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G a l e c t i n - 3 , a - g a l a c t o s i d e b i n d i n g l e c t i n , a c t s a s a s t r o n g p r o - i n f l a m m a t o r y s i g n a l . M a n y i m m u n o m o d u l a t o r y d r u g s a f f e c t s i g n a l i n g p a t h w a y s t h a t c o m p r i s e t r a n s c r i p t i o n f a c t o r s i n v o l v e d i n regulation of galectin-3 gene (LGALS3) expression. The aim of this study was to investigate the effects of non-steroidal anti-inflammatory drugs aspirin and indomethacin on the expression of galectin-3, both on mRNA and protein levels. The human monocytic cell line THP-1 was exposed to various therapeutic concentrations of aspirin and indomethacin for 72 hours. Relative RT-PCR method and the GeneScan analysis software were used for assessing the galectin-3 mRNA level and chemiluminescent-western blot analysis was used for measuring the galectin-3 level. The results showed that galectin-3 is a new target molecule of NSAIDs, which cause reduction of both gene and protein expression of galectin-3, but the intensity and time-course of the changes strongly depend on the kind and concentration of the drugs.
Phone: +385 1 4818 757; Fax: +385 1 4856 201; E-mail: sanjad(pharma.hr
Keywords: aspirin, galectin-3, indomethacin, LGALS3
*Author to whom all correspondence should be addressed. (E-mail: sanjad(pharma.hr)
K e y w o r d s :
a s p i r i n , g a l e c t i n - 3 , i n d o m e t h a c i n , L G A L S 3
A b s t r a c t
G a l e c t i n - 3 , a - g a l a c t o s i d e b i n d i n g l e c t i n , a c t s a s a s t r o n g p r o - i n f l a m m a t o r y s i g n a l . M a n y i m m u n o m o d u l a t o r y d r u g s a f f e c t s i g n a l i n g p a t h w a y s t h a t c o m p r i s e t r a n s c r i p t i o n f a c t o r s i n v o l v e d i n r egulation of galectin-3 gene (LGALS3) expression. The aim of our study was to investigate the effects of non-steroidal anti-inflammatory drugs aspirin and indomethacin on the expression of galectin-3, both on mRNA and protein level. The human monocytic cell line THP-1 was exposed to various therapeutic concentrations of aspirin and indomethacin during 72 hours. Relative RT-PCR method and GeneScan analysis software were used for assessing galectin-3 mRNA level and chemiluminescent-western blot analysis was used for measuring galectin-3 level. The results showed that galectin-3 is a new target molecule of NSAIDs, that cause reduction of both gene and protein expression of galectin-3, but the intensity and time-course of the changes strongly depend on the kind and the concentration of the drugs.
INTRODUCTION
Galectin-3 is a (32 kDa (-galactoside-binding lectin found widely in epithelial, ADDIN EN.CITE Hikita20006101112143815162000Dec 11Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 31235-46Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.Hikita, C.Vijayakumar, S.Takito, J.Erdjument-Bromage, H.Tempst, P.Al-Awqati, Q.J Cell BiolAnimalsAntigens, Differentiation/*metabolismCell DifferentiationCell PolarityColon/cytologyEpithelial Cells/*cytology/metabolismExtracellular Matrix/*metabolismExtracellular Matrix Proteins/*metabolismGalectin 3Protein BindingRatsReceptors, Cell SurfaceReceptors, Immunologic/*metabolismSignal TransductionSupport, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=111214381 immune ADDIN EN.CITE Ochieng200433014758076197-92004Extracellular functions of galectin-3527-35Department of Biochemistry, Meharry Medical College, Nashville, TN 37208, USA. jochieng@mmc.eduOchieng, J.Furtak, V.Lukyanov, P.Glycoconj Jhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=147580762 and tumor cells. ADDIN EN.CITE Bresalier1996670881312356191996Oct 1Colon cancer mucin: a new ligand for the beta-galactoside-binding protein galectin-34354-7Department of Medicine, Henry Ford Health Sciences Center, Detroit, Michigan 48202, USA.Bresalier, R. S.Byrd, J. C.Wang, L.Raz, A.Cancer ResAdenocarcinoma/*metabolism/pathologyAntigens, Differentiation/genetics/*metabolismCarbohydrate SequenceColonic Neoplasms/*metabolism/pathologyComparative StudyGalactosides/*metabolismGalectin 3GlycosylationHumanLectins/genetics/*metabolismLigandsMolecular Sequence DataMucins/chemistry/*metabolismNeoplasm MetastasisNeoplasm Proteins/chemistry/genetics/*metabolismPolysaccharides/*metabolismRecombinant Proteins/metabolismSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.Tumor Cells, Culturedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=88131233 Galectin-3 can be found in the cytoplasm, nucleus, as well as on the cell surface. Inside the cell, galectin-3 can act as a factor in pre-mRNA splicing ADDIN EN.CITE Wang200416501523825116731-22004Jul 6Nucleocytoplasmic lectins75-93Department of Biochemistry and Molecular Biology, Michigan State University, 501 Biochemistry Building, East Lansing, MI 48824, USA. wangj@msu.eduWang, J. L.Gray, R. M.Haudek, K. C.Patterson, R. J.Biochim Biophys ActaAnimalsAnnexins/metabolismCell Nucleus/*metabolismCytoplasm/*metabolismGalectins/metabolismHumanLectins/*metabolismLigandsSupport, Non-U.S. Gov'tSupport, U.S. Gov't, Non-P.H.S.Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=152382514 and as a regulator of the cell cycle ADDIN EN.CITE Kim1999174414801046362159161999Aug 15Cell cycle arrest and inhibition of anoikis by galectin-3 in human breast epithelial cells4148-54Department of Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA. hrckim@med.wayne.eduKim, H. R.Lin, H. M.Biliran, H.Raz, A.Cancer ResAntigens, Differentiation/*pharmacologyApoptosis/*drug effectsBreast/*pathologyCell Adhesion/drug effectsCell Cycle/*drug effectsCell LineEpithelial Cells/*drug effects/pathologyFemaleGalectin 3HumanSupport, U.S. Gov't, Non-P.H.S.Support, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=104636215 and apoptosis. ADDIN EN.CITE Akahani1997370939374857231997Dec 1Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family5272-6Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Detroit, Michigan 48201, USA.Akahani, S.Nangia-Makker, P.Inohara, H.Kim, H. R.Raz, A.Cancer ResAmino Acid SequenceAntigens, Differentiation/*biosynthesis*Apoptosis/drug effectsBreast NeoplasmsCell SurvivalCisplatin/pharmacologyConserved SequenceDNA FragmentationFemaleGalectin 3HumanKineticsMembrane Glycoproteins/biosynthesisMolecular Sequence DataPoly(ADP-ribose) Polymerases/metabolismProto-Oncogene Proteins/biosynthesis/chemistryProto-Oncogene Proteins c-bcl-2/*biosynthesis/chemistrySequence AlignmentSequence Homology, Amino AcidSupport, Non-U.S. Gov'tSupport, U.S. Gov't, Non-P.H.S.Support, U.S. Gov't, P.H.S.Tumor Cells, Culturedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=93937486 However, the precise mechanisms of these actions are still elusive. Galectin-3, secreted in the extracellular space by an unknown mechanism, can bind numerous cell-surface or extracellular matrix ligands and consequently mediate important biological processes such as cell-cell and cell-matrix adhesion ADDIN EN.CITE Hughes2001120115223968372001JulGalectins as modulators of cell adhesion667-76National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. chughes@nimr.mrc.ac.ukHughes, R. C.BiochimieAnimalsCell Adhesion/*physiologyCell Adhesion Molecules/*genetics/physiologyCell CommunicationExtracellular Matrix/physiologyGalectinsGlycoproteins/physiologyHemagglutinins/chemistry/classification/*physiologyHumanMiceMice, KnockoutModels, BiologicalModels, Molecularhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=115223967 and cell signaling. ADDIN EN.CITE Ochieng200433014758076197-92004Extracellular functions of galectin-3527-35Department of Biochemistry, Meharry Medical College, Nashville, TN 37208, USA. jochieng@mmc.eduOchieng, J.Furtak, V.Lukyanov, P.Glycoconj Jhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=147580762 Upon the binding to the specific structures of surface glycoconjugates, galectin-3 molecules are able to oligomerize through their N-terminal domains and cause cross-linking of their surface ligands. ADDIN EN.CITE Barboni1999340106197091671999JulKinetic measurements of binding of galectin 3 to a laminin substratum365-73National Institute for Medical Research, Mill Hill, London, UK.Barboni, E. A.Bawumia, S.Hughes, R. C.Glycoconj JAmino Acid SequenceAnimalsAntigens, Differentiation/*chemistry/geneticsCattleGalectin 