Rev. bras. farmacogn. vol.25 número3

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w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Fucoidan

induces

G1

arrest

of

the

cell

cycle

in

EJ

human

bladder

cancer

cells

through

down-regulation

of

pRB

phosphorylation

Hye

Young

Park

a,1

,

Il-Whan

Choi

b,1

,

Gi-Young

Kim

c

,

Byung

Woo

Kim

d,e

,

Wun-Jae

Kim

f

,

Yung

Hyun

Choi

a,e,∗

a_Department_of_{Biochemistry,}_Dongeui_University_College_of_Korean_Medicine,_Busan_614-052,_Republic_of_Korea b_Department_of_{Microbiology,}_College_of_Medicine,_Inje_University,_Busan_608-756,_Republic_of_Korea

c_Laboratory_of_{Immunobiology,}_Department_of_Marine_Life_Sciences,_Jeju_National_University,_Jeju_690-756,_Republic_of_Korea

d_Department_of_Life_Science_and_{Biotechnology,}_College_of_Natural_Sciences_&_Human_Ecology,_Dongeui_University,_Busan_614-714,_Republic_of_Korea e_Blue-Bio_Industry_RIC_and_Anti-Aging_Research_Center,_Dongeui_University,_Busan_614-714,_Republic_of_Korea

f_Department_of_Urology,_College_of_Medicine,_Chungbuk_National_University,_Cheongju_362-763,_Republic_of_Korea

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30December2014 Accepted22March2015 Availableonline25April2015

Keywords:

Fucoidan EJcells G1arrest Apoptosis pRB

a

b

s

t

r

a

c

t

Fucoidan,asulfatedpolysaccharidefoundinmarinealgaeandbrownseaweeds,hasbeenshowntoinhibit theinvitrogrowthofhumancancercells.ThisstudywasconductedinculturedhumanbladdercancerEJ cellstoelucidatethepossiblemechanismsbywhichfucoidanexertsitsanti-proliferativeactivity,which untilnowhasremainedpoorlyunderstood.FucoidantreatmentofEJcellsresultedindose-dependent inhibitionofcellgrowthandinducedapoptoticcelldeath.Flowcytometricanalysisrevealedthatfucoidan ledtoG1arrestincellcycleprogression.Itwasassociatedwithdown-regulationofcyclinD1,cyclinE, andcyclin-dependent-kinases(Cdks)inaconcentration-dependentmanner,withoutanychangeinCdk inhibitors,suchasp21andp27.Furthermore,dephosphorylationofretinoblastomaprotein(pRB)bythis compoundwasassociatedwithenhancedbindingofpRBwiththetranscriptionfactorsE2F-1andE2F-4. Overall,ourresultsdemonstratethatfucoidanpossessesanticanceractivitypotentialagainstbladder cancercellsbyinhibitingpRBphosphorylation.

Introduction

Bladdercanceris anyofseveraltypes ofmalignancy arising fromtheepithelial lining ofthe urinary bladder.In theUnited States,bladdercanceristhefourthmostcommontypeofcancer inmenandtheninthmostcommoncancerinwomen,although theincidenceofbladdercancerinAsiaismuchlower(Kakehietal., 2010;Abdollahetal.,2013).Despiterecentadvancesinsurgicaland chemotherapeuticprocedures,long-termsurvivalratesarepoor, andthemostcommoncauseofmortalityisrecurrencewith metas-tasis(Racioppietal.,2012).Therefore,itisimportanttodevelop othereffectivestrategiestoimprovethesurvivalrateforbladder cancerpatients.

Several epidemiological studies have revealed that dietary intakeofmarinealgaeand seaweedsareprotective againstthe riskofvarioustypesofmalignancies(Kimetal.,2011;Parkand

∗ Correspondingauthor.

E-mail:choiyh@deu.ac.kr(Y.H.Choi). 1_These_authors_contributed_equally_to_this_work.

