FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2013165612> ?p ?o ?g. }

• W2013165612 endingPage "47775" @default.
• W2013165612 startingPage "47762" @default.
• W2013165612 abstract "Sulindac, a non-steroidal anti-inflammatory prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Sulindac sulfide, but not sulindac sulfone, inhibits cyclooxygenase (COX) enzyme activities, yet both derivatives have growth inhibitory effects on colon cancer cells. Microarray analysis was used to detect COX-independent effects of sulindac on gene expression in human colorectal cells. Spermidine/sperm-ine N1-acetyltransferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was induced by clinically relevant sulindac sulfone concentrations. Northern blots confirmed increased SSAT RNA levels in these colon cancer cells. Deletion analysis and mutational studies were done to map the sulindac sulfone-dependent response sequences in the SSAT 5′-flanking sequences. This led us to the identification of two peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the SSAT gene. PPRE-2, at +48 bases relative to the transcription start site, is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPARγ in the Caco-2 cells as shown by transfection and gel shift experiments. PPRE-1, at–323 bases relative to the start site, is not required for the induction of SSAT by sulindac sulfone but can be bound by both PPARδ and PPARγ. Sulindac sulfone reduced cellular polyamine contents in the absence but not in the presence of verapamil, an inhibitor of the export of monoacetyl diamines, inhibited cell proliferation and induced apoptosis. The induced apoptosis could be partially rescued by exogenous putrescine. These data suggest that apoptosis induced by sulindac sulfone is mediated, in part, by the COX-independent, PPAR-dependent transcriptional activation of SSAT, leading to reduced tissue polyamine contents in human colon cancer cells. Sulindac, a non-steroidal anti-inflammatory prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Sulindac sulfide, but not sulindac sulfone, inhibits cyclooxygenase (COX) enzyme activities, yet both derivatives have growth inhibitory effects on colon cancer cells. Microarray analysis was used to detect COX-independent effects of sulindac on gene expression in human colorectal cells. Spermidine/sperm-ine N1-acetyltransferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was induced by clinically relevant sulindac sulfone concentrations. Northern blots confirmed increased SSAT RNA levels in these colon cancer cells. Deletion analysis and mutational studies were done to map the sulindac sulfone-dependent response sequences in the SSAT 5′-flanking sequences. This led us to the identification of two peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the SSAT gene. PPRE-2, at +48 bases relative to the transcription start site, is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPARγ in the Caco-2 cells as shown by transfection and gel shift experiments. PPRE-1, at–323 bases relative to the start site, is not required for the induction of SSAT by sulindac sulfone but can be bound by both PPARδ and PPARγ. Sulindac sulfone reduced cellular polyamine contents in the absence but not in the presence of verapamil, an inhibitor of the export of monoacetyl diamines, inhibited cell proliferation and induced apoptosis. The induced apoptosis could be partially rescued by exogenous putrescine. These data suggest that apoptosis induced by sulindac sulfone is mediated, in part, by the COX-independent, PPAR-dependent transcriptional activation of SSAT, leading to reduced tissue polyamine contents in human colon cancer cells. Numerous epidemiological, animal, and in vitro studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) 1The abbreviations used are: NSAIDnon-steroidal anti-inflammatory drugCOXcyclooxygenaseSSATspermidine/spermine N1-acetyltransferaseODCornithine decarboxylasePPARperoxisome proliferator-activated receptorPPREPPAR response elementRXRretinoid X receptorPGprostaglandinGAPDHglyceraldehyde-3-phosphate dehydrogenasecPGIcarbaprostacyclin.1The abbreviations used are: NSAIDnon-steroidal anti-inflammatory drugCOXcyclooxygenaseSSATspermidine/spermine N1-acetyltransferaseODCornithine decarboxylasePPARperoxisome proliferator-activated receptorPPREPPAR response elementRXRretinoid X receptorPGprostaglandinGAPDHglyceraldehyde-3-phosphate dehydrogenasecPGIcarbaprostacyclin. have antitumorigenic activities against colorectal cancer (1Patten E.J. DeLong M.J. Cancer Lett. 1999; 147: 95-100Crossref PubMed Scopus (16) Google Scholar, 2Newmark H.L. Bertagnolli M.M. Gastroenterology. 