Oudineta, Lior Pytowskia, Jean-Baptiste Bérardb, Elodie Nicolaub, Valérie Thiérya, Isabelle Lanneluca, Laureen Beaugearda, Grégoire Pruniera, Jackson Roberto Guedes Da Silva Almeidac, Laurent Picota, ∗

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Zeaxanthin

from

Porphyridium

purpureum

induces

apoptosis

in

human

melanoma

cells

expressing

the

oncogenic

BRAF

V600E

mutation

and

sensitizes

them

to

the

BRAF

inhibitor

vemurafenib

Camille

Juin

a

_,

_Raimundo

_Gonc¸

_alves

_de

_Oliveira

_Junior

a,c

_,

_Audrey

_Fleury

a

_,

_Chloé

_Oudinet

a

_,

Lior

Pytowski

a

_,

_{Jean-Baptiste}

_Bérard

b

_,

_Elodie

_Nicolau

b

_,

_Valérie

_Thiéry

a

_,

_Isabelle

_Lanneluc

a

_,

Laureen

Beaugeard

a

_,

_Grégoire

_Prunier

a

_,

_Jackson

_Roberto

_Guedes

_Da

_Silva

_Almeida

c

_,

_Laurent

_Picot

a,∗

a_LIttoral_{ENvironnement}_Et_Sociétés_UMR_7266,_Centre_National_de_la_Recherche_{Scientifique,}_University_of_La_Rochelle,_La_Rochelle,_France

b_Laboratoire_{Biotechnologies}_et_Ressources_{Marines/Laboratoire}_Physiologie_et_{Biotechnologie}_des_Algues,_Institut_Franc¸_ais_de_Recherche_pour_{l’Exploitation}_de_la_Mer,_Nantes,_France c_Center_for_Studies_and_Research_on_Medicinal_Plants,_Federal_University_of_San_Francisco_Valley,_Petrolina,_PE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16January2018 Accepted30May2018 Availableonline15June2018

Keywords: Cancer Carotenoid Melanoma Microalgae Phytoplankton Zeaxanthin

a

b

s

t

r

a

c

t

Zeaxanthin,anabundantcarotenoidpresentinfruits,vegetablesandalgaewasreportedtoexert antipro-liferativeactivityandinduceapoptosisinhumanuvealmelanomacells.Italsoinhibiteduvealmelanoma tumorgrowthandcellmigrationinnudemicexenograftmodels.Herewereportthatzeaxanthin puri-fiedfromtherhodophytePorphyridiumpurpureum(Bory)K.M.Drew&R.Ross,Porphyridiaceae,promotes apoptosisintheA2058humanmelanomacelllineexpressingtheoncogenicBRAFV600Emutation. Zeax-anthin40␮M(IC50)inducedchromatincondensation,nuclearblebbing,hypodiploidy,accumulationof cellsinsub-G1phase,DNAinternucleosomalfragmentationandactivationofcaspase-3.Westernblot analysisrevealedthatzeaxanthininducedup-regulationofthepro-apoptoticfactorsBimandBidand inhibitionofNF-␬Btransactivation.Additionally,zeaxanthinsensitizedA2058melanomacellsinvitro tothecytotoxicactivityofvemurafenib,aBRAFinhibitorwidelyusedfortheclinicalmanagementof melanoma,suggestingitspotentialinterestasdietaryadjuvantincreasingmelanomacellssensitivityto chemotherapy.

Introduction

Melanomas accountfor lessthan 2%of skincancersbut are

responsible for 80% of theirmortality (Armstrong and Kricker,

2001;Grange,2005;LeiterandGarbe,2008;MacKieetal.,2009).

Melanomacellsarecharacterizedbymutationsthatconferthem

a strong resistance to anticancer drugs-induced apoptosis and

selectiveadvantagesforcellsurvival,proliferationandmetastasis

(Locatellietal.,2013).Particularly,mutationsintheBRAF

onco-genarefoundin70%ofmalignantmelanoma(Daviesetal.,2002;

Haluskaetal.,2006;Dankortetal.,2009;Dutton-Regesteretal.,

2012;JangandAtkins, 2014)and leadtoover-activationofthe

MAPkinasepathwaythatstimulatescellproliferation.Most

anti-cancerdrugsonlydelaytheearlygrowthofmelanomatumorsbut

failtoprovidealong-termcurebecauseoftherapidacquisition

ofdrugresistance(Locatelliet al.,2013;Spagnoloet al.,2014).

∗ Correspondingauthor.

E-mail:laurent.picot@univ-lr.fr(L.Picot).

