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• W2087753442 abstract "Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E2 (PGE2) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE2. Treatment with indomethacin, or COX-selective inhibitors, prevents PGE2 biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE2 or the EP4 receptor-selective agonist PGE1-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE2-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE2, suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers. Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E2 (PGE2) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE2. Treatment with indomethacin, or COX-selective inhibitors, prevents PGE2 biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE2 or the EP4 receptor-selective agonist PGE1-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE2-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE2, suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers. The identification of cyclooxygenase (COX) 1The abbreviations used are: COX, cyclooxygenase; PGE2, prostaglandin E2; PI3K, phosphatidylinositol 3-kinase; EP, Prostaglandin Receptor; ERK, extracellular signal-regulated kinase; AA, arachidonic acid; YAMC, young adult mouse colon cells.1The abbreviations used are: COX, cyclooxygenase; PGE2, prostaglandin E2; PI3K, phosphatidylinositol 3-kinase; EP, Prostaglandin Receptor; ERK, extracellular signal-regulated kinase; AA, arachidonic acid; YAMC, young adult mouse colon cells. as the target for non-steroidal anti-inflammatory drugs (NSAIDs) led to new understandings of its pathophysiological role and of the mechanisms of action of these drugs (1Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (288) Google Scholar, 2Turini M.E. DuBois R.N. Annu. Rev. Med. 2002; 53: 35-57Crossref PubMed Scopus (548) Google Scholar). The discovery of COX2, an isoform that, although catalytically identical to COX1, shows inducible tissue-selective expression, suggested physiological and pathophysiological roles for the constitutively expressed (COX1) and inducible (COX2) isoforms, respectively (1Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (288) Google Scholar, 2Turini M.E. DuBois R.N. Annu. Rev. Med. 2002; 53: 35-57Crossref PubMed Scopus (548) Google Scholar, 3Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar, 4He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). Studies showing a correlation between NSAIDs and decreased colon cancer incidence, and the demonstration of up-regulated COX2 expression in colon carcinoma, suggested a role for COX2 in the pathophysiology of colon cancer and created new paradigms for the study of the role of prostanoids in cancer (1Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (288) Google Scholar, 2Turini M.E. DuBois R.N. Annu. Rev. Med. 2002; 53: 35-57Crossref PubMed Scopus (548) Google Scholar, 3Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar). Nevertheless, despite extensive supporting evidence, direct links between the anti-tumor effects of NSAIDs and COX inhibition are yet to be established (4He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 5Chan T.A. Morin P.J. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 681-686Crossref PubMed Scopus (568) Google Scholar, 6Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Rigas B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (596) Google Scholar, 7Piazza G.A. Alberts D.S. Hixson L.J. Paranka N.S. Li H. Finn T. Bogert C. Guillen J.M. Brendel K. Gross P.H. Sperl G. Ritchie J. Burt R.W. Ellsworth L. Ahnen D.J. Pamukcu R. Cancer Res. 1997; 57: 2909-2915PubMed Google Scholar, 8Raz A. Biochem. Pharmacol. 2002; 63: 343-347Crossref PubMed Scopus (74) Google Scholar, 9Rigas B. Shiff S.J. Med. Hypotheses. 2000; 54: 210-215Crossref PubMed Scopus (78) Google Scholar). The potential for non-COX-dependent anti-tumor effects of NSAIDs (6Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Rigas B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (596) Google Scholar, 7Piazza G.A. Alberts D.S. Hixson L.J. Paranka N.S. Li H. Finn T. Bogert C. Guillen J.M. Brendel K. Gross P.H. Sperl G. Ritchie J. Burt R.W. Ellsworth L. Ahnen D.J. Pamukcu R. Cancer Res. 1997; 57: 2909-2915PubMed Google Scholar, 8Raz A. Biochem. Pharmacol. 2002; 63: 343-347Crossref PubMed Scopus (74) Google Scholar, 9Rigas B. Shiff S.J. Med. Hypotheses. 