3HamstersHumanKineticsLaminin/*chemistryMembrane Glycoproteins/*chemistryMiceMolecular Sequence DataMutationProtein BindingProtein Structure, TertiarySupport, Non-U.S. Gov'tSurface Plasmon Resonancehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=106197098 It has been suggested that through such cross-linking galectin-3 initiates signal transduction cascades (involved in several immune responses), thereby triggering i) an oxidative burst in monocytes and neutrophils; ADDIN EN.CITE Yamaoka1995800789722815471995Apr 1A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils3479-87Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.Yamaoka, A.Kuwabara, I.Frigeri, L. G.Liu, F. T.J ImmunolAntigens, Differentiation/*physiologyCarbohydrates/physiologyChromatography, AffinityElectrophoresis, Polyacrylamide GelGalectin 3HumanImmunoblottingIn VitroLectins/*physiologyNeutrophils/*metabolismProtein Binding/physiologyStructure-Activity RelationshipSuperoxides/*metabolismSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7897228Karlsson199893095584029191998May 1Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils3430-8The Phagocyte Research Laboratory, the Department of Medical Microbiology and Immunology, University of Goteborg, Guldhedsgatan 10, S-413 46 Goteborg, Sweden.Karlsson, A.Follin, P.Leffler, H.Dahlgren, C.BloodAlkaline Phosphatase/metabolismAntigens, Differentiation/*physiologyCells, CulturedEnzyme ActivationExudates and Transudates/cytologyGalectin 3HumanMacrophage-1 Antigen/metabolismNADPH Oxidase/*metabolismNeutrophil ActivationNeutrophils/*enzymologyReceptors, Cell Surface/metabolismReceptors, Complement 3b/metabolismRespiratory Burst/drug effectsSignal TransductionSupport, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=95584029,10 ii) augmentation of LPS-induced release of IL-1 in monocytes; ADDIN EN.CITE Jeng199446078903094231994OctAn endogenous lectin, galectin-3 (epsilon BP/Mac-2), potentiates IL-1 production by human monocytes113-6Department of Medical Research, Taichung Veterans General Hospital, Taiwan, ROC.Jeng, K. C.Frigeri, L. G.Liu, F. T.Immunol LettAntigens, Differentiation/metabolism/*physiologyGalectin 3HumanIn VitroInterleukin-1/*biosynthesisLectins/*physiologyMonocytes/*immunology/metabolismProtein Binding/physiologySupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=789030911 iii) initiation of degranulation in mast cells; ADDIN EN.CITE Frigeri1993830834757432301993Aug 3Epsilon BP, a beta-galactoside-binding animal lectin, recognizes IgE receptor (Fc epsilon RI) and activates mast cells7644-9Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.Frigeri, L. G.Zuberi, R. I.Liu, F. T.BiochemistryAnimalsAntigens, Differentiation/*metabolism/pharmacologyBinding SitesCells, CulturedCross-Linking ReagentsGalectin 3Lectins/*metabolismLeukemia, Basophilic, AcuteMast Cells/*drug effects/immunologyMembrane Glycoproteins/*metabolismPeritoneal Cavity/cytologyRatsReceptors, IgE/*metabolismSerotonin/secretionSupport, U.S. Gov't, P.H.S.Tumor Cells, Culturedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=834757412 iv) chemoattraction of monocytes and macrophages; ADDIN EN.CITE Sano20008901092530216542000Aug 15Human galectin-3 is a novel chemoattractant for monocytes and macrophages2156-64Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.Sano, H.Hsu, D. K.Yu, L.Apgar, J. R.Kuwabara, I.Yamanaka, T.Hirashima, M.Liu, F. T.J ImmunolAnimalsAntigens, Differentiation/administration & dosage/metabolism/*physiologyCalcium/metabolismCell Migration InhibitionCell Movement/immunologyCells, CulturedChemotactic Factors/administration & dosage/metabolism/*physiologyChemotaxis, Leukocyte/immunologyComparative StudyDiffusion Chambers, CultureDose-Response Relationship, ImmunologicGalectin 3HumanInjections, IntradermalIntracellular Fluid/metabolismKineticsMacrophages/*immunologyMacrophages, Alveolar/immunologyMiceMice, Inbred BALB CMonocyte Chemoattractant Protein-1/physiologyMonocytes/*immunology/metabolism/pathologyPertussis ToxinProtein Structure, TertiaryReceptors, Chemokine/physiologySignal Transduction/immunologySkin/immunology/pathologySupport, U.S. Gov't, P.H.S.Virulence Factors, Bordetella/pharmacologyhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1092530213 v) induction of leukocyte recruitment; ADDIN EN.CITE Sato200443014758083197-92004Seeing strangers or announcing "danger": galectin-3 in two models of innate immunity583-91Glycobiology Laboratory, Research Centre for Infectious Diseases Laval University, Medical Centre, Centre Hospitalier Universitaire de Quebec, Ste-Foy, Quebec, Canada. Sachiko.Sato@crchul.ulaval.caSato, S.Nieminen, J.Glycoconj Jhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1475808314 vi) selective down-regulation of IL-5 expression; ADDIN EN.CITE Cortegano2000790107045221032000MarInteraction between galectin-3 and FcgammaRII induces down-regulation of IL-5 gene: implication of the promoter sequence IL-5REIII237-42Immunology Department, Fundacion Jimenez Diaz, 28040 Madrid, SpainCortegano, I.Pozo, V.Cardaba, B.Arrieta, I.Gallardo, S.Rojo, M.Aceituno, E.Takai, T.Verbeek, S.Palomino, P.Liu, F. T.Lahoz, C.GlycobiologyAnimalsAntigens, Differentiation/metabolism/*physiologyDown-RegulationFlow CytometryGalectin 3*Gene Expression RegulationHumanIn VitroInterleukin-5/*genetics/metabolismMiceMice, Inbred C57BLOligonucleotides/metabolism*Promoter Regions (Genetics)RNA, Messenger/biosynthesis/metabolismReceptors, IgG/metabolism/*physiologyRecombinant Fusion Proteins/metabolismSupport, Non-U.S. Gov'tTranscription Factors/metabolismhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1070452215 vii) modulation of lymphocyte T activation. ADDIN EN.CITE Demetriou200110001121786440968212001Feb 8Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation733-9Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.Demetriou, M.Granovsky, M.Quaggin, S.Dennis, J. W.NatureAnimalsAntigens, Differentiation/metabolism*AutoimmunityGalectin 3GlycosylationHumanHypersensitivity, Delayed*Lymphocyte ActivationMajor Histocompatibility ComplexMiceN-Acetylglucosaminyltransferases/deficiency/*metabolismPolysaccharides/metabolismReceptors, Antigen, T-Cell/metabolismSignal TransductionSupport, Non-U.S. Gov'tT-Lymphocytes/*immunologyhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1121786416
Galectin-3 is coded by a single gene (LGALS3). The promoter region contains five Sp1 binding sites (GC boxes), which is a common feature of housekeeping genes. However, LGALS3 is considered to be an immediate early gene due to the presence of binding sites for several other transcription factors, such as AP-1, AP-4, NF-(B and CREB. ADDIN EN.CITE Kadrofske1998150943957734911998Jan 1The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter7-20Department of Biochemistry, Michigan State University, East Lansing 48824, USA.Kadrofske, M. M.Openo, K. P.Wang, J. L.Arch Biochem BiophysAntigens, Differentiation/*geneticsBase SequenceGalectin 3*Genome, HumanHumanLectins/geneticsMolecular Sequence DataPromoter Regions (Genetics)/*geneticsSequence AlignmentSequence AnalysisSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=943957717 Galectin-3 expression seems to be affected by various stimuli, ADDIN EN.CITE Dumic2000200010817217553-42000Mar-AprTransfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages261-6Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia.Dumic, J.Lauc, G.Hadzija, M.Flogel, M.Z Naturforsch [C]AnimalsAntigens, Differentiation/*metabolismCells, CulturedGalectin 3Macrophages, Peritoneal/*metabolismMaleMiceMice, Inbred BALB CRatsSubcellular Fractions/metabolismSupport, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10817217Dumic2000198010938585332000MayGalectin-3 decreases in mice exposed to immobilization stress241-6Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovacica 1, 10000 Zagreb, Croatia.Dumic, J.Barisic, K.Flogel, M.Lauc, G.StressAging/*physiologyAnimalsAntigens, Differentiation/*metabolismCell LineGalectin 3Glioblastoma/metabolism/pathologyHeat-Shock Response/physiologyImmunoblottingLiver/metabolismMacrophages, Peritoneal/metabolismMiceMice, Inbred C3HMice, Inbred StrainsRestraint, PhysicalSpleen/metabolismStress/*metabolismSupport, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1093858518,19 but the signaling pathways ruling these reactions have not been well studied.