Additionally,melanomacellsdisplaypronounced

neoangiogene-sis and a highability toescape immune cell that explain why

the5-yearsurvivalrateformetastaticmelanomarangesfrom5

to10%,withamediansurvivaloflessthan8months(Marneros,

2009;Mathieuetal.,2012).Brainmetastasisarepresentin75%

ofadvancedmelanomapatientsandconstituteamajorcauseof

mortalitybecauseofthelowpermeabilityoftheblood-brain

bar-riertochemotherapeuticdrugs(Halletal.,2000).Thesearchfor

cytostatic,antimetastaticandantiangiogenicmoleculesinplants

and algaehas establishedthat carotenoidshave a great

poten-tialasnaturalantimelanomacompounds(Hashimotoetal.,2011;

Tanakaetal.,2012;Firdousetal.,2010;Gagezetal.,2012;Baudelet

etal.,2013; Reboul,2013;Chungetal.,2013; Kimetal.,2013;

Xuetal.,2015;Luetal.,2015;Chenetal.,2017).Thesepigments

havenooraltoxicity,areresorbedbyenterocytes,transportedin

bloodafterperosconsumption(Burrietal.,2001;Hashimotoetal.,

2011;Reboul,2013)and canintegratecellmembranes(Reboul,

2013;Oliveira-Junioretal.,2016)andreachtumorcellswherethey

exertcytotoxic,cytostatic,antimetastatic,anti-inflammatoryand

antiangiogenicactivities(Sugawaraetal.,2006;Gagezetal.,2012).

https://doi.org/10.1016/j.bjp.2018.05.009

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4 µm

Fig.1. PorphyridiumpurpureumstrainCCAP1380.3.©RaymondKaas.IFREMER.

Fucoxanthin,acarotenoidpresentinbrownmicroalgaeand

sea-weedsinhibitsmelanomacellsandtumorgrowthinvitroandinvivo

(Chungetal.,2013;Kimetal.,2013).Italsolimitsmelanoma

metas-tasisinmurinemodels(Chungetal.,2013;Kumaretal.,2013),

suggestingthatitalsohasaclinicalefficacyinhumans.Werecently

demonstratedthatzeaxanthin(1),anabundantcarotenoidfoundin

variousdietarysources(corn,spinach,saffron,seaweeds,

microal-gae) inhibitsthe in vitrogrowth of the highly invasivehuman

melanomacelllineA2058(Baudeletetal.,2013).Zeaxanthinalso

inducedapoptosisintwohumanuvealmelanomacelllines(SP6.5

andC918)withoutimpairingthecellviabilityofnoncanceruveal

melanocytes(Bietal.,2013;Xuetal.,2015).Zeaxanthin-induced

apoptosiswasassociated toa decreaseintheexpressionofthe

antiapoptoticproteins Bcl-2 and Bcl-xL and an increase in the

expressionoftheproapoptoticproteinsBakandBax(Bietal.,2013).

Zeaxanthinalsoevokedthereleaseofmitochondrialcytochromec

inthecytosolandcaspase-9and-3activation(Bietal.,2013).In

thepresentreport,weperformedadditionalexperimentstofurther

elucidatethemolecularmechanismsofzeaxanthinpro-aptoptotic

activityin melanoma cells and assessed its ability to sensitize

melanomacellstoaBRAFinhibitorusedtotreatclinicalmelanoma.

WeselectedthehighlyinvasiveA2058humanmelanomacellline

asarelevantclinicalmodelexpressingtheV600EBRAFoncogenic

mutation(Dutton-Regesteretal.,2012).

Materialandmethods

Microalgaeculture,harvestandfreeze-drying

Porphyridium purpureum (Bory) K.M.Drew & R.Ross CCAP

1380.3 (bangiophyceae, rhodophyte) (Fig. 1) was grown at

120␮molm−2_s−1_irradiance._Cells_were_grown_in_four_units_of

50-lcolumn photobioreactorswith35‰ _salinity_seawater_enriched

byWalnemedium(Walne,1966;Juinetal.,2015).Batchcultures

weremaintainedat20◦_C_under_continuous_light_provided_by

flu-orescentlamps(PhilipsTLD58W865)andbubbledwith0.22␮m

filteredaircontaining3%CO2(v/v).Microalgaewereharvestedafter

12–16daysofgrowthandseparatedfromtheculturemediumbya

two-stepprocess.Firststepusedaclarifierseparator(Clara20,Alfa

LavalCorporateAB,Sweden)at100lh−1_,₉₀₀₀

×g,atroom

tem-perature.Steptwousedasoftcentrifugationat4000×g,20mn,at

4◦_C_to_separate_the_slurry._Algal_paste_was_freeze-dried_at₋₅₅◦_C

andP<1hPa,onafreeze-dryerequippedwithaHetoLyoPro3000

condenserandaHetocoolingtrap(Thermo,France).

PurificationandcharacterizationofPorphyridiumpurpureum zeaxanthin

Porphyridiumpurpureumpigmentswereextractedin ethanol

using a mixer mill extraction process developed in our group

(Seriveetal.,2012).Zeaxanthin(1)identificationwasconfirmed

afterseparationbyanalytical RP-HPLC(Zapataetal.,2000)and

cross-checkanalysisofitspolarity,absorptionspectrum,maximal

absorptionwavelengths,bandIII/IIratioandfragmentation

pro-fileinUPLC-MSE₍_Roy_et_al.,_2011;_Baudelet_et_al.,_2013;_Juin_et_al.,

2015),in comparisonwithstandardzeaxanthin (Sigma–Aldrich,

France). Purezeaxanthin wasthencollectedbypreparative

RP-HPLC (Pasquet et al., 2011)in glass vials, dried under reduced

pressure in a Buchi R-210 rotatory evaporatorat 40◦_C _(Buchi,

France)andstoredat−80◦_C_before_use_in_cell_culture_experiments.