2000; 54: 210-215Crossref PubMed Scopus (78) Google Scholar), as well as limited evidence available identifying a specific prostanoid(s) as the mediator responsible for the anti-tumor effects of the COX inhibitors (5Chan T.A. Morin P.J. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 681-686Crossref PubMed Scopus (568) Google Scholar), raised questions regarding the mechanism of action of NSAIDs and of the role of arachidonic acid, prostanoids, and COXs in human cancer (4He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 5Chan T.A. Morin P.J. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 681-686Crossref PubMed Scopus (568) Google Scholar, 6Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Rigas B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (596) Google Scholar, 7Piazza G.A. Alberts D.S. Hixson L.J. Paranka N.S. Li H. Finn T. Bogert C. Guillen J.M. Brendel K. Gross P.H. Sperl G. Ritchie J. Burt R.W. Ellsworth L. Ahnen D.J. Pamukcu R. Cancer Res. 1997; 57: 2909-2915PubMed Google Scholar, 8Raz A. Biochem. Pharmacol. 2002; 63: 343-347Crossref PubMed Scopus (74) Google Scholar, 9Rigas B. Shiff S.J. Med. Hypotheses. 2000; 54: 210-215Crossref PubMed Scopus (78) Google Scholar). During studies of arachidonic acid metabolism in mouse colon carcinoma (CT26) cells, we observed the rapid biosynthesis of prostaglandin E2 (PGE2). This, and the powerful tumorigenic activity of CT26 cells, prompted us to characterize the role of PGE2 in tumor growth and development. We show here a direct correlation between COX inhibition, reduced PGE2 biosynthesis, and marked reductions in CT26 cell growth in vitro, as well as tumorigenicity in vivo. These data, together with the finding that the anti-proliferative effects of NSAIDs treatment can be selectively reverted by PGE2 via the Prostaglandin Receptor EP4-mediated phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinase (ERK) activation, provide a direct functional link between PGE2-induced cell growth and EP4-mediated ERK signaling. Arachidonic Acid (AA) Metabolism—Serum-free CT26 cells (a gift of Dr. R. Xiang, Scripps Research Institute, San Diego, CA) or young adult mouse colon cells (YAMC) (a gift of Dr. B. Whitehead, Vanderbilt University, Nashville, TN) were incubated with [1-14C]AA (30 μm, final concentration) at 37 °C. After 20 min, the metabolites present in cells and media were extracted, resolved, and quantified by reversed-phase high performance liquid chromatography (10Capdevila J.H. Morrow J.D. Belosludtsev Y.Y. Beauchamp D.R. DuBois R.N. Falck J.R. Biochemistry. 1995; 34: 3325-3337Crossref PubMed Scopus (105) Google Scholar). For inhibition studies, cells were incubated with varying concentrations of indomethacin, NS398 (both from BioMol, Plymouth Meeting, PA) or SC560 (a gift from Monsanto/Searle, Chesterfield, MO) for 2 h prior to AA addition. The eluted products with the retention time of PGE2 were collected and characterized by gas chromatography/mass spectrometry (GC/MS) (10Capdevila J.H. Morrow J.D. Belosludtsev Y.Y. Beauchamp D.R. DuBois R.N. Falck J.R. Biochemistry. 1995; 34: 3325-3337Crossref PubMed Scopus (105) Google Scholar, 11Morrow J.D. Harris T.M. Roberts II, L.J. Anal. Biochem. 1990; 184: 1-10Crossref PubMed Scopus (410) Google Scholar). Proliferation Assays—CT26 cells (5 × 103/96-well plates) were plated in Dulbecco's modified Eagle's medium containing 2% fetal calf serum with or without 10 μm indomethacin, 5 μm SC560, or 5 μm NS398. To determine the mitogenic activity of PGE2, cells were incubated with or without COX inhibitors in the presence of PGE2 (0-5 μm). To determine the major prostanoid and the receptor involved in the control of CT26 cell proliferation, cells were cultured with or without 10 μm indomethacin added alone or in combination with butaprost, sulprostone, PGE1-OH, cicaprost, PGD2, PGF2α, or PGJ2 (1 μm each) (Cayman Chemicals, Ann Arbor, MI). To determine the intracellular pathways involved in the control of CT26 cell proliferation, cells were cultured with 10 μm indomethacin and 1 μm PGE2 or PGE1-OH in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor PD98059 (5 μm), the P38 mitogen-activated protein kinase (MAPK) inhibitor (1 μm), and the PI3K inhibitor wortmannin (100 nm) (all from Calbiochem). Two days after, the medium was replaced with fresh media containing [3H]thymidine (10 μCi/ml), and the cells were incubated for another 48 h. Cells were then processed as described (12Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (348) Google Scholar). Three to four independent experiments with quadruplicate samples were performed. Manual cell counts paralleled the results of [3H]thymidine incorporation (not shown). Primary Tumor Growth—Experiments were performed according to institutional animal care guidelines. Male BALB/c mice (5 weeks old; 18-20 g of body weight) were given four dorsal subcutaneous injections of CT26 cells (5 × 105/site injection) as described (13Pozzi A. LeVine W.F. Gardner H.A. Oncogene. 