1998; 115: 1036Abstract Full Text Full Text PDF PubMed Google Scholar, 3Gupta R.A. DuBois R.N. Gastroenterology. 1998; 114: 1095-1098Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 4Taketo M.M. J. Natl. Cancer Inst. 1998; 90: 1529-1536Crossref PubMed Scopus (492) Google Scholar). Sulindac, an NSAID, inhibits colorectal carcinogenesis in rodent models (5Rao C.V. Rivenson A. Simi B. Zang E. Kelloff G. Steele V. Reddy B.S. Cancer Res. 1995; 55: 1464-1472PubMed Google Scholar, 6Boolbol S.K. Dannenberg A.J. Chadburn A. Martucci C. Guo X.J. Ramonetti J.T. Abreu-Goris M. Newmark H.L. Lipkin M.L. DeCosse J.J. Bertagnolli M.M. Cancer Res. 1996; 56: 2556-2560PubMed Google Scholar) and causes regression of adenomas (7Giardiello F.M. Hamilton S.R. Krush A.J. Piantadosi S. Hylind L.M. Celano P. Booker S.V. Robinson C.R. Offerhaus G.J. N. Engl. J. Med. 1993; 328: 1313-1316Crossref PubMed Scopus (1540) Google Scholar, 8Cruz-Correa M. Hylind L.M. Romans K.E. Booker S.V. Giardiello F.M. Gastroenterology. 2002; 122: 641-645Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) in patients with familial adenomatous polyposis coli. NSAIDs work by inhibiting cyclooxygenases (COXs) of which there are at least two distinct forms, COX-1 and COX-2. Physiologically sulindac is metabolized into sulfide- or sulfone-containing derivatives. The sulfide derivative inhibits colon carcinogenesis by inhibiting COX-1 and COX-2 enzyme activities (9Goldberg Y. Nassif I.I. Pittas A. Tsai L.L. Dynlacht B.D. Rigas B. Shiff S.J. Oncogene. 1996; 12: 893-901PubMed Google Scholar). However, sulindac sulfone also inhibits chemical carcinogenesis in rodents but by a mechanism that cannot be explained solely by the inhibition of prostaglandin synthesis (10Hanif 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 (593) Google Scholar, 11Chiu C.H. McEntee M.F. Whelan J. Cancer Res. 1997; 57: 4267-4273PubMed Google Scholar), yet both derivatives inhibit growth and induce apoptosis in a variety of human tumor-derived cell lines (12Piazza 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, 13Lim J.T. Piazza G.A. Han E.K. Delohery T.M. Li H. Finn T.S. Buttyan R. Yamamoto H. Sperl G.J. Brendel K. Gross P.H. Pamukcu R. Weinstein I.B. Biochem. Pharmacol. 1999; 58: 1097-1107Crossref PubMed Scopus (189) Google Scholar). Sulindac sulfone, at clinically relevant concentrations ranging from 35 μm (in humans) to around 150 μm (in mice), has been shown to have chemopreventive effects on colon cancer (12Piazza 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, 14Stoner G.D. Budd G.T. Ganapathi R. DeYoung B. Kresty L.A. Nitert M. Fryer B. Church J.M. Provencher K. Pamukcu R. Piazza G. Hawk E. Kelloff G. Elson P. van Stolk R.U. Adv. Exp. Med. Biol. 1999; 470: 45-53Crossref PubMed Google Scholar, 15van Stolk R. Stoner G. Hayton W.L. Chan K. DeYoung B. Kresty L. Kemmenoe B.H. Elson P. Rybicki L. Church J. Provencher K. McLain D. Hawk E. Fryer B. Kelloff G. Ganapathi R. Budd G.T. Clin. Cancer Res. 2000; 6: 78-89PubMed Google Scholar, 16Charalambous D. O'Brien P.E. J. Gastroenterol. Hepatol. 1996; 11: 307-310Crossref PubMed Scopus (38) Google Scholar). non-steroidal anti-inflammatory drug cyclooxygenase spermidine/spermine N1-acetyltransferase ornithine decarboxylase peroxisome proliferator-activated receptor PPAR response element retinoid X receptor prostaglandin glyceraldehyde-3-phosphate dehydrogenase carbaprostacyclin. non-steroidal anti-inflammatory drug cyclooxygenase spermidine/spermine N1-acetyltransferase ornithine decarboxylase peroxisome proliferator-activated receptor PPAR response element retinoid X receptor prostaglandin glyceraldehyde-3-phosphate dehydrogenase carbaprostacyclin. One of the COX-independent mechanisms of action of sulindac and its metabolites is to act as ligands for peroxisome proliferator-activated receptors (PPARs). PPARs are nuclear hormone receptors that bind to sequence-specific DNA response elements known as PPREs as a heterodimer with RXRα and can regulate gene expression. There are three PPAR isotypes, α, γ, and δ, present in humans. There is evidence that arachidonic acid metabolites like 15-deoxy-Δ12,14-PGJ2 can serve as activating ligands for PPARs (17Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2701) Google Scholar, 18Gupta R.A. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13275-13280Crossref PubMed Scopus (356) Google Scholar). Further PPARγ can act as a potential tumor suppressor (19Kohno H. Yoshitani S. Takashima S. Okumura A. Hosokawa M. Yamaguchi N. Tanaka T. Jpn. J. Cancer Res. 2001; 92: 396-403Crossref PubMed Scopus (56) Google Scholar, 20Sarraf P. Mueller E. Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (922) Google Scholar, 21DuBois R.N. Gupta R. Brockman J. Reddy B.S. Krakow S.L. Lazar M.A. Carcinogenesis. 1998; 19: 49-53Crossref PubMed Scopus (234) Google Scholar), while PPARδ can act as a potential oncogene (22He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar, 23Park B.H. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2598-2603Crossref PubMed Scopus (223) Google Scholar) in colon cancer. Sulindac can bind PPARγ and PPARδ as their ligands and lead to their activation, which can have either a positive or a negative effect on gene transcription (22He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar, 24Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. J. Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). NSAIDs, like piroxicam, aspirin, and indomethacin, have been shown to exert their chemopreventive action by affecting the polyamine metabolism in colorectal cancer (25Turchanowa L. Dauletbaev N. Milovic V. Stein J. Eur. J. Clin. Investig. 2001; 31: 887-893Crossref PubMed Scopus (38) Google Scholar, 26Carbone P.P. Douglas J.A. Larson P.O. Verma A.K. Blair I.A. Pomplun M. Tutsch K.D. Cancer Epidemiol. Biomark. Prev. 1998; 7: 907-912PubMed Google Scholar, 27Martinez M.E. O'Brien T.G. Fultz K.E. Babbar N. Yerushalmi H. Qu N. Guo Y. Boorman D. Einspahr J. Alberts D.S. Gerner E.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7859-7864Crossref PubMed Scopus (149) Google Scholar). The polyamines putrescine, spermidine, and spermine are abundant polycations in eukaryotic cells that are often elevated in neoplastic cells when compared with normal cells and tissues (28Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar). The polyamine levels are tightly regulated by the biosynthetic enzyme ornithine decarboxylase (ODC) and the catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in cells. High levels of polyamines lead to rapid proliferation (29Meyskens Jr., F.L. Gerner E.W. J. Cell Biochem. Suppl. 1995; 22: 126-131Crossref PubMed Scopus (39) Google Scholar), while lower levels of polyamines have been shown to promote apoptosis (30Scorcioni F. Corti A. Davalli P. Astancolle S. Bettuzzi S. Biochem. J. 2001; 354: 217-223Crossref PubMed Scopus (35) Google Scholar, 31Li L. Rao J.N. Bass B.L. Wang J.Y. Am. J. Physiol. 2001; 280: G992-G1004Crossref PubMed Google Scholar) and inhibit cell growth (32Vujcic S. Halmekyto M. Diegelman P. Gan G. Kramer D.L. Janne J. Porter C.W. J. Biol. Chem. 2000; 275: 38319-38328Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Because of the effects of the polyamines on apoptosis and proliferation, regulation of the expression of these enzymes has been an area of intense research (33Wang Y. Xiao L. Thiagalingam A. Nelkin B.D. Casero Jr., R.A. J. Biol. Chem. 1998; 273: 34623-34630Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 34Wang Y. Devereux W. Stewart T.M. Casero Jr., R.A. J. Biol. Chem. 1999; 274: 22095-22101Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 35Wang Y. Devereux W. Stewart T.M. Casero Jr., R.A. Biochem. J. 2001; 355: 45-49Crossref PubMed Scopus (56) Google Scholar, 36Farriol M. Segovia-Silvestre T. Castellanos J.M. Venereo Y. Orta X. Nutrition. 