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Acta
Histochemica
j ou rn a l h o m e p a g e :w w w . e l s e v i e r . d e / a c t h i s
Melatonin
action
in
tumor
skeletal
muscle
cells:
an
ultrastructural
study
S.
Burattini
a,∗,
M.
Battistelli
a,
S.
Codenotti
b,
E.
Falcieri
a,
A.
Fanzani
b,1,
S.
Salucci
a,1aDepartmentofBiomolecularSciences(DiSB),UrbinoUniversityCarloBo,ViaCa’LeSuore2,61029Urbino,Italy bDepartmentofMolecularandTranslationalMedicine,BresciaUniversity,VialeEuropa11,25123Brescia,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received14October2015
Receivedinrevisedform19February2016 Accepted19February2016 Keywords: Apoptosis Melatonin Rhabdomyosarcoma
a
b
s
t
r
a
c
t
Melatonin(Mel),orN-acetyl-5-methoxytryptamine,isacircadianhormonethatcandiffusethroughall thebiologicalmembranesthankstoitsamphiphilicstructure,alsoovercomingtheblood–brainbarrier andplacenta.AlthoughMelhasbeenreportedtoexhibitstrongantioxidantpropertiesinhealthytissues, studiescarriedoutontumorculturesgaveadifferentpictureofitsaction,oftendescribingMelaseffective totriggerthecelldeathoftumorcellsbyenhancingoxidativestress.
Basedonthispremise, hereMeleffectwasinvestigatedusingatumorcelllinerepresentativeof thehumanalveolarrhabdomyosarcoma(ARMS),themostfrequentsofttissuesarcomaaffecting child-hood.Forthispurpose,Melwasgiveneitherdissolvedinethanol(EtOH)ordimethylsulfoxide(DMSO) atdifferentconcentrationsandtimeexposures.Cellviabilityassaysandultrastructuralobservations demonstratedthatMelwasabletoinduceadose-andtime-dependentcelldeathindependentlyonthe dissolutionsolvent.Microscopyanalyseshighlightedthepresenceofvariousapoptoticandnecrotic pat-ternscorrelatingwiththeincreasingMeldoseandtimeofexposure.ThesefindingssuggestthatMel, triggeringapoptosisinARMScells,couldbeconsideredasapromisingdrugforfuturemultitargeted therapies.
©2016PublishedbyElsevierGmbH.
1. Introduction
Humanrhabdomyosarcoma(RMS)isthemostcommon
pedi-atricsofttissuesarcomathatarisesfrommesenchymalprecursors
withthepotentialtodifferentiateintoskeletalmusclecells,but
failingtocompletemyogenesisbecauseofthepresenceof
chromo-somalaberrations(Zanolaetal.,2012).RMSareaggressivetumors
whose current chemotherapy is often unsuccessful(Dantonello
etal.,2015),especiallyforthealveolarrhabdomyosarcoma(ARMS)
thatischaracterizedbyachromosomaltranslocationthat
generat-ingthePax3-Foxo1fusionproteinconfersamoreaggressivetumor
phenotypeandresistancetoradiationtherapy(Jothietal.,2013).
Currently,theemploymentofamultimodaltherapyhasincreased
thesurvivalrateforpatientswithlocalizeddiseaseto70%albeit
withsignificanttoxicity(Soundararajanetal.,2012).Despitethat,
∗ Correspondingauthor.Fax:+390722304244. E-mailaddresses:sabrina.burattini@uniurb.it
(S.Burattini),michela.battistelli@uniurb.it(M.Battistelli),silvia.codenotti@unibs.it (S.Codenotti),elisabetta.falcieri@uniurb.it(E.Falcieri),alessandro.fanzani@unibs.it (A.Fanzani),sara.salucci@uniurb.it(S.Salucci).
1 Theseauthorsequallycontributedtothiswork.
thetherapysuccessforhighriskpatientsisabout20–30%(Lietal.,
2013),suggestingtheneedofmoreeffectivedrugs.