2002; 21: 272-281Crossref PubMed Scopus (135) Google Scholar). In some experiments, mice injected with CT26 cells were treated with indomethacin (2.5 mg/kg body weight, intraperitoneal injection daily), NS398 (2.5 mg/kg/body weight, intraperitoneal injection every other day), or SC560 (30 μg/ml in drinking water) starting at the time of, or 1 week after, cell injection; tumors were harvested after 14 days of growth (see Fig. 4A). For recovery experiments (see Fig. 5A), mice received indomethacin at the time of, or 1 week after, cell injection. Two weeks after cell injection, indomethacin treatment was stopped, and the animals were kept for an additional 10 days. Tumors (24 days total growth) were then harvested, and their volumes were measured and expressed as mm3 of total tumor volume/mouse.Fig. 5Effects of COX inhibitors on tumor uptake and development. A, schematic representation of the in vivo experimental protocol. CT26 cells (5 × 105 cells/mice) were injected into BALB/c mice (10 mice/group), and the animals were sacrificed 2 weeks after injection. Tumor-bearing mice were divided into three groups: untreated (c, control); treated for 1 week with COX inhibitors starting 1 week after cell injection (a, 1 week); and treated for 2 weeks with COX inhibitors starting at the time of cell injection (b, 2 weeks). B, gross appearance of tumors derived from CT26 cells injected as described in A. C, total tumor volume was measured as described under “Materials and Methods.” Values represent the average of total tumor volume/mouse calculated from three experiments, using 10 mice/experiment. D, PGE2 levels in tumor extracts. PGE2 levels were determined as described under “Materials and Methods” and expressed as ng/mg total protein. Values are the average of 10 tumors/treatment. Differences between mice untreated and treated with COX inhibitors (*) or between mice treated with COX inhibitors for 1 (a) versus 2 (b) weeks (**) were significant with p < 0.05.View Large Image Figure ViewerDownload (PPT) Measurement of PGE2 and cAMP Levels—Tumors or cells were homogenized and lipids were extracted with methanol containing 4 ng of [2H4]PGE2 (Cayman Chemicals). PGE2 levels were then quantified as described (14DuBois R.N. Awad J. Morrow J. Roberts II, L.J. Bishop P.R. J. Clin. Invest. 1994; 93: 493-498Crossref PubMed Scopus (377) Google Scholar, 15Reese J. Paria B.C. Brown N. Zhao X. Morrow J.D. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9759-9764Crossref PubMed Scopus (119) Google Scholar). CT26 cells were incubated for 16 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 10 μm indomethacin, followed by serum-free medium containing 10 μm indomethacin and 0.5 mm isobutylmethylxanthine for 30 min. Cells were then incubated for 10 min with PGE2, PGE1-OH, butaprost, or forskolin (1 μm each), washed with PBS, and after adding HCl (0.1 m final concentration), the cells were scraped and lysed; cAMP levels (ng/mg total protein) were determined by enzyme-linked immunosorbent assay (cAMP kit, Cayman Chemicals), according to the manufacturer's instructions (16Hata A.N. Zent R. Breyer M.D. Breyer R.M. J. Pharmacol. Exp. Ther. 2003; 306: 463-470Crossref PubMed Scopus (63) Google Scholar). RT-PCR Analysis—Total RNA was purified from CT26 cells or mouse kidneys using TRIzol reagent (Invitrogen). RNA samples were reverse-transcribed using a SuperScript II™ kit and oligo(dT) (12-18 bp). cDNAs were amplified using the following EP-subtype selective primers: EP1 (710 bp) sense, 5′-cacccaggctccccaatacatctg-3′, anti-sense, 5′-ggagggtggctgtggctgaag3′; EP2 (507 bp) sense, 5′-ccggggttctggggaatc-3′, antisense, 5′-gtgcatgcgaatgaggttgag-3′; EP3α (617 bp) and β (528 bp) sense, 5′-cgccgtctcgcagtc-3′, antisense, 5′-tgtgtcgtcttgcccccg-3′; EP3γ (690 bp) sense, 5′-cgccgtctcgcagtc-3′, antisense, 5′-tgtggcttcattccttgccca-3′; EP4 (407 bp) sense, 5′-actgaccttctgggcaccttg-3′, antisense, 5′-tccttcctcatccttgccacc-3′. Immunohistochemistry—Immunohistochemistry on frozen tumor sections (7 μm each) was done using rat anti-mouse CD31 (1:100, PharMingen) or rabbit anti-mouse PCNA (1:100, Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated goat secondary antibody to rat or rabbit IgG (1:200, Jackson ImmunoResearch) and Sigma Fast diaminobenzidine chromogenic tablets (Sigma). CD31-positive structures were then imaged and processed as described (12Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (348) Google Scholar). Tumor vascularity was expressed as a percentage of area occupied by CD-31-positive structures per microscopic field. The proliferation index within tumors was expressed as (number of PCNA-positive cells/total number of cells per microscopic field) × 100. Apoptosis within tumors was evaluated by staining frozen sections with the Dead End™ colorimetric terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) system (Promega) using diaminobenzidine as the chromogenic substrate. The apoptotic index was expressed as (number of TUNEL-positive cells/total number of cells per microscopic field) × 100. Five images/tumor were evaluated, with a total of 10 tumors/treatment. Western Blot Analysis—To evaluate ERK and Akt phosphorylation, CT26 cells were cultured for 24 h in serum-free medium before treatment with PGE2, PGE1-OH, or butaprost (1 μm each) for 15 min. The cells were then washed with PBS, scraped, suspended in 50 mm HEPES, pH 7.5, 150 mm NaCl, 1% Triton X-100, and centrifuged for 10 min at 14,000 rpm. Cell lysates were resolved by 10% SDS/PAGE (50 μg of total protein/lane) and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with a rabbit anti-phospho ERK or rabbit anti-phospho Akt antibody (both from Cell Signaling Technology). Immunoreactive proteins were visualized using a peroxidase-conjugated goat anti-rabbit and an ECL kit (Pierce). Total ERK and Akt content was verified by stripping the membranes in 50 mm Tris-HCl, pH 6.5, containing 2% SDS and 0.4% β-mercaptoethanol for 1 h at 55 °C and re-probing them with a rabbit anti-ERK or rabbit anti-Akt antibody (both from Cell Signaling Technology). To evaluate the effects of PD98059, p38 MAPK inhibitor, or wortmannin (all from Calbiochem) upon the phosphorylation levels of ERK, p38, and PI3K/Akt, respectively, cells were cultured in the presence of 2% fetal calf serum (to mimic the conditions used for proliferation; see above) with 10 μm indomethacin and the different inhibitors mentioned above in the presence or absence of PGE2 or PGE1-OH. Four days later, cells lysates were prepared and analyzed as indicated above. Levels of p38 phosphorylation were detected by using a rabbit anti-phospho p38 antibody (Cell Signaling Technology). COX expression in the CT26 or YAMC cell lysates (50 μg of protein/lane) was analyzed as above by using either a rabbit anti-COX2 (Cayman Chemicals) or a goat anti-COX1 (Santa Cruz Biotechnology). CT26 Cells Biosynthesize PGE2—To explore the enzymology of AA metabolism by CT26 cells, we incubated the cells with radiolabeled AA and compared their AA metabolite profiles with those generated by cultured YAMC cells. As shown in Fig. 1A, although YAMC do not metabolize exogenous AA, CT26 cells actively oxidized the fatty acid to a product (98% of total metabolism) with the high performance liquid chromatography retention time of authentic PGE2 (16.5 min). Subsequent chromatographic and GC/MS analyses confirmed that CT26 cells metabolized AA to PGE2 (19 ± 5 nmol/min/mg of cell protein) as the only detectable product (Fig. 1A). Western blot analysis demonstrated the presence of both COX1 and COX2 in lysates isolated from CT26 cells, whereas only COX1 was clearly evident in YAMC cells (Fig. 1B). It is interesting that despite similar levels of COX1 expression in these two cell types, YAMC are unable to support prostanoid biosynthesis (Fig. 1B). The fact that most of the AA is recovered (Fig. 1A) indicates that in non-tumorigenic YAMC cells, COX activity is under the control of additional, yet to be characterized regulatory mechanisms. To dissect the roles of COX1 and COX2 in PGE2 formation by CT26 cells, we incubated them with AA in the presence of either a nonspecific COX inhibitor (indomethacin) or a COX1-specific (SC560) or COX2-specific (NS398) inhibitor. As shown in Fig. 1C, 10 