Fucan effect on CHO cell proliferation and migration

(1)

ContentslistsavailableatSciVerseScienceDirect

Carbohydrate

Polymers

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Fucan

effect

on

CHO

cell

proliferation

and

migration

Leonardo

Thiago

Duarte

Barreto

Nobre

a,b

_,

_Arthur

_Anthunes

_Jacome

_Vidal

a

_,

Jailma

Almeida-Lima

a

_,

_Ruth

_Medeiros

_Oliveira

a

_,

_Edgar

_Jean

_{Paredes-Gamero}

b

_,

Valquiria

Pereira

Medeiros

b

_,

_Edvaldo

_Silva

_Trindade

c

_,

_Celia

_Regina

_Cavichiolo

_Franco

c

_,

Helena

Bonciani

Nader

b

_,

_Hugo

_Alexandre

_Oliveira

_Rocha

a,∗

a_Departamento_de_Bioquímica,_Universidade_Federal_do_Rio_Grande_do_Norte_–_UFRN,_Av._Senador_Salgado_Filho,_3000,_Lagoa_Nova,_59072-970_Natal,_RN, Brazil

b_Disciplina_de_Biologia_Molecular,_Depto._de_Bioquímica,_Universidade_Federal_de_São_Paulo_–_UNIFESP,_Rua_Tres_de_maio,_100,₄◦_andar,_Vila_Clementino, SãoPaulo,SP,Brazil

c_Departamento_de_Biologia_Celular,_Universidade_Federal_do_Paraná_–_UFPR,_Curitiba,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received20March2013

Receivedinrevisedform17May2013 Accepted19May2013

Available online 25 May 2013

Keywords: Fucoidan Antiproliferative Cancer MAPKpathway

a

b

s

t

r

a

c

t

Fucanisatermusedtodenominatesulfatedl-fucoserichpolysaccharides.Here,aheterofucan,named fucanB,wasextractedfromtheSpatoglossumschröederiseaweed.This21.5kDagalactofucaninhibited CHO-K1proliferationandmigrationwhenfibronectinwasthesubstrate.FucanBderivativesrevealed thatsucheffectsdependontheirdegreeofsulfation.FucanBdidnotinducecelldeath,butpromotedG1 cellcyclearrest.WesternblottingandflowcytometryanalysissuggestthatfucanBbindstofibronectin andactivatesintegrin,mainlyintegrin␣5␤1,whichinducesFAK/RAS/MEK/ERKactivation.FAKactivation inhibitsCHO-K1migrationonfibronectinandERKblockscellcycleprogression.Thisstudyindicatesthat fucanBcouldbeappliedindevelopingnewantitumordrugs.

1. Introduction

Sulfatedfucanscompriseafamilyofpolydisperse polysaccha-ridescomposedofsulfatedl-fucoseandheterofucans,alsoknown

asfucoidans.Theyarenotwidespreadinnature,occurringonlyin brownseaweedandtunicates(Pomin,2010),therefore,seaweeds areitsmostimportantsource.Eachseaweedsynthesizesitsown fucanpossessing uniquestructuralcharacteristics,whichreflect intheirbiological,pharmacologicalandbiotechnological propri-eties.Furthermore,thesestructuralcharacteristicscanbealtered bybioticandabioticfactorstowhichtheseaweedisexposed,as wellasextractionandpurificationmethodsusedintheattempt oftheirobtention.Recentreviewsregardingseaweedfucanshave beenrestrictedtotheirstructuralcharacteristicsandprimary bio-logical/pharmacologicalactivities(Jiao,Yu,Zhang,&Ewart,2011;

Li,Lu,Wei,&Zhao,2008).

Severalfucansshowedantiproliferativeactivityagainst differ-enttypesofcellsbyblockingtheirpassageintheG1phaseofthecell

cycle(Moreauetal.,2006).However,inmostcases,the antiprolif-erativeactivityofseaweedfucansisassociatedwiththeirability toinduceapoptosisin severalcell lines.Apoptosisinduction is

∗Correspondingauthor.

E-mailaddresses:hugo@cb.ufrn.br,hugo-alexandre@uol.com.br(H.A.O.Rocha).

oftenrelated tocaspaseactivation(Aisaetal.,2005;Athukorala

etal.,2009;Yamasaki-Miyamoto,Yamasaki,Tachibana,&Yamada,

2009);however,otherproteinsinvolvedincellsurvivalpathways maybeaffectedbythepresenceofsulfatedpolysaccharides(SP) intheculturemedium.Arecentlypublishedstudyreporteda het-erofucan,extractedfromtheseaweedSargassumfilipendula,with pro-apoptotic activityin which such compound inhibited HeLa (adenocarcinomacervicalcell)cellproliferationbyinducing apo-ptosisthroughacaspase-independentmechanismwhereapoptosis is promoted mainly by mitochondrial release of an apoptosis-inducingfactor(AIF)intocytosol(Costaetal.,2011).

Thebrownseaweed,Spatoglossumschröederi,synthesizesthree heterofucansknownasfucanA(Almeida-Limaetal.,2011),fucanB andfucanC(Rocha,Bezerra,etal.,2005;Rocha,Moraes,etal.,2005). The21.5kDafucanBwaspurifiedanditsstructurewassuggested tocontainacentralcorecomposedof␤-(1-4)linkedgalactose, par-tiallysulfatedatC3.25%ofgalactoseresiduesarelinkedinC2to atetrasaccharideformedof3-sulfated␣-(1-4)linkedfucose,and oneortwononsulfated␤-(1-4)linkedxylose,atthereducingand nonreducingend,respectively(Rocha,Moraes,etal.,2005).Usinga biotinylatedfucanBasaprobe,itwasreportedthatitbinds specif-icallytofibronectin,therebyhinderingCHOcellsadhesiontothis matrixmolecule(Rocha,Franco,etal.,2005),yet,its antiprolifer-ativeactivitywasnotevaluated.Therefore,theaimofthepresent studywastoevaluateitsonCHOcellproliferation,cellcycleand

(2)

98 (2013) 224–232 225

cellmigration usingseveralsuitablemethods.Thisinvestigation mayprovideevidenceforthedevelopmentofapossiblealternative chemotherapeutictreatmentforcancer.