Over thepast years, Mel,besideits strongand documented
antioxidanteffects (Saluccietal.,2014b;Ganieetal.,2015),has
been shown to act as an anticancer drug in different tumors
(Sánchez-Hidalgoetal.,2012;Batistaetal.,2014;Codenottietal.,
2015),forexampleviainductionofthemitochondrialapoptosis
(Bejaranoetal.,2009)orattenuationoftelomeraseactivitybothin
vivoandinvitro(Leon-Blancoetal.,2003).
Numerousstudieshavedescribeditsanticanceractioninsolid
tumorsand inleukemiathroughinductionoftheapoptoticcell
death,leadingtoreplacementofneoplasticcellswithhealthycells
(Sánchez-Hidalgo etal., 2012;Chuffa etal.,2015).Additionally,
Kubatkaetal.(2001)demonstratedtheanti-tumoractionofMelin
breastcancerinvivo,andthiseffectwasthenconfirmedinclinical
studiesshowingitsprotectiveeffectfrommammary
carcinogen-esis(Nowfaretal.,2002)andinotherneoplasticdiseases(Hong etal.,2014;Wuetal.,2015).
Herewehaveevaluatedtheultrastructuralchangesoccurring
inthehumanalveolarRH30cellsuponadministrationofMel
dis-solvedeitherinEtOH(Saluccietal.,2014a)orDMSO(Jardim-Perassi
etal.,2014)atdifferentdosesandexposuretimes.
http://dx.doi.org/10.1016/j.acthis.2016.02.004 0065-1281/©2016PublishedbyElsevierGmbH.
S.Burattinietal./ActaHistochemica118(2016)278–285 279 2. Materialsandmethods
2.1. Cellculture
Human RH30 cell line, purchased from the European
Col-lection of Cell Cultures (ECACC; Salisbury, UK), was routinely
tested for the expression of the Pax3-Foxo1 chimeric protein
byboth genesequencingand westernblotanalysisusing
poly-clonalanti-Foxo1(H-128)antibody(Rabbit,sc-11350,SantaCruz
Biotecnology, Dallas, USA). Cells were also routinely tested for
mycoplasmacontaminationusingtheLookOutMycoplasmaPCR
DetectionKit (MP0035 Lookout,Sigma), according tothe
man-ufacturer’sinstructions.RH30cellswereculturedinRPMI1640,
supplementedwith10%heat-inactivatedfetalbovineserum,2mM
glutamine,1%antibiotics(PenicillinandStreptomycin)and
main-tained at37◦Cin humidified airwith5% CO2 (Codenotti etal.,
2015).
Mel(Sigma,St.Louis,MO,USA)wasdissolvedin1%DMSOor1%
EtOHattheconcentrationof100mM.After1or2mMMel
treat-ment,thecellbehaviorwasmonitoredbymeansoftheinverted
microscopy.TrypanBlueexclusionassaywasemployedto
quan-tifycellviability.Therelativenumberofdeadandlivingcellswas
obtainedcountingthenumber of stained(dead)and unstained
(live)cellsusingaNeubauerchamberatrevertedmicroscope.
Stu-dent’st-testwasappliedforstatisticalanalysestocompareresults
obtainedincontrolversustreatedsamples;P<0.05wasconsidered
assignificancethreshold.
2.2. Scanningelectronmicroscopy(SEM)
RH30cells werecultured oncoverslipsin Petridishes. After
washing with0.1Mphosphate buffer, adherent and suspended
cells were fixed with 2.5% glutaraldehyde in 0.1M phosphate
bufferfor1h.Thesuspendedcellpopulationwasdepositatedon
polylysine-coatedcoverslipsandthesampleswerepost-fixedwith
1%osmiumtetroxide(OsO4)in 0.1Mphosphatebuffersolution
(PBS)for1h.Afteralcoholdehydration,thesampleswere
criti-calpointdried,goldsputteredandobservedusingaPhilips515
scanningelectronmicroscope(Codenottietal.,2015).
2.3. Transmissionelectronmicroscopy(TEM)
RH30-treatedcellswerewashedandfixedwith2.5%
glutaralde-hydein0.1MPBSfor15min.Thecellswerescrapedandcentrifuged
at300xgfor10min.Thepelletswerefixedin2.5%
glutaralde-hydeforanadditional30min.Thesuspendedcellswerecollected
inEppendorf,centrifugedandfixedfor45mininglutaraldehyde.