Cellculture

A2058(ATCC® CRL-11147TM_,_LGC _ATCC_Standards,_France) _is

amelanomacelllineestablishedfrommetastaticcellsremoved

fromthelymphnodeofa43yearsoldmalecaucasianpatient.It

constitutesaclinicallyrelevantmodeltoassessthecytotoxicityof

newantimelanomadrugsasitcombineshighinvasive,metastatic

andchemoresistancepotentialswithagenemutationprofileoften

encounteredinhumanmelanomas(V600EmutationinBRAFand

mutationsinthePTENandP53genes)(Dankortetal.,2009).Cells

wereroutinelygrownasmonolayers,at37◦_C_in_a_5%_CO₂_–95%_air

humidifiedatmosphere,inDMEM(Fischerscientific,France)

sup-plementedwith10%heat-inactivated(56◦_C,₃₀_min)_FCS_(Dutscher,

France)towhichwereaddedpenicillin100Uml−1 _and

strepto-mycin100␮gml−1_.

DeterminationofzeaxanthinIC50inA2058melanomacells

Purifiedzeaxanthin(1)wassolubilizedinDMSOat6mM(stock

solution)anddilutedincellculturemediumtoobtain5–60␮M

solutions.ThefinalDMSOconcentrationinthecellculturemedium

waslower than 1%,tested as a negative control and validated

asanoncytotoxicconcentration.Theantiproliferativeactivityof

zeaxanthinwasdeterminedusingtheMTTassay(Sigma–Aldrich,

France)aspreviouslydescribed(Puteyetal.,2007;Baudeletetal.,

2013;Hedidietal.,2016).IC50wasdeterminedusingthefreeGraph

padPrismsoftwareusingthe“sigmoidaldoseresponse”(variable

slope)function.

Nuclearmembranemodification,chromatincondensationand DNAfragmentation

Sub-confluent A2058 melanoma cells were trypsinized and

2×105_cells_were_seeded_in_6-well_plates,_in_a_final_volume_of₃_ml_of

controlmediumorculturemediumcontainingzeaxanthin40␮M

(IC50)orstaurosporine2␮M.Cellsweregrownfor72hat37◦Cand

washedinPBS0.1MpH7.4,beforebeingfixedwithformaldehyde

3%for30minat37◦_C._Cells_were_then_rinsed_in_PBS,_{permeabilized}

withTritonX-1001%inPBSandstainedwithDAPI2␮gml−1_for

1hat37◦_C._Cells_were_rinsed,_mounted_on_glass_microscope_slides

andobservedusingaLeicaepifluorescencemicroscopeequipped

withanepifluorescenceAfilterblock(excitation340–360nm)and

(3)

AnnexinV-Cy3and6-CFDAdetectionassay

ApoptosiswasevaluatedbyusingdoublestainingwithAnnexin

V-Cy3(red) and6-carboxyfluoresceindiacetate (6-CFDA,green)

(Sigma–Aldrich®,France).Cells(5×103_cells/well)_were_incubated

inconventionalcultureconditionsfor24h.Then,cellsweretreated

withzeaxanthin (IC50,40␮M) for72handstaurosporine

(posi-tivecontrol,1␮M)for24h.CellswerefurtherwashedwithPBS,

suspendedinbindingbufferandstainedwithAnnexinVand

6-CFDAsolutionfor10min.DAPIsolutionwasalsoaddedtothewells

forDNAlabelling.Finally,cellswereobservedunderfluorescence

microscope(ZEISSAxioObserver).

Flowcytometricdetectionofapoptoticcells

Melanoma cells were grown in control culture medium or

treatedwithzeaxanthin40␮Mfor72hbeforebeingstainedfor

30minat37◦_C_in_PBS_containing_propidium_iodide_(PI₁₀₀_␮_g_ml−1₎

andRnaseA(100␮gml−1₎_(Molecular_Probes,_France)._Cells_were

washedandsuspendedin1mlPBSbeforebeinganalyzedusing

aFACSCantollfluxcytometer(BDBiosciences,France)equipped

withanaircooledblueLASER(=488nm,20mW).Lightdiffusion

parameters(forwardandlateralscatterlights)wereoptimizedto

definethesizethresholdexcludingcellulardebrisandcellclusters

forsingle-cellfluorescenceanalysis.PIfluorescencewasmeasured

usingaFL3filter(—— 670—— nm)andanalyzedusingtheBDFACSDiva

Software(BDBiosciences,France).Distributionofmelanomacells

inthedifferentcellcyclephasesandhypodiploidywasdetermined

accordingtotheirDNAcontentasmeasuredbythefluorescence

intensityofPI:Diploidcells(2n):G0/G1phase;Replicativecells

(2n<DNAcontent<4n):SPhase;Tetraploidcells(4n):G2/Mphase;

hypodiploidcells(DNAcontent<2n):apoptoticSub-G1Phase.

