Hyaluronan Constitutively Regulates Activation of COX-2-mediated Cell Survival Activity in Intestinal Epithelial and Colon Carcinoma Cells
2008; Elsevier BV; Volume: 283; Issue: 21 Linguagem: Inglês
10.1074/jbc.m703811200
ISSN1083-351X
AutoresSuniti Misra, Lina M. Obeid, Yusuf A. Hannun, Susumu Minamisawa, Franklin G. Berger, Roger R. Markwald, Bryan P. Toole, Shibnath Ghatak,
Tópico(s)Fibroblast Growth Factor Research
ResumoHyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, β-catenin, and cyclooxygenase-2/prostaglandin E2 in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 → phosphoinositide 3-kinase/AKT → β-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E2-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer. Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, β-catenin, and cyclooxygenase-2/prostaglandin E2 in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 → phosphoinositide 3-kinase/AKT → β-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E2-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer. Cyclooxygenase (COX), 3The abbreviations used are: COX, cyclooxygenase; HA, hyaluronan; O-HA, HA oligomers; RTK, receptor tyrosine kinase; PGE2, prostaglandin E2; EMT, epithelial-mesenchymal transition; CA-, constitutively active; DN-, dominant negative; siRNA, small interfering RNA; PI3K, phosphoinositide 3-kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; EGFR, epidermal growth factor receptor; RTK, receptor tyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase. or prostaglandin G/H synthetase, which catalyzes the rate-limiting step in the synthesis of prostaglandins and other eicosanoids from arachidonic acid, is considered a promising target for cancer prevention and therapy. Of the two COX isoforms, COX-1 is constitutively present in tissues, whereas COX-2 is inducible. COX-1 is expressed in normal intestine and remains unchanged in intestinal tumors. In contrast, COX-2 is undetectable in normal intestine but is elevated in up to 85% of colorectal adenocarcinoma cells by a variety of pro-inflammatory stimuli and growth factors (1Sheng H. Shao J. Dixon D.A. Williams C.S. Prescott S.M. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 2000; 275: 6628-6635Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 2Wu R. Abramson A.L. Shikowitz M.J. Dannenberg A.J. Steinberg B.M. Clin. Cancer Res. 2005; 11: 6155-6161Crossref PubMed Scopus (55) Google Scholar). COX-2 overexpression can occur as a consequence of EGFR-induced phosphoinositide 3-kinase (PI3K) signaling in papillomas (2Wu R. Abramson A.L. Shikowitz M.J. Dannenberg A.J. Steinberg B.M. Clin. Cancer Res. 2005; 11: 6155-6161Crossref PubMed Scopus (55) Google Scholar). In contrast, in cells transformed by mutant-activated Ras overexpression, increased EGFR signaling and transforming growth factor-β overproduction do not account for increased COX-2 expression (3Du J. Jiang B. Barnard J. Neoplasia. 2005; 7: 761-770Crossref PubMed Scopus (8) Google Scholar). Recent evidence indicates that elevated expression of COX-2 is required for increased invasiveness, cellular adhesion, and inhibition of apoptosis in colon cancer. Increasing evidence suggests that COX-2 and COX-2-derived prostaglandin E2 (PGE2) are also involved in the control of angiogenesis (4Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar). Hyaluronan (HA), a multifunctional anionic polysaccharide, is composed of 2,000–25,000 disaccharide units of glucuronic acid and N-acetylglucosamine (105 to 107 Da). HA has a structural role in many connective tissues and is associated with the pericellular matrix surrounding proliferating and motile cells in normal and pathological systems, where it has both structural and signaling functions (5Markwald R.R. Fitzharris T.P. Bank H. Bernanke D.H. Dev. Biol. 1978; 62: 292-316Crossref PubMed Scopus (111) Google Scholar, 6Toole B.P. Semin. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (441) Google Scholar, 7Toole B.P. Glycobiology. 2002; 12: R37-R42Crossref PubMed Scopus (183) Google Scholar, 8Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (450) Google Scholar, 9Turley E.A. Noble P.W. Bourguignon L.Y. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 10Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 11Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar). Moreover, a large body of literature indicates that high levels of HA exacerbate chronic inflammation in Crohn disease (12de la Motte C.A. Hascall V.C. Calabro A. Yen-Lieberman B. Strong S.A. J. Biol. Chem. 1999; 274: 30747-30755Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 13Majors A.K. Austin R.C. de la Motte C.A. Pyeritz R.E. Hascall V.C. Kessler S.P. Sen G. Strong S.A. J. Biol. Chem. 2003; 278: 47223-47231Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), in bleomycin-induced lung inflammation (14Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Pure E. Henson P.M. Noble P.W. Science. 2002; 296: 155-158Crossref PubMed Scopus (575) Google Scholar), in diabetic neuropathy (15Wang A. Hascall V.C. J. Biol. Chem. 2004; 279: 10279-10285Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), in atherosclerosis (16Chai S. Chai Q. Danielsen C.C. Hjorth P. Nyengaard J.R. Ledet T. Yamaguchi Y. Rasmussen L.M. Wogensen L. Circ. Res. 2005; 96: 583-591Crossref PubMed Scopus (112) Google Scholar), and in graft-versus-host disease (17Milinkovic M. Antin J.H. Hergrueter C.A. Underhill C.B. Sackstein R. Blood. 2004; 103: 740-742Crossref PubMed Scopus (34) Google Scholar). Recent evidence associates HA with malignant cell activities in vivo and in vitro (11Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar, 18Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). HA levels are elevated and predictive of malignant progression in several human cancers (19Anttila M.A. Tammi R.H. Tammi M.I. Syrjanen K.J. Saarikoski S.V. Kosma V.M. Cancer Res. 2000; 60: 150-155PubMed Google Scholar, 20Lipponen P. Aaltomaa S. Tammi R. Tammi M. Agren U. Kosma V. Eur. J. Cancer. 2001; 37: 849-856Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), although squamous adenocarcinomas have a low percentage of HA-positive cells (21Kosunen A. Ropponen K. Kellokoski J. Pukkila M. Virtaniemi J. Valtonen H. Kumpulainen E. Johansson R. Tammi R. Tammi M. Nuutinen J. Kosma V.M. Oral. Oncol. 2004; 40: 257-263Crossref PubMed Scopus (58) Google Scholar). Also, despite the fact that high levels of stromal HA predict tumor progression (10Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar), recent data suggest that stromal HA, but not tumor cell-produced HA, inhibits rather than promotes tumor progression (22Lopez J.I. Camenisch T.D. Stevens M.V. Sands B.J. McDonald J. Schroeder J.A. Cancer Res. 2005; 65: 6755-6763Crossref PubMed Scopus (143) Google Scholar). This is most likely because HA turnover may be important in tumor malignancy, i.e. high levels of HA coupled with degradation to smaller sized HA fragments may be required for induction of pro-malignant signaling (23Itano N. Sawai T. Atsumi F. Miyaishi O. Taniguchi S. Kannagi R. Hamaguchi M. Kimata K. J. Biol. Chem. 2004; 279: 18679-18687Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 24Lokeshwar V.B. Cerwinka W.H. Isoyama T. Lokeshwar B.L. Cancer Res. 2005; 65: 7782-7789Crossref PubMed Scopus (105) Google Scholar, 25Simpson M.A. Am. J. Pathol. 2006; 169: 247-257Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Manipulations of HA levels in cells and perturbation of endogenous HA-CD44 protein interactions in animal models have directly implicated HA in tumor progression (10Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Rat colon adenocarcinomas have high levels of HA, and HA enhances colorectal tumor cell proliferation and motility in vitro and in vivo (26Kim H.R. Wheeler M.A. Wilson C.M. Iida J. Eng D. Simpson M.A. McCarthy J.B. Bullard K.M. Cancer Res. 2004; 64: 4569-4576Crossref PubMed Scopus (114) Google Scholar, 27Laurich C. Wheeler M.A. Iida J. Neudauer C.L. McCarthy J.B. Bullard K.M. J. Surg. Res. 2004; 122: 70-74Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). HA-CD44 interaction influences growth, adhesion, and invasion of colon carcinoma cells (26Kim H.R. Wheeler M.A. Wilson C.M. Iida J. Eng D. Simpson M.A. McCarthy J.B. Bullard K.M. Cancer Res. 2004; 64: 4569-4576Crossref PubMed Scopus (114) Google Scholar, 27Laurich C. Wheeler M.A. Iida J. Neudauer C.L. McCarthy J.B. Bullard K.M. J. Surg. Res. 2004; 122: 70-74Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 28Bourguignon L.Y. Gunja-Smith Z. Iida N. Zhu H.B. Young L.J. Muller W.J. Cardiff R.D. J. Cell. Physiol. 