Thesampleswerepost-fixedin1%OsO4for1h,alcoholdehydrated
andembeddedinaraldite.Thinsectionswerestainedwithuranyl
acetateandleadcitrateandanalyzedusingaPhilipsCM10
trans-missionelectronmicroscope(Saluccietal.,2014b).
2.4. Acridineorange(AO)andpropidiumiodide(PI)nuclei stainingconfocallaserscanningmicroscope(CLSM)
Cells werefixed with4% paraformaldehydein PBS (pH 7.4)
for30min,andsuspendedcellsweredepositedonpoly-lysinated
coverslips in Petri dishes. Adherent and suspendend samples
werewashedtwiceusingPBSandthen pre-treatedwithRNase
A10g/mL inPBSfor 30min.AfterPBSwashing sampleswere
exposedtoanequalmixtureofPI(1g/mL,LifeTechnologies)and
AO(1g/mL,LifeTechnologies)dilutedinPBSatroomtemperature
(protectedfromlight)for10minandthenobservedatCLSM(Leica
MicrosystemsCMSGmbH);FICTandPIexcitationwereat488and
500nm,respectively,andtheiremissionsignalsweredetectedat
617and525nm.CLSMimagesarepresentedasmaximumintensity
projectionorsingle-planeimages(Saluccietal.,2014b).
2.5. Westernblotting
Proteinhomogenateswereobtainedbyharvestingcellsinacold
lysisbuffer,composedby20mMTris–HCl(pH7.6),1%NonidetP40,
0.5%sodiumdeoxycholate,0.1%SDS,50mMNaClandacocktail
ofproteaseinhibitors(Roche)plusphosphataseinhibitors(1mM
Na3VO4and4mMNaF).
Bradford reagent assay has been used to calculate protein
concentration. Equal amount of protein samples were
sepa-ratedbySDS-PAGEunderreducingconditionsandtransferredto
polyvinylidinefluoridemembranes.Samplewereincubatedwith
specific primaryantibodies, Rabbitanti-Bax, polyclonal (Rabbit,
sc-526,SantaCruzBiotecnology,Dallas,USA)orRabbit
anti-Bcl-2, polyclonal (Rabbit, sc-492, Santa Cruz Biotecnology, Dallas,
USA) followed by peroxidase-conjugated secondary antibodies
(anti-mouseIgGfromSantaCruzBiotechnology,Dallas,USA;
anti-rabbitIgGfromThermoScientific,Erembodegem,Belgium).The
immunocomplexeswerevisualizedusingtheenhanced
chemilu-minescencereagent(GeneSpin,Milan,Italy)andimmune-reactive
bandswerequantifiedusingdensitometryanalyses(SoftwareGel
ProAnalyzer,version4)(Codenottietal.,2015).
2.6. Insilicoanalysis
The primary mouse tumor cultures fromRMS-bearing mice
werepreviouslyobtainedbyKellerandcolleagues(Rubinetal.,
2011)andutilizedforgeneexpressionanalysisbyemployingthe
microarrayapproach(IlluminaMouseRef-8BeadChip,v1.1).Data
setsweredepositedintheNCBIGeneExpressionOmnibusdatabase
with the accession number GSE22520. To analyze Mel
recep-torexpression,datawereprocessedusingthePartek® Genomics
Suite software version 6.6 (Partek, St. Louis, USA). Briefly, the
microarrayrawdatasetwerereprocessedbythebackground
cor-rection, normalization and summarization of probe intensities,
using the robust multiarray average analysis to determine the
specifichybridizingsignalfor each probe set.Afterbackground
correction,thedataweretransformedinbase-2logarithm.
Qual-itycontrolwasalsoperformedtoidentifythepresenceofarrays
withpoorqualityandevaluatewhetherbatcheffectsignificantly
affectedthedata.
A one-way analysis of variance (ANOVA) was performed to
determinedifferentiallyexpressedgenesinmousealveolarRMS
incomparisontonormalmusclesamples.Foldchange>2and
p-values<0.05wereusedascriteriatoevaluatesignificantdifference
ingeneexpression.
