance between pairs of groups was assessed using the paired samples t-test; multiple comparisons were performed by ANOVA test. A p value <0.05 was considered significant. Fig. 1A shows illustrative HPLC chromatograms of the AAc profile of FaDu and dermal fibroblasts regarding prostanoids after incubation with [14C]labeled-AAc. Both cells produced PGE2 as the major prostanoid, and fibroblasts additionally produced PGI2 (determined as its stable hydrolysis product 6-oxo-PGF1α). When we analyzed the release of PGE2 by cocultures of FaDu and fibroblasts, we found that production of PGE2 was significantly higher than the production by cells incubated alone and was higher than the sum of the individual production of FaDu plus fibroblasts. This indicated that coculture of both cell types causes a synergistic effect on PGE2 biosynthesis (Fig. 1B). We then explored the effect of the coculture on the expression of the enzymes involved in PGE2 biosynthesis. We examined the mutual influence of each cell type in the expression of COX-1, COX-2, mPGES-1, mPGES-2, and cPGES. Both cell types expressed all the enzymes, but COX-1, mPGES-2, and cPGES were not modified in the coculture samples when compared with cells incubated individually (not shown). In contrast, COX-2 was upregulated in FaDu and fibroblasts after coculture, whereas mPGES-1 was only appreciably upregulated in the fibroblasts (Fig. 1C). However, coculture caused much more COX-2 upregulation in fibroblasts than in FaDu. We next explored the effect of FaDu-CM, and human recombinant IL-1β as positive control, on the release of prostanoids by fibroblasts. FaDu-CM, like IL-1β, induced the release of PGE2 and PGI2 in a time-dependent manner (Fig. 2). PGE2 accumulated in the culture medium was about 5-fold the amount of 6-oxo-PGF1α. Fig. 3 shows the effect of conditioned medium from two head and neck tumor cell lines on the expression of COX-2 and mPGES-1 in dermal fibroblasts analyzed in terms of mRNA and protein. FaDu-CM and SCC-25-CM time dependently induced mPGES-1 mRNA levels in the fibroblasts, whereas only FaDu-CM was able to significantly induce COX-2 mRNA levels (Fig. 3A). Analysis of the proteins showed similar results. Only FaDu-CM increased COX-2 protein levels, whereas both FaDu-CM and SCC-25-CM time-dependently upregulated mPGES-1 in terms of protein. Tumor cell-induced mPGES-1 expression was delayed compared with COX-2 (Fig. 3). In addition, the transcription inhibitor actinomycin-D totally suppressed the effect of tumor cells on COX-2 and mPGES-1 expression (not shown). To explore other activities potentially re
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