1998; 176: 206-215Crossref PubMed Scopus (245) Google Scholar). We and others have shown that interaction of HA with CD44 induces formation of complexes containing CD44 and ErbB2 or EGFR in a variety of tumor cells (28Bourguignon L.Y. Gunja-Smith Z. Iida N. Zhu H.B. Young L.J. Muller W.J. Cardiff R.D. J. Cell. Physiol. 1998; 176: 206-215Crossref PubMed Scopus (245) Google Scholar, 29Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). We have shown recently that inhibition of constitutive HA-tumor cell interactions in malignant colon and other carcinoma cells by HA oligomers (O-HA), soluble HA-binding proteins (soluble CD44), or CD44 siRNA suppresses constitutive ErbB2 or ligand-dependent activation of several receptor tyrosine kinases (RTKs), e.g. IGF1Rβ, platelet-derived growth factor receptor-β, EGFR, and c-MET (30Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). On the other hand, we showed that these RTKs are activated in phenotypically normal intestinal epithelial HIEC6 cells by experimentally increasing HA production (29Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 30Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We also showed that O-HA not only compete for interaction of endogenous HA with CD44 but also affect HA production. We conclude that HA serves a general function in RTK activation in these and other cancer cells, thus leading to increased activity of cell survival and proliferation signaling pathways and increased oncogenic properties (29Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 30Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Recent studies have demonstrated that exogenous addition of HA induces COX-2 through interaction with CD44 in endothelial cells (31Murphy J.F. Lennon F. Steele C. Kelleher D. Fitzgerald D. Long A.C. FASEB J. 2005; 19: 446-448Crossref PubMed Scopus (60) Google Scholar) and that COX-2 overexpression is a proximal mediator of CD44-dependent invasion in human non-small cell lung cancer and human renal cell carcinoma cells (32Chen Q. Shinohara N. Abe T. Harabayashi T. Nonomura K. J. Urol. 2004; 172: 2153-2157Crossref PubMed Scopus (22) Google Scholar, 33Dohadwala M. Luo J. Zhu L. Lin Y. Dougherty G.J. Sharma S. Huang M. Pold M. Batra R.K. Dubinett S.M. J. Biol. Chem. 2001; 276: 20809-20812Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Most studies of the role of HA in the malignant properties of various cancer cells have addressed the effect of exogenously added HA rather than the function of endogenous tumor cell-produced HA (34Okada H. Yoshida J. Sokabe M. Wakabayashi T. Hagiwara M. Int. J. Cancer. 1996; 66: 255-260Crossref PubMed Scopus (78) Google Scholar, 35Bourguignon L.Y. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). However, exogenously added HA does not necessarily mimic the effects of endogenously produced HA (11Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar, 29Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 36Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1484) Google Scholar), which carries out many biological functions through interaction with its receptor CD44 (37Edward M. Br. J. Dermatol. 2001; 144: 465-470Crossref PubMed Scopus (20) Google Scholar, 38Edward M. Gillan C. Micha D. Tammi R.H. Carcinogenesis. 