3. Results
ToanalyzewhetherMelmight affectcellsurvival in human
alveolarRH30cells,TrypanBlueexclusionassaywasperformed,
revealingthatMeldissolvedeitherinEtOHorDMSOsignificantly
reducedtheviabilityofRH30cellsinatimeanddosedependent
manner(Fig.1A).
Insupportofthisevidence,SEM(Fig.1)andTEM(Figs.2and3)
analysesconfirmedthatMelinducedcelldeathofRH30cellsina
dose-andtime-dependentmannerandregardlessofthe
dissolu-tionsolvent.Inparticular,whilecontroladherentcellsappearedto
befusiformorstar-shaped(1B),1mMMel-treatedcellsshoweda
roundshapemorphologyalreadyafter24hoftreatment(1C,1H).
Thiseffectwasevenincreasedafter48h(1D,1I)or72h(1E,1L)of
Meltreatmentandaccompaniedbythemembraneblebbing
Fig.1.Trypanblueexclusionassay.Histogramshowingthepercentagesofliving(darkgreycolumn)anddead(lightgreycolumn)cells,underthedifferenttreatment conditions(A).Dataarefromthreeindependentexperimentsandareshownasmeanofvaluepercentage±standarddeviation.AllMeltreatmentsarestatisticallysignificant versuscontrolcondition(T-test,p<0.05).
SEManalysisofcontroluntreatedcells(B)versusMel-treatedcells(C-O);indetail,representativepicturesshowingcellstreatedwith1mMMel(inEtOH)for24-48–72h(C, D,Erespectively)and2mMMel(inEtOH)for24–48(F,Grespectively)or,alternatively,with1mMMel(inDMSO)for24-48–72h(H,I,L,respectively)and2mMMel(in DMSO)for24(M)-48h(N,O).Bars:10mB-O.
S.Burattinietal./ActaHistochemica118(2016)278–285 281
Fig.2. TEManalysisofcontroluntreatedcells(A)versusMel-treatedRH30cells(inEtOH)(B–G);indetail,representativepicturesshowingcellstreatedwith1mMMelfor 24h(B,C),48h(D,insetD),72h(E)and2mMMelfor24–48h(F,Grespectively)Bars:A,B,G,F,insetD,0.5m;C,insetG,0.25m;D,E,insetF,1m.
thediffusecelldamageoccurringafter24h(1F,1M)and48h(1G,
1N,1O)wascharacterizedbyroundingandblebbedcells.
TEMobservations(Fig.2)showedcontrolcellshavingan
elon-gatedshape andintactsubcellularstructures(2A).Instead,after
1mM Mel administration for 24h, cells showed altered
mito-chondria(2B)and autophagic vacuoles (2C).Occasionally, early
apoptoticnuclearfeatureswereobserved(datanotshown).After
48and72h,mostofthetreatedcellsappearedtobeinlate
apo-ptosis(2D,2E),whereassomeofthemshowedahighnumberof
autophagic-likestructures(inset2D).
Increasing Mel concentration up to 2mM for 24 and 48h
triggereddifferentcellular alterations, includinga diffuse
cyto-plasmicvacuolization(2F),chromatincondensation(insetF)and
autophagic features (inset G). In particular, cells in secondary
necrosiswereobservedafter2mMexposurefor48h(2G).
Con-sistentwiththeseresults,theeffectsproducedonRH30cellsby
MeldissolvedinDMSOweresimilar(Fig.3).Earlyandlate
apop-toticpatternscouldbeobservedafter1mMMelat24h(3A),48h
(3B)and72h(3C).Secondarynecrosisaswellasautophagic
fea-turesappearedafter2mMMeltreatmentat24h(3D,insetD,3E)
and48h(3F).
CLSM evaluation of RH30 cells exposed to Mel dissolvedin
DMSOwascarriedoutbymeansofAO/PIdoublestaining(Fig.4).