2. Materialsandmethods

2.1. FucanBextraction

ThemarinealgaeS.schroederiwascollectedontheseashoreof NisiaFloresta,RN,Brazil.Immediatelyaftercollection,thealgae wasdriedat50◦_C _under_ventilation_and_grounded_in_a_blender. Theseaweedwasthentreatedwithacetonetoeliminatelipidsand pigments.100gofdefatted,dried,andpowderedalgaewere sus-pendedin500mLof0.25MNaCl,andthepHwasadjustedto8.0 withNaOH.Prolav750(3mg/mL)(ProzynBiosolutions,SãoPaulo, SP,Brazil),amixtureofalkalineproteases,wasaddedtothemixture forproteolyticdigestion.After18hofincubationat60◦_C_with con-stantagitation,themixturewasfilteredandthefiltratefractionated byacetoneprecipitationasfollows:0.5volumeofice-coldacetone wasaddedtothesolutionundergentleagitationandmaintained at4◦_C_for₂₄_h._The_precipitate_formed_was_collected_by centrifuga-tion(10,000×g,20min),driedundervacuum,dissolvedindistilled water,andanalyzed.Theoperationwasrepeatedbyadding0.6, 0.7,0.9,1.1,1.3,and2.0volumesofacetonetothesupernatant. Thefractionprecipitatedwith0.9volumeofacetonecontainsthe sulfatedfucanBused in thepresent work.This polysaccharide wasfurtherpurified byionexchangechromatography(Lewatite fromBayer,SãoPaulo,Brazil)withincreasingconcentrationsof NaCl(0.25–3.0M).Theeluateswereprecipitatedwith2volumesof methanol(18h,4◦_C)_being_the_precipitates_collected_by centrifu-gation(10,000×g,15min),dried,anddissolvedindistilledwater forfurtheranalysis.

2.2. Agarosegelelectrophoresis

Agarosegelelectrophoresisoftheacidicpolysaccharideswas performed in 0.6% agarose gel (7.5cm×10cm×0.2cm thick) prepared in 0.05M 1,3-diaminopropane acetate buffer pH 9.0, as previously described (Dietrich & Dietrich, 1976). Aliquots of the polysaccharides (about 50␮g) were applied to the gel and subjected to electrophoresis. The gel was fixed with 0.1% cetyltrimethylammonium bromide solution for 2h, dried, and stainedfor15minwith0.1%toluidinebluein 1%aceticacidin 50%ethanol.Thegelwas,then,destainedwiththesamesolution withoutthedye.

2.3. Chemicalanalysisandmonosaccharidecomposition

Totalsugars,sulfate,proteinandglucuronicacidwere deter-minedaspreviouslydescribed(Rocha,Moraes,etal.,2005).

Thepolysaccharideswerehydrolyzedwith2MHClfor2hat 100◦_C_and_sugar_composition_was_determined_using_a_LichroCART® 250-4column(250mm×40mm)packedwithLichrospher® ₁₀₀ NH2(5␮m)coupledtoaLaChromElite®_HPLC_system_from VWR-Hitachi equipped witha refractive index detector (RI detector modelL-2490).The samplemass usedwas0.2mg andanalysis timewas25min.Thechromatogram wascompared tothoseof sugar references:arabinose, fructose, fucose,galactose,glucose, glucosamine,mannose,andxylose.

ThemolecularweightofthefucanswasdeterminedbyHPLC in0.2MNaCl,0.5%ethanol,usingaGF-250column(AsahipakGF series,AsahiChemicalIndustryCo.,Yakoo,Japan).Thecolumnwas calibratedwithstandardglycosaminoglycans,asdescribedearlier

(Rocha,Moraes,etal.,2005).

2.4. Fouriertransformedinfraredspectroscopy(FT-IR)

Sulfatedpolysaccharides(10mg)weremixedthoroughlywith drypotassium bromide.A pelletwaspreparedand theinfrared spectrumbetween500and4000cm−1_was_measured_on_a

Thermo-NicoletNexus470ESPFT-IRspectrometer.Thirty-twoscanswith aresolutionof4cm−1_were_averaged_and_referenced_against_air.

2.5. Unspecificfucandesulfation

Polysaccharide desulfation was performed by solvolysis in dimethylsulfoxideasusedpreviouslyfordesulfationofasulfated fucans(Rocha,Moraes,etal.,2005).Usingdifferentreactiontimes, 1hand5h,twodesulfatedfucanB;FucB2.0M1handFucB2.0M 5h,containing15.0%and7.6%ofsulfategroupsrespectively,were obtained.

2.6. Cellculture

Chinesehamsterovary(CHO)cells(ATCCCCL-61)weredonated byProf.JeffreyD.Esko(GlycobiologyResearchandtrainingCenter, UCSD,SanDiego,California,USA).ThecellsweregrowninF-12 medium(Invitrogen,Carlsbad,CA,USA)supplementedwith10% fetalbovineserum–FBS(Cutilab,Campinas,SP,Brazil)and peni-cillin(100U/mL),andstreptomycin(0.1mg/mL)(Sigma-Aldrich,St Louis,MO,USA),andmaintainedat37◦_C_in_2.5%_CO

2.

2.7. MTTassay

ForMTTassay,cellswereseededinto96-wellplatesata den-sity of 5×103_cells/well _and _allowed _to _attach _overnight _in _a

300␮Loffreshmedium,incubatedat37◦_C,_2.5%_CO

2.Cellswere,

then,incubatedfor24hat37◦_C_and_2.5%_CO₂ _in_the_presence_of differentconcentrations(0.1–0.5mg/mL)ofFucB.After incuba-tion,tracesofsulfatedpolysaccharidefractionswereremovedby washingthecellstwicewith200␮LPBSandapplying100␮Lof freshmedium,aswellas10␮Lof12mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)dissolvedinPBS,to determinetheeffectofalgalsulfatedpolysaccharidesoncell viabil-ity.Cellswere,then,incubatedfor4hat37◦_C,_2.5%_CO₂_._In_order_to solubilizetheproductofMTTcleavage,100␮Lofisopropanol con-taining0.04MHClwasaddedtoeachwellandthoroughlymixed usingamultichannelpipette.Within1hofHCl–isopropanol addi-tion,absorbanceat570nmwasmeasuredusingaMultiskanAscent MicroplateReader(ThermoLabsystems,Franklin,MA).