2001; 17: 934-938Crossref PubMed Scopus (24) Google Scholar, 37Milovic V. Stein J. Odera G. Gilani S. Murphy G.M. Cancer Lett. 2000; 154: 195-200Crossref PubMed Scopus (21) Google Scholar, 38Celano P. Berchtold C.M. Giardiello F.M. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 1989; 165: 384-390Crossref PubMed Scopus (39) Google Scholar). The effects of one of the NSAIDs, indomethacin, on polyamine metabolism involve suppression of ODC and induction of SSAT, thereby decreasing polyamine pools in colon cancer cells (25Turchanowa L. Dauletbaev N. Milovic V. Stein J. Eur. J. Clin. Investig. 2001; 31: 887-893Crossref PubMed Scopus (38) Google Scholar). However, it is not known how NSAIDs induce SSAT nor has a role for polyamines in NSAID actions, such as induction of apoptosis, been investigated. In this study, we investigated the effects of sulindac sulfone on SSAT and polyamine metabolism and asked whether polyamines were involved in sulindac sulfone-induced apoptosis of colon cancer cells. All cell culture reagents, DNA-modifying enzymes, TRIzol® reagent (Total RNA Isolation reagent), and LipofectAMINE reagent were purchased from Invitrogen. Ciglitazone and Wy-14643 were purchased from Biomol Research Laboratories. GW9662 and cPGI was purchased from Cayman Chemical. Sulindac sulfone was purchased from ICN Biomedicals, Inc. The anti-PPAR antibody PA3-820 was purchased from Affinity Bioreagents, Inc. Verapamil was purchased from Sigma. Expression plasmids pSG5-xPPARγ, pSG5-xPPARδ, and pSG5-xPPARα were generously given by Dr. Liliane Michalik (Institute de Biologie Animale, Lausanne, Switzerland). The expression plasmid pCMX-hRXRαKpn was kindly provided by Dr. Ronald Evans (The Salk Institute for Biological Studies, La Jolla, CA). Full-SSAT-luc, having a 3.493-kb-long 5′-flanking sequence of the human SSAT gene was cloned into a promoterless pGL2-basic vector (Promega, Madison, WI) as previously reported (39Xiao L. Celano P. Mank A.R. Griffin C. Jabs E.W. Hawkins A.L. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 1992; 187: 1493-1502Crossref PubMed Scopus (43) Google Scholar). 197-SSAT-luc, having 283 nucleotides of the 5′-flanking region of SSAT promoter, was made from Full-SSAT-luc using polymerase chain reaction and subcloned into pGL2-basic vector. PPRE3-tk-Luc reporter construct, having three tandem repeats of PPRE 5′ to the luciferase gene, was a gift from Dr. Ronald Evans. 48ppre-SSAT-luc was made by cloning 100 nucleotides of the 5′-flanking region of SSAT promoter into pGL2-basic vector. Δ48ppre-SSAT-luc has the PPRE at +48 replaced by a random sequence from the SSAT 5′ promoter region, which has no putative transcription factor binding sites as determined by TRANSFAC data base analysis. The Caco-2 cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA) at passage 12 and was maintained in minimum essential α-medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. The human colon cancer cell line HCT-116 was maintained as a monolayer culture in McCoy's 5A medium supplemented with 10% fetal bovine serum plus 1% penicillin/streptomycin solution. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. All cell culture supplies were from Invitrogen. Transient transfections were performed using LipofectAMINE reagent according to the manufacturer's protocol. Briefly, 5 × 105 cells were seeded in a 6-well plate and cultured in normal medium for 24 h. Each well was transfected with 1 μg of firefly luciferase reporter construct along with 0.2 μg of pCMV-β-galactosidase expression plasmid, which acted as a transfection efficiency control. After 6 h of incubation with LipofectAMINE-DNA complex, cells were supplemented with complete medium having 20% fetal bovine serum and 2% penicillin/streptomycin solution and grown overnight. Afterward the medium was removed, and cells were refed with medium along with various concentrations of sulindac sulfone or its vehicle, dimethyl sulfoxide (Me2SO), for 48 h. For PPAR studies, triple transient transfections were performed using the same protocol as above. Each well was transfected with 1 μg of firefly luciferase reporter construct with or without 0.5 μg of expression plasmid for xPPARγ, xPPARδ, or xPPARα and hRXRα. 