DNAinternucleosomalfragmentation

Aftertreatment withcontrol culture medium or zeaxanthin

40␮M,106 _melanoma_cells_were_washed_with_PBS_and_lysed_in

400␮l lysis buffer (Tris–HCl 10mM pH 8 NaCl 150mM, EDTA

40mM,SDS1%,proteinaseK0.2mgml−1₎_for₃_h_at₅₆◦_C._The_cell

lysatewascentrifuged(11,000×g,15min,4◦_C)_and_the_DNA

con-tainedinthesupernatantwasextractedfor15minusingamixof

phenol/chloroform/isoamylalcohol(PCI)(25/24/1,v/v/v)atpH9.

Themixwascentrifuged(11,000×g,15min,4◦_C),_and_the_aqueous

phasewascollectedtoprecipitateDNAusingsodiumacetate3M

in1mlabsoluteethanol(1night,−20◦_C)._The_precipitated_DNA

wascentrifugated(11,000×g,30min,4◦_C),_the_supernatant_was

discardedandthepelletwasdried5minat60◦_C._The_DNA

pel-letwassuspended in50␮lUltrapure watercontainingRNaseA

100␮gml−1 _for ₃₀_min_at₃₇◦_C. _Twenty_microliters_of_the_DNA

extractwereloadedandseparatedonanagarose/tris-borate-EDTA

1%gelfor30minat100V.Gelswerestainedwithethidium

bro-mide,andobservedusingaUVtransilluminator.

Caspase-3colorimetricassay

Caspase-3 activation was quantified using a commercial

assay based on the hydrolysis of Ac-DEVD-pNA (CASP3C kit,

Sigma–Aldrich,France).

Western-blot

A2058cells wereincubatedincontrolculturemedium orin

thepresenceofzeaxanthin(1)40␮Mfor72h.Thecellswere

col-lectedand lysedin a lysis buffer(HEPES 50mM pH7.4 CHAPS

5mMDTT5mM).Totalproteinswereseparatedby10%SDS-PAGE

and then transferred to a nitrocellulose membrane. The

mem-braneswereblockedwith5%(w/v)nonfatdrymilkinTBSfor1h

andchangedtoanappropriatedilutionofspecificprimary

anti-bodies against Bid,Bim,Bak,Bcl-xL, I␬B␣,NF-␬Bp65,p-NF-␬B

(Ser536),IKK␣,IKK␤and␤-actin(Ozyme,France)inmilkovernight

at4◦_C._The_secondary_antibodies_were_horseraddish

peroxidase-conjugatedanti-rabbitIgG(1:5000).Signalsweredetectedusing

theChemiDocTM_imaging_system_(Biorad,_France).

SensitizationofA2058melanomacellstotheBRAFinhibitor Vemurafenib

VemurafenibwasobtainedfromSelleckchem,France,diluted

toa10mMstocksolutioninPBS0.1MpH7.4,beforefurther

dilu-tionincellculturemedium.A2058cellswereincubatedfor72h

incontrolculturemediumorinthepresenceofzeaxanthin40␮M,

Vemurafenib(0.1,1and5␮M)orinamixofzeaxanthin40␮Mand

Vemurafenib(0.1,1and5␮M).Antiproliferativeactivitywas

cal-culatedusingtheMTTassayandpotentiationoftheVemurafenib

antiproliferativeactivitybyzeaxanthinwasexpressedasthe

per-centageofgrowthinhibitionincreaseascomparedtoVemurafenib

alone.

Statistics

Antiproliferativeactivityofzeaxanthinwasexpressedas

per-centagegrowthinhibition±SEMfromthreeindependentassays.

The normal distribution of absorbance data in control and

treated cells was demonstrated using the Hartley’s Fmax test

to confirm homogeneity of absorbances variances. The

statis-tical significance of proliferation differences between control

and treated cells was then investigated by an unpaired

Stu-dent’s t test, using a free online calculator developed by

Institut Pierre Louis d’Epidémiologie et de Santé publique

UMR S 1136 INSERM University Pierre et Marie Curie Paris

(http://marne.u707.jussieu.fr/biostatgv/?module=tests).

Results

PurificationofzeaxanthinfromPorphyridiumpurpureum

RP-HPLCoftheethanolicextractofP.purpureumgavea

chro-matogram containingeightmajor peaksat436nm(Fig.2).The

firstpigmentelutingasamajorpeakat24.582minwasidentified

aszeaxanthin(1)asitpresentedamedianpolaritywithmaximal

absorptionwavelengthsat452and481nm(Fig.3),aIII:IIbandratio

of29.03%(Fig.3),ahighresolutionmolecularweightof568.4294

(Fig.4)andaMSE_{fragmentation}_pattern_{characteristic}_of

zeaxan-thin(Fig.4)(Juinetal.,2015).