2.8. BrdUincorporation

Cells were seeded into 96-well plates at a density of 5×103_cells/well_and_allowed_to_attach_overnight_in₃₀₀_␮_L_of_fresh

mediumandincubatedat37◦_C,_2.5%_CO₂_._In_some_cases_the_cells wereaddedtofibronectinorvitronectincoatedwells.After12h, themediumwasremovedandthefucanBinF12mediumwas added toa final concentration of 0.1–0.5mg/mL and incubated for24h,at37◦_C_and _2.5%_CO

2.Afterincubation,tracesoffucan

Bwereremovedbywashingthecellstwicewith200␮LPBSand theBrdUincorporationwasdeterminedaccordingtothe manufac-turer’sinstruction(BrdUcellproliferationassaykit–CellSignaling, Danvers,MA,USA).

2.9. AnnexinV-FITC/pidoublestainingandanalysisbyflow cytometry

(3)

226 98 (2013) 224–232

Cellsweregrownin6-wellplatesuntiltheyreachedconfluence of2×105_cells/mL_when _they_were_stimulated_to_enter _G

0 in a

mediumwithoutserumfor 48h. Next,cells weretoexitG0 by

addingF-12medium supplemented10%FBS,inthepresenceof fucanB(0.5mg/mL).Anegativecontrolwaspreparedwithoutthe presenceoffucanB.After24h,thecellswereharvestedusing2mM EDTAPBS,collectedandwashedwithcoldPBS.Thesupernatant wasdiscardedandcellswereresuspendedin200␮Lof1×binding buffer.5␮LofAnnexinV-FITCand1␮LofPIsolution100␮g/mL wasaddedin100␮Lofcellsuspension.Cellswereincubatedfor 15minatroomtemperatureandkeptunderlightprotection.After theincubationtime,400␮LofbindingbufferforannexinV(1×) wasaddedandcellswhichwere,then,analyzedbyflowcytometry (flowcytometerFACSCANTOII,BDBiosciences),measuring fluo-rescenceemissionat530–575nmforannexinVand630/22nmfor PI.FlowJosoftwarev.7.6.3(TreeStar,Inc.,CA,USA)wasusedfor dataanalysis.

2.10. Expressionof˛5integrinsubunit

CHO-K1cellsweregrowninthepresenceoffucanB(0.5mg/mL) for24h,as describedabove.Cells wereremovedfromthe bot-tleswith2mMEDTAPBSfor10min,and106_cells_were_suspended

andwashed3timeswithPBScontaining1%BSA.Thecell suspen-sionswere,then,incubatedwithMABanti-␣5antibody(2␮g/mL) inPBS+1%BSAfor1hat4◦_C,_washed_with_PBS_and_incubated_with thesecondaryantibody(fluoresceinisothiocyanate-conjugategoat antimouseIgG)for30minat4◦_C._Cells_were_washed₃_times_with theFACSsolution,asrecommendedbythemanufacturer. Anal-ysesof40,000eventswereperformedusingaFACSCANTO flow cytometer(BectonDickinsonImmunocytometrySystem,CA,USA).

2.11. EffectoffucanBontheCHOcellssulfated glycosaminoglycanssynthesis

FucanBeffectonthesynthesisofheparansulfate and chon-droitin sulfate by CHO-K1 cells was performed essentially as previouslydescribed(Rocha,Bezerra,etal.,2005).Cellproteinwas estimatedbyaCoomassieBluemethod.Alltheexperimentswere performedintriplicateforeachdatapoint.Thebarsofthefigures indicatethe±SD.

2.12. Westernblotting

CHOcellsat80%confluencewereincubatedwithfucanBand washed after 12, 24 and 48hin ice-cold PBS. They were then scrapedinto200␮Llysisbuffer[50mMTris–HCl(pH7.4),1%Tween 20,0.25%sodiumdeoxycholate,150mMNaCl,1mMEDTA,1mM Na3VO4,1mMNaF],andproteaseinhibitors(1mg/mLaprotinin,

10mg/mLleupeptinand1mM4-(2-aminoethyl)benzenesulfonyl fluoride)and,then,keptonicefor2h.Proteinextractswerecleared by centrifugation and protein concentrations were determined usinga BCAprotein assaykit(Pierce, USA), withbovineserum albuminasstandard.Anequalvolumeofsodiumdodecylsulfate (SDS)gelloadingbuffer[100mMTris–HCl(pH6.8),200mM dithio-threitol(DTT),4%SDS,0.1%bromophenolblueand20%glycerol] wasaddedtosamples,whichweresubsequentlyboiledfor10min. Fromeachsample,50␮gofproteinwasloadedontoSDS-PAGEand blottedontoPVDFmembranes(Millipore,Bedford,MA,USA). Mem-braneswereblockedin1%fat-freedriedmilkor2%bovineserum albumininTris–bufferedsaline(TBS),with0.05%Tween20(TBST), andincubatedovernightat4◦_C_with_the_appropriate_primary anti-bodyat1:1000dilution.AfterwashinginTBST,membraneswere incubatedwithanti-rabbithorseradishperoxidase-conjugated sec-ondaryantibodies,at1:2000 dilution,inblockingbufferfor1h. Intensityofthespecificimmunoreactivebandswasdetectedby

enhanced chemiluminescence (ECL),according tothe manufac-turer’sprotocol(KirkegaardandPerryLaboratories),quantifiedby densitometryandexpressedasaratioofactin.

2.13. Motilityassay

Themotilityassaywasconductedasdescribedby(Francoetal., 2009).Briefly,haptotacticmotilityexperimentswereperformed usingtranswellmotilitychambers(8␮mporesize)(CostarCorp.). Theundersurfaceofthepolycarbonatemembranewascoatedwith fibronectin(10␮g/mLinPBS).Aninsolublesolid-phasegradient wasestablishedbyfloatingthefilters(40minat37◦_C),_which_were thenblockedwith1%BSA (1h, 37◦_C)._Cells₍₅_×₁₀5₎_were

sus-pendedin100␮LofF-12mediumandaddedtotheupperchamber inthepresenceorabsence(control)ofdifferentconcentrationsof fucanB,incubatedat37◦_C_in_a_humid_atmosphere_of_2.5%_CO₂_for 5h.Followingincubation,cellsontheuppersurfacewereremoved usingacotton-tippedapplicator.Membraneswerefixedwith2% formaldehyde for10minand stainedwith1%toluidine bluein Boraxfor10min.Thecellswhichmigratedwerevisualizedusing aninvertedmicroscope(Nikon,TE-300)andcounted.