0.2 μg of pCMV-β-galactosidase expression plasmid was cotransfected into each well for transfection efficiency. After 6 h of incubation with LipofectAMINE-DNA complex, cells were supplemented with complete medium having 20% fetal bovine serum and 2% penicillin/streptomycin solution and grown overnight. On the following day, the medium was removed, and cells were refed with medium along with the appropriate PPAR activator or its vehicle, Me2SO, for 48 h. All transfections were performed in triplicates unless stated differently. All transfected cells were washed once with phosphate-buffered saline and lysed, and luciferase activities were measured using 10 μl of cell extract and 50 μl of luciferase reagent (Promega). β-Galactosidase activity was measured using the β-galactosidase assay kit (Invitrogen) according to the manufacturer's protocol. Probe Preparation—The microarray chip was made as described in detail before (40Watts G.S. Futscher B.W. Isett R. Gleason-Guzman M. Kunkel M.W. Salmon S.E. J. Pharmacol. Exp. Ther. 2001; 299: 434-441PubMed Google Scholar). The chip has ∼5,300 human genes; >3,000 are known genes, and the remainder are expressed sequence tags as determined by UniGene. A list of the clones on the arrays is available upon request. Target Preparation—Microarray analysis was done as described before (41Crowley-Weber C.L. Payne C.M. Gleason-Guzman M. Watts G.S. Futscher B. Waltmire C.N. Crowley C. Dvorakova K. Bernstein C. Craven M. Garewal H. Bernstein H. Carcinogenesis. 2002; 23: 2063-2080Crossref PubMed Scopus (78) Google Scholar). In short, the RNeasy total RNA kit (Qiagen, Valencia, CA) and protocol was used to isolate total RNA from the Caco-2 cells treated with either vehicle or sulindac sulfone. Fluorescent first strand cDNA was made using the Micromax Direct cDNA microarray system (PerkinElmer Life Sciences) following the manufacturer's protocols. cDNA from sulindac sulfone-treated cells were Cy5 (Amersham Biosciences)-labeled, while vehicle-treated cells were Cy3 (Amersham Biosciences)-labeled. Labeled cDNA from two reactions (one Cy3-labeled and one Cy5-labeled) was combined and purified on a Microcon-50 column, lyophilized, resuspended in hybridization buffer (2× SSC, 0.1% SDS, 100 ng/μl Cot1 DNA, 100 ng/μl oligo(dA)), denatured by boiling for 2.5 min, and added to a denatured (2-min boil, slide in double distilled water, plunge into room temperature ethanol, spin dry at 500 × g) microarray. A coverslip (22 × 22 mm) was applied, and the array was placed in a hybridization chamber (catalog number HYB-03, GeneMachines) at 62 °C for 18 h. Following hybridization, slides were washed and then scanned for Cy3 and Cy5 fluorescence using an Axon GenePix 4000 microarray reader (Axon Instruments, Foster City, CA) and quantitated using GenePix software. The analysis was done three independent times with the RNA of sulindac sulfone-treated Caco-2 cells. Total RNA was obtained from cells by extraction using TRIzol reagent and used for Northern blotting as described previously (42Taylor M.T. Lawson K.R. Ignatenko N.A. Marek S.E. Stringer D.E. Skovan B.A. Gerner E.W. Cancer Res. 2000; 60: 6607-6610PubMed Google Scholar, 43Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). Membranes were hybridized with 32P-labeled cDNA encoding for human SSAT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as the ratio of the integrated densities of 32P-labeled hybridization bands for the SSAT and GAPDH genes. cDNAs for xPPARα, xPPARγ, xPPARδ, and hRXRα were transcribed and translated in vitro from the pSG5-xPPARα, pSG5-xPPARγ, pSG5-xPPARδ, and pCMX-hRXRαKpn plasmids, respectively. The TnT coupled reticulocyte lysate system (Promega) was used according to the manufacturer's instructions. Translation products were verified by SDS-polyacrylamide gel electrophoresis. Nuclear extracts were prepared from Caco-2 cells essentially as described previously (44Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). To study the binding of nuclear hormone receptors to the putative PPRE, two double-stranded oligonucleotides, PPRE-2 and PPRE-1, spanning nucleotides +35 to +70 and –304 to –336, respectively, of the SSAT 5′ sequence, were 32P-labeled with polynucleotide kinase (Promega). A 15-μl