ExposuretozeaxanthininhibitstheproliferationofA2058 melanomacells

A 72h exposure to increasing concentrations of zeaxanthin

[0–60␮M]inducedadose-dependentreductioninthenumberof

A2058cellsascomparedtotheuntreatedcontrol,reaching60%

growthinhibition.TheIC50valueofzeaxanthinwasdeterminedas

40␮MusingthefreeGraphpadPrismsoftware“sigmoidaldose

response”(variableslope)function(Fig.5).

Exposuretozeaxanthinevokedcytotoxicityandnuclear fragmentationinA2058cells

A2058 cells incubated in control medium showed a regular

epithelialshape,exceptformitoticcellsexhibitingaroundshape

(4)

mAU

300 400

200

100

0

5 10 15 20 25 30 35

36.925

31.845

33.143

26.584

24.582

32.622

25.607

31.453

min Zeaxanthin Chloroph

yll

α

Chloroph

yll

α

allomers

Chloroph

yll

α

epimer

β,β

-carotene

Fig.2. RP-HPLCChromatogramofPorphyridiumpurpureumethanolextractat436nmobtainedusingtheVanHeukelemandThomasanalysis(VanHeukelemandThomas, 2001).ThepigmentprofilewasidenticaltothatpreviouslyreportedinSeriveetal.(2017).

Norm

80

60

40

20

0

300 400 452 481500 600

Band II

Band III

III:II band ratio=29.03%

700 nm

100

Fig.3.Identificationofthepigmentelutingat24.582minaszeaxanthin(1)basedonabsorptionspectrum,maximalabsorbancewavelengthsandIII:IIbandratio.

controlcellswasroundandshowednosignofDNAcondensation,

blebbingorshrinkage(Fig.6B).Thetreatmentwithstaurosporine

2␮M(nonselectivekinasesinhibitor,controlapoptosisinducer)

evokedahighcytotoxicityevidencedbycellshrinkage(Fig.6C)and

DNAcondensationinthenucleus(Fig.6D).A72htreatmentwith

zeaxanthin40␮Mhalfloweredthecelldensity,evokedrounding

ofthecells(Fig.6E)andnuclearfragmentation(Fig.6F),suggesting

ablockadeinthecellcycleandapoptosisinduction.

ZeaxanthininducesapoptosisinA2058cells

Toevaluatetheeffectofzeaxanthinoncelldeath,double

flu-orescencestainingwithAnnexinVand6-CFDAwasperformedto

differentiateliveandapoptoticcells.6-CFDAisusedtomeasure

viability.Inthissense,livecellswillonlystainwith6-CF(green),

cellsinearlyapoptosiswillstainbothwithAnnexinV(red)and

6-CF(green),andcellsinlateapoptosiswillonlystainwithAnnexin

VandDAPI.After72hoftreatment,zeaxanthin40␮Mincreased

thenumber ofAnnexinVand 6-CFDAdouble-stainedcells, and

AnnexinVandDAPIstainedcellscomparedtocontrol,indicating

enhancementofapoptosis(Fig.7).Asimilarresultwasobserved

forstaurosporine(1␮M),aknownpro-apoptoticagent.

Exposuretozeaxanthinevokedcaspase-3activation,

internucleosomalfragmentationandhypopolyploidyinA2058 cells

Activationofcaspase-3isconsideredasacentraleventfor

inte-gratingpro-apoptoticstimuliandactivatingdownstreameffector

caspasesandDNAsesduringcancercellapoptosis.Thebasal

activ-ityofcaspase-3waslowinA2058cellsgrownfor72hincontrol

cellculturemedium (Fig.8).Treatmentwithzeaxanthin 40␮M

for72hinducedaverysignificantincreaseincaspase-3activity

(Studentttest,p<0.01),asdemonstratedbyhydrolysisofthe

spe-cificchromogenicsubstrateAc-DEVD-pNA(Fig.8).Treatmentwith

staurosporine2␮m,usedasapositivecontrolforcaspase-3

acti-vation,inducedahighlysignificantincreaseincaspase-3activity

(Studentttest,p<0.001).

Asone of the main consequences of caspase-3 activation is

thesubsequentDNAfragmentationbycaspase-activatedDNases,

(5)

100

100 476.3641

477.3517

568.4217

567.4088

569.4084 569.4305 568.4294

1:TOF MS ES+ 9.42e5

5.91e4 2: TOF MS ES+

0

0 440

440

480

500

520

540

560

580

600

600 m/z

m/z 460

460

%

Fig.4. MSE_{fragmentation}_pattern_of_zeaxanthin₍₁₎_isolated_from_Porphyridium_purpureum.