2.14. Statisticalanalysis

Alldatawereexpressedasmean±standarddeviation. Statis-ticalanalysiswasdonebyone-wayANOVAusingthePrisma5.01 software.Tukeypost-testswereperformedformultiplegroup com-parison.Inallcases,statisticalsignificancewassetatP<0.05.

3. Resultsanddiscussion

3.1. PurificationandcharacterizationoffucanB

Fucans from S. schröederi were extracted using proteolysis and submittedtoprecipitation withdifferentconcentrations of acetone.Thefractionsunderwentelectrophoresisonagarosegel (Fig.1A),indicatingthatthefractionobtainedwith90%ofacetone (v/v),denominatedF0.9,wasafucanB-richfraction,aspreviously reported(Rocha,Moraes,etal.,2005).ThefucanBfromfraction F0.9wasfurtherpurifiedusingionexchangechromatographyona Lewatiteresin.Sulfatedpolysaccharideswereseparatedintofour newfractionselutedwith1.0,1.5,2.0,and3.0MNaCl.Thesewere namedF1.0M,F1.5M,F2.0M,andF3.0M,respectively.The com-poundsweresubmittedtoelectrophoresisonagarosegeland,as showninFig.1B,onlyasinglebandwasobtainedinF1.0M,F1.5M, andF2.0M.Electrophoreticmobilitiesofthesebandscorrespond tofucanBelectrophoreticmobility,aspreviouslyreported(Rocha,

Moraes,etal.,2005).Thisindicatesthatweobtainedthreefractions

offucanB.FractionF3.0MwasrichinfucanC(datanotshown). Thechemical composition of thedifferentfucanBfractions, F1.0M,F1.5MandF2.0M,isshowninTable1.Allofthemare com-posedofxylose,fucose,galactose,sulfateandatraceofglucuronic acid.ThemonosaccharidemolarratiosofF1.0M,F1.5M,F2.0Mare verysimilar.Themaindifferenceintheirchemicalcompositionis theproportionofsulfate,whichrangedfrom14.1%(F1.0M)to19.0 (F2.0M).

FT-IRspectroscopyisaneasy-to-usetechniqueandprovidesfast results.Itisalsoapowerfultoolforshowingsimilaritiesbetween molecules.Table2illustratesthemainsignalsobservedintheFT-IR spectraoffucanBfractionsfromS.schröederi.

Characteristic sulfate absorptions wereidentified in the FT-IR spectra of fucan B fractions: signals around 1257cm−1 _are

causedprimarilybyasymmetricS Ostretchingvibration(Pielesz

&Binias, 2010), while those atapproximately 1043–1046cm−1

(4)

98 (2013) 224–232 227

Fig.1. (A)Electrophoresisin0.05Mdiaminopropaneacetatebuffer,pH9.0,offractionsobtainedbyacetoneprecipitation.(B)FucansBobtainedthroughionexchange chromatography.About5␮L(50␮g)ofeachpolysaccharidewasappliedinagarosegelpreparedindiaminopropaneacetatebufferandsubjectedtoelectrophoresis,as describedinSection2.F0.5;F0.6;F0.7;F0.9;F1.1;F1.3;F2.0–fractionsobtainedbyacetoneprecipitation.F1.0M;F1.5M;F2.0M–fucanBfractionsobtainedfromion exchangechromatography.Or.–origin.

Table1

ChemicalcompositionoffucansfrombrownseaweedS.schröederi.

Fucans Yield(%) Sulfate(%) Protein(%) Molarratio

Fucose Galactose Mannose Xylose Glucuronicacid

F1.0M 32.9 14.1 nd 1.0 0.5 0.1 0.3 >0.1

F1.5M 18.1 17.8 nd 1.0 1.9 0.0 0.5 >0.1

F2.0M 38.8 19.0 nd 1.0 2.0 0.0 0.5 >0.1

FucB2.0M1h – 15.0 nd 1.0 2.0 0.0 0.5 >0.1

FucB2.0M5h – 7.5 nd 1.0 1.8 0.0 0.5 >0.1

nd–notdetected;FucB2.0M1handFucB2.0M5h–desulfatedfucansB(seeSection2)

confirmingthepresenceofasignificantamountofsulfateinthe polysaccharides. The bandat 848cm−1 _is _caused _by _the

bend-ingvibrationofC O SofsulfategroupsattheaxialC-4position

(Pielesz&Binias,2010).TheIRfeaturesat583cm−1_are_the_result_of

symmetricO S Odeformationofsulfates(Synytsyaetal.,2010).In addition,allfractionsshowedsignalsat3446–3447cm−1_from_the

stretchingvibrationofO H(Camaraetal.,2011).Thebandaround 1632–1645cm−1_was_caused_by_the_carboxyl_group_of_uronic_acid,

whichoverlapswithsignalsofH2Odeformation(Camara etal.,

2011; Pielesz &Binias, 2010). The bandat about 1420cm−1 _is

causedbysymmetricCH2deformationofgalactoseandasymmetric

deformationofCH3fromfucose(Pielesz&Binias,2010).

Themolecularweight offucanBwascalculated as21.0kDa (F1.0M),21.5kDa(F1.5M)and21.5(F2.0M)usingHPLCanalysis.

Overall,thechemistry,FT-IRspectrum,andmolecularweight ofthefucanBF2.0Misolatedherewerethesameasthosealready reportedforfucanB(Rocha,Moraes,etal.,2005),suggestingthat weobtainedthesamefucanBaspreviouslydescribed.Inaddition,

Table2

IRspectrumdataoffucanBfractionsfromthebrownseaweedS.schröederi.

Sulfatedpolysaccharides IRsignals(cm−1₎

F1.0M 3446;2034;1632;1420;1257;1043;848;582 F1.5M 3445;2031;1645;1421;1257;1045;848;583 F2.0M 3447;2031;1645;1420;1257;1045;848;583

datashowedthatF1.0MandF1.5MareBfucanswithlesssulfate content.