reaction containing 0.5 ng of PPRE probe and 5 μg of nuclear extract or 0.5–1 μl of in vitro translation reaction was incubated for 20 min at 25 °C and 15 min at 4 °C in a buffer containing 20 mm HEPES (pH 8), 60 mm KCl, 1 mm dithiothreitol, 10% glycerol, and 2 μg of poly(dI-dC). The DNA-protein complexes were resolved from the free probe by electrophoresis at 4 °C on a 5% polyacrylamide gel in 1× Tris borate-EDTA buffer, pH 8. Double-stranded oligonucleotides composed of the following sequences were used for gel shift analysis: PPRE-2 (w), 5′-AGAAAAGAGCAAGGTCACTTGTCGGGGGGCTG-3′; PPRE-1 (w2), 5′-CCGTCACTCGCCGAGGTTCCTTGGGTCATGGTGCC-3′; PPRE-2mut (m), 5′-AGAAAAcAGtAAttgaACTTGgtGGGGGGCTG-3′; PPRE-1mut (m2), 5′-CGTCACTCGtttgGGTgaCGGGTCgTGGTGCC-3′. The PPRE sequence is underlined, and the mutated bases are shown in lowercase letters. Western blots were done for COX-2 as described elsewhere (42Taylor M.T. Lawson K.R. Ignatenko N.A. Marek S.E. Stringer D.E. Skovan B.A. Gerner E.W. Cancer Res. 2000; 60: 6607-6610PubMed Google Scholar). COX-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:5,000 dilutions, respectively. All Western blots were repeated three times, and a representative blot was chosen for presentation. Caco-2 cells were seeded at a concentration of 1 × 106 cells/100-mm culture plate. The cells were grown for 24 h before they were refed with a new medium and treated with sulindac sulfone, 1 mm putrescine, both, or the vehicle Me2SO. The cells were then harvested after 0, 1, 2, 4, and 6 days postdrug exposure. Cells were removed from the monolayer by treatment with trypsin (∼1,500 units/ml, Calbiochem)-EDTA (0.7 mm) and counted using a hemocytometer. A sample of the cell suspension was combined in a 1:1 volume ratio with trypan blue dye (Invitrogen), and at least two independently prepared suspensions were counted (two counts each) on a hemocytometer. Viability was determined by the percentage of cells able to exclude the trypan blue dye. Caco-2 cells were seeded at a concentration of 1 × 106 cells/100-mm culture plate. Cells were grown for 24 h before they were refed with a new media and treated with sulindac sulfone, 1 mm putrescine, both, or vehicle. The cells were then harvested after 0, 1, 2, 4, and 6 days postdrug exposure. Cells were removed from the monolayer by treatment with trypsin and counted using a hemocytometer. 5 × 105 cells were pelleted down for the apoptosis staining. The procedure for staining with the ApoAlert® Annexin V kit (Clontech) was based on the manufacturer's protocol. Briefly, the cells were resuspended in 200 μlof kit 1× binding buffer. To each tube, 5 μl of the Annexin V/fluorescein isothiocyanate binding buffer (20 μg/ml in Tris-NaCl) and 10 μl of the kit propidium iodide (50 μg/ml in 1× binding buffer) were added. Each tube was gently mixed and incubated for 15 min at room temperature in the dark. The volume was then brought up to 500 μl by adding 1× binding buffer. Cells were analyzed using a BD Biosciences FACScan flow cytometer. For enzyme activities, cells were grown overnight and then treated with various concentrations of sulindac sulfone or its vehicle. Cells were harvested after 48 h of treatment and washed in cold phosphate-buffered saline. The radiochemical assay of the SSAT activity was performed by estimation of labeled N1-acetylspermidine synthesized from [14C]acetyl-CoA and unlabeled spermidine as described elsewhere (45Ignatenko N.A. Gerner E.W. Cell Growth Differ. 1996; 7: 481-486PubMed Google Scholar). The ODC enzyme activity was measured by evaluating the release of 14CO2 from l-[14C]ornithine as described elsewhere (46Fultz K.E. Gerner E.W. Mol. Carcinog. 2002; 34: 10-18Crossref PubMed Scopus (40) Google Scholar). The -fold change was calculated by dividing the enzyme activity for the sample by the vehicle. The enzyme assays were done in triplicates. Cell extracts were prepared in 0.1 n HCl (4 × 107 cells/900 μl). After sonication, the preparation was adjusted to 0.2 n HClO4, and the supernatant was analyzed by reverse-phase high performance liquid chromatography with 1,7-diaminoheptane as an internal standard (47Seiler N. Knodgen B. J. Chromatogr. 