The immune response can be modulated by the action of non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs, along with their analgesic, anti-pyretic and anti-inflammatory uses, are now being also considered for prevention of cardiovascular diseases, cancer and treatment of human immunodeficiency virus. ADDIN EN.CITE Hull20031510129275712942003AugActivity of the non-steroidal anti-inflammatory drug indomethacin against colorectal cancer309-20Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds, LS9 7TF, UK. M.A.Hull@leeds.ac.ukHull, M. A.Gardner, S. H.Hawcroft, G.Cancer Treat RevAnimalsAnti-Inflammatory Agents, Non-Steroidal/*pharmacology/therapeutic useAntineoplastic Agents/*pharmacology/therapeutic useAntineoplastic Combined Chemotherapy Protocols/pharmacologyColorectal Neoplasms/*drug therapy/metabolism/*prevention & controlCyclooxygenase Inhibitors/pharmacologyHumanIn VitroIndomethacin/*pharmacology/therapeutic useReceptors, Cytoplasmic and Nuclear/drug effects/metabolismSupport, Non-U.S. Gov'tTranscription Factors/drug effects/metabolismhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12927571Kim20021520124858699732002NovThe role of mitogen-activated protein kinases and their relationship with NF-kappaB and PPARgamma in indomethacin-Induced apoptosis of colon cancer cells241-5Department of Internal Medicine and Institute of Gastroenterology, Brain Korea 21 Project for Medical Science, College of Medicine, Yonsei University, Seoul, Korea.Kim, T. I.Jin, S. H.Kang, E. H.Shin, S. K.Choi, K. Y.Kim, W. H.Ann N Y Acad SciApoptosis/*drug effectsColonic Neoplasms/*pathologyHumanIndomethacin/*pharmacologyMAP Kinase Signaling System/drug effects/*physiologyMitogen-Activated Protein Kinases/metabolismNF-kappa B/*metabolismReceptors, Cytoplasmic and Nuclear/*metabolismSupport, Non-U.S. Gov'tTranscription Factors/*metabolismTumor Cells, Culturedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1248586920,21 Although many of the NSAIDs' pharmacological actions are related to their ability to inhibit prostaglandin biosynthesis, some of their beneficial therapeutic effects are not completely understood. These effects are mediated through inhibition of certain transcription factors (such as NF-(B and AP-1) and modulation of the transcriptional activation. ADDIN EN.CITE Lewis1999134010419850341999AugNew targets for anti-inflammatory drugs489-94Signal Pharmaceuticals Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA. alewis@signalpharm.comLewis, A. J.Manning, A. M.Curr Opin Chem BiolAnimalsAnti-Inflammatory Agents, Non-Steroidal/*pharmacologyCa(2+)-Calmodulin Dependent Protein Kinase/drug effectsCytokines/*drug effectsHumanNF-kappa B/drug effects/metabolismTranscription Factors/*drug effectshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10419850Tegeder200115401164123315122001OctCyclooxygenase-independent actions of cyclooxygenase inhibitors2057-72Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt, 60590 Frankfurt am Main, Germany.Tegeder, I.Pfeilschifter, J.Geisslinger, G.Faseb JAnimalsAnti-Inflammatory Agents, Non-Steroidal/pharmacologyCyclooxygenase Inhibitors/*pharmacologyDNA-Binding Proteins/antagonists & inhibitorsGrowth Substances/biosynthesisHeat-Shock Response/drug effectsIsoenzymes/metabolismMAP Kinase Signaling System/drug effectsMiceModels, BiologicalNF-kappa B/antagonists & inhibitorsPregnancy Proteins/biosynthesisProstaglandin-Endoperoxide Synthase/metabolismReceptors, Cytoplasmic and Nuclear/antagonists & inhibitorsRibosomal Protein S6 Kinases/antagonists & inhibitorsSupport, Non-U.S. Gov'tTrans-Activators/antagonists & inhibitorsTranscription Factor AP-1/antagonists & inhibitorshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1164123322,23 Differences in the cyclooxygenase-independent mechanism may result in a specific use of these drugs in individual therapy, because additional effects may either enhance the efficacy or reduce the toxicity of particular compounds. Since NSAIDs may affect transcription factors NF-(B and AP-1, which were shown to be involved in the regulation of galectin-3, the important factor in many immune reactions, in this study we investigated the effects of two most frequently used NSAIDs (aspirin and indomethacin), applied in different therapeutic ranges, on the expression of LGALS3 and galectin-3 in the monocytic cell line THP-1.
EXPERIMENTAL
Materials
All chemicals were of analytical grade and, if not stated otherwise, were purchased from Sigma (St. Louis, MO, USA). Indomethacin was obtained from Fluka (St. Louis, MO, USA). Aspirin and indomethacin were initially dissolved in DMSO and subsequently diluted with culture medium to the final concentration prior to use.
Immobilon PVDF-membranes were purchased from Millipore Corp. (Bedford, MA, USA), CellTiter 96(AQueous One Solution Cell Proliferation Assay from Promega (Madison, WI, USA), ECL Western blot detection kit from Amersham Biosciences (Cardiff, UK).
M u r i n e a n t i - - a c t i n a n t i b o d i e s a n d H R P - l a b e l e d g o a t a n t i - m o u s e I g M a n t i b o d i e s w e r e f r o m O n c o g e n e R e s e a r c h P r o d u c t s ( D a r m s t a d t , G e r m a n y ) , h y b r i d o m a c e l l l i n e T I B - 1 6 6 t h a t p r o d u c e s r a t m o n o c l o n a l a n t i - h u m a n - g a l e c t i n - 3 a n t i b o d i e s ( M 3 / 3 8 ) w a s f r o m A T C C ( M a n a s s as, VA, USA) while HRP-labeled goat anti-rat IgG antibodies were from Sigma (St. Louis, MO, USA).
Cell Culture
Human acute monocytic leukemia suspension cell line THP-1 was obtained from ATCC (Manassas, VA, USA). Cells were asynchronously grown (37 C, 5 % CO2, relative humidity 95 %) in RPMI 1640 medium supplemented with fetal bovine serum (10 %), penicillin G (60 g / m L ) , s t r e p t o m y c i n - s u l p h a t e ( 1 0 0 g / m L ) , a m p h o t e r i c i n B ( 2 5 0 n g / m L ) a n d p l a s m o c i n ( 2 . 5 g / m L ) ( a l l p u r c h a s e d f r o m G i b c o , G r a n d I s l a n d , N Y , U S A ) a n d s u b c u l t u r e d e v e r y 2 - 3 d a y s t o m a i n t a i n a c e l l c o n c e n t r a t i o n b e t w e e n 1 x 1 0 5 a n d 1 x 1 0 6 c e l l s / m L . V i a b l e c ells and the cell count were assessed with trypan blue (0.04 %) exclusion dye using a hemocytometer.
Treatment of THP-1 Cells with Aspirin and Indomethacin
Aspirin (Sigma) and indomethacin (Fluka) were initially dissolved in dimethyl-sulphoxide (DMSO) and subsequently added to the culture medium to the final concentration (0.1, 0.5, 1 and 2 mmol dm-3 aspirin; 0.1, 1, 5 and 10 m o l d m - 3 i n d o m e t h a c i n ; D M S O d" 0 . 2 % ) p r i o r t o u s e . A f t e r a d d i t i o n o f t h e m e d i u m c o n t a i n i n g t e s t c o m p o u n d s , c e l l s ( 2 x 1 0 5 / m L ) w e r e c u l t i v a t e d i n 1 7 5 m m 2 t i s s u e c u l t u r e f l a s k s ( N u n c l o n , R o s k i l d e , D e n m a r k ) f o r t h e n e x t 1 , 3 , 5 , 2 4 , 4 8 a n d 7 2 h o u r s .
T h e c e l l s cultivated in 0.2 % DMSO during the same period of time served as controls.
Cell Viability MTS Test
A cell viability and potential cytotoxic effect of test compounds was investigated using the CellTiter 96(AQueous One Solution Cell Proliferation Assay. Fifty microliters of the cell culture (1 x 106 cells/mL) or fifty microliters of RPMI 1640 medium were t r a n s f e r r e d i n t o t h e w e l l s o f a 9 6 - m i c r o w e l l p l a t e . T h e t e s t c o m p o u n d s w e r e s e r i a l l y d i l u t e d a n d 5 0 L o f 2 - f o l d d i l u t i o n w a s a d d e d i n t o e a c h w e l l t o o b t a i n t h e f i n a l c o n c e n t r a t i o n o f a s p i r i n o f 0 . 1 , 0 . 5 , 1 a n d 2 m m o l d m - 3 , i n d o m e t h a c i n 0 . 1 , 1 , 5 a n d 1 0 m o l d m - 3 a n d D M S O 0 . 2 % . T h e c o n t r o l s c o n t a i n e d t e s t m o d e l c e l l s , c u l t u r e m e d i u m b u t n o t e s t c o m p o u n d s . T h e c e l l s w e r e i n c u b a t e d f o r 3 d a y s b e f o r e 2 0 L M T S w a s a d d e d a n d w e r e a d d i t i o n a l l y i n c u b a t e d i n t h e d a r k a t 3 7 C f o r t h e f o l l o w i n g 2 h o u r s . A D D I N E N .CITE Mosmann198321206606682651-21983Dec 16Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays55-63Mosmann, T.J Immunol MethodsAnimalsCell LineCell SurvivalColorimetry/*methodsConcanavalin A/pharmacologyCytotoxicity Tests, Immunologic/*methodsDose-Response Relationship, ImmunologicInterleukin-2/physiologyLipopolysaccharides/pharmacology*Lymphocyte ActivationLymphocytes/*immunology/physiologyLymphoma/immunologyMacrophage ActivationMiceMice, Inbred BALB CTetrazolium Salts/diagnostic useThiazoles/diagnostic usehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=660668224 The absorbance was read at the wavelength of 490 nm using the microplate reader Victor2-1420 Multilabel Counter (PerkinElmer, Wellesley, MA, USA).