2005; 26: 1215-1223Crossref PubMed Scopus (54) Google Scholar, 39Tammi R. Rilla K. Pienimaki J.P. MacCallum D.K. Hogg M. Luukkonen M. Hascall V.C. Tammi M. J. Biol. Chem. 2001; 276: 35111-35122Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 40Kultti A. Rilla K. Tiihonen R. Spicer A.P. Tammi R.H. Tammi M.I. J. Biol. Chem. 2006; 281: 15821-15828Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this study, we demonstrate that increased cell-autonomous HA production in stably transfected intestinal epithelial cells is sufficient to maintain the hallmark characteristics of cancer cells, such as cell proliferation and survival, anchorage-independent growth, and induction of elements of epithelial mesenchymal transitions (EMT) (41Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar). Moreover, constitutive HA-CD44 interaction mediates an ErbB2 → PI3K/AKT → β-catenin signaling axis that induces COX-2 expression and activity in colon carcinoma cells. We also show that increased expression of HAS2 induces this pathway in phenotypically normal intestinal epithelial HIEC6 cells. By defining this HA-dependent COX-2/PGE2-associated signaling pathway, we conclude that HA may account for the "constitutively" increased pathologic expression of COX-2 in colon cancer cells. Materials—HA oligomers (O-HA) were prepared as described previously (18Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). HA polymer (200 kDa) was from ICN Biomedicals. Antibodies against human CD44 (HCAM), vimentin, cytokeratin, β-catenin, glyceraldehyde-3-phosphate dehydrogenase, and goat polyclonal antibody against COX-2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against ErbB2 and p-ErbB2 were from Upstate Biotechnology; rabbit polyclonal antibodies against AKT and p-AKT were from Cell Signaling Technology, Inc. (Danvers, MA); rabbit polyclonal antibody to β-galactosidase (ab616) was from Abcam Inc.(Cambridge, MA); antibody against β-actin, β-tubulin, COX-2 inhibitor NS398, and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium salt, and chondroitinase ABC were from Sigma. The secondary antibodies used were horseradish peroxidase-conjugated with anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG from Amersham Biosciences, and horseradish peroxidase-anti-biotin antibody was purchased from Zymed Laboratories Inc.. Biotinylated HA-binding protein, chitin oligosaccharide mixture was from North Star Bioproducts (Cape Cod, MA). Human recombinant epidermal growth factor was purchased fromR&D Systems. Enhanced chemiluminescence reagents (Western Lightning Chemiluminescence Reagent Plus) were from PerkinElmer Life Sciences. AG825 (an ErbB2 inhibitor) was from EMD Biosciences (La Jolla, CA). DMEM (high glucose), RPMI 1640 medium, Ham's F-12, penicillin/streptomycin, glutamine, and Versene were from Invitrogen. Fetal bovine serum was purchased from Atlanta Biologicals. Phosphate-buffered saline (PBS) was from Cambrex. Plasmids for dominant-negative (DN) AKT, pUSEamp myr-p110 PI3K (constitutively active (CA) PI3K), and pUSEamp plasmid were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Dominant-negative p110 PI3K was a gift from Dr. L. Cantley (Brigham and Womens Hospital, Boston) and subcloned into pUSEamp. pSV2-ErbB2 (V659E) CA-ErbB2 was a gift from Dr. J. Schlegel, Freiburg, Germany. p-IRES2-antisense (As)-COX-2 plasmid was a gift from Dr. Michael C. Archer, Toronto, Ontario, Canada. pIRES2 was from Clontech. The mouse HAS2 cDNA construct pCI-neo-HAS2 was obtained from Dr. A. Spicer (University of California, Davis). pCI-neo and pSV-β-galactosidase plasmids were from Promega. A DN mutant of AKT (T308A, S473A)-adenovirus (DNAKT-Ad) (42Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) and β-galactosidase-adenovirus (β-gal-Ad) were gifts from Dr. K. Walsh (Boston University). Unless specified, all other reagents were the highest grade from Sigma. Cell Culture—HCA7 (colony 29) human colon carcinoma cells (European Collection of Cell Cultures, UK) were maintained in DMEM, 10% fetal bovine serum, 2 mm glutamine, and 11 mg/ml pyruvate. The HIEC-6 human intestinal cell line (J.-F. Beaulieu, University of Sherbrook, Quebec, Canada) was maintained in DMEM (high glucose), 4% fetal bovine serum, 20 mm HEPES buffer (pH 7.4), 50 units/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml insulin, and 5 ng/ml human recombinant epidermal growth factor (43Perreault N. Jean-Francois B. Exp. Cell Res. 1996; 224: 354-364Crossref PubMed Scopus (137) Google Scholar), and used between the 15th and 17th passage in this study. Cell lines were grown at 37 °C in 5% CO2. Cell Proliferation Assay—Cell proliferation was determined by using cell titer 96R Aqueous One Solution cell proliferation assay kit (Promega) according to the manufacturer's instructions. Cells (5,000/well) were plated in a 96-well tissue culture plate, grown for 72 h, and assayed for cell proliferation by adding 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt, a tetrazolium compound. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (was bioreduced by the cells into a formazan product that is soluble in the medium, and the absorbance at 490 nm in 96-wells was measured by an ELISA plate reader. Anchorage-independent Growth Assay—This assay was performed in 6-well plates following the procedure as described (44Fukazawa H. Mizuno S. Uehara Y. Anal. Biochem. 1995; 228: 83-90Crossref PubMed Scopus (104) Google Scholar). Briefly, the wells were coated with 0.6% agarose in PBS. The single-cell suspension was prepared in PBS and suspended in RPMI containing 5% fetal bovine serum. A 3-ml cell suspension containing 12,000 cells was mixed with 300 μl of 3% agarose, and 1 ml was layered onto 0.6% solidified agarose and allowed to solidify. Culture medium (3 ml) was added on the top layer of each well and changed every 72 h until colonies were visible (within 7–10 days) under an inverted microscope. The colonies were stained with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium salt at 1.0 mg/ml (45Schaeffer W.I. Friend K. Cancer Lett. 1976; 1: 259-262Crossref PubMed Scopus (55) Google Scholar). The cell clusters appeared as deep brick-red against a colorless background and were counted under the microscope and photographed in a digital camera. RNA Silencing and HAS2 Adenovirus, β-Galactosidase Adenovirus, and DN-AKT Adenovirus Infection—Human CD44 siRNA (46Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) and β-catenin siRNA (47Hwang J.T. Ha J. Park O.J. Biochem. Biophys. Res. Commun. 2005; 332: 433-440Crossref PubMed Scopus (158) Google Scholar) were prepared by Dharmacon. The siRNA transfection was carried out at 100 pmol using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Cells were transfected with the siRNA in 6-well plates with cells at 70–90% confluence. The cells were then incubated at 37 °C in 5% CO2 for 24 h, replated in 150-mm dishes, and allowed to grow for 48 h in complete medium. The recombinant HAS2 adenovirus (HAS2-Ad) was produced, and β-galactosidase-adenovirus (β-gal-Ad), DN-AKT was amplified at Tufts University and used as described previously (29Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 46Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). To validate transfection efficiency of various cDNAs used in this study, we co-transfected HIEC6 cells, HIEC6-HAS2-Ad cells, and HCA7 cells with pSV-β-galactosidase plasmid. Preparation of Cell Lysates—Cells were washed twice in cold PBS after treatment, harvested with Versene, and then washed twice in cold PBS. The cell pellet was lysed in buffer containing 1% Nonidet P-40, 0.5 mm EGTA, 5 mm sodium orthovanadate, 10% (v/v) glycerol, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 50 mm HEPES (pH 7.5), and then stored as described previously (30Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Protein concentration in the cell lysate was determined by Folin's phenol reagent (48Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Treatment with ErbB2 Inhibitor AG825 and COX-2 Inhibitor NS398—For specific inhibition of ErbB2, cells were preincubated in a serum-free complete medium for 24 h and then in 10 mm NaN3 and 10 mm deoxyglucose in glucose-free RPMI medium for 30 min to slow metabolism, after which they were treated with 0.0–2.0 μm AG825 (49Osherov N. Gazit A. Gilon C. Levitzki A. J. Biol. Chem. 1993; 268: 11134-11142Abstract Full Text PDF PubMed Google Scholar) for 2 h. For selective inhibition of COX-2, cells were preincubated in a serum-free complete medium for 24 h and then treated with NS398 (5 μm) (50Crew T.E. Elder D.J. Paraskeva C. Carcinogenesis. 2000; 21: 69-77Crossref PubMed Scopus (78) Google Scholar). After treatment the cells were washed in PBS, lysed, and then immunoblotted. SDS-PAGE—Western blotting of cell lysates was carried out as described earlier (30Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Intensity of the bands was quantified by densitometry using ImageJ (NIH) program. HA Assay by ELISA—HA released in serum-free culture media collected after 24 h of incubation was assayed using an ELISA-like assay (51Gordon L.B. Harten I.A. Calabro A. Sugumaran G. Csoka A.B. Brown W.T. Hascall V. Toole B.P. Hum. Genet. 