Article start

Phytomedicine23(2016)550–557ContentslistsavailableatScienceDirectPhytomedicinejournalhomepage:www.elsevier.com/locate/phymedIsololiolide,acarotenoidmetaboliteisolatedfromthebrownalgaCystoseiratamariscifolia,iscytotoxicandabletoinduceapoptosisinhepatocarcinomacellsthroughcaspase-3activation,decreasedBcl-2levels,increasedp53expressionandPARPcleavageCatarinaVizetto-Duartea,LuísaCustódioa,KatkamN.Gangadhara,JoãoHenriqueG.Lagob,CatarinaDiasc, Ana MartaMatosc,NunoNengc, José Manuel FlorêncioNogueirac,LuísaBarreiraa, FernandoAlbericiod,e,f,AmeliaP.Rauterc,JoãoVarelaa,aCentreofMarineSciences,UniversityofAlgarve,FacultyofSciencesandTechnology,Ed.7,CampusofGambelas,Faro,PortugalbInstituteofEnvironmental,ChemicalandPharmaceuticalSciences,FederalUniversityofSaoPaulo,09972-270,SaoPaulo,BrazilcCenterofChemistryandBiochemistry,DepartmentofChemistryandBiochemistry,FacultyofSciencesUniversityofLisbon,CampoGrande,Ed.C8,Piso5,1749-016Lisbon,PortugaldInstituteforResearchinBiomedicine,BarcelonaSciencePark,BaldiriReixac10,08028,Barcelona,SpaineCIBER-BBN,NetworkingCentreonBioengineering,BiomaterialsandNanomedicine,BarcelonaSciencePark,BaldiriReixac10,08028Barcelona,SpainfUniversityofBarcelona,DepartmentofOrganicChemistry,Martí iFranqués1-11,08028Barcelona,SpainarticleinfoArticlehistory:Received30January2016Accepted9February2016Keywords:MarinenaturalproductCystoseiraIsololiolideCarotenoidmetaboliteCellcycleApoptosisabstractBackground:Brownmacroalgaehaveattractedattentionbecausetheydisplayawiderangeofbiologicalactivities,includingantitumoralproperties.InthisstudyweisolatedisololiolidefromCystoseiratamarisci-foliaforthefirsttime.Purpose:Toexaminethetherapeuticalpotentialofisololiolideagainsttumorcelllines.Methods/Studydesign:Thestructureofthecompoundwasestablishedandconfirmedby1Dand2DNMRaswellasHRMSspectralanalysis.Theinvitrocytotoxicitywasanalyzedbycolorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromideassayintumoralaswellasinnon-tumoralcelllines.Cellcyclearrestandinductionofapoptosiswereassessedbyflowcytometry.Alterationofexpres-sionlevelsinproteinsimportantintheapoptoticcascadewasanalyzedbywesternblotting.Results:IsololiolidewasisolatedforthefirsttimefromthebrownmacroalgaC.tamariscifolia.Isololiolideexhibitedsignificantcytotoxicactivityagainstthreehumantumoralcelllines,namelyhepatocarcinomaHepG2cells,whereasnocytotoxicitywasfoundinnon-malignantMRC-5andHFF-1humanfibroblasts.IsololiolidecompletelydisruptedtheHepG2normalcellcycleandinducedsignificantapoptosis.More-over,westernblotanalysisshowedthatisololiolidealteredtheexpressionofproteinsthatareimportantintheapoptoticcascade,increasingPARPcleavageandp53expressionwhiledecreasingprocaspase-3andBcl-2levels.Conclusion:IsololiolideisolatedfromC.tamariscifoliaisabletoexertaselectivecytotoxicactivityonhepatocarcinomaHepG2cellsaswellasinduceapoptosisthroughthemodulationofapoptosis-relatedproteins.© 2016 Elsevier GmbH. All rights reserved.Abbreviations:1Dand2DNMR,one-dimensionalandtwo-dimensionalnuclearmagneticresonance;1Hand13CNMR,protonandcarbon-13nuclearmagneticres-onance;ANOVA,analysisofvariance;Bak,Bcl-2homologousantagonistkiller;Bax,Bcl-2-associatedXprotein;Bcl-2,B-celllymphoma2;CDCl3,deuteratedchloro-form;CNT,compound-inducedcytotoxicityonnon-tumoralcells;CT,compound-inducedcytotoxicityontumoralcells;DEPT,distortionlessenhancementbypolarizationtransfer;DMEM,Dulbecco’sModifiedEagle’smedium;DMSO,dimethylsulfoxide;ECL,enhancedchemiluminescence;EDTA,ethylenediaminetetraaceticacid;EtOAc,ethylacetate;FBS,fetalbovineserum;FITC,fluoresceinisothiocyanate;HCC,hepatocellularcarcinoma;HPLC,highperformanceliquidchromatography;HRESIMS,high-resolutionelectrosprayionizationmassspectrometry;HRMS,highresolutionmassspectrometry;HRP,horseradishperoxidase;HSD,honestsignificantdifference;IC50,halfmaximalinhibitoryconcentration;m/z,mass-to-chargeratio;MeOH,methanol;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide;NP-40,nonidetP-40;p53,protein53;PARP,poly(ADP-ribose)polymerase;PI,propidiumiodide;RPMI,RoswellParkMemorialInstitutemedium;SARs,structure-activityrelationships;SDS,sodiumdodecylsulfate;SEM,standarderrorofmean;SiO2,silica;TLC,thinlayerchromatography;TMS,tetramethylsilane;T-TBS,tween-trisbufferedsaline;WHO,WorldHealthOrganization.Correspondingauthor.Tel.:+351289800051;fax:+351289800051.E-mailaddress:jvarela@ualg.pt,joaocvarela@gmail.com(J. Varela).http://dx.doi.org/10.1016/j.phymed.2016.02.0080944-7113/© 2016 Elsevier GmbH. All rights reserved.