Sample Collection
After the pre-determined incubation time, aliquots of the cell culture containing 4 x 106 cells (for protein analysis) and 1 x 106 cells (for RNA analysis) were centrifuged at 600 ( g for 10 min. The cell pellet (4 x 106 cells) was washed twice with ice-cold PBS, resuspended in 0.15 mL lysis buffer (1 % Triton X-100 in 5 mmol dm-3 Tris/HCl, pH=8.0, 15 mmol dm-3 NaCl, 2 mmol dm-3 PMSF) and additionally homogenized by sonication with an ultrasound disintegrator (MSE, 100 W, London, England) for 2 min (amplitude 5 (m, ( = 22 kHz).
Protein concentrations in cell homogenates were determined by the method of BCA. ADDIN EN.CITE Smith19852030384370515011985OctMeasurement of protein using bicinchoninic acid76-85Smith, 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 BiochemHydrogen-Ion ConcentrationIndicators and ReagentsProteins/*analysis*QuinolinesSolubilitySpectrophotometryhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=384370525 Total RNA was extracted from 1 x 106 cells as described afterwards.
Western-blot Analysis
SDS-polyacrylamide gel electrophoresis was performed in 12 % polyacrylamide gels according to Laemmli. ADDIN EN.CITE Laemmli1970201054320632272591970Aug 15Cleavage of structural proteins during the assembly of the head of bacteriophage T4680-5Laemmli, U. K.NatureAutoradiographyCarbon IsotopesColiphages/*metabolismElectrophoresis, DiscGenes, StructuralGenetics, MicrobialKineticsViral Proteins/*metabolismhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=543206326 Separated proteins were transferred onto PVDF-membrane using the semi-dry transfer technique. The part of the membrane containing proteins smaller than (40 kDa was incubated overnight with rat M3/38 anti-galectin-3 antibodies (culture supernatant from h y b r i d o m a c e l l l i n e T I B 1 6 6 ) d i l u t e d 1 : 2 0 0 i n T B S . T h e p a r t o f t h e m e m b r a n e c o n t a i n i n g p r o t e i n s b i g g e r t h a n ( 4 0 k D a w a s i n c u b a t e d o v e r n i g h t w i t h m u r i n e a n t i - - a c t i n a n t i b o d i e s d i l u t e d 1 : 2 5 0 0 i n T B S . A f t e r o n e h o u r o f i n c u b a t i o n w i t h H R P - l a b e l e d s e c o n d a r y a n t i b o d i e s ( g o a t a n t i - r a t I g G d i l u t e d 1 : 1 0 0 0 0 i n T B S o r g o a t a n t i - m o u s e I g M d i l u t e d 1 : 1 0 0 0 0 i n T B S ) g a l e c t i n - 3 a n d - a c t i n w e r e v i s u a l i z e d w i t h t h e E C L W e s t e r n b l o t d e t e c t i o n k i t ( A m e r s h a m , C a r d i f f , U K ) . Q u a n t i f i c a t i o n o f t h e b a n d i n t e n s i t y w a s p e r f o r m e d u s i n g t h e S c i o n I m a g e A n a l y s i s s o f t w a r e , M i c r o s o f t . T h e l e v e l o f g a l e c t i n - 3 e x p r e s s i o n i n e a c h s a m p l e w a s n o r m a l i z e d t o t h e l e v e l o f - a c t i n .
R N A I s o l a t i o n a n d R T - P C R
T o t a l R N A w a s e x t r a c t e d f r o m 1 0 6 c e l l s w i t h t h e R N e a s y M i n i K i t ( Q i a g e n , G m b H , H i l d e n , G ermany) following the manufacturer's protocol.
Using the 6000 Nano LabChipKit, analysis of RNA quality and measurement of RNA concentration were performed on an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), according to the manufacturer's instructions.
Total RNA ((0. 8 g ) w a s r e v e r s e t r a n s c r i b e d u s i n g o l i g o - d T p r i m e r s ( 2 . 5 m o l d m - 3 ) a n d m u r i n e l e u k e m i a v i r u s ( M u L V ) r e v e r s e t r a n s c r i p t a s e ( 2 . 5 U / L ) , i n a f i n a l v o l u m e o f 2 0 l c o n t a i n i n g 1 m m o l d m - 3 d N T P s , 5 m m o l d m - 3 M g C l 2 a n d P C R b u f f e r ( 5 0 m m o l d m - 3 K C l , 1 0 m m o l d m - 3 T r i s / H C l , p H = 8 . 3 ) d u r i n g 1 - h o u r i n c u b a t i o n a t 3 7 C . T h e s a m p l e s w e r e h e a t e d f o r 5 m i n a t 9 5 C a n d c o o l e d d o w n t o 4 C .
T h e P C R u s i n g P l a t i n u m T a q p o l y m e r a s e ( 0 . 5 U ) w a s p e r f o r m e d i n a f i n a l v o l u m e o f 1 0 l c o n t a i n i n g 2 l o f c D N A i n a G e n e A m p P C R S y s t e m 2 7 0 0 ( A p p l i e d B i o s y s t e m s , F o s t e r C i t y , C A , U S A ) . O t h e r r e a c t i o n c o n s t i t u e n t s i n c l u d e d M g C l 2 ( 1 . 5 m m o l d m - 3 ) , d N T P s ( 0 . 2 m m o l d m - 3 ) , P C R b u f f e r a n d 3 ' a n d 5 ' p r i m e r s ( 0 . 5 m o l d m - 3 e a c h ) . T h e P C R w a s c o n d u c t e d i n 2 1 c y c l e s f o r g a l e c t i n - 3 a n d 1 4 c y c l e s f o r - a c t i n u n d e r t h e f o l l o w i n g c o n d i t i o n s : 1 m i n o f d e n a t u r a t i o n a t 9 4 C , 1 m i n o f a n n e a l i n g a t 5 8 C f o r g a l e c t i n - 3 o r a t 6 3 C f o r - a c t i n a n d 1 m i n o f e x t e n s i o n a t 7 2 C , f o l l o w e d b y t h e f i n a l e x t e n s i o n f o r 5 m i n a t 7 2 C a n d c o o l i n g d o w n t o 4 C . T h e f o l l o w i n g p r i m e r p a i r s w e r e u s e d : f o r g a l e c t i n - 3 : T G C T G A T A A C A A T T C T G G G C ( s e n s e ) a n d T A A C C C G A T G A T T G T A C T G C ( a n t i s e n s e ) , f o r - a c t i n : C A T G T G C A A G G C C G G C T T C G ( s e n s e ) a n d G A A G G T G T G G T G C C A G A T T T ( a n t i s e n s e ) . S e n s e p r i m e r s w e r e l a b e l e d w i t h A B I f l u o r e s c e n c e d y e s 6 - F A M o n the 5' end.
Sequencing Analysis
To confirm that the PCR products were derived from targeted mRNA, the products were purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and the nucleotide sequences were analyzed with the DNA Sequencing Big Dye Terminator v3.0 kit on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, USA), according to the manufacturer's instructions.
GeneScan Method
The same volume of RT-PCR amplicons for galectin-3 and - a c t i n ( 0 . 5 l e a c h ) w e r e p o o l e d t o g e t h e r i n o n e t u b e . G S 5 0 0 T A M R A - l a b e l e d s i z e m a r k e r ( 0 . 5 l ) a n d f o r m a m i d e ( 1 1 . 5 l ) w e r e a d d e d t o t h e s a m e t u b e . R T - P C R a m p l i c o n s w e r e r e s o l v e d b y c a p i l l a r y e l e c t r o p h o r e s i s , a n d f l u o r e s c e n c e d e t e c t i o n w i t h a v i r t u a l A filter was performed on an ABI Prism 310 Genetic Analyzer. The runs were executed with the module GS STR POP 4 (1 ml) A with 3 s and 15 kV injection and run voltage, 60 C constant temperature, 24 min running time, using polymer POP 4 and the running buffer Genetic Analyzer. GeneScan 2.1 software was then used to analyze the RT-PCR amplicons, with accurate sizing and quantification of the peak areas.