3.2. AntiproliferativeactivityoffucansBagainstCHOcells

InordertoevaluatetheantiproliferativeeffectoffucanB,we

selectedwild-typeCHO-K1andmutantCHO-745cells,sincethe

glycosaminoglycans(GAG)producedbymutantcellswereabout

5%ofthosesynthesizedbyCHO-K1(Francoetal.,2009).However, thisdeclineinGAGdidnotaffectCHO-745proliferation,whichis similartothatoftheCHO-K1cells(Francoetal.,2001).Therefore, sincetheeffectoffucanBwassimilarinbothcelllines,wecanrule outthepossibilitythatthisoccursduetorepulsionbetweenfucans andGAGcontentofthecellsurface.Ourdatashowedthatfucans B1.0M,1.5Mand2.0Marethesamecompound,withdifferent degreesofsulfation(Tables1and 2).AsshowedinFig.2,fucan B2.0Mdisplayedadose-dependenteffect,reachingsaturationat around0.4mg/mLforbothcelllines.Thus,usingaconcentrationof 0.4mg/mL,wecomparedtheantiproliferativeeffectofthreefucans B.Fig.2alsodemonstratesthat,regardlessofthedegreeofsulfation, allfucansBexhibitantiproliferativeactivityatthesameextension. Duringthetreatmentperiod,nocellmorphologyalterationswere observed.SincefucanB2.0Mshowedthehighestyield(Table1),it wasselectedforsubsequenttestingandfromhereafteritisreferred toasfucanBorFucB.

(5)

228 98 (2013) 224–232

Fig.2. (A)EffectoffucanBonCHOcellproliferation.Theresultsarepresentedas percentageofcontrolcellsnormalizedto100%intheabsenceoffucanB(mean±SD offourdeterminations).(B)EffectoffucanBfractionsanddesulfatedfucansB(FucB 2.0M1heFucB2.0M5h)onCHO-K1proliferation.(C)EffectoffucanBonCHO-K1 growingoncoatedsurfacewithextracellularmatrixproteins.Therateofcell prolif-erationinhibitionwasdeterminedusingBrdU.Resultsareexpressedaspercentage ofcontrolcellsnormalizedto100%intheabsenceofpolysaccharide(mean±SDof fourdeterminations).***P<0.001comparedtocontrol.a_P_<_0.05_compared_to_other concentrations.b_P_<_0.01_compared_to_other_{concentrations.}c_P_<_0.001_compared_to otherconcentrations.d_P_<_0.001_compared_to_other_fucans.

position,typeofsugarandglycosidicbranching.Structural analy-sesofoligofucansobtainedfromfucoidanofAscophyllumnodosum showedthatanticoagulantactivityrequiressulfationatallO-2and someO-3positions offucoseresidues,whereassulfationatO-4 seemsunnecessary.Inaddition,3-and/or4-linkedfucoseresidues arealwayspresent(Chevolotetal.,1999).However,thereareno dataregardingsulfationpositionforantiproliferativefucans.When fucansfromA.nodosumweredesulfated,theylostanticoagulant activity,althoughtheirabilitytoinhibittumorcellproliferationwas notaffected(Haroun-Bouhedja,Ellouali,Sinquin,&Boisson-Vidal, 2000).Inotherwords,thedegreeofsulfationisnotasimportantto Ascophyllumfucanantiproliferativeactivityassulfationposition.

Inordertodeterminetheroleofsulfategroupsinthe antipro-liferativeeffectoffucanB,weuseddesulfatedfucanB2.0M1h andFucB2.0M5h(see Section2),containing15%and 7.5%of sulfategroups,respectively.Dataindicated(Fig.2B)thatnoneof thedesulfatedpolysaccharidesdisplayedantiproliferativeactivity. ThisisaninterestingresultbecauseFucB1.0Mcontains14.1% sul-fategroups,lessthanfucanB2.0M1h.Nevertheless,thesmaller amountofsulfatedidnotaltertheantiproliferativeactivityofFucB 1.0MincomparisontootherfucansB.Sincethedesulfationmethod appliedinthisstudyremovesthesulfategroupsnonspecifically, specificrelationshipbetweensulfatepositionandbioactivity can-notbemade.Inaddition,whenwecomparedthemonosaccharide compositionofdesulfatedfucanswiththenativefucanwedidnot findanydifferencebetweenthem(Table1).Thedataindicatethat thesulfationposition,asobservedforAscophyllumfucans,ismore importantthanthedegreeofsulfationforfucanB antiprolifera-tiveactivity.Furtherinvestigationusingmorespecificdesulfation methodswillhelptodeterminetheroleofsulfationpositionon fucanBantiproliferativeactivity.

Fucans can bind several growth factors and also affect cell metabolism(Irhimeh,Fitton,Ko,Lowenthal,&Nordon,2011).Thus, fucanBwasincubatedwithCHO-K1cellsanddifferentFBS con-centrationstoidentifywhethertheeffectoffucansdependsonthe interactionoffucanandFBSmolecules.Wefoundthattheamount ofFBS didnotaffectfucanBantiproliferativeactivity(datanot shown).

(6)

98 (2013) 224–232 229

3.3. FucanBbindsfibronectinandinhibitsCHOcellproliferation

Previously,confocalmicroscopyand flow cytometryshowed that FucBbinds the extracellular matrix of CHOcells, but not theCHO cellsurface (Rocha,Bezerra, et al., 2005).In addition, fucansfromA.nodosumareabletobindfibronectinandaffectthe cytoskeletonofMDA-MB-231cells.Infact,thereare,atleast,four bindingsitesalongthefibronectinchainforfucans,twoofwhich arealsoheparin-bindingsites(Liuetal.,2005).Thus,inorderto identifywhethertheFucBmoleculartargetisfoundinthe extra-cellularmatrix,CHO-K1cellswereseededintowellscoatedwith fibronectin(FN),collagenI,collagenIV,laminin(LN)orvitronectin (VN).FucanB(from0.01mg/mLto1mg/mL)wasthenaddedand cellswerestimulatedtogrow.

Whencellswerecoated ontoLN orcollagens,FucBdidnot showed antiproliferative activity (datanot shown), although it acted as an antiproliferative molecule when VN and particu-larly FN were used as substrate for cell proliferation (Fig. 2C). Indeed, Fuc B inhibits around 60% of CHO-K1 cell prolifera-tion, which is greater than the effect observed when using

plastic assubstrate.DataindicatethatFucBbindsextracellular matrixproteins,mainlyFN,andactsasanantiproliferative com-pound.

FNcontainsabindingsiteforproteoglycan-mediatedcell

adhe-sion(Dreyfussetal.,2009),aswellasanArg-Gly-Asp(RGD)motif,

whichefficientlyactsasthebindingsiteforintegrin-mediatedcell adhesion(Kim,Turnbull,&Guimond,2011).The␣5␤1integrinand cellsurfaceproteoglycans,suchassyndecanIV,arethemajorFN adhesionreceptors.Thesecooperateinsignalingevents,including cellproliferationandcelldeath.Moreover,wepreviously demon-stratedthatthe␣5␤1integrininCHOcellsisahybridproteoglycan, containingboth heparanandchondroitin sulfatechains(Franco

etal.,2009).