Pezzuto,2013;Ahmedetal.,2014).Brownseaweedsareusedas an importanthealthcare foodand a pharmaceutical product in Asiancountries.Fucoidanisamajorsulfatedpolysaccharidefound inbrownseaweed;ithasbeenwellcharacterizedandisknown tohave variousbiologicalfunctions,includingantioxidant, anti-inflammatory,andanticancereffects(Lietal.,2008;Sennietal., 2011;Fitton,2011;Wangetal.,2012;Senthilkumaretal.,2013; ThomasandKim,2014).Wereportedrecentlythatfucoidanmay offer substantial therapeutic potential for treatment of inflam-matoryandneurodegenerativediseasesthatareaccompaniedby microglial activation (Park et al., 2011a). In addition, fucoidan suppresses cancercell proliferation and inhibitsthe growth of transplantedtumorxenograftsbyinducing apoptosisand/orby blockingabnormalcellcycleprogressionattheG1orG2/Mphase (Riouetal.,1996;Fukahorietal.,2008;Parketal.,2011b,2013; Zhangetal.,2011,2013;Hsuetal.,2013;Xueetal.,2013;Yang etal.,2013;Parketal.,2014;Banafaetal.,2013;Chenetal.,2014; SenthilkumarandKim,2014;Choetal.,2014).However,thiseffect isselectiveforcancercells,asnormalcelllinesareresistanttocell cyclearrestand apoptosisbyfucoidan.Moreover,fucoidanalso inhibitsmigrationandinvasionofhighlymetastaticcancercells

http://dx.doi.org/10.1016/j.bjp.2015.03.011

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viadown-regulationofmatrixmetalloproteinasesandinhibitionof thephosphoinositide3-kinase/Aktandnuclearfactor-kBsignaling pathways(Saitohetal.,2009;Leeetal.,2012;Wangetal.,2014; SenthilkumarandKim,2014).However,themolecularmechanisms ofitsanti-proliferativeactionsonhumanbladdercarcinomacell growthhave not yetbeen examined.Thus, thepurposeof this studywastoinvestigatetheeffectsoffucoidanoncell prolifera-tionofthehumanbladdercarcinomacelllineEJandtoexplorethe potentialmechanismsoftheeffects.Ourdataindicatethatfucoidan inhibitedthegrowthofEJcellsinaconcentration-dependent man-ner,arrestingthemintheG1phaseoftheircellcycleandinducing apoptosis.

Materialsandmethods

Cellculture

EJcells werepurchased fromAmericanTypeCulture Collec-tion (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 medium(GIBCO-BRL,Gaithersburg,MD)supplementedwith10% fetal bovine serum (FBS) and 2mM of l-glutamine and

peni-cillin/streptomycin.Thecellswereculturedinanincubatorwith 5%CO2at37◦C.FucoidanwaspurchasedfromSigma-Aldrich(St.

Louis,MO,USA)anddissolvedinphosphate-bufferedsaline(PBS) asastocksolutionata200mg/mlconcentration,andthestock solu-tionwasthendilutedwiththemediumtothedesiredconcentration priortouse.

MTTassay

Measurementofcellviabilitywasdeterminedusingthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich)assay,whichisbasedontheconversionofMTTto MTT-formazanbymitochondria.Inbrief,cells(2×104_cells/well)

wereseededin24-wellplatesandexposedtofucoidanfor48h. Aftertreatment,5mg/mlMTTsolutionwasadded,followedby3h incubationat37◦_C_in_the_dark,_and_the_media_was_then_removed.

The formazan precipitate was dissolved in dimethyl sulfoxide (Sigma-Aldrich), and absorbance of the formazan product was measured at a wavelength of 540nm with an enzyme-linked immunosorbentassay(ELISA)reader(MolecularDevices, Sunny-vale,CA,USA)(Leeetal.,2014).Forthemorphologicalstudy,cells werephotographeddirectlyusing aninverted microscope(Carl Zeiss,Oberkochen,Germany).

Flowcytometryanalysis

Exponentiallygrowingcellswerecomparedtocellstreatedwith variousconcentrationsoffucoidanfor48h.Aftertreatmentwith fucoidan,thecellswerecollectedandfixedin70%ethanolat4◦_C

for30min,and theDNAcontentofcells wasstainedwith pro-pidiumiodide(PI)usingaDNAstainingkit(CycleTESTPLUSKit, BectonDickinson,SanJose,CA,USA)inaccordancewiththe manu-facturer’sinstructions.ThecellswerethensubjectedtoaFACScan flowcytometer(BectonDickinson)withthepercentagesofcellsin differentphasesofthecellcyclecalculatedfromDNAhistograms. Cellswithsub-G1DNAcontentwereconsideredapoptotic cells (Kimetal.,2014).