Statistical Analysis
Results are shown as means ( SD. Differences between various treatments were tested with unpaired Student's t-tests; p values < 0.05 were considered significant.
RESULTS
To investigate the effects of NSAIDs on the expression of galectin-3 on both the mRNA and protein levels, we exposed the monocytic cell line THP-1 to the various therapeutic concentrations of aspirin (0.1 mmol dm-3, 0.5 mmol dm-3, 1mmol dm-3 and 2 mmol dm-3) and indomethacin (0.1 m o l d m - 3 , 1 m o l d m - 3 , 5 m o l d m - 3 a n d 1 0 m o l d m - 3 ) . M e a s u r e m e n t s o f c e l l v i a b i l i t y a n d m e t a b o l i c a c t i v i t y w i t h t r y p a n e x c l u s i o n d y e a n d t h e M T S t e s t s h o w e d t h a t n o n e o f t h e a p p l i e d c o n c e n t r a t i o n s o f a s p i r i n o r i n d o m e t h a c i n d u r i n g 7 2 h o u r s o f c u l t i v a t i o n affected the cellular metabolism either in a cytotoxic or a proliferative manner. Cultivation of THP-1 cells in the presence of 0.2 % DMSO, which was the final DMSO concentration after dilution of the test compound dissolved in DMSO, did not change the metabolic status of cells either (data not shown).
The relative amount of mRNA for galectin-3 (i.e., relative expression of LGALS3) was determined by the GeneScan Analysis of RT-PCR amplicons for galectin-3 and - a c t i n . T h e o p t i m i z e d m e t h o d i n c l u d e s a m p l i f i c a t i o n o f g a l e c t i n - 3 c D N A d u r i n g 2 1 c y c l e s o f P C R a n d a m p l i f i c a t i o n o f - a c t i n c D N A d u r i n g 1 4 c y c l e s o f P C R . S u b s e q u e n t q u a n t i f i c a t i o n w a s f e a s i b l e u n d e r t h e s e c o n d i t i o n s b e c a u s e p o l y m e r a s e c h a i n r e a c t i o n s w e r e still in the exponential phase when the reactions were stopped (Fig.Figure 1). Sequencing analysis of the PCR products showed the high specificity and accuracy of the PCR reactions; the synthesized PCR products were the only products of the PCR reactions and their sequences corresponded entirely to the parts of galectin-3 or - a c t i n m R N A .
C u l t i v a t i o n o f T H P - 1 c e l l s f o r 7 2 h o u r s d i d n o t a f f e c t t h e e x p r e s s i o n o f L G A L S 3 o r t h e e x p r e s s i o n o f t h e c o r r e s p o n d i n g p r o t e i n , g a l e c t i n - 3 . T h e b a s a l l e v e l s o f t h e e x p r e s s i o n o f b o t h L G A L S 3 a n d g a l e c t i n - 3 w e r e a l s o m a i n t a i n e d d u r i n g 7 2 h o u r s of cultivation in 0.2 % DMSO/medium (data not shown).
The lowest applied concentration of aspirin (0.1 mmol dm-3) did not affect the expression level of LGALS3 during the first three hours of cultivation. A decrease was observed after 5 hours of cultivation, though it became statistically significant 24 hours after the beginning of the treatment. The level of mRNA continued to fall and it reached (31.1 ( 1.4) % of the initial value after 72 hours of cultivation (Fig.Figure 2, ribbed bars). When THP-1 cells were exposed to medium (0.5 mmol dm-3 and 1 mmol dm-3) and high (2 mmol dm-3) therapeutic concentrations of ASA, the level of mRNA for galectin-3 dropped sharply already after the first hour of cultivation (to (59.6 ( 7.6) %, (52.2 (1.7) % and (45 ( 3.4) % of the initial value, respectively, Fig.Figure 2). Prolonged cultivation caused a further decrease of the galectin-3 mRNA level in a dose dependent manner. At the end of the treatment (after 72 hours) the expression of LGALS3 dropped to (25.6 ( 4.8) %, (15.5 ( 0.5) % and (12.3 ( 0.04) % of the expression observed in the control cells, respectively (Fig.Figure 2).
Expression of galectin-3 was measured by the chemiluminescent-western blot analysis. Incubation of the cells for 24 hours with aspirin in all applied concentrations did not affect the level of galectin-3 expression, except in the case of the highest ASA concentration applied (2 mmol dm-3), which caused a 20 % decrease of galectin-3 expression, compared to the control cells. The same intensity of decrease was observed when the cells were cultivated for 48 hours with 1 mM ASA, while at the same time 2 mM ASA caused a further decline of galectin-3 expression (to (71 ( 3) % of the initial value). The inhibitory effect of ASA on galectin-3 expression observed after 72 hours of cultivation correlates with the applied ASA concentration, varying from approximately 30 % (0.1 mmol dm-3 ASA) to 40 % of inhibition (2 mmol dm-3 ASA) (Fig.Figure 3).
Expression of LGALS3 was also attenuated during the cultivati o n o f T H P - 1 c e l l s i n t h e p r e s e n c e o f t h e r a p e u t i c d o s e s o f i n d o m e t h a c i n , t h o u g h t h e p a t t e r n o f i n h i b i t i o n w a s s o m e w h a t d i f f e r e n t f r o m t h e p a t t e r n o b s e r v e d w h e n c e l l s w e r e t r e a t e d w i t h a s p i r i n . A l l t h e a p p l i e d i n d o m e t h a c i n c o n c e n t r a t i o n s ( 0 . 1 , 1 , 5 a n d 1 0 mol dm-3) caused a decrease of galectin-3 mRNA level already after the first hour of cultivation (ranging from (66.7 ( 4.2) to (34.7 ( 1.8) % of the initial value, depending on the applied concentration). The intensity of inhibition continued to increase with time, and it depended more on time than on the applied dose. After 72 hours of cultivation, the level of LGALS3 expression ranged from 28 to 8 % of the initial value, depending on the applied indomethacin concentration (Fig.Figure 4).
The basal level of g a l e c t i n - 3 d i d n o t c h a n g e s i g n i f i c a n t l y a f t e r t h e f i r s t 2 4 h o u r s o f c u l t i v a t i o n o f T H P - 1 c e l l s w i t h i n d o m e t h a c i n ( c o n c e n t r a t i o n r a n g e 0 . 1 t o 1 0 m o l d m - 3 ) . A l t h o u g h 4 8 h o u r s o f c u l t i v a t i o n w i t h 0 . 1 m o l d m - 3 I n d c a u s e d a r e d u c t i o n o f t h e g a l e c t i n - 3 l e v e l t o ( 6 9 . 0 ( 5 . 1 ) % , a n d c u l t i v a t i o n w i t h 5 m o l d m - 3 I n d c a u s e d a r e d u c t i o n t o ( 8 0 . 5 ( 2 . 7 ) % o f t h e i n i t i a l v a l u e , 1 m o l d m - 3 I n d a n d 1 0 m o l d m - 3 I n d d i d n o t a f f e c t t h e e x p r e s s i o n o f t h e l e c t i n . S u r p r i s i n g l y , a f t e r 7 2 h o f i n c u b a t i o n , t h e l o w e r c o n c e n t r ation of indomethacin had a stronger inhibitory effect on galectin-3 than higher doses the inhibition varied from approximately 50 to 20 %, respectively (Fig.Figure 5).
DISCUSSION
The importance of non-steroidal anti-inflammatory drugs in modern medicine and pharmacy has stimulated an impressive amount of scientific studies on the effects of NSAIDs on their cellular and molecular targets in the last few decades. Recent findings indicating that NSAIDs affect the activity of cellular transcription fact o r s a n d m o d u l a t e t r a n s c r i p t i o n a l a c t i v a t i o n p r o m p t e d a n e n o r m o u s i n t e r e s t i n g e n e e x p r e s s i o n a s a n e w t a r g e t o f N S A I D s .
G a l e c t i n - 3 , a - g a l a c t o s i d e b i n d i n g l e c t i n , h a s r e c e n t l y e m e r g e d a s a n e w p o w e r f u l c e l l u l a r m o d u l a t o r a t i n f l a m m a t o r y f o c i . Y e t , u n t i l now there were no data about the effects of NSAIDs on the expression of either galectin-3 or any other lectin. In the presented study, we have demonstrated for the first time that non-steroidal anti-inflammatory drugs aspirin and indomethacin, applied in clinically relevant doses, effectively inhibited galectin-3 expression at the mRNA level, and to a lesser extent at its protein level in the monocytic cell line THP-1.