TheantiproliferativeeffectoffucanBmayberelatedtoreduce FNcellsurfacereceptors.Toruleoutthispossibility,CHO-K1cells weregrownfor24hinthepresenceorabsence(control)ofFucB. Next,weappliedflowcytometryandfoundnodifferencebetween theexpressionof␣5integrinsubunitonCHOcellsgrowninthe presenceorabsenceofFucBfor24h(Fig.3A).Anothersetof exper-imentsshowedthatlevelsofheparanandchondroitinsulfateof

(7)

230 98 (2013) 224–232

Fig.5. (A)FlowcytometryanalysisofapoptoticdeathofCHO-K1cellsbyfucanB.OnerepresentativeFACSassayofthreeindependentexperimentsispresented.Annexin−/PI− (Q4),viablecells;Annexin+/PI−(Q3),cellsundergoingapoptosis;Annexin+/PI+(Q2),cellsinend-stageapoptosisoralreadydead;Anexin−/PI+(Q1),cellsinnecrosis.(B) EffectoffucanBonCHO-K1cellcycle.CHO-K1cellsweregrownto50%confluence,treatedfor24hwithfucanB,harvestedandfixedwithparaformaldehyde,thenstained withPIandanalyzedusingflowcytometry.

CHO-K1cellsdidnotchangewhenthesecellswereincubatedwith FucB(Fig.3B).

3.4. FucanBbindsFNandactiveMAPKs

Cell-matrixcommunicationoccursmainlythroughthebinding ofmatrixproteinstoreceptorspresentonthecellsurface,andinthe caseofthefibronectin,itoccurswithmembraneintegrins.Thus,the nextstepwastoinvestigatewhichproteinsresponsiblefor intracel-lularsignalingcouldbeaffectedbyFucB.Thefirsttobeinvestigated wasfocaladhesionkinase(FAK),sinceitiscloselyassociatedwith integrins(Telcietal.,2008).Westernblotanalysisdemonstrated thattreatingCHOcellswithFucBinducedsuperiorexpressionof focaladhesionkinase(FAK)(Fig.4AandB).Thehighestlevelof expressionofFAKoccurredwith12hofexposuretoFucB,which wasdecreasedtomatchthecontrolat48h.

ActivatingFAKleadstoactivationofseveralintracellular pro-teins(Telcietal.,2008),amongthem,proteinkinaseC(PKC).After exposuretoFucB,cellsexhibitedactivationofPKC(Fig.4CandD). However,whenthecellswereincubatedwithFucBandaspecific PKCinhibitor,theantiproliferativeeffectofFucBwasnot signifi-cantlyaffected,despiteshowingatendencytodecrease(Fig.4E).

FAKcanpromoteactivationoftheRasprotein,leadingto activa-tionoftheMAPK/ERKpathway,animportantcellsurvivalpathway. Thus,usingWesternblotanalysis,wefoundthattreatingCHOcells

withFucBinducesactivationofRas(Fig.4FandG).Asobserved withFAK,thehighestlevelsofRASactivationwereobservedafter 12hofexposuretofucan.Following48hoftreatmentwithFucB, RASlevelsbecamesimilartothoserecordedincellsnotexposedto FucB.

ThenextproteinanalyzedwasERK.Fig.4HandIshowsthat cellsexposedtoFucBexhibitedthesamepatternofERKexpression observedforFAKandRAS.AhighamountofERKwasrecordedafter 12hofincubationwithFucB,whichdecreasedtime-dependently. RASoccasionally regulatestheactivityofdownstream effec-torssuchasMEKandERK.Therefore,aspecificMEKinhibitorwas usedtoassesswhethertheantiproliferativeeffectofFucBwas dependentonthisprotein.CHOcellswereexposedtoFucBandthe MEKinhibitor(PD98059)for24h.Next,cellproliferationwas ana-lyzedusingBrdUincorporation.DatademonstratedthattheMEK inhibitorabolishedtheFucBeffect(Fig.4J).

3.5. EffectoffucanBonCHOcellcycle

Data showed that the antiproliferative effect of fucan B is dependent on activation of the FAK/RAS/MEK/ERK cellular signalingroute.Severalstudiesshowthatfucanscaninducecell deathbystimulatingthispathway(Kimetal.,2011;Kim,Lee,Choi,

&Moon,2010;Li etal.,2008; Liuet al.,2005;Nabeyrat, Jones,

(8)

98 (2013) 224–232 231

Fig.6. (A)MotilityonfibronectinofCHO-K1cellsexposedtofucanB.Cellsontheothersideofthemembranewerestainedwith1%toluidineblueandcounted.Resultsare expressedasmean±SDofthreedeterminations.(B)CHO-K1transwellmigrationat40␮g/mLand200␮g/mLoffucanB.Cellsshowpurpleordarkbluecolor.

insightonthemechanismofFucBinducedcelldeath,cellswere incubatedwithFucB,stainedwithannexin-V(apoptosisindicator) andiodidepropidium(necrosisindicator)andsubmittedtoflow cytometryanalysis.AsseeninFig.5A,FucBdidnotincreasethe percentageofiodidepropidiumorannexinV-positivecellsin com-parisontothecontrol.ThisdatasuggestthatFucBdoesnotinduce celldeath.

InordertoestablishtheeffectofFucBonCHOcellcycle pro-gression,flowcytometryanalysiswasperformedoncellstreated withFucB.Administrationoffucanpromoteda20%increaseinthe proportionofcellsintheG1phaseandacorrespondingdecrease

intheproportionofcellsintheG2/Mphase(Fig.5B).Otherfucans

inhibitcellproliferationbyblockingthecellcycle.Forinstance, fucansfromtheseaweedsTurbinariaornata(Deslandesetal.,2000) andBifurcariabifurcatainhibitlungcarcinoma(NSCLC-N6) prolif-erationbyblockingitspassageintheG1 phaseofthecellcycle

(Moreauetal.,2006).Thiseffectwasalsoobservedwithsulfated

fucansfromtheseaweedA.nodosum,whichinhibitproliferationof NSCLC-N6celllinesbyblockingcellgrowthintheG1phase(Riou

etal.,1996).However,themolecularmechanismsofthesefucans asblockingcellcyclecompoundsareyettobedetermined.