μm indomethacin blocked all cellular AA oxidation and PGE2 biosynthesis almost completely. On the other hand, only partial inhibition of PGE2 biosynthesis was achieved at 5 μm SC560 and 10 μm NS398 (70 and 85% inhibition, respectively), and the extent of inhibition did not increase with higher doses. All three inhibitors blocked AA metabolism and PGE2 formation without changing the metabolite profile of the enzyme (Fig. 1C). These results show that in CT26 cells, COX1 and COX2 are capable of metabolizing added AA to PGE2. To determine whether CT26 cells metabolize endogenous pools of AA and the role of the COX isoforms in PGE2 formation, we quantified (by GC/MS) the levels of endogenous PGE2 in CT26 cells. In the absence of external stimuli, CT26 cells produce significant amounts of PGE2 (58.54 ± 2.9 ng/mg of total protein, n = 10), most of which is secreted into the media (>90% of the total). Endogenous PGE2 synthesis is inhibited by 10 μm indomethacin (7.5 ± 0.9 ng/mg total protein, n = 8), 5 μm NS398 (14.95 ± 1.5 ng/mg total protein, n = 5), or 5 μm SC560 (16.1 ± 3.3 ng/mg total protein, n = 5). PGE2 Induces CT26 Cell Growth—Because PGE2 has been implicated in cell survival (17Hoshino T. Tsutsumi S. Tomisato W. Hwang H.J. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 12752-12758Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and CT26 cells generate high levels of PGE2 (Fig. 1A), we determined the effect of COX inhibitors on cell proliferation. All three COX inhibitors decreased CT26 cell growth in a dose-dependent manner (Fig. 2, A-C), with maximal inhibition obtained between 5-10 μm SC560 or NS398 and 5-20 μm indomethacin, all doses shown to block PGE2 formation (Fig. 1C). Thus, growth of CT26 cells is equally sensitive to COX1 or COX2 inhibition, and the source of PGE2 is not a determining factor for its mitogenic activity. To determine whether exogenous PGE2 could rescue the cells from COX inhibition, CT26 cells were grown in the presence of 10 μm indomethacin, 5 μm SC560, or 5 μm NS398 added alone or in combination with PGE2 (ranging from 0 to 5 μm) (Fig. 2, D-F). Exogenous PGE2 rescued the growth of cells treated with the COX inhibitors to the levels observed in cells exposed to PGE2 alone, demonstrating that PGE2 formation was sufficient to rescue cell proliferation and that COX was the target for the inhibitors. The lack of a PGE2 effect upon the growth of cells incubated in the absence of inhibitors (Fig. 2, D-F) suggested that its endogenous levels are sufficient to support growth under steady-state conditions. To determine the selectivity of the PGE2 mitogenic activity, cells were incubated in the presence of 10 μm indomethacin together with PGE2, PGD2, PGF2α, PGJ2, or cicaprost (a prostacyclin receptor agonist) (1 μm each), and their ability to rescue cell growth from COX inhibition was evaluated. Only PGE2 restored proliferation in indomethacin-treated cells to levels similar to those of untreated controls (Fig. 3A), showing that its effects were selective. CT26 Cell Growth Is Associated with EP4 Receptor-mediated Akt/ERK Activation—Many of the cellular functions of PGE2 are mediated by a family of G protein-coupled receptors, namely EP1, EP2, EP3, and EP4 (18Breyer R.M. Bagdassarian C.K. Myers S.A. Breyer M.D. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 661-690Crossref PubMed Scopus (850) Google Scholar), of which CT26 cells express the EP1, EP2, and EP4 subtypes (Fig. 3B). To characterize the role of these receptors in PGE2-induced mitogenesis, cells were incubated with 10 μm indomethacin, and the ability of PGE2, sulprostone (an EP1 and EP3 agonist), butaprost (an EP2 agonist), or PGE1-OH (an EP4 agonist) (1 μm each) to rescue proliferation from COX inhibition was evaluated. Only PGE1-OH mimicked PGE2 in restoring cell growth to levels similar to those of indomethacin-free controls (Fig. 3C), indicating that the EP4 receptor mediates the effects of PGE2. To characterize the EP4-mediated signaling pathways involved in the PGE2 mitogenic activity, we determined the effects of PGE2 on intracellular levels of cAMP, known to increase after EP2 and/or EP4 activation (18Breyer R.M. Bagdassarian C.K. Myers S.A. Breyer M.D. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 661-690Crossref PubMed Scopus (850) Google Scholar). Incubation of cells with forskolin, an activator of adenylate cyclase, caused nearly 3-fold increases in cAMP levels when compared with untreated cells, but no significant changes were observed with PGE2, PGE1-OH, or butaprost (Fig. 4A). It has been proposed that PGE2 stimulation of the EP4 receptors leads to phosphorylation of ERK through a PI3K-dependent mechanism (19Fujino H. Xu W. Regan J.W. J. Biol. Chem. 2003; 278: 12151-12156Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). For this reason, we determined the ability of PGE2 and PGE1-OH to activate these two kinases in CT26 cells. Incubation of cells with PGE2 (1 μm) markedly increased ERK and Akt (a PI3K substrate) phosphorylation (Fig. 4B). Similarly, phosphorylation of these two kinases was also observed after treatment with PGE1-OH, but not butaprost (used as the negative control), indicating that the EP4 receptor mediates the effects of PGE2. In addition, our data parallels the finding that phosphorylation of Akt is observed in CT26 cells after PGE2 stimulation (20Yasumaru M. Tsuji S. Tsujii M. Irie T. Komori M. Kimura A. Nishida T. Kakiuchi Y. Kawai N. Murata H. Horimoto M. Sasaki Y. Hayashi N. Kawano S. Hori M. Cancer Res. 2003; 63: 6726-6734PubMed Google Scholar). To confirm that the EP4-mediated phosphorylation of PI3K/ERK was necessary to support CT26 cell proliferation, cells were incubated with 10 μm indomethacin in the presence of PD98059 (a MEK1/ERK inhibitor), wortmannin (a PI3K inhibitor), and a p38 MAPK inhibitor (used as the negative control), and the ability of PGE2 or PGE1-OH (1 μm each) to rescue cell proliferation was evaluated. As shown in Fig. 4C, neither PGE2 nor PGE1-OH was able to rescue the proliferation of indomethacin-treated CT26 cells when the ERK or PI3K pathways were inhibited. In contrast, both prostanoids were able to rescue indomethacin-treated cells cultured in the presence of a p38 kinase inhibitor (Fig. 4C) or PKA or PKC inhibitors (not shown), suggesting that these kinases are not involved in the EP4-mediated cell proliferation. Cell lysates derived from cells incubated as described above were analyzed by Western blot analysis. As shown in Fig. 4D, phosphorylation of ERK, Akt, or p38 was completely inhibited after the addition of the specific inhibitors, and no changes in phosphorylation were observed after the addition of PGE2 or PGE1-OH. Effects of COX Inhibition on Tumor Growth—It has been shown recently that CT26-derived tumors express both COX1 and COX2 (20Yasumaru M. Tsuji S. Tsujii M. Irie T. Komori M. Kimura A. Nishida T. Kakiuchi Y. Kawai N. Murata H. Horimoto M. Sasaki Y. Hayashi N. Kawano S. Hori M. Cancer Res. 2003; 63: 6726-6734PubMed Google Scholar), suggesting that they could be sensitive to both COX1- and/or COX2-specific inhibition. For this reason, we determined the effects of COX inhibition on the size and number of tumors derived from the injection of CT26 cells in BALB/c mice (13Pozzi A. LeVine W.F. Gardner H.A. Oncogene. 2002; 21: 272-281Crossref PubMed Scopus (135) Google Scholar). Mice were treated at the time of cell injection or 1 week after cell injection with indomethacin (Fig. 5A), and tumor growth was compared with untreated control mice. Mice were sacrificed 14 days after cell injection, and tumor number and volume were evaluated. Indomethacin caused dramatic decreases in tumor number (Fig. 5B) and volume (Fig. 5C), and its anti-tumorigenic effects appeared to be independent of the time of treatment initiation (Fig. 5, B and C). Indomethacin, administered 1 week after tumor cell injection, arrested tumor growth at levels similar to those observed in control mice sacrificed 1 week after tumor cell injection (not shown). Thus, depending upon the time of treatment initiation, indomethacin can both prevent tumor uptake and development and inhibit tumor growth. To determine whether the effects of indomethacin on tumor growth correlated with COX inhibition and a resultant decrease in PGE2 levels, endogenous PGE2 levels in tumors from untreated or indomethacin-treated mice were evaluated by GC/MS. Indomethacin treatment caused dramatic reductions in tumor PGE2 levels, thus providing a strong demonstration of a direct link between the biological response to the inhibitor, reductions in COX activity, and PGE2 biosynthesis (Fig. 5D). To identify the COX isoform involved in tumor growth, tumor-bearing mice were treated with SC560 or NS398 using a protoc" @default.
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