The promotor region of LGALS3 gene, whose sequence was determined by Kadrofske et al. ADDIN EN.CITE Kadrofske1998150943957734911998Jan 1The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter7-20Department of Biochemistry, Michigan State University, East Lansing 48824, USA.Kadrofske, M. M.Openo, K. P.Wang, J. L.Arch Biochem BiophysAntigens, Differentiation/*geneticsBase SequenceGalectin 3*Genome, HumanHumanLectins/geneticsMolecular Sequence DataPromoter Regions (Genetics)/*geneticsSequence AlignmentSequence AnalysisSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=943957717 in 1998, contains binding sites for several transcription factors. Previously, we showed that galectin-3 expression is influenced by various stimuli, ADDIN EN.CITE Dumic2000200010817217553-42000Mar-AprTransfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages261-6Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia.Dumic, J.Lauc, G.Hadzija, M.Flogel, M.Z Naturforsch [C]AnimalsAntigens, Differentiation/*metabolismCells, CulturedGalectin 3Macrophages, Peritoneal/*metabolismMaleMiceMice, Inbred BALB CRatsSubcellular Fractions/metabolismSupport, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10817217Dumic2000198010938585332000MayGalectin-3 decreases in mice exposed to immobilization stress241-6Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovacica 1, 10000 Zagreb, Croatia.Dumic, J.Barisic, K.Flogel, M.Lauc, G.StressAging/*physiologyAnimalsAntigens, Differentiation/*metabolismCell LineGalectin 3Glioblastoma/metabolism/pathologyHeat-Shock Response/physiologyImmunoblottingLiver/metabolismMacrophages, Peritoneal/metabolismMiceMice, Inbred C3HMice, Inbred StrainsRestraint, PhysicalSpleen/metabolismStress/*metabolismSupport, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1093858518,19, and that the involvement of transcription factors AP-1 and NF-(B depends on the type of the stimuli. ADDIN EN.CITE Dumic20001970108784451032000Expression of galectin-3 in cells exposed to stress-roles of jun and NF-kappaB149-58Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovavica 1, 10000 Zagreb, Croatia.Dumic, J.Lauc, G.Flogel, M.Cell Physiol BiochemAlkylating Agents/pharmacologyAntigens, Differentiation/*geneticsGalectin 3Gene Expression RegulationHeat-Shock ResponseHumanNF-kappa B/*metabolismProto-Oncogene Proteins c-jun/*metabolismSupport, Non-U.S. Gov'tTrypsin/pharmacologyTumor Cells, CulturedUltraviolet Rayshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1087844527 NSAIDs comprise a large class of diverse drugs, classified in two main groups weak and strong NSAIDs. Mechanisms of action of these drugs, especially as far as their influence on the signal transduction pathway is concerned, are somewhat different. The observed time- and dose-dependent suppression of LGALS3 expression caused by aspirin, the most frequently used weak NSAID, might be a consequence of aspirin's ability to inhibit either the transcription factor AP-1 ADDIN EN.CITE Dong199715709092536272151997Apr 11Inhibition of activator protein 1 activity and neoplastic transformation by aspirin9962-70The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.zgdong@wolf.co.netDong, Z.Huang, C.Brown, R. E.Ma, W. Y.J Biol ChemAnimalsAntineoplastic Agents/*pharmacologyAntineoplastic Agents, Hormonal/pharmacologyAspirin/*pharmacologyCa(2+)-Calmodulin Dependent Protein Kinase/antagonists & inhibitorsCarcinogens/pharmacologyCell AdhesionCell Transformation, Neoplastic/*drug effectsCyclooxygenase Inhibitors/pharmacologyDiethylstilbestrol/pharmacologyGenes, junGenes, rasHydrogen-Ion ConcentrationIndomethacin/pharmacologyMice*Mitogen-Activated Protein KinasesPhosphorylationSodium Salicylate/pharmacologySupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.Tetradecanoylphorbol Acetate/pharmacologyTranscription Factor AP-1/*antagonists & inhibitorsTumor Cells, Culturedp42 MAP Kinasehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=909253628 or NF-(B. ADDIN EN.CITE Tegeder200115401164123315122001OctCyclooxygenase-independent actions of cyclooxygenase inhibitors2057-72Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt, 60590 Frankfurt am Main, Germany.Tegeder, I.Pfeilschifter, J.Geisslinger, G.Faseb JAnimalsAnti-Inflammatory Agents, Non-Steroidal/pharmacologyCyclooxygenase Inhibitors/*pharmacologyDNA-Binding Proteins/antagonists & inhibitorsGrowth Substances/biosynthesisHeat-Shock Response/drug effectsIsoenzymes/metabolismMAP Kinase Signaling System/drug effectsMiceModels, BiologicalNF-kappa B/antagonists & inhibitorsPregnancy Proteins/biosynthesisProstaglandin-Endoperoxide Synthase/metabolismReceptors, Cytoplasmic and Nuclear/antagonists & inhibitorsRibosomal Protein S6 Kinases/antagonists & inhibitorsSupport, Non-U.S. Gov'tTrans-Activators/antagonists & inhibitorsTranscription Factor AP-1/antagonists & inhibitorshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1164123323 According to the literature data, indomethacin, the most commonly applied strong NSAID, does not affect directly the activity of AP-1 or NF-(B, but activates the transcription factor PPAR(. The activation of PPAR( may, on the other hand, cause inhibition of NF-(B activity through the process of transrepression. ADDIN EN.CITE Shimada20041610147595125581-32004Jan 30PPARgamma mediates NSAIDs-induced upregulation of TFF2 expression in gastric epithelial cells33-8Department of Gastroenterology and Institute for Medical Science, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. tshimada@dokkyomed.ac.jpShimada, T.Koitabashi, A.Fujii, Y.Hashimoto, T.Hosaka, K.Tabei, K.Namatame, T.Yoneda, M.Hiraishi, H.Terano, A.FEBS LettAnilides/pharmacologyAnti-Inflammatory Agents, Non-Steroidal/*pharmacologyAspirin/pharmacologyCell Line, TumorDose-Response Relationship, DrugEnzyme Inhibitors/pharmacologyEpithelial Cells/drug effects/*metabolismGene Expression Regulation, NeoplasticGenes, ReporterHumanIndomethacin/pharmacologyPeptides/*metabolismRNA, Messenger/metabolismReceptors, Cytoplasmic and Nuclear/genetics/*metabolismReverse Transcriptase Polymerase Chain ReactionStomach Neoplasms/pathologyTrans-Activation (Genetics)/drug effectsTranscription Factors/genetics/*metabolismTranscription, Genetic/drug effectsUp-Regulation/*drug effectshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1475951229 The other possibility is that the inhibitory effect of NSAIDs on LGALS3 expression is mediated through some other molecules whose expression is affected by NSAIDs (such as TNF(, IL-4 and IL-8) through the complex network of signal transduction pathways.
Under the influence of NSAIDs, changes in galectin-3 expression did not follow the pattern of changes of the mRNA level. Discrepancy between the changes in mRNA and protein expression levels is not rare and was previously reported by many other authors. ADDIN EN.CITE Andersson200020801109774812122000DecSignalling to translational activation of tumour necrosis factor-alpha expression in human THP-1 cells1784-7Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, S-221 00, Lund, Sweden.Andersson, K.Sundler, R.CytokineAnisomycin/pharmacologyBlotting, NorthernBlotting, WesternButadienes/pharmacologyCalcium/metabolismCell LineCycloheximide/pharmacologyDactinomycin/pharmacologyDose-Response Relationship, DrugEnzyme Inhibitors/pharmacologyFlavonoids/pharmacologyHumanImidazoles/pharmacologyIonophores/pharmacologyMAP Kinase Signaling SystemMitogen-Activated Protein Kinases/metabolismMonocytes/metabolismNitriles/pharmacologyProtein Synthesis Inhibitors/pharmacologyPyridines/pharmacologyRNA, Messenger/metabolism*Signal TransductionSupport, Non-U.S. Gov'tTetradecanoylphorbol Acetate/pharmacologyTime FactorsTranslation, GeneticTumor Necrosis Factor/biosynthesis/*metabolismp42 MAP Kinase/metabolismhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11097748Lorenz2003209012833524362003JunFrom transcriptome to proteome: differentially expressed proteins identified in synovial tissue of patients suffering from rheumatoid arthritis and osteoarthritis by an initial screen with a panel of 791 antibodies991-1002Institute of Immunology, Rostock, Germany.Lorenz, P.Ruschpler, P.Koczan, D.Stiehl, P.Thiesen, H. J.ProteomicsAntibodies/metabolismArthritis, Rheumatoid/*metabolism/pathologyBlotting, WesternComparative StudyFemaleGene Expression ProfilingHumanMiddle AgedOligonucleotide Array Sequence AnalysisOsteoarthritis/*metabolismProteins/analysis/*metabolismProteome/*metabolismSupport, Non-U.S. Gov'tSynovial Membrane/*metabolism/pathologyTranscription, Genetichttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12833524Shipp2002210011786909812002JanDiffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning68-74Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. margaret_shipp@dfci.harvard.eduShipp, M. A.Ross, K. N.Tamayo, P.Weng, A. P.Kutok, J. L.Aguiar, R. C.Gaasenbeek, M.Angelo, M.Reich, M.Pinkus, G. S.Ray, T. S.Koval, M. A.Last, K. W.Norton, A.Lister, T. A.Mesirov, J.Neuberg, D. S.Lander, E. S.Aster, J. C.Golub, T. R.Nat MedAntineoplastic Combined Chemotherapy Protocols*Artificial IntelligenceCyclophosphamideDoxorubicinGene Expression Profiling/*methodsHumanLymphoma, B-Cell/*diagnosis/drug therapy/mortalityLymphoma, Large-Cell, Diffuse/*diagnosis/drug therapy/mortalityOligonucleotide Array Sequence AnalysisPredictive Value of TestsPrednisoneSupport, Non-U.S. Gov'tTreatment OutcomeVincristinehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1178690930-32 As far as this study is concerned, they could be a result of an enhanced translation rate and/or reduced degradation rate (turnover). On the other hand, enhanced binding of previously secreted galectin-3 on its membrane receptors, whose expression might also be modulated as a result of NSAIDs application, could also be the cause of a sustained protein level.