3.6. EffectoffucanBonCHOcellmigration

Integrinsinteractwiththeextracellularmatrix,initiating sev-eralintracellularpathwaysthatplayimportantrolesduringmany processes,includingcellproliferation,differentiationand

migra-tion(Schaller,2010).TheFAKpathwayisinvolvedintheregulation

ofcellmotility.PublisheddataregardingtheroleofFAKincell migration are intriguing, given that activationof this molecule istypicallyassociatedwithincreasedmigration(Schaller,2010). However,severalstudiesshowthatphosphorylationofFAKalso inhibits migration (Borensztajn, Peppelenbosch, & Spek, 2010;

Schaller, 2010).Since FucBincreases FAKphosphorylation,we

investigatedwhetherFucBcouldregulatetheCHO-K1cell migra-tiononfibronectin.Fig.6indicatesthatFucBinhibitscellmigration dose-dependently,reachingsaturationataround0.1mg/mL. How-ever,whenlamininwasthesubstrate,noeffectwasobserveduntil 1mg/mLofFucB(datanotshown).

Anumberofstudieshaveshownthatfucansinhibitendothelial cellmigrationbybindingtoseveralgrowthfactors(Girauxetal.,

1998;Lakeetal.,2006).However,sincewedidnotusefetalbovine

seruminthecellmigratorytest,thispossibilitywasruledout.Thus, wehypothesizedthatFucBbindsfibronectinandactivates integ-rin,leadingtodecreasedcellmigration.Thiseffectisrelatedtothe abilityofthepolysaccharidetopromotephosphorylationofFAK.

4. Conclusion

ThisstudyevaluatedthefucanBeffectonCHO-K1cell prolifer-ationandmigration.FucanBexhibitedadose-dependenteffect, which depended on its degree of sulfation. Data suggest that fucanBbinds fibronectinand activatesintegrin, which induces FAK/RAS/MEK/ERKactivation.ERKblocks cellcycleprogression. FAKactivationalsoinhibitsCHO-K1migrationonfibronectin.This informationcouldhelpthedevelopmentofnewantitumoragents. Furthermore,ourdataindicatethatfurtherstudiesareneededto evaluatetheinvivoeffectoffucanB.

Acknowledgments

(9)

232 98 (2013) 224–232

References

Aisa,Y.,Miyakawa,Y.,Nakazato,T.,Shibata,H.,Saito,K.,Ikeda,Y.,etal.(2005). FucoidaninducesapoptosisofhumanHS-sultancellsaccompaniedby activa-tionofcaspase-3anddown-regulationofERKpathways.AmericanJournalof Hematology,78(1),7–14.

Almeida-Lima,J.,Dantas-Santos, N.,Gomes,D. L.,Cordeiro,S.L.,Sabry,D. A., Costa,L.S.,etal.(2011).Evaluationofacuteandsubchronictoxicityofa non-anticoagulant,butantithromboticalgalheterofucanfromthe Spatoglos-sumschroederi in Wistar rats.Revista Brasileira DeFarmacognosia, 21(4), 674–679.

Athukorala,Y.A.,Jee,G.N.,Kim,Y.,Kim,G.,Ha,S.,Kang,J.,etal.(2009). Antiprolif-erativeactivityofsulfatedpolysaccharideisolatedfromanenzymaticdigest ofEckloniacavaontheU-937cellline.JournalofAppliedPhycology,21(5), 307–314.

Borensztajn,K.,Peppelenbosch,M.P.,&Spek,C.A.(2010).CoagulationFactorXa inhibitscancercellmigrationviaLIMK1-mediatedcofilininactivation. Throm-bosisResearch,125(6),e323–e328.

Camara,R.B.,Costa,L.S.,Fidelis,G.P.,Nobre,L.T.,Dantas-Santos,N.,Cordeiro,S.L., etal.(2011).HeterofucansfromthebrownseaweedCanistrocarpuscervicornis withanticoagulantandantioxidantactivities.MarineDrugs,9(1),124–138. Chevolot,L.,Foucault,A.,Chaubet,F.,Kervarec,N.,Sinquin,C.,Fisher,A.M.,etal.

(1999).Furtherdataonthestructureofbrownseaweedfucans:Relationships withanticoagulantactivity.CarbohydrateResearch,319(1–4),154–165. Costa,L.S.,Telles,C.B.,Oliveira,R.M.,Nobre,L.T.,Dantas-Santos,N.,Camara,R.B.,

etal.(2011).HeterofucanfromSargassumfilipendulainducesapoptosisinHeLa cells.MarineDrugs,9(4),603–614.

Deslandes,E.,Pondaven, P.,Auperin,T.,Roussakis,C.,Guézennec,C.,Stiger,J., etal. (2000).Preliminarystudy oftheinvitroantiproliferativeeffectofa hydroethanolicextractfromthesubtropicalseaweedTurbinariaornata(Turner) J.Agardhonahumannon-small-cellbronchopulmonarycarcinomaline (NSCLC-N6).JournalofAppliedPhycology,12(3),257–262.

Dietrich, C. P., & Dietrich, S. M. (1976). Electrophoretic behaviour of acidic mucopolysaccharides in diamine buffers. Analytical Biochemistry, 70(2), 645–647.

Dreyfuss,J.L.,Regatieri,C.V.,Jarrouge,T.R.,Cavalheiro,R.P.,Sampaio,L.O.,&Nader, H.B.(2009).Heparansulfateproteoglycans:Structure,proteininteractionsand cellsignaling.AnaisdaAcademiaBrasileiradeCiencias,81(3),409–429. Franco,C.R.,Rocha,H.A.,Trindade,E.S.,Santos,I.A.,Leite,E.L.,Veiga,S.S.,etal.

(2001).Heparansulfateandcontrolofcelldivision:Adhesionandproliferation ofmutantCHO-745cellslackingxylosyltransferase.BrazilianJournalofMedical andBiologicalResearch,34(8),971–975.

Franco,C.R.,Trindade,E.S.,Rocha,H.A.,daSilveira,R.B.,Paludo,K.S.,Chammas,R., etal.(2009).Glycosaminoglycanchainsfromalpha5beta1integrinareinvolved infibronectin-dependentcellmigration.BiochemistryandCellBiology-Biochimie etBiologieCellulaire,87(4),677–686.