60

40

20

0

0 20 40 60

Zeaxanthin concentration (µM)

(% control,2000 cells g

ro

wn f

or 72h)

Gro

wth inhibtion of A2058 melanoma cells

Fig.5. GrowthinhibitionofA2058cellinthepresenceofzeaxanthin(1).A2058 cellsweregrownfor72hinacellculturemediumcontainingincreasing concen-trationsofzeaxanthin.Theantiproliferativeactivityofzeaxanthinwasobservedfor concentrationssuperiorto5␮MandIC50wasdeterminedas40␮M.

fragmentationinA2058cellstreatedwithzeaxanthin.Evaluationof

cellcycleprogressionbyflowcytometryrevealedtheappearance

ofasub-G1cellpopulationafterexposuretozeaxanthin40␮M,

characteristicofdyingcells(Fig.9).Quantificationusingtheflux

cytometersoftwareindicatedthat2.9±0.4%ofcontrolcellswerein

sub-G1phaseincomparisonwith23.6±3.7%inzeaxanthin-treated

cells.

Agarose gel electrophoresis confirmed the internucleosomal

fragmentation ofDNA extracted from A2058cells treated with

zeaxanthin40␮Morstaurosporine2␮M(Fig.10),demonstrating

theactivationofcaspase-activatedDNasebyzeaxanthin.

Zeaxanthinstimulatestheexpressionofthepro-apoptoticfactors BimandBidandinhibitsthenucleartranslocationofNF-Bin A2058melanomacells

It was previously reported that zeaxanthin decreased the

expression of antiapoptotic proteins (Bcl-2 and Bcl-xL) and

increasedtheexpressionofproapoptoticproteins(BakandBax)

in zeaxanthin-treated uveal melanoma cells (Bi et al., 2013).

To complete the identification of signaling pathways involved

in zeaxanthin-induced apoptosis of melanoma cells (Bi et al.,

2013), theexpression of pro-apoptotic,anti-apoptotic and

pro-inflammatoryfactorswereinvestigatedbywestern-blotanalysis.

Zeaxanthin 40␮M respectively induced a high and moderate

expression increase of the pro-apoptotic factors Bim and Bid

(Fig.11)thatcouldbeinvolvedintheonsetofapoptosis.The

expres-sionofthepro-apoptoticfactorBakwasunchanged.Thelevelof

phosphorylatedNF-␬Bp65(Ser536)wasdecreasedwhileI␬B␣and

Ikk␣wereup-regulated,indicatingthatzeaxanthininhibitedthe

nucleartranslocationofNF-␬Bandsubsequentlydown-regulated

theexpressionofpro-inflammatorygenes.Ikk␤and

unphosphory-latedNF-kBexpressionswereunchanged(Fig.11).

Zeaxanthinpotentiatestheantiproliferativeactivityof VemurafenibinA2058melanomacells

Toassessthecapacityofzeaxanthintopotentiatethegrowth

(6)

A

B

D

F

20 µm 20 µm 20 µm

E

C

100 µM

Fig.6.CytotoxicityandnuclearfragmentationinA2058cellsexposedtozeaxanthin40␮M.A2058cellsweregrownfor72hinacontrolcellculturemedium(AandB)orin amediumcontainingstaurosporine2␮M(CandD)orzeaxanthin40␮M(EandF).Treatmentwithstaurosporinevokedcellroundingandchromatincondensation(Cand D)whilezeaxanthinevokedcellrounding(E),chromatincondensationandnuclearfragmentation(F).

6-CFDA

Staurospor

ine

z

eaxanthin

Control (A2058)

Annexin V DAPI Merge

Fig.7.ZeaxanthininducesapoptosisofA2058melanomacells.AnnexinV(red)and 6-CFDA(green)doublestainingofapoptoticcellswasexaminedbyfluorescence microscopy.A2058cellsinearlyapoptosisshowedbothgreenandredstains;A2058 cellsinlateapoptosisshowedredandbluestains;andcontrol(untreated)cells stainedgreenonly.Cellstreatedwithzeaxanthin(40␮M,for72h)orStaurosporine (1␮M,for24h)wereconsideredinearlyorlateapoptosiscomparedtocontrol.Scale bar:50␮m.

clinicaltreatmentofmetastaticmelanoma,A2058melanomacells

weretreatedfor72hwithzeaxanthin40␮M,vemurafenib5␮Mor

thecombinationofbothmolecules.ControlA2058cellsexhibited

aregularepithelialmorphologyandbecamesub-confluentin72h

(Fig.12A),withahighproportionofmitoticcellsindicatingahigh

proliferationrate.Zeaxanthin40␮Minduceda reductionincell

density,cellshrinkage,DNAcondensationevidencedbythe

obser-vationofnucleargranulationsand appearanceofapoptoticcells

(blackarrows)(Fig.12B).Vemurafenib5␮Mhadadrasticeffect

onA2058cellproliferationandmorphology,evidencedbyalow

celldensity,cellbodyshrinkage,cellthinningandDNA

conden-sationwiththepresenceof nucleargranulations(Fig.12C).The

morphologyofcellstreatedwiththecombinationofzeaxanthin

and vemurafenibwas similarto thatobserved withthe

vemu-rafenibtreatmentalone (Fig.12D). As compared tothecontrol

cellculturemedium,zeaxanthin40␮Minduced44.1±6.4%growth

inhibition, vemurafenib 0.1␮M10.40±2.12, 1␮M 32.74±2.71,

and5␮M67.3±7.7%growthinhibitionand thecombination of

both48.38±3.98,58.48±3.80and74.5±6.2%growthinhibition,

respectively.Thuszeaxanthin40␮Minduceda37.98,25.75and

10.7%increaseoftheantiproliferativeactivityofvemurafenib0.1,

1and5␮M,respectively(Fig.12E).Thisvaluewasintherangeof

calculatedstandarddeviationsofantiproliferativeactivities.