Maintenance of an unchanged level of galectin-3 during the first 24 hours of exposure to NSAIDs compared to the strong decrease of mRNA expression observed in that period suggests the necessity and the importance of galectin-3 for a proper monocyte function. It seems that these cells require a particular amount of galectin-3 and somehow they succeed in preserving it despite the strong reduction of its mRNA. However, after the prolonged exposure to NSAIDs (48 and 72 hours), the galectin-3 level significantly decreased. The observed changes in LGALS3 and galectin-3 expression should be considered in the sense of multiple roles of galectin-3 that were shown to be involved in transcription control, in pre-mRNA splicing and to be an anti-apoptotic factor. Additionally, galectin-3, secreted in extracellular space affects the physiology of many various cells. A prolonged therapy with NSAIDs is frequently used for the prevention of cardiovascular diseases and since recently as a supportive cancer treatment. Thus, the observed reduction of galectin-3 expression due to the prolonged NSAIDs' application can modulate numerous biological processes in which galectin-3 is involved. For instance, according to very recent data, ADDIN EN.CITE Shimura200421301537493964182004Sep 15Galectin-3, a novel binding partner of beta-catenin6363-7Karmanos Cancer Institute, Wayne State University Medical School, Detroit, Michigan 48201, USA.Shimura, T.Takenaka, Y.Tsutsumi, S.Hogan, V.Kikuchi, A.Raz, A.Cancer ResBreast Neoplasms/genetics/metabolismCell Line, TumorCytoskeletal Proteins/genetics/*metabolismDNA-Binding Proteins/genetics/*metabolismGalectin 3/genetics/*metabolismGene Expression Regulation, NeoplasticGenes, bcl-1HCT116 CellsHT29 CellsHumansPlasmids/geneticsPromoter Regions (Genetics)Protein BindingProtein Structure, TertiaryResearch Support, U.S. Gov't, P.H.S.Trans-Activation (Genetics)Trans-Activators/genetics/*metabolismTranscription Factors/genetics/*metabolismTransfectionUp-Regulationhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15374939Fukumori200421201515008764102004May 15Endogenous galectin-3 determines the routing of CD95 apoptotic signaling pathways3376-9Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.Fukumori, T.Takenaka, Y.Oka, N.Yoshii, T.Hogan, V.Inohara, H.Kanayama, H. O.Kim, H. R.Raz, A.Cancer ResAntigens, CD95/metabolism/*physiologyApoptosis/*physiologyB-Lymphocytes/physiologyCaspases/metabolismCell LineGalectin 3/genetics/metabolism/*physiologyHumansJurkat CellsResearch Support, U.S. Gov't, P.H.S.Signal Transduction/physiologyT-Lymphocytes/physiologyTransfectionhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1515008733,34 extracellular galectin-3 was shown to be one of the key molecules engaged in the control of apoptotic signaling pathways, so the changes in its expression can modulate apoptosis, an extremely important process especially for tumor and immune cells.
Taken together, the obtained data suggest that besides the numerous already known effects of NSAIDs therapy, the effects of NSAIDs on galectin-3 expression and the consequences that can be provoked should be inevitably considered in terms of a better understanding of the mechanisms of NSAIDs action not only in monocytes but rather in all cells expressing galectin-3. In summary, the results obtained in this study have established galectin-3 as a new target molecule of NSAIDs and at the same time they represent a valuable contribution for the understanding of the effects of immunomodulatory drugs.
Acknowledgement.
This work was supported by grant 0006611 from the Ministry of Science, Education and Sports of the Republic of Croatia.
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7 7 7 7 7 7 7 7 7 8 8 X8 Y8 Z8 M M M M ;N >N N N 7O h Ch Dh Gh \h ]h vh wh h h h h h h h h h h h h h įcTۯcTįT hA{ h2S; CJ aJ mH sH 6H hFEfhA{ hy CJ aJ mH sH FEf* 1hA{ hp CJ OJ QJ aJ mH sH FEf*,hA{ hp 5CJ aJ mH sH FEf* )hA{ hp CJ aJ mH sH FEf*,H hFEfhA{ hy 6CJ aJ mH sH ,hA{ hp 6CJ aJ mH sH FEf* h_ 6CJ aJ mH sH h h h h h h h h h h h Mi Ni \i ]i ^i _i `i hi ii ji ni i ƯƘcƯLLƘ ,H hGEfhA{ hy 6CJ aJ mH sH 6H hFEfhA{ hy CJ aJ mH sH FEf* 1hA{ hp CJ OJ QJ aJ mH sH FEf*,hA{ hp 5CJ aJ mH sH FEf* ,hA{ hp 6CJ aJ mH sH FEf* )hA{ hp CJ aJ mH sH FEf* hA{ h2S; CJ aJ mH sH )H hFEfhA{ hy CJ aJ mH sH i i i i i i i i i i i i i i @j Aj Nj Oj Pj \j ]j `j pj sj tj }a}R? $hA{ hp CJ OJ QJ aJ mH sH hA{ hp CJ aJ mH sH 6H hGEfhA{ hy CJ aJ mH sH GEf* ,hA{ hp 5CJ aJ mH sH GEf* ,H hGEfhA{ hy 6CJ aJ mH sH ,hA{ hp 6CJ aJ mH sH GEf* hA{ h2S; CJ aJ mH sH 1hA{ hp CJ OJ QJ aJ mH sH GEf*)hA{ hp CJ aJ mH sH GEf* tj j j j j j j j j j j j j j j j j k k k k k k k 'k l 8l >l @l Xl Zl \l hl jl vvjU )H hHEfhA{ hy CJ aJ mH sH h_ CJ aJ mH sH ,H hGEfhA{ hy 6CJ aJ mH sH h_ 6CJ aJ mH sH $hA{ hp CJ OJ QJ aJ mH sH hA{ hp 5CJ aJ mH sH hA{ hp 6CJ aJ mH sH hA{ h2S; CJ aJ mH sH )H hGEfhA{ hy CJ aJ mH sH hA{ hp CJ aJ mH sH !jl nl l l l l l l l l l l l l l l l m m m m ˲˦˦ˊ{dMdMd ,H hHEfhA{ hy 6CJ aJ mH sH ,hA{ hp 6CJ aJ mH sH HEf* hA{ h2S; CJ aJ mH sH 6H hHEfhA{ hy CJ aJ mH sH HEf* h_ CJ aJ mH sH 1hA{ hp CJ OJ QJ aJ mH sH HEf*)hA{ hp CJ aJ mH sH HEf* hA{ hp CJ aJ mH sH hA{ hp 5CJ aJ mH sH m m @m Fm Hm m m m m m m m m m m m m m m n n n ŲŝŎ~g~gG~g~g~g > hA{ hp hy 6CJ aJ cH dh dh dhHEfmH sH ,H hHEfhA{ hy 6CJ aJ mH sH hA{ hp 6CJ aJ mH sH hA{ h2S; CJ aJ mH sH )H hHEfhA{ hy CJ aJ mH sH $hA{ hp CJ OJ QJ aJ mH sH hA{ hp CJ aJ mH sH )hA{ hp CJ aJ mH sH HEf*,hA{ hp 5CJ aJ mH sH HEf*n n n n 4n 5n En Fn Gn Sn Tn Xn Yn Zn ]n pn qn n n n n n n n n n n n n kNkk 9H hIEfhA{ hy 6CJ aJ mH sH IEf*,hA{ hp 6CJ aJ mH sH IEf* ,H hIEfhA{ hy 6CJ aJ mH sH hA{ hp 6CJ aJ mH sH hA{ h2S; CJ aJ mH sH )H hIEfhA{ hy CJ aJ mH sH $hA{ hp CJ OJ QJ aJ mH sH hA{ hp CJ aJ mH sH hA{ hp 5CJ aJ mH sH n n n n n n n n n n n n n n n n n n o ꕀkk^G0G ,hA{ hp 6CJ aJ mH sH IEf* ,H hIEfhA{ hy 6CJ aJ mH sH h_ 6CJ aJ mH sH )H hJEfhA{ hy CJ aJ mH sH )H hIEfhA{ hy CJ aJ mH sH H hA{ hp hy CJ aJ cH dh dh dhJEfmH sH IEf* 1hA{ hp CJ OJ QJ aJ mH sH IEf*,hA{ hp 5CJ aJ mH sH IEf* )hA{ hp CJ aJ mH sH IEf* o o o o o !o "o *o +o 3o 4o 7o Ho Io o o o o o ѼѥwbFb/,hA{ hp 6CJ aJ mH sH JEf* 6H hJEfhA{ hy CJ aJ mH sH JEf* )hA{ hp CJ aJ mH sH JEf*,hA{ hp 5CJ aJ mH sH IEf* ,H hIEfhA{ hy 6CJ aJ mH sH ,hA{ hp 6CJ aJ mH sH IEf* )H hJEfhA{ hy CJ aJ mH sH )hA{ hp CJ aJ mH sH IEf*1hA{ hp CJ OJ QJ aJ mH sH IEf* o o o o o o o o p " һn_]_J_5_ )H hJEfhA{ hy CJ aJ mH sH $hA{ hp CJ OJ QJ aJ mH sH U hA{ hp CJ aJ mH sH ,hA{ hp 5CJ aJ mH sH KEf* )hA{ hp CJ aJ mH sH KEf*AH hJEfhA{ hy 6CJ aJ mHsH KEf*mH sH ,hA{ hp 6CJ aJ mH sH KEf* ,hA{ hp 6CJ aJ mH sH JEf* ,H hJEfhA{ hy 6CJ aJ mH sH . U . K . L a e m m l i , N a t u r e 2 2 7 ( 1 9 7 0 ) 6 8 0 - 6 8 5 .