1980; 221: 227-235Crossref PubMed Scopus (289) Google Scholar). Protein was determined by BCA assay (48Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18349) Google Scholar). All transient transfection experiments were performed in triplicates and were repeated at least three times. Cell growth assays, apoptosis assays, and Northern blots were done at least three times. Representative experiments or mean values ± S.D. are shown. Statistical differences were determined by Student's t test. A p value of <0.05 was considered significant. Sulindac Sulfone Leads to Cell Growth Inhibition and Induction of Cell Death in Caco-2 Cells—Sulindac and its derivatives have been shown to either inhibit cell proliferation or induce apoptosis in colon cancer cells (49Akashi H. Han H.J. Iizaka M. Nakamura Y. Int. J. Cancer. 2000; 88: 873-880Crossref PubMed Scopus (35) Google Scholar, 50Goluboff E.T. Shabsigh A. Saidi J.A. Weinstein I.B. Mitra N. Heitjan D. Piazza G.A. Pamukcu R. Buttyan R. Olsson C.A. Urology. 1999; 53: 440-445Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 51Rahman M.A. Dhar D.K. Masunaga R. Yamanoi A. Kohno H. Nagasue N. Cancer Res. 2000; 60: 2085-2089PubMed Google Scholar). We wanted to see whether sulindac sulfone, at doses that are clinically relevant, have any growth-suppressive effects on Caco-2 cells. We did cell counting using a hemocytometer to estimate cell proliferation and found that sulindac sulfone at concentrations of 50 μm and above have a statistically significant effect on the inhibition of cell proliferation after 6 days (Fig. 1A). By doing trypan blue cell staining, which stains for dead cells, we found that sulindac sulfone at concentrations of 150 μm and above have a significant effect on inducing cell death (Fig. 1B). Sulindac Sulfone Induces SSAT Leading to Decreased Polyamine Levels in Caco-2 Cells—DNA microarray analysis of 5,300 genes with the cDNA prepared from total RNA from the Caco-2 human colorectal cancer cells treated with 600 μm sulindac sulfone, which is the dose necessary to reduce colony formation by 50% (IC50 dose), showed altered expression of several genes compared with the control-treated Caco-2 cells. One of these genes was SSAT whose expression was induced 3.94 ± 0.64-fold in treated Caco-2 cells as compared with the controls (p < 0.06). The induction in SSAT mRNA expression was confirmed by Northern analysis (Fig. 2A). We next wanted to determine whether clinically relevant concentrations of sulindac sulfone have any effect on SSAT expression. We found that sulindac sulfone at concentrations of 100 μm and above led to an induction in SSAT mRNA (data not shown). To determine whether this induction of SSAT expression is at the level of SSAT transcription, we did transient transfection experiments using the Full-SSAT-luc reporter promoter construct, which has 3.53 kb of the SSAT 5′ promoter flanking region in front of the luciferase gene, and then treated the cells with various concentrations of sulindac sulfone. Sulindac sulfone at concentrations of 100 μm or greater led to an induction in the Full-SSAT-luc promoter activity in the Caco-2 cells after 48 h of incubation (Fig. 2B). These data suggest that sulindac sulfone increases the SSAT gene expression at the level of transcription. Next we wanted to see whether this induction in SSAT promoter activity and SSAT RNA affects t" @default.
• W2013165612 created "2016-06-24" @default.
• W2013165612 creator A5016178832 @default.
• W2013165612 creator A5034763642 @default.
• W2013165612 creator A5076964907 @default.
• W2013165612 creator A5090466629 @default.
• W2013165612 date "2003-11-01" @default.
• W2013165612 modified "2023-10-07" @default.
• W2013165612 title "Cyclooxygenase-independent Induction of Apoptosis by Sulindac Sulfone Is Mediated by Polyamines in Colon Cancer" @default.
• W2013165612 cites W1580875936 @default.
• W2013165612 cites W1964288594 @default.
• W2013165612 cites W1965844660 @default.
• W2013165612 cites W1979201752 @default.
• W2013165612 cites W1980124508 @default.
• W2013165612 cites W1980235624 @default.
• W2013165612 cites W1984029898 @default.
• W2013165612 cites W1988146667 @default.
• W2013165612 cites W1993243592 @default.