Morphologicalobservationofnuclearchange

Afterculturewithvariousconcentrationsoffucoidan,cellswere washed twicewith PBS and fixedwith 3.7% paraformaldehyde (Sigma-Aldrich)inPBSfor10minatroomtemperature.Thefixed cellswerewashedtwicewithPBSandstainedwith 4,6-diamidino-2-phenylindole(DAPI,Sigma-Aldrich)solutionfor10minatroom

Table1

OligonucleotidesusedinRT-PCR.

Genename Sequence

CyclinD1 Sense 5′-TGG-ATG-CTG-GAG-GTC-TGC-GAG-GAA-3′

Antisense 5′_{-GGC-TTC-GAT-CTG-CTC-CTG-GCA-GGC-3}′

CyclinE Sense 5′-AGT-TCT-CGG-CTC-GCT-CCA-GGA-AGA-3′

Antisense 5′_{-TCT-TGT-GTC-GCC-ATA-TAC-CGG-TCA-3}′

Cdk2 Sense 5

′_-GCT_TTC_TGC_CAT_TCT_CAT_CG-3′

Antisense 5′_-GTC_CCC_AGA_GTC_CGA_AAG_AT-3′

Cdk4 Sense 5

′_{-ACG-GGT-GTA-AGT-GCC-ATC-TG-3}′

Antisense 5′_{-TGG-TGT-CGG-TGC-CTA-TGG-GA-3}′

Cdk6 Sense 5

′_{-CGA-ATG-CGT-GGC-GGA-GAT-C-3}′

Antisense 5′_{-CCA-CTG-AGG-TTA-GAG-CCA-TC-3}′

p21 Sense 5′-CTCAGAGGAGGCGCCATG-3′

Antisense 5′_-GGG_CGG_ATT_AGG_GCT_TCC-3′

p27 Sense 5′-AAG-CAC-TGC-CGG-GAT-ATG-GA-3′

Antisense 5′_{-AAC-CCA-GCC-TGA-TTG-TCT-GAC-3}′

GAPDH Sense 5

′_{-CGG-AGT-CAA-CGG-ATT-TGG-TCG-TAT-3}′

Antisense 5′_{-AGC-CTT-CTC-CAT-GGT-GGT-GAA-GAC-3}′

temperature.ThecellswerewashedtwomoretimeswithPBS. Cov-erslipsweremountedonglassslidesandanalyzedbyfluorescence microscopyusingaZeissAxiophotmicroscope(CarlZeiss).

Annexin-Vstaining

Toanalyzeapoptosis,thecellsweretreatedwiththeindicated concentrationsoffucoidanfor48handresuspendedinan annexin-Vbindingbuffercontaining10mMHEPES/NaOH(pH7.4),140mM NaCl,and2.5mMCaCl2.Aliquotsofthecellswereincubatedwith annexin-V fluorescein isothiocyanate(FITC, R&D Systems, Min-neapolis, MN, USA), mixed, and incubated for 15min at room temperatureinthedark.PIataconcentrationof5␮g/mlwasadded todistinguishnecrotic cells.Theapoptoticcells (AnnexinV+_/PI− cells)weremeasuredbyafluorescence-activatedcellsorter analy-sisinaFACScanflowcytometer(ParkandHan,2014).

RNAextractionandreversetranscription-polymerasechain reaction(PCR)

TotalRNAwasisolatedusinganRNeasyminikit(Qiagen,LaJolla, CA,USA)andprimedwithrandomhexamerstosynthesize comple-mentaryDNAusingAMVreversetranscpriptase(AmershamCorp., ArlingtonHeights,IL,USA)followingthemanufacturer’s instruc-tions.PCRwasperformedinaMastercycler(Eppendorf,Hamburg, Germany)withtheindicatedprimers,whichwerepurchasedfrom Bioneer(Seoul,RepublicofKorea),inTable1.Theconditionsforthe PCRreactionswere1×(94◦_C_for₃_min),_35X₍₉₄◦_C_for₄₅_s;₅₈◦_C_for

45s;and72◦_C_for₁_min),_and₁_×₍₇₂◦_C_for₁₀_min)._The

amplifica-tionproductsobtainedbyPCRwereelectrophoreticallyseparated ona1.0%agarosegelandvisualizedbyethidiumbromide(EtBr, Sigma-Aldrich)staining(Gongetal.,2014).In aparallel experi-ment,glyceraldehyde-3-phosphatedehydrogenase(GAPDH)was usedasaninternalcontrol.