2 7 . J . D u m i , G . L a u c , a n d M . F l g e l , C e l l P h y s i o l . B i o c h e m . 1 0 ( 2 0 0 0 ) 1 4 9 - 1 5 8 .
2 8 . Z . D o n g , C . H u a n g , R . E . B r o w n , a n d W . Y . M a , J . B i o l . C h e m . 2 7 2 ( 1 9 9 7 ) 9 9 6 2 - 9 9 7 0 .
2 9 . T . S h i m a d a , A . K o i t a b a s h i , Y . F u j i i , T . Hashimoto, K. Hosaka, K. Tabei, T. Namatame, M. Yoneda, H. Hiraishi, and A. Terano, FEBS Lett. 558 (2004) 33-38.
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Legends
Fig.Figure 1.
GeneScan electrophoretogram display of RT-PCR amplicons.
Total RNA was isolated from THP-1 cells. After reverse transcription using oligo-dT primers, 21 cycles of PCR were performed u s i n g f l u o r e s c e n t p r i m e r s f o r g a l e c t i n - 3 a n d 1 4 c y c l e s o f P C R u s i n g f l u o r e s c e n t p r i m e r s f o r - a c t i n . R T - P C R a m p l i c o n s w e r e p o o l e d a n d s e p a r a t e d b y c a p i l l a r y e l e c t r o p h o r e s i s o n a n A b i P r i s m 3 1 0 G e n e t i c A n a l y z e r . T h e y - a x i s i s t h e r e l a t i v e i n t e n s i t y o f t h e fluorescence and the x-axis represents the designate size of the fragments in the base pair (bp), as shown at the bottom of the electrophoretogram.
Fig.Figure 2.
Aspirin inhibits expression of LGALS3 in THP-1 cells.
THP-1 cells were cultivated for 1, 3, 5, 24, 48 and 72 hours with 0.1, 0.5, 1 and 2 mmol dm-3 aspirin. Following the total RNA isolation and reverse transcription, 21 cycles of PCR were performed using fluorescent primers for galectin-3 and 14 cycles of PCR using fluorescent primers for - a c t i n . R T - P C R a m p l i c o n s w e r e p o o l e d a n d s e p a r a t e d b y c a p i l l a r y e l e c t r o p h o r e s i s o n a n A b i P r i s m 3 1 0 G e n e t i c A n a l y z e r . R e s u l t s o f t h e t w o i n d e p e n d e n t e x p e r i m e n t s p e r f o r m e d i n d u p l i c a t e w e r e c a l c u l a t e d u s i n g t h e G e n e S c a n 2 . 1 A n a l y s i s s o f t w a r e a n d e x p r e s s e d a s g a l e c t i n - 3 / - a c t i n R T - P C R a m p l i c o n p e a k a r e a s c o m p a r e d t o t h e c o n t r o l ( x ( S D ) . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( p < 0 . 0 5 c o m p a r e d t o t h e c o n t r o l s ) a r e m a r k e d w i t h a s t e r i s k s .
F i g . F i g u r e 3 .
A s p i r i n a f f e c t s g a l e c t i n - 3 e x p r e s s i o n i n T H P - 1 c e l l s .
Immunoblot analysis of galectin-3 in THP-1 cell homogenates cultivated for 1, 3, 5, 24, 48 and 72 hours in a) 0.1 mmol dm-3 ASA, b) 0.5 mmol dm-3 ASA, c) 1 mmol dm-3 ASA, d) 2 mmol dm-3 ASA (5 (g of proteins/well). e) Results of the densitometric analysis of the two independent experiments performed in duplicate are expressed as the relative level of galectin-3/(-actin compared to the control (x ( SD). Statistically significant differences (p < 0.05 compared to the control) are marked with asterisks.
F i g . F i g u r e 4 .
I n d o m e t h a c i n i n h i b i t s e x p r e s s i o n o f L G A L S 3 i n T H P - 1 c e l l s .
T H P - 1 c e l l s w e r e c u l t i v a t e d f o r 1 , 3 , 5 , 2 4 , 4 8 a n d 7 2 h o u r s w i t h 0 . 1 , 1 , 5 a n d 1 0 m o l d m - 3 i n d o m e t h a c i n . F o l l o w i n g t h e t o t a l R N A i s o l a t i o n a n d r e v e r s e t r a n s c r i p t i o n , 2 1 c y c l e s o f P C R w e r e p e r f o r m e d u s i n g f l u o r e s c e n t p r i m e r s f o r g a l e c t i n - 3 a n d 1 4 c y c l e s o f P C R u s i n g f l u o r e s c e n t p r i m e r s f o r - a c t i n . R T - P C R a m p l i c o n s w e r e p o o l e d a n d s e p a r a t e d b y c a p i l l a r y e l e c t r o p h o r e s i s o n a n A b i P r i s m 3 1 0 G e n e t i c A n a l y z e r . R e s u l t s o f t h e t w o i n d e p e n d e n t e x p e r i m e n t s p e r f o r m e d i n d u p l i c a t e w e r e c a l c u l a t e d u s i n g t h e G e n e S c a n 2 . 1 A n a l y s i s s o f t w a r e a n d e x p r e s s e d a s g a l e c t i n - 3 / - a c t i n R T - P C R a m p l i c o n p e a k a r e a s c o m p a r e d t o t h e c o n t r o l ( x ( S D ) . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( p < 0 . 0 5 c o m p a r e d t o t h e c o n t r o l s ) a r e m a r k e d w i t h a s t e r i s k s .
F i g . F i g u r e 5 .
I n d o m e t h a c i n a f f e c t s g a l e c t i n - 3 e x p r e s s i o n i n T H P - 1 c e l l s .
I m m u n o b l o t a n a l y s i s o f g a l e c t i n - 3 i n T H P - 1 c e l l h o m o g e n a t e s c u l t i v a t e d f o r 1 , 3 , 5 , 2 4 , 4 8 a n d 7 2 h o u r s i n a ) 0 . 1 m o l d m - 3 I n d , b ) 1 m o l d m - 3 I n d , c ) 5 m o l d m - 3 I n d , d ) 1 0 m o l d m - 3 I n d ( 5 g o f p r o t e i n s / w e l l ) . e ) R e s u l t s o f t h e d e n s i t o m e t r i c a n a l y s i s o f t h e t w o i n d e p e n d e n t e x p e r i m e n t s p e r f o r m e d i n d u p l i c a t e a r e e x p r e s s e d a s t h e r e l a t i v e l e v e l o f g a l e c t i n - 3 / ( - a c t i n c o m p a r e d t o t h e c o n t r o l ( x ( S D ) . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( p < 0 . 0 5 c o m p a r e d t o t h e c o n t r o l ) a r e m a r k e d w i t h a s t e r i s k s .
D a b e l i e t a l . , F i g . F i g u r e 1 .
D a b e l i e t a l . , F i g . F i g u r e 2 .
D a b e l i c e t a l . , F i g . F i g u r e 3 .
D a b e l i e t a l . , F i g . F i g u r e 4 .
D a b e l i e t a l . , F i g . F i g u r e 5 .
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