Giraux,J.L.,Matou,S.,Bros,A.,Tapon-Bretaudiere,J.,Letourneur,D.,&Fischer, A.M.(1998).Modulationofhumanendothelialcellproliferationand migra-tion by fucoidan and heparin. European Journal of Cell Biology, 77(4), 352–359.

Haroun-Bouhedja,F.,Ellouali,M.,Sinquin,C.,&Boisson-Vidal,C.(2000).Relationship betweensulfategroupsandbiologicalactivitiesoffucans.ThrombosisResearch, 100(5),453–459.

Irhimeh,M.R.,Fitton,J.H.,Ko,K.H.,Lowenthal,R.M.,&Nordon,R.E.(2011). For-mationofanadherenthematopoieticexpansioncultureusingfucoidan.Annals ofHematology,90(9),1005–1015.

Jiao,G.L.,Yu,G.L.,Zhang,J.Z.,&Ewart,H.S.(2011).ChemicalStructuresand BioactivitiesofSulfatedPolysaccharidesfromMarineAlgae.MarineDrugs,9(2), 196–223.

Kim,S.H.,Turnbull,J.,&Guimond,S.(2011).Extracellularmatrixandcellsignalling: Thedynamiccooperationofintegrin,proteoglycanandgrowthfactorreceptor. JournalofEndocrinology,209(2),139–151.

Kim,W.J.,Lee,S.J.,Choi,Y.D.,&Moon,S.K.(2010).Decursininhibitsgrowth ofhumanbladderandcoloncancercellsviaapoptosis,G1-phasecellcycle arrestandextracellularsignal-regulatedkinaseactivation.InternationalJournal ofMolecularMedicine,25(4),635–641.

Lake,A.C.,Vassy,R.,DiBenedetto,M.,Lavigne,D.,LeVisage,C.,Perret,G.Y.,etal. (2006).LowmolecularweightfucoidanincreasesVEGF165-inducedendothelial cellmigrationbyenhancingVEGF165bindingtoVEGFR-2andNRP1.Journalof BiologicalChemistry,281(49),37844–37852.

Li,B.,Lu,F.,Wei,X.J.,&Zhao,R.X.(2008).Fucoidan:Structureandbioactivity. Molecules,13(8),1671–1695.

Liu,J.M.,Bignon,J.,Haroun-Bouhedja,F.,Bittoun,P.,Vassy,J.,Fermandjian,S.,etal. (2005).Inhibitoryeffectoffucoidanontheadhesionofadenocarcinomacellsto fibronectin.AnticancerResearch,25(3B),2129–2133.

Moreau,D.,Thomas-Guyon,H.,Jacquot,C.,Jugé,M.,Culioli,G.,Ortalo-Magné,A.,etal. (2006).AnextractfromthebrownalgaBifurcariabifurcatainducesirreversible arrestofcellproliferationinanon-small-cellbronchopulmonarycarcinomaline. JournalofAppliedPhycology,18(1),87–93.

Nabeyrat,E.,Jones,G.E.,Fenwick,P.S.,Barnes,P.J.,&Donnelly,L.E.(2003). Mitogen-activatedproteinkinasesmediateperoxynitrite-inducedcelldeathinhuman bronchialepithelialcells.AmericanJournalofPhysiology–LungCellularand MolecularPhysiology,284(6),L1112–L1120.

Pielesz,A.,&Binias,W.(2010).CelluloseacetatemembraneelectrophoresisandFTIR spectroscopyasmethodsofidentifyingafucoidaninFucusvesiculosusLinnaeus. CarbohydrateResearch,345(18),2676–2682.

Pomin,V.H.(2010).Structuralandfunctionalinsightsintosulfatedgalactans:A systematicreview.GlycoconjugateJournal,27(1),1–12.

Riou,D.,Colliec-Jouault,S.,PinczonduSel,D.,Bosch,S.,Siavoshian,S.,LeBert,V., etal.(1996).Antitumorandantiproliferativeeffectsofafucanextractedfrom ascophyllumnodosumagainstanon-small-cellbronchopulmonarycarcinoma line.AnticancerResearch,16(3A),1213–1218.

Rocha,H.A.,Bezerra,L.C.,deAlbuquerque,I.R.,Costa,L.S.,Guerra,C.M.,deAbreu, L.D.,etal.(2005).AxylogalactofucanfromthebrownseaweedSpatoglossum schroederistimulatesthesynthesisofanantithromboticheparansulfatefrom endothelialcells.PlantaMedica,71(4),379–381.

Rocha,H.A.,Franco,C.R.,Trindade,E.S.,Veiga,S.S.,Leite,E.L.,Nader,H.B.,etal. (2005).FucaninhibitsChinesehamsterovarycell(CHO)adhesiontofibronectin bybindingtotheextracellularmatrix.PlantaMedica,71(7),628–633. Rocha,H.A.,Moraes,F.A.,Trindade,E.S.,Franco,C.R.,Torquato,R.J.,Veiga,S.S.,etal.

(2005).Structuralandhemostaticactivitiesofasulfatedgalactofucanfromthe brownalgaSpatoglossumschroederi.Anidealantithromboticagent?Journalof BiologicalChemistry,280(50),41278–41288.

Schaller,M.D.(2010).CellularfunctionsofFAKkinases:Insightintomolecular mechanismsandnovelfunctions.JournalofCellScience,123(Pt7),1007–1013. Synytsya,A.,Kim,W.,Kim,S.,Pohl,R.,Synytsya,A.,Kvasniˇcka,F.,etal.(2010).

Struc-tureandantitumouractivityoffucoidanisolatedfromsporophyllofKorean brownseaweedUndariapinnatifida.CarbohydratePolymers,81,41–48. Telci,D.,Wang,Z.,Li,X.,Verderio,E.A.,Humphries,M.J.,Baccarini,M.,etal.(2008).

Fibronectin-tissuetransglutaminasematrixrescuesRGD-impairedcell adhe-sionthroughsyndecan-4andbeta1integrinco-signaling.JournalofBiological Chemistry,283(30),20937–20947.

Yamasaki-Miyamoto,Y.,Yamasaki,M.,Tachibana,H.,&Yamada,K.(2009).Fucoidan inducesapoptosisthroughactivationofcaspase-8onhumanbreastcancer MCF-7cells.JournalofAgriculturalandFoodChemistry,57(18),8677–8682. Zhang,Z.,Wang,F.,Wang,X.,Liu,X.,Hou,Y.,&Zhang,Q.(2010).Extractionofthe