Totalproteinextraction,immunoprecipitation,andWesternblot analysis

For isolation of total protein fractions, the cells were har-vested and washedoncewithice-cold PBS,and lysed withcell lysis buffer[20mMTris pH7.5,150mM NaCl,1%Triton X-100, 2.5mM sodiumpyrophosphate, 1mM EDTA,0.5g/mlleupeptin, 1% Na3CO4, 1mM phenylmethane-sulfonyl fluoride], and

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120

A

B

100

80

60

Cell viability (%)

Cell numbers

40

20

0

0 50

Fucoidan (µg/ml)

DNA contents

G1 G2/M

47.4 79.4

17.1 8.5

91.1 92.7% of G1

% of G2/M % of S

2.8 4.5 3.0

5.9 29.8

22.8

S

Fucoidan (µg/ml)

100 150

0 50 100 150

Fig.1. InhibitionofcellviabilityandinductionofG1arrestbyfucoidantreatment inhumanbladdercancerEJcells.(A)Cellsweretreatedwiththeindicated concen-trationsoffucoidanfor48h.Thecellswereharvested,andthepercentageofcell viabilitywascalculatedwithanMTTassay.Dataarepresentedasmean±SDin trip-licate.SignificancewasdeterminedbytheStudent’st-test(*p<0.05vs.untreated control).(B)Thecellswerecollected,fixed,andstainedwithPIforflowcytometry analysis.DNAcontentisrepresentedonthex-axis,andthenumberofcellscounted isrepresentedonthey-axis.Eachpointrepresentsthemeanoftwoindependent experiments.

assay (Bio-Rad Lab., Hercules, CA, USA), following the proce-duredescribedbythemanufacturer.Forimmunoprecipitation,cell extractswereincubatedwithimmunoprecipitatingantibodiesin extractionbufferfor1hat4◦_C,_and_then_the_{immunocomplexes}

wereprecipitatedwithproteinA-Sepharosebeads(Sigma-Aldrich). ForWesternblotassay,thetotalproteinsandimmunoprecipitated proteinswere subjected to electrophoresis on sodium dodecyl sulphate(SDS)–polyacrylamide gelsandtransferred to nitrocel-lulose membranes (Schleicher & Schuell, Keene, NH, USA) by electroblotting.Blotsweresubsequentlyblockedwith5%non-fat milkandprobedwiththedesiredantibodiesfor1hat4◦_C,

incu-bated withdiluted enzyme-linked secondary antibodyat room temperaturefor afurther1h, and thenvisualizedbyenhanced chemiluminescence(ECL,Amersham)accordingtorecommended procedures.Eextracellular-regulatedkinase(ERK)wasusedasan internal control. The primary antibodies were purchased from SantaCruzBiotechnologyInc.(SantaCruz,CA,USA). Peroxidase-labeleddonkeyantirabbitimmunoglobulinandperoxidase-labeled sheepantimouse immunoglobulin were purchased from Amer-sham.

Statisticalanalysis

Thedatawereexpressedasmeans±SD fortriplicate experi-ments.StatisticalanalyseswereperformedusingaStudent’st-test. Apvalue<0.05wasconsideredstatisticallysignificant.

Results

FucoidaninhibitscellviabilityandinducesG1arrestinEJcells

Todeterminewhetherfucoidaninfluencedthecellviabilityof theEJcells,thecellswereculturedinthepresenceoffucoidanfor 48h,andthecellviabilitywasdeterminedwithanMTTassay.As showninFig.1A,fucoidanreducedtheviabilityoftheEJcellsin aconcentration-dependentmanner.Thecellcyclephase distribu-tionofexponentiallygrowingEJcellswasnextcomparedtocells

Fucoidan (µg/ml)

Apoptotic cells (%)