• W2013165612 cites W1999364273 @default.
• W2013165612 cites W2005674716 @default.
• W2013165612 cites W2007454357 @default.
• W2013165612 cites W2013679434 @default.
• W2013165612 cites W2014769149 @default.
• W2013165612 cites W2018425946 @default.
• W2013165612 cites W2019390040 @default.
• W2013165612 cites W2021797210 @default.
• W2013165612 cites W2026307857 @default.
• W2013165612 cites W2029758835 @default.
• W2013165612 cites W2052555886 @default.
• W2013165612 cites W2052653126 @default.
• W2013165612 cites W2063200033 @default.
• W2013165612 cites W2069550275 @default.
• W2013165612 cites W2087224686 @default.
• W2013165612 cites W2088404163 @default.
• W2013165612 cites W2091321756 @default.
• W2013165612 cites W2105847503 @default.
• W2013165612 cites W2111929519 @default.
• W2013165612 cites W2114597279 @default.
• W2013165612 cites W2114864434 @default.
• W2013165612 cites W2117384086 @default.
• W2013165612 cites W2117580825 @default.
• W2013165612 cites W2129251988 @default.
• W2013165612 cites W2131840121 @default.
• W2013165612 cites W2167740392 @default.
• W2013165612 cites W2168972147 @default.
• W2013165612 cites W2209336445 @default.
• W2013165612 cites W2336215030 @default.
• W2013165612 cites W4232534952 @default.
• W2013165612 cites W4248767187 @default.
• W2013165612 cites W4251194026 @default.
• W2013165612 cites W4294216491 @default.
• W2013165612 cites W78238624 @default.
• W2013165612 doi "https://doi.org/10.1074/jbc.m307265200" @default.
• W2013165612 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14506281" @default.
• W2013165612 hasPublicationYear "2003" @default.
• W2013165612 type Work @default.
• W2013165612 sameAs 2013165612 @default.
• W2013165612 citedByCount "132" @default.
• W2013165612 countsByYear W20131656122012 @default.
• W2013165612 countsByYear W20131656122013 @default.
• W2013165612 countsByYear W20131656122014 @default.
• W2013165612 countsByYear W20131656122015 @default.
• W2013165612 countsByYear W20131656122016 @default.
• W2013165612 countsByYear W20131656122017 @default.
• W2013165612 countsByYear W20131656122018 @default.
• W2013165612 countsByYear W20131656122019 @default.
• W2013165612 countsByYear W20131656122020 @default.
• W2013165612 countsByYear W20131656122021 @default.
• W2013165612 countsByYear W20131656122022 @default.
• W2013165612 countsByYear W20131656122023 @default.
• W2013165612 crossrefType "journal-article" @default.
• W2013165612 hasAuthorship W2013165612A5016178832 @default.
• W2013165612 hasAuthorship W2013165612A5034763642 @default.
• W2013165612 hasAuthorship W2013165612A5076964907 @default.
• W2013165612 hasAuthorship W2013165612A5090466629 @default.
• W2013165612 hasBestOaLocation W20131656121 @default.
• W2013165612 hasConcept C121608353 @default.
• W2013165612 hasConcept C126322002 @default.
• W2013165612 hasConcept C181199279 @default.
• W2013165612 hasConcept C185592680 @default.
• W2013165612 hasConcept C188027245 @default.
• W2013165612 hasConcept C190283241 @default.
• W2013165612 hasConcept C2776241388 @default.
• W2013165612 hasConcept C2778484676 @default.
• W2013165612 hasConcept C2778806918 @default.
• W2013165612 hasConcept C2779689624 @default.
• W2013165612 hasConcept C502942594 @default.
• W2013165612 hasConcept C526805850 @default.
• W2013165612 hasConcept C55493867 @default.
• W2013165612 hasConcept C71924100 @default.
• W2013165612 hasConcept C98274493 @default.
• W2013165612 hasConceptScore W2013165612C121608353 @default.
• W2013165612 hasConceptScore W2013165612C126322002 @default.
• W2013165612 hasConceptScore W2013165612C181199279 @default.
• W2013165612 hasConceptScore W2013165612C185592680 @default.
• W2013165612 hasConceptScore W2013165612C188027245 @default.
• W2013165612 hasConceptScore W2013165612C190283241 @default.
• W2013165612 hasConceptScore W2013165612C2776241388 @default.

• next