InFig4A,theuniformgreenlabelingindicativeofcellularhealthy
structureswasvisibleincontrolcondition.AfterAOandPInuclear
staining,treatedcells(1mMMelfor24h,Fig.4B)showedagreen
stainsimilartocontrols,althoughdisplayingamorerounded
mor-phologycomparedtountreatedcells.After48and72h,thenumber
ofapoptoticcellsincreased(Fig.4C,insetCandD)andsomecells
withorangeareasduetoPIpermeabilitywereobserved(Fig.4C).
Cellstreated with2mMMelfor 24 h(Fig.4E, insetE)showed
orangenucleiduetolateapoptoticeventsandnecrosis.
Inaccordancewiththeobservedapoptoticresponseafter1mM
Fig.3.TEManalysisofMel–treatedRH30cells(inDMSO)(A–F);indetail,representativepicturesshowingcellstreatedwith1mMMelfor24–48–72h(A,B,Crespectively) and2mMMelfor24(D,E)and48h(F)Bars:A,C,E,1m;B,F,0.5m;insetB,D,0.25m;insetD,2m.
Fig.4.CLSManalysisofcontroluntreated(A)versusMel-treatedRH30cells(inDMSO)afterAO/PIdoublestaining(B–E).Indetail,representativepicturesshowingcells treatedwith1mMMelfor24–48–72h(B,C,Drespectively)and2mMMelfor24h(E).Bars:A–E,10m.(Forinterpretationofthereferencestocolorinthetext,thereader isreferredtothewebversionofthisarticle.)
WesternBlotanalysisshowingexpressionlevelsofBaxandBcl-2invehicle-treatedversus1mMMel-treatedRH30cellsattheindicatedtime-points(F).Tubulinwasused asproteinloadingcontrol.
bothanincreaseinthepro-apoptoticBaxexpressionandadecrease
inthelevelsofanti-apoptoticBcl-2incomparisontountreatedcells
(Fig.4F),thereforeconfirmingthatMelinducedapoptosisinRH30
cells.
Finally,aninsilicoanalysisofmicroarraysdatageneratedfrom
primarymouseRMStumorcultures(Kelleretal.,2004;Kellerand
Capecchi,2005)revealedthatARMStumorscouldbesensitiveto
S.Burattinietal./ActaHistochemica118(2016)278–285 283
Fig.5.DotplotgraphsandrelativemeanhistogramsrepresentingthetranscriptionallevelsofMT1(ILMN1256388probenumber)andMT2(ILMN2770987probenumber), asdetectedinsilicobyperformingacomparisonbetweentwentyARMSsamplesandfourskeletalmusclesamples.pvaluewasnotsignificant.
receptorsMT1and MT2weredetectableinARMS subsets with
expressionlevelscomparabletothoseobservedinskeletalmuscle
(Fig.5).
4. Discussion
Epidemiologicalstudiescarriedout overthepastyears have
indicated that patients affected by some types of cancer have
reducedbloodylevelsofMelcomparedwithhealthypeople(Mills
etal.,2005;Lee,2006).Sincethen,thispinealneurohormoneMel
hasbeenrepeatedlydescribedasapotentialanti-cancer
molecu-lartoolgivenitsdemonstratedpro-apoptoticeffectandoncostatic
action towards differentcancercell lines (Bizzarriet al., 2013;
Perdomoetal.,2013;Rodriguezetal.,2013;Hongetal.,2014),
thereforecontributingtocancerprotection(Hrusheskyetal.,2009;
Maoetal.,2010;Bukowska,2011;Santoroetal.,2012).
Basedonthispremise,herewefocusedontheultrastructural
effectsinducedbyMelonahumanARMScelllinecarryingaunique
molecularsignaturefoundamongsarcomas,thePax3-Foxo1fusion
proteinarisingfroma specificchromosomal translocation(Jothi
etal.,2013).RMSneoplasmisthemostcommonsofttissuesarcoma
ofchildhood.AlthoughaggressivetreatmentofRMScouldprovide
long-termbenefit,resistancetoprolongedtherapiesisarecurrent
problem(Fayeetal.,2015).Thelastdecadehasseenan
extraordi-naryincreaseofstudiesonRMSforunderstandinghowapoptotic
mechanismsmaycontributeoncancereradication.Inthisregard,
theintegrityofapoptosispathwaysinRMSconstitutesacritical
determinantofthesensitivitytomostcurrenttreatmentstrategies
(Fulda,2013).