0

A

B

C

50 100 150

0 0 5 10 15 20 25 30

50 100 150

Fig.2. InductionofapoptosisbyfucoidantreatmentinEJcells.Thecellsweretreated withvariousconcentrationsoffucoidanfor48h.(A)Themorphologicalchangesof thecellswereimagedusinganinvertedmicroscope(originalmagnification,200×). (B)ThecellswerefixedandstainedwithDAPIsolution.After10minincubationat roomtemperature,thestainednucleiwereobservedwithafluorescentmicroscope (originalmagnification,400×).(C)Thecellswereharvestedfordeterminingthe per-centageofannexin-Vpositive/PInegative(apoptoticcells).Thedataareexpressedas themean±SDofthreeindependentexperiments.Thesignificancewasdetermined bytheStudent’st-test(*p<0.05vs.untreatedcontrol).

treatedwithfucoidan.Comparedwiththeuntreatedcontrol,the fucoidan-treatedcellsaccumulatedintheG1phaseofthecellcycle inaconcentration-dependentmanner(Fig.1B),andtherewasa decreaseinthenumberofcellsintheSandG2/Mphases.Taken together,theseresultssuggestthatthegrowthinhibitoryeffectof fucoidanonEJcellsistheresultofitblockingtheG1phase.

FucoidaninducesapoptosisinEJcells

Asfucoidaninducedmorphologicalchanges,suchasmembrane blebbinganda reductionincellvolumeunder thesame condi-tions(Fig.2A),weassessed theeffectoffucoidantreatmenton theapoptoticcharacteristicsoftheEJcellstodeterminewhether apoptosiswasinvolvedinfucoidan-mediatedantiproliferation[0].

Fig.2Brevealsthatnucleicmorphologicalchangesofapoptosisas indicatedbyrepresentativeimagesofcondensedandfragmented nucleiwereclearlyevidentinfucoidan-treatedEJcellsincontrast totheuntreatedcells.Toquantifyfucoidan-inducedapoptosis,the EJcellstreatedwithfucoidanwerestainedwithannexinV-FITCand analyzedbyflowcytometry.AsshowninFig.2C,fucoidanincreased theannexin-V-FITCstainedapoptoticpopulationofcells,andthese effectsweredosedependent.Theseobservationsindicatethatthe inhibitionofcellviabilityobservedinresponsetofucoidanisalso associatedwiththeinductionofapoptosis.

Effectoffucoidanontheexpressionofcellcyclecheckpoint regulators

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0

1 0.6 0.5 0.4

1 0.9 0.9 0.8

1 0.9 0.9 1

1 0.9 0.8 0.5

1 0.8 0.5 0.4

1 0.9 0.8 0.7

1 0.8 0.7 0.4

1 0.7 0.6 0.3

1 1 1 1

50

Fucoidan (µg/ml)

A

B

Cycline D1

100 150 0 50

Fucoidan (µg/ml)

100 150

Cycline E

Cdk2

Cdk4

Cdk6

GAPDH

Cycline D1

Cycline E

Cdk2

Cdk4

Cdk6

ERK

Fig.3.Down-regulationofG1-associatedcyclinsandCdksbyfucoidantreatmentinEJcells.(A)Aftertreatmentwithvariousconcentrationsoffucoidanfor48h,thecells werecollected,andtotalRNAwasisolatedandreverse-transcribed.TheresultingcDNAswerethensubjectedtoPCRwiththeindicatedprimers,andthereactionproducts wereseparatedin1.0%agarosegelandvisualizedbyEtBrstaining.GAPDHwasusedasaninternalcontrol.(B)Thecelllysateswereseparatedandequalamountsoftotal celllysatesweresubjectedtoSDS–polyacrylamidegels,transferred,andprobedwiththeindicatedantibodies.ERKwasusedasaninternalcontrol.Therelativeratiosof expressionintheresultsoftheWesternblottingwerepresentedatthebottomofeachoftheresultsasrelativevaluesoftheGAPDHandERKexpression,respectively.

andthenRT-PCRandWesternblotanalysiswereperformed.As illustratedinFig.3,fucoidansuppressedtheexpressionof biomark-ers,suchascyclinD1,cyclinE,cyclin-dependent-kinase(Cdk)2, Cdk4,andCdk6,in thetransitionoftheG1totheSphasein a concentration-dependentmanner.However,fucoidandidnot sig-nificantlyaffectthelevelsofCdkinhibitors,suchasp21andp27 (Fig.4).