Discussion

Advancedmelanomahaveabadprognosisasmostmolecules

usedincancerchemotherapyareineffectiveinkillingmetastatic

melanomacellswhichareconstitutivelyoradaptativelyresistant

to pro-apoptotic drugs. The developmentof targeted therapies

usingBRAFinhibitorshassignificantlyimprovedthetreatmentof

metastaticmelanomasastheV600EBRAFoncogenicmutationis

foundinmorethan70%ofclinicalcases.Howevermostpatients

eventuallydevelopresistancemechanismsthatultimatelyleadto

therapeuticimpasses. In this view,many researchprojectsaim

toidentifynaturalmoleculeswithcytostatic,antimetastaticand

(7)

8

7

5 6

4

3

2

1

∗∗

∗∗∗

0

Control Zeaxanthin Staurosporine

40 µM 2 µM

A

C-DEVD-pNA h

ydrolysis per 10

6 cells (

µ

mol.min

-1)

Fig.8. Zeaxanthininducescaspase-3activationinA2058melanomacells.A2058cellsweregrownfor72hinacontrolcellculturemediumorinamediumcontaining zeaxanthin40␮Morstaurosporine2␮M(positivecontrolforapoptosisinduction).

900

700

800

600

500

400

300

200

100

102 ₁₀3 ₁₀4 ₁₀5

0

Control cell culture medium

Zeaxanthin 40 µM

IP fluorescence

900

700

800

600

500

400

300

200

100

102 ₁₀3 ₁₀4 ₁₀5

0

Number of A2058 cells

Sub-G1

G0

G0 G1 S

G1 S G2/M

G2/M

Fig.9.CellcycleanalysisofA2058cellsgrownfor72hincontrolcellculturemedium(A)orcellculturemediumcontainingzeaxanthin40␮M(B).Themajorityofcontrolor treatedcellswereinthequiescence(G0)orpre-replicative(G1)phases.Zeaxanthininducedtheappearanceofasub-G1peak,characteristicofhypodiploidiccellsundergoing celldeaths.

andcouldbeusedtopotentiatetheefficiencyofchemotherapy

andimmunotherapyandslowtheemergenceofresistance

mech-anisms(Craggetal., 1997;Caltagirone etal.,2000; Nilesetal.,

2003;Mesquitaetal.,2009;VanGoietsenovenetal.,2010;Pasquet

etal.,2011;Ahmadetal.,2013;Zhangetal.,2014;Alqathamaand

Prieto,2015;Mirzaeietal.,2016).Manycarotenoidsmeetallthese

activities,astheydisplayhighcytotoxicityintumorcellsfrom

var-ioushistologicalorigins,includingchemoresistantmelanomacell

lines,andexertsignificantantitumoralactivityinvivoby

(8)

10000 pb

MW - _Zea stau

3000 pb

1000 pb

500 pb

Fig.10.AgarosegelelectrophoresisofDNAextractedfromA2058cellsincubatedfor 72hintheabsence(−)orpresenceofzeaxanthin40␮M(zea)orstaurosporine2␮M (stau).Observationofasmearinthezeaxanthinandstaurosporinelanesrevealed theactivationofcaspase-activatedDNasesandinternucleosomalfragmentationof DNAinapoptoticcells.

Kumaretal.,2013;Chenetal.,2017).Moreover,mostcarotenoids

havenooraltoxicityanddonotweakentheimmunesystem(Chew

and Park, 2004;Pechinskii and Kuregyan,2014; Ghodratizadeh

etal.,2014).Microalgaeconstituteanoptimalsourcetoproduce

carotenoidsforpharmaceuticalapplicationsastheycombinethe

advantagesofsynthesizingthewidechemodiversityofcarotenoids,

withhighproductionyields,withouttheneedoffreshwater,

agri-culturalsurfacesorpesticidestobegrown(Mimounietal.,2012).

Selection of hyper-producing strains combined tooptimization

oftheirgrowthconditions and purificationprocesses allowthe

recoveryofhighamountsofcarotenoidsdevoidofchemicalsor

endotoxins.Inthepresentreport,wedemonstratethat

zeaxan-thin,anabundantcarotenoid presentinmicroalgae,thatcanbe

easily obtained in high amounts from the rhodophyte P.