Inthisworkwehavefirstconfirmedpreviousfindingsshowing
the ability of Mel administered at pharmacological
concentra-tions(Rodriguezetal.,2013)intriggeringcelldeathofRMScells
(Codenottietal.,2015);moreover,sincethedissolution solvent
representsanimportantvariablepotentiallyinfluencingtheMel
action,typicallytheabsoluteEtOH(Saluccietal.,2014a;Salucci
etal.,2014),herewe haveestablishedthatboth thesesolvents
didnotinfluencetheefficacyofneurohormoneactivityasdeath
inducerinRH30cellline.
IncreasingbothMeldosesandexposuretimeleadtolate
apo-ptosisandsecondarynecrosisaconsistentnumberofRH30cells.
In this regard, ourultrastructural analysesrevealed that 1mM
Meldoseforupto48and72hrepresentedtheidealconditions
fortriggeringclassicalapoptosis-related morphologicaland
bio-chemicalchanges,suchassurfaceblebbing,chromatinmargination
andcondensation,mitochondriaalteration,cytoplasmic
vacuoliza-tion, autophagic vacuoles accompanied by Bax activation and
Bcl-2downregulation;inthiscase,RH30cellsmaintaineda
mor-phological plasma membrane integrity and necrotic cells were
detectedonlyoccasionally.Ontheotherside,secondarynecrosis
andnecroticcelldeathappearedtobethemajordeathresponseat
veryhighMeldose(2mM).Fromatherapeuticpointofview,killing
cancercellsviaapoptosisratherthannecrosiscouldbeconsidered
amorepreferableway,giventhattheappearanceofnecrosisinvivo
mightbeaccompaniedbyaninflammatoryresponsetypically
asso-ciatedtoamoreunfavorableoutcome.Hence,theoptimizationof
dosagesforinvivotrialsshouldtakeintoaccountthesepreliminary
invitroobservations.
ThemechanismsbywhichMelinfluencesapoptosishavenot
beenyetfullyclarified(Sainzetal.,2003;Wölfleretal.,2001;Gong
etal.,2003),althoughseveralreportshaveputforwardtheability
ofMeltoelicitapoptoticeffectsthroughchangesintheoxidative
status.Inthisregard,acorrelationbetweentheincreaseinROS
productionand theinductionofMel-drivenapoptosishasbeen
describedindifferentcelllines(Büyükavcietal.,2006).
Melaction isclassicallymediatedbytheinteractionwithits
membrane receptors(Radognaet al., 2009), i.e., MT1and MT2,
whichseemedtobeexpressedalsoinanumberofmouseARMS
tumors,asinferredthroughaninsilicoanalysisthatstillawaits
furtherinvitroandinvivoconfirmation.Hence,thepro-apoptotic
MeleffectonRH30cellscouldbereceptordependent,althoughit
isalsopossiblethathighdosesofMelmayfavoritssimple
diffu-sionacrossthecellmembraneinareceptor-independentmanner.
Onthewhole, understandingwhetherMelactionin ARMScells
maybe mediated or not (orat least partially)by its receptors
willbeimportantforidentifyingthepathwaysunderlyingits
pro-apoptoticaction;inaddition,thesynthesisandselectionofmimetic
compoundswithincreasedactivitycouldprovideafurther
phar-macologicaltoolforovercomingthegrowthofthissolidtumor.
5. Conclusions
ThedatareportedinthisworkindicatethatMel,when
adminis-teredatpharmacologicaldoses,canprofoundlyaffectcellsurvival
inRMS,themostfrequentmyogenicsofttissuesarcomaaffecting
childrenandadolescents(Ognjanovicetal.,2009;MeyerandSpunt,
2004).Inparticular,Melwasabletotriggeranapoptoticprogram,
independentlyonthedissolutionsolvent,thereforerepresentinga
promisingdrugforcounteractingRMStumorprogression.
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