0

1 0.9 1 1

50 Fucoidan (µg/ml)

A

p21

p27

100 150

GAPDH

0

1 0.9 0.5 0.3

1 0.9 1 1.1

B

p21

p27

100 150

ERK

Fig.4. EffectsoffucoidanonthelevelsofCdkinhibitorsinEJcells.(A)Thecells weretreatedwithdifferentconcentrationsoffucoidanfor48h.TotalRNAwas iso-latedandreverse-transcribed,andtheresultingcDNAsweresubjectedtoPCR.The reactionproductsweresubjectedtoelectrophoresisin1.0%agarosegeland visual-izedbyEtBrstaining.GAPDHwasusedasaninternalcontrol.(B)Afterincubation withfucoidanunderthesameconditions,thecelllysateswereseparated,andequal amountsoftotalcelllysatesweresubjectedtoSDS–polyacrylamidegels,transferred, andprobedwithantibodiesagainstp21andp27.ERKwasusedasaninternal con-trol.TherelativeratiosofexpressionintheresultsoftheWesternblottingwere presentedatthebottomofeachoftheresultsasrelativevaluesoftheGAPDHand ERKexpression,respectively.

Fucoidandown-regulatespRBphosphorylationandincreasesthe bindingofpRBandE2Fs

As D-type cyclins and cyclin E-stimulatedCdk activity con-verges in hyperphosphorylation of the retinoblastoma protein (pRB), the effect of fucoidan on the phosphorylation status of pRBwasinvestigatedbyWesternblotanalysis.Fucoidancauseda remarkabledecreaseintotallevelsofpRBexpressionandchanged fromahyperphosphorylatedformtoahypophosphorylatedform (Fig.5A).Furthermore,co-immunoprecipitationanalysisindicated thatthereappearedtobenoassociationbetweenpRBandE2Fs, suchasE2F-1andE2F-4,intheuntreatedcontrolcells[0]. How-ever,therewasastrongincreaseinthebindingofpRBandE2Fs inthefucoidan-treatedEJcells(Fig.5B),suggestingthatfucoidan inhibitsthereleaseofE2FsproteinfrompRB.

Discussion

Although fucoidan exerts various pharmacological activities, such as anti-inflammatory, antioxidant, and anticancer effects (Riouetal.,1996;Lietal.,2008;Fukahorietal.,2008;Saitohetal., 2009;Fitton,2011;Sennietal.,2011;Parketal.,2011b,2013;Zhang etal.,2011,2013;Wangetal.,2012;Leeetal.,2012;Liuetal.,2012; Hsuetal.,2013;Xueetal.,2013;Yangetal.,2013;Senthilkumar etal.,2013;Parketal.,2014;Banafaetal.,2013;Chenetal.,2014; SenthilkumarandKim,2014;Wangetal.,2014),theanti-cancer activityoffucoidaninhumanbladdercancercellshasrarelybeen reported.Therefore,weelucidatedtheabilityoffucoidantoinhibit thegrowthofhumanbladdercancerEJcells.Inthisstudy,wefound thatfucoidanshowsconsiderablepotentialtoinhibitthe prolifer-ationofEJcellsviacellcyclearrestintheG1phaseandapoptosis induction.

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0

1 0.8 0.5 0.2

1 0.9 0.5 0.5

1

1 3.1

– +

1 4.8

0.9 0.8 0.5

A

pRB

E2F-1

100 150

E2F-4

ERK

FCD (150 µg/ml)

IP : E2F-1

IP : E2F-4

B

pRB

Fig.5. InductionofhypophosphorylationofpRBandenhancedassociationofpRB andE2FsbyfucoidantreatmentinEJcells.(A)Aftertreatmentwithvarious concen-trationsoffucoidanfor48h,thetotalcelllysateswerepreparedandseparatedby electrophoresisonan8%or10%SDS–polyacrylamidegel.Westernblottingwasthen performedusinganti-pRB,anti-E2F-1,andanti-E2F-4antibodies.ERKwasusedasan internalcontrol.(B)Thecellswereincubatedwithorwithout150␮g/mloffucoidan for48h,andthenequalamountsofproteins(0.5mgofprotein)were immunoprecip-itatedwithanti-E2F-1oranti-E2F-4antibody.Immunocomplexeswereseparated by8%SDS–polyacrylamidegelelectrophoresis,transferredtoanitrocellulose mem-brane,andprobedwithanti-pRBantibody.ProteinsweredetectedbyECLdetection. TherelativeratiosofexpressionintheresultsoftheWesternblottingwere pre-sentedatthebottomofeachoftheresultsasrelativevaluesoftheERKexpression oruntreatedcontrol,respectively.