C.Vizetto-Duarteetal./Phytomedicine23(2016)550–557551IntroductionCancerisamajorpublichealthproblemwithanestimatedprevalenceofabout3%inEurope,increasingto15%atoldage.Moreover,cancerrelateddeathsareestimatedtoincreasetoover11millionin2030(WHO,2010).Hepatocellularcarcinoma(HCC)isthethirdleadingcauseofcancer-relateddeathworldwide,af-terlungandstomachcancer(Ferencietal.2010).ThecurrenttherapeuticsusedforHCCtreatmentinvolvessurgicalresection,transplantationand/orsystemicchemotherapy;however,surgeryandtransplantationmaynotbeappropriateformanypatientsandchemotherapyoftenfails(Liuetal.2014).Chemotherapyisalsoconstrainedbyitstoxicity,significantresistancetoavailablechemotherapeuticagentsandsideeffects,includingneutropeniaandmyelosuppression(Chauetal.2006).Currentstudiesinvolvedindevelopingeffectivecancerpreventionapproacheshavefocusedontheuseofbioactivenaturalagentsthatmayhavelessadverseeffectsandcanexertselectivecytoxicityagainstcancercells(Ghateetal.2014).Thechemicalandbiologicaldiversityofthemarineenviron-mentisimmeasurableandthereforeisanextraordinaryresourceforthediscoveryofnovelanticancerdrugs.Brownalgaearearichsourceofsecondarymetabolitesdisplayingawidevarietyofbioactivitieswithimportantfeaturesforpharmaceuticalpur-poses.Cystoseiratamariscifoliahasdemonstratedinterestingbi-ologicalactivitiessuchasantibacterial,antifungal,antiprotozoal,celldivisioninhibition,anti-inflammatory,antioxidantandcyto-toxicproperties(Bennamaraetal.1999;Spavierietal.2010;Lopesetal.2012;Andradeetal.2013).ThesepropertieshavebeenascribedtothepresenceofdifferentclassesofmoleculesthatwereidentifiedinC.tamariscifolia,suchasphlorotannins(fu-cophloroethol,fucodiphloroethol,fucotriphloroethol,7-phloroeckol,phlorofucofuroeckolandbieckol/dieckol),phloroglucinol,proline,β-sitosterol,fucosterol,anddiversefattyacids(Ferreresetal.2012;Andradeetal.2013;Vizetto-Duarteetal.2015).AsC.tamarisci-foliaextractshavepreviouslydemonstratedcytotoxicpotential,inthisstudywedescribetheidentificationofisololiolide,aknowncarotenoidmetabolite,asaselectivecytotoxiccompoundthatwasisolatedfromthebrownmacroalgaC.tamariscifoliaforthefirsttime.HereweshowevidencethatexposureofhepatocarcinomaHepG2cellstoisololiolideisassociatedwithchangesintheex-pressionofp53,PARP,Bcl-2andprocaspase-3.TheseresultsmightexplainthedramaticsuppressionoftheSphaseaswellasthein-ductionofapoptosiscausedbythismonoterpene.MaterialandmethodsChemicalsandreagentsHexaneandethylacetatewerepurchasedfromProlabo(VWRInternational,Leuven,Belgium).Merck(Darmstadt,Germany)sup-plieddimethylsulfoxide(DMSO).RoswellParkMemorialInstitutemedium(RPMI),Dulbecco’sModifiedEagle’smedium(DMEM),fe-talbovineserum(FBS),L-glutamineandpenicillin/streptomycinwereobtainedfromLonzaIbérica(Barcelona,Spain).3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)wasobtainedfromCalbiochem.Primaryantibodiesforpoly(ADP-ribose)polymerase(PARP),p53,Bcl-2,procaspase-3,actinandre-spectivesecondaryantibodieswerefromSantaCruzBiotechnol-ogyInc.,Heidelberg,Germany.FITC-conjugatedannexinV/pro-pidiumiodide(PI)assaykitwasacquiredfromCaymanChemi-calCompany,USA.Silicagel(Merck,40–63μmmesh)wasusedforcolumnchromatographicseparation,whilesilicagel60PF254(Merck)wasusedforanalytical(0.25mm)TLC.CDCl3(Aldrich)wasusedassolventfor1Hand13CNMRspectraacquisitionandTMS(Aldrich)wasusedasinternalstandard.1Dand2DNMRspec-trawererecordedatBrukerDigitalAvance800MHzspectrometer.AdditionalreagentsandnecessarysolventswerepurchasedfromVWRInternational(Leuven,Belgium).SamplingCystoseiratamariscifoliawascollectedinthemiddle/lowerinter-tidalareas,duringthelowtide,betweenMayandSeptember2012onthePortuguesecoast.Biomasswasrinsedwithdistilledwaterandmacroscopicepiphytesandextraneousmatterwerecarefullyremoved.IdentificationofspecimenswasmadebyDrAschwinEn-gelen(CentreofMarineSciences,UniversityofAlgarve,Portugal)andDrJavierCremadesUgarte(FacultadedeCiencias,UniversityofACoruña)andavoucherspecimenofC.tamariscifolia(codenum-berMB016)wasdepositedattheCentreofMarineSciences,Uni-versityofAlgarve.Sampleswerefreeze-driedandstoredat–20°Cuntiltheextractionprocedure.ExtractionBiomasswasmixedwithhexane(1:10,w/v)andhomogenizedfor2minusingadisperserIKAT10BUltra-Turraxatroomtem-perature(RT).Thetubeswerethenvortexedfor1min,centrifuged(5000g,10min,RT)andthesupernatantsrecovered.Theextractionprocedurewasrepeated3timesandthesupernatantscombinedandfiltered.Theextractwasdriedat40ºCundervacuumanddis-solvedinDMSOforbiologicalactivitiesscreeningorintheade-quatesolventforchemicalcharacterization,aliquotedandstored(–20°C).IsolationandelucidationofisololiolideC.tamariscifoliahexaneextract(9g)wasfractionatedbycol-umnchromatography(2.5cm×18cm)oversilicagel(SiO2)usingincreasingamountsofEtOAcinhexane(9:1;85:15;4:1;75:25;7:3;3:2;1:1)andincreasingamountsofMeOHinEtOAc(9:1;8:1;5:1;2:1;1:1),MeOH(100%)andH2O(100%)aseluents.Thisprocedureafforded57fractions,whichwereanalyzedbyTLCandpooledtogetherin21groups(A–U).Fraction14(70mg)wasre-fractionatedoverSiO2elutedwithhexane(100%);hexane/EtOAc(9:1,8:2,7.5:2.5,7:3,6.5:3.5,6:4,5.5:4.5,1:1,4:6),EtOAc(100%)andMeOH(100%)toafford151fractionswhichwerepooledto-getherin9groupsafterTLCanalysis.Group6–8,obtainedfromthehexane/EtOAcelution(6:4through1:1),waspurifiedbyre-versephasepreparativeHPLCtoafford3mgofisololiolide.Isololiolide:Paleyellowoil;1HNMR(800MHz,CDCl3,TMS,ppm)δ5.71(1H,s,H-7),4.21(1H,m,H-3),2.55(2H,brd,J=2.4Hz,H-4),2.03(1H,brd,J=2.4Hz,H-2),1.59(3H,s,H-11),1.23(3H,s,H-10),1.21(3H,s,H-9).13CNMRδ(200MHz,CDCl3,TMS,ppm):181.2(C-6),171.5(C-8),113.3(C-7),86.4(C-5),65.1(C-3),49.8(C-2),47.9(C-4),35.0(C-1),29.9(C-9),25.6(C-11),25.1(C-10);HRESIMSm/z219.0993[M+Na]+(calctoC11H16O3Na219.0997).CellcultureHepG2cells(humanhepatocellularcarcinoma)weremain-tainedinRPMI-1640culturemediasupplementedwithglucose(1000mg/ml),10%FBS,L-glutamine(2mM),penicillin(50U/ml)andstreptomycin(50μg/ml).MRC-5andHFF-1humanfibrob-lasts,AGShumangastriccancer,HCT-15humancoloncancercellsweregrowninDMEMculturemediasupplementedwithglucose(1000mg/ml),10%FBS,L-glutamine(2mM),penicillin(50U/ml)andstreptomycin(50μg/ml).Celllinesweregrowninanincuba-torat37ºCand5.0%CO2inhumidifiedatmosphere.