2003; 113: 178-187Crossref PubMed Google Scholar). All incubations were performed at room temperature. Briefly, 4-fold 1:2 dilutions of medium were mixed with an equal volume of biotinylated HA-binding protein (1 μg/ml stock) in a total volume of 100 μl and incubated for 1 h. Control (buffer only) and various concentrations of standard HA were incubated similarly. Each dilution was added in triplicate. The mixture (100 μl) was added to HA-coated wells in a Maxisorp plate (Nunc) and incubated 1 h. After washing, horseradish peroxidase-anti-biotin antibody at 1:5000 dilution (100 μl) was added. After 1 h, the plate was washed, and 100 μl of substrate solution (containing 7 mg of o-phenylenediamine and 5 μl of 30% hydrogen peroxide in 12 ml of citrate (0.05 m) phosphate (0.1 m) buffer (pH 5.0)) was added and incubated with shaking in the dark for 5 min. The reaction was stopped by adding 50 μl of sulfuric acid (2 m). The plate was read at 490 nm in an ELISA plate reader. The concentration of HA in the medium was calculated from a standard curve. Caspase-3 Assay—We used a caspase-3 assay kit from Biovision Research Products (Mountain View, CA). The assay was performed according to the manufacturer's instructions (18Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Cell lysates were prepared in the supplied lysis buffer, and extracts containing 200 μg of protein were incubated with DEVD-p-nitroanilide as substrate. The enzyme activity was determined by measuring the absorbance of the colored product, p-nitroanilide, at 405 nm spectrophotometrically. Preparation of Stable Transfectants—All transfections were carried out using an AMAXA Nucleofector II following instructions from the manufacturer. The HAS2 stable transfectant of HIEC6 (HIEC6-HAS2) and vector transfectant (HIEC6-v) clones were prepared using linearized pCIneo-Has2 construct and pCI-neo plasmid and selected in the presence of geneticin at 500 μg/ml (Invitrogen). Immunohistochemistry—HIEC6-v and HIEC6-HAS2 cells were plated on polylysine-coated coverslips and incubated at 37 °C. The next day, cells were fixed, permeabilized, and allowed to react with antibodies (1:1000 dilution) against vimentin, cytokeratin, and β-catenin and then counterstained with secondary antibodies conjugated with Texas Red (1:2000 dilution). After washing, the coverslips were attached to glass slides using a drop of antifade solution. The outer rim of the coverslips were closed with clear fingernail polish and stored at 4 °C until analysis. PGE2 Assay—PGE2 released in cultured media of treated and untreated HIEC6-v clones, HIEC6-HAS2 clones, and HCA7 cells was measured by competitive enzyme immunoassay (EIA kit, Cayman Chemical) following the manufacturer's instructions. Briefly, dilutions of culture media were incubated with a fixed amount of PG-acetylcholinesterase conjugate (tracer) in wells coated with PG antiserum. Because of the competition between tracer and PGE2 in the media, the amount of tracer bound to PG-antiserum is inversely proportional to the PGE2 concentration of the mixture in the well. The bound esterase activity was determined by adding Ellman's reagent containing the substrate and measuring the absorbance at 412 nm. A standard curve was prepared by using different amounts of PGE2 in medium without serum. The amount of PGE2 was calculated by comparing the absorbance value with the standard. Increased HA Production Induces Activation of Cell Survival Properties and EMT in Human Intestinal Epithelial HIEC6 Cells—Previously, we showed that increased HA production stimulates cell survival activities leading to increased drug resistance and anchorage-independent growth in breast carcinoma cells (46Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 52Misra S. Ghatak S. Zoltan-Jones A. Toole B.P. J. Biol. Chem. 2003; 278: 25285-25288Abstract Full T
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