inhibitors,includingp21andp27(Dobashietal.,2003;Paternot etal.,2010).Inthepresent study,theresultsfromRT-PCR and immunoblottinganalysisclearlydemonstratedthatthelevelsof cyclinD1 and cyclin E expression weremarkedly inhibited by fucoidantreatmentatbothtranscriptionalandtranslational lev-els(Fig.3).Inaddition,fucoidan-inducedG1arrestwascorrelated withthedown-regulationofCdks,suchasCdk2,Cdk4,andCdk6. Although,arecentstudyindicatedthatfucoidandown-regulated cyclinE,Cdk2,Cdk4resultinginG0/G1arrestthroughbindingof Cdkinhibitorp21toCdk2andCdk4incellstreatedwithfucoidanin humanbladdercancer5637cells(Choetal.,2014),theexpression ofCdkinhibitorssuchasp21andp27wasremainedunchangedin fucoidan-treatedEJcells(Fig.4).

Ontheotherhand,pRBasatumorsuppressorisalso impor-tantforcellcycleprogressionduringtheG1toSphasetransition. DephosphorylationofpRBinhibitscellcycleprogressionby inter-acting with transcription factors of the E2F family, whereas phosphorylationofpRBresultsininductionofcellcycle progres-sionthroughthebreakingofpRB/E2Fcomplexes(Dobashietal., 2003;Paternotetal.,2010).Therefore,ifthelevelsofeitherprotein aredecreasedortheassociationbetweentheirrespectivebindings partnersisdiminished,aconcomitantdecreaseinthedegreeofpRB phosphorylationwouldbeexpected.In conjunctionwith down-regulationoftotallevelsofpRB,thedatashowedthattreatment withfucoidaninhibitedthephosphorylationofpRBandenhanced theassociationofpRBandE2Fs(Fig.5).TheroleofCdkinhibitors

hasattractedattentionintheinductionofG1phasearrest,which isrelatedtotherestrictionoftheproliferationofcancercellsvia fucoidantreatment.However,accordingtoBooetal.(2012), restric-tionofE2FexpressionplaysanimportantroleininducingG1cell cyclearrestinPC-3prostaticcancercells,alongwiththeincreased expressionof Cdkinhibitor p21.Moreover,a recentstudy con-ductedinourlabrevealedthatphosphorylationinhibitionofpRB wassimultaneouslyinvolvedinadditiontotheexpressionincrease ofCdkinhibitorp21inT24bladdercancercells’G1arrestbrought aboutbyfucoidan(Parketal.,2014).Hence,evenifcancercells’G1 arrestresultingfromfucoidantreatmentdiffersaccordingtotypes ofcancercells,itcanatleastbesaidthatexpressionincreaseof Cdkinhibitorssuchasp21andphosphorylationinhibitionplayan importantfactorsinG1arrest.

Conclusions

In conclusion, the resultspresented here demonstrate that: (i) reduced survival of EJ cells after exposure to fucoidan is associatedwithG1phase cellcyclearrestand apoptosis induc-tion;(ii)fucoidaninhibitscellcycleprogressionintheG1phase bydecreasingG1-related cyclins and Cdks;(iii) treatmentwith fucoidanresultsinincreasedbindingofpRBtoE2Fs.Thesenovel phenomenahavenotbeenpreviouslydescribedinbladdercancer cellsandsuggestthatfucoidanandrelatedcompoundsmayhave significantpotentialinthedevelopmentofcancertreatments.

Authors’contributions

HYP and IWC contributed in running the laboratory work, analysisofthedataand draftedthepaper.GYK, BWKandWJK contributedtocritical readingof themanuscript.WJKand YHC designed the study, supervised the laboratory work and con-tributedtocriticalreadingofthemanuscript.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbyBlue-BioIndustryRegional Innova-tionCenter(RIC08-06-07)atDongeuiUniversityasaRICprogram underMinistryofTrade,Industry&EnergyandBusancityandthe NationalResearchFoundationofKorea(NRF)grantfundedbythe Koreagovernment(MSIP)(No.NRF-2014R1A2A1A09006983).

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