pur-pureum,inducesapoptosisinhumanmelanomacells expressing

theoncogenicBRAFV600Emutationandpotentiatesthe

antipro-liferativeactivityofvemurafenib,aBRAFinhibitorusedinpatients

withadvancedmetastaticmelanomas.Zeaxanthin(1)was

previ-ouslyreportedtohavenooraltoxicityanddecreasetheincidence

of variouscancers afteroral ingestion(Thurnhamand Howard,

2013;Xuetal.,2013).Itwasalsoreportedtoexertpro-apoptotic

activity in humanuveal melanoma cells (SP6.5 and C918) and

limit uveal melanoma invasivity without impairing the

viabil-ity of non canceruveal melanocytes (Xu et al.,2015; Bi et al.,

2013).InA2058melanomacells,zeaxanthinIC50wasdetermined

as40␮M,aconcentrationinducingcellrounding,chromatin

con-densation,nuclearfragmentation,hypodiploidy,cellapoptosisin

early and late stages, accumulation of cells in sub-G1 phase,

DNAinternucleosomalfragmentationand activationof

caspase-3.Zeaxanthin-inducedapoptosiswasaccompaniedbyinhibition

ofNF-␬Bandup-regulationofthepro-apoptoticfactorsBimand

Bid,demonstratingtheinvolvementofthemitochondrialsignaling

pathwayinapoptosistriggering,aspreviouslyreportedinuveal

melanomamodels(Bietal.,2013).Theobservationthat

zeaxan-thinwasabletomoderatelypotentiatetheinvitroantiproliferative

activityofvemurafenibisapromisingresultofourstudyasit

sug-gestsitspotentialinterestasanutritionaladjuvantincreasingthe

sensitivityoftumorcellstoBRAFinhibitors.Bypotentiatingthe

antiproliferativeeffectofBRAFinhibitors,zeaxanthinmayallowto

decreaseBRAFinhibitorseffectivedoses,limittheiradverseeffects

inpatientsanddelaytheemergenceofresistancemechanisms(Chu

etal.,2012;Zimmeretal.,2012;Anforthetal.,2015;Welshand

Corrie,2015).Themolecularmechanismsinvolvedinthisincrease

ofsensitivityaswellaspreclinicalandclinicalrelevanceof

com-bining carotenoidswithBRAFinhibitorswillhave tobefurther

Ctrl

ß-action

ß-action pNF-kB NF-kB kDa

kDa

37

37 87 85 37 39 70 70

30

25

20

(Ser 536)

IkBα

Ikkα

Ikkβ

β-action Bcl-XL

Bim

Bak

Bid

Ctrl

Zea Zea

Zea

(9)

A

B

C

D

E

90

80

70

60

50

40

30

20

10

0

Control Zea

+ 37.98%

+ 25.74%

+ 10.70%

% Gro

wth inhibition at 72 h

(2000 A2058 cells)

Vemurafenib

Vemurafenib Vemurafenib

5 µM 1 µM

0.1 µM

Zea Zea Zea

50 µm

Fig.12. ZeaxanthinsensitizesA2058melanomacellstotheBRAFinhibitorvemurafenib.TwothousandsA2058melanomacellsweregrownfor72hincontrolcellculture medium(A),mediumcontainingzeaxanthin40␮M(B),vemurafenib5␮M(C),oramixofzeaxanthin40␮Mandvemurafenib5␮M(D).Zeaxanthininduceda37.98,25.75 and10.70%increaseoftheantiproliferativeactivityofvemurafenib0.1,1and5␮M,respectively(E).

explored,asthisstrategycouldextendthedurationof

chemother-apyefficiency.In summary,thisstudyconfirms thepotentialof

zeaxanthintolimitthegrowthofchemoresistantmelanomacells,

confirmsthemajorinterestofphytoplanktoncarotenoidsas

natu-ralanticancermoleculesdevoidoforaltoxicity,andsuggestsforthe

firsttimetheinterestofcombiningacarotenoidtoBRAFinhibitors

topotentiatetheirchemotherapeuticefficiencyinmelanomacells.

Authors’contributionandresponsibility

JBBandENproducedP.purpureumbiomass.CJ,RGOJ,AF,CO,LPy,

IL,GPandLPperformedthepigmentextraction,purification,cell

culture,western-blotandapoptosisexperiments.CJperformedthe

HRMSanalysis.LBandCJperformedthefluxcytometryanalysis.LP

designedtheexperiments,interpretedthedata,directedthestudy

andwrotethemanuscriptincollaborationwithVTandJRGDSA.LP

takesresponsibilityfortheintegrityofthework,frominceptionto

finishedarticle.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor

thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat

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Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearegratefultotheFrenchcancerleague(Comité17dela

LigueNationalecontreleCancer)forfinancialsupportandtothe

Poitou-CharentesregionforCJ’sPhDgrant.Wealsothankthe

“Can-céropôleGrandOuest,axeValorisationdesproduitsdelameren

cancérologie”forscientificsupport.LPisgratefultotheBrazilian

SocietyofPharmacognosyforitskindinvitationtotheXth_Brazilian

SymposiumofPharmacognosyinPetrolinainSeptember2015.We

thankDrRaymondKaasfromIFREMERforhiskindpermissionto

usethemicrophotographyofPorphyridiumpurpureumandAntoine

Bonnet(PlatformfortheHighResolutionAnalysisofBiomolecules,

UniversityofLaRochelle,France)forexcellenttechnicalassistance

withHRMS.

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