In vitro effect of 5-fluorouracil and paclitaxel on Echinococcus granulosus larvae and cells

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Acta Tropica

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a

In vitro effect of 5-fluorouracil and paclitaxel on Echinococcus granulosus larvae and cells

P.E. Pensel

, C. Albani

, G. Ullio Gamboa

, J.P. Benoit

, M.C. Elissondo

aLaboratoriodeZoonosisParasitarias,Fac.CienciasExactasyNaturales,UniversidadNacionaldeMardelPlata,CONICET,MardelPlata,Argentina

bLaboratoriodeFarmacotecnia,Fac.CienciasQuímicas,UniversidadNacionaldeCórdoba,UNITEFA,Córdoba,Argentina

cConsejoNacionaldeInvestigacionesCientíficasyTécnicas(CONICET),BuenosAires,Argentina

dINSERMU1066,MINT—MicroetNanomédecinesBiomimétiques,IBS-CHUAngers,49933AngersCedex9,France

a r t i c l e i n f o

Articlehistory:

Received26March2014

Receivedinrevisedform27June2014 Accepted23July2014

Availableonline1August2014

Keywords:

Cystechinococcosis Echinococcusgranulosus Hydatidcysts 5-Fluorouracilo Paclitaxel

a b s t r a c t

HumancysticechinococcosisisazoonosiscausedbythemetacestodestageofthetapewormEchinococcus granulosus.Althoughbenzimidazolecompoundssuchasalbendazoleandmebendazolehavebeenthe cornerstoneofchemotherapyforthedisease,thereisoftennocompleterecoveryaftertreatment.Hence, insearchingfornoveltreatmentoptions,weexaminedtheinvitroefficaciesof5-fluorouracil(5-FU)and paclitaxel(PTX)againstE.granulosusgerminalcells,protoscolecesandcysts.5-FUorPTXinhibitedthe growthofE.granulosuscellsinatimedependentmanner.Althoughbothtreatmentshadaprotoscolicidal effect,5-FUhadaconsiderablystrongereffectthanPTX.5-FUproducedadose-andtime-dependent effect,provokingthecompletelossofviabilityafter24daysofincubation.Moreover,cystsdidnotdevelop followingtheinoculationoftreatedprotoscolecesintomice.ThelossofviabilitywasslowerinPTXtreated protoscoleces,reachingtoapproximately60%after30days.Theresultsoftheinvitrotreatmentwith5- FUandPTXweresimilarinsecondarymurinecysts.TheemploymentofSEMandTEMallowedusto examine,atanultrastructurallevel,theeffectsinducedby5-FUandPTXonE.granulosusgerminalcells, protoscolecesandmurinecysts.Inconclusion,thedataobtainedclearlydemonstratedthat5-FUandPTX atclinicallyachievableconcentrationsinhibitthesurvivaloflarvalcells,protoscolecesandmetacestodes.

Invivostudiestotesttheantiparasiticactivitiesof5-FUandPTXarecurrentlybeingundertakenonthe murinemodelofcysticechinococcosis.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Cystic echinococcosis (CE)caused by the larval stageof the tapewormEchinococcusgranulosusisachronic,complex,andstill neglectedparasiticinfectiondisease(Brunettietal.,2011).Human orungulateintermediatehosts(sheep,goats,pigs,cattle,horses orcamels)becomeinfectedafteringestionofeggspassedintothe environmentwithfaecesfromdefinitivehosts(dogsandwolves).

Theoutcomeofinfectionisthedevelopmentoffluid-filledcysts mainlyintheliverandthelungs,althoughotherorganscanalsobe affected(Budkeetal.,2013).

The WHO-IWGE classification providesthe basis for choos- ing basically four treatment and management options for CE: surgery, percutaneous sterilization, chemotherapy with

∗Correspondingauthorat:LaboratoriodeZoonosisParasitarias,FacultaddeCien- ciasExactasyNaturales,UniversidadNacionaldeMardelPlata(UNMdP),Funes 3250,7600MardelPlata,Argentina.Tel.:+542234752426;fax:+542234753150.

E-mailaddresses:c.elissondo@gmail.com,mceliss@mdp.edu.ar(M.C.Elissondo).

benzimidazolesandobservation(watchandwait)forinactive,clin- icallysilentcysts(Brunettietal.,2011).Eachofthesetherapeutic toolshaslimitationsdependingontheindividualcase.Theevi- dencesupportinganyofthesemodalitiesfromcarefullydesigned clinicalstudiesisinsufficientandthechoiceoftreatmentoptions remainscontroversial(Stojkovicetal.,2009).

Medical treatmentis indicatedwhen surgicalremoval isnot appropriateforpatientswithmultiplecystsintwoormoreorgans, forpreventionof secondaryechinococcosisaftersurgery andin somecasesforpresurgicaltreatment(Pawlowskietal.,2001).The benzimidazole(BZ)compounds–albendazole(ABZ)andmeben- dazole(MBZ)–havebeenthecornerstoneofchemotherapyforCE (McManusetal.,2012).

Severalfactorsaffecttheresponsetomedicaltreatment,suchas sizeandageofthecysts,thicknessofhostderivedconnectivetis- sue,calcification,cystcomplicationwithmultiplecompartments ordaughtercysts,abilityofthedrugtopenetratethecystwalland persistenceofadequatelevelof drugoritsactivemetaboliteat thesiteofparasitelocation(Ceballosetal.,2008;Hemphilland Müller,2009).ApproximatelyathirdofpatientstreatedwithBZ http://dx.doi.org/10.1016/j.actatropica.2014.07.013

0001-706X/©2014ElsevierB.V.Allrightsreserved.

drugshavebeencured,30–50%developsomeevidenceofathera- peuticresponsewhilebetween20and40%ofcasesdonotrespond favorably(MoroandSchantz,2009).Moreover,BZsometimesacts exclusivelyasaparasitostaticagent,andrelapsesafterchemother- apyhavebeenreported(Stamatakosetal.,2009).

Praziquantel(PZQ),aheterocyclicpyrazinoisoquinolinederiva- tive,hasbeenproposedtobeusedalongsideBZinCE-patients.

Nevertheless,furtherstudiesarerequiredtodeterminewhether there are significantbenefitsfrom a combination therapy with ABZandPZQovermonotherapywithABZ(Bygott andChiodini, 2009).Withregardtothesedifficulties,thedevelopmentofanew therapeuticdrugforhumantreatmentofcysticechinococcosisis necessary.

There are a number of similarities between cancer cells and some parasites (Klinkert and Heussler, 2006). Particu- larly,Echinococcusmetacestodesexhibittumor-likeproperties,as reflectedbytheirseeminglyunlimited growthand proliferation potential,andtheirabilitiestomodulatetheimmuneresponseand toformmetastases(Hemphilletal.,2007).Consequently,several drugsinhibitingproliferationofcancercellshavebeenassayedon Echinococcusmetacestodesandprotoscoleces(HemerandBrehm, 2012;Hübneretal.,2010;Küsteretal.,2012;Lianceetal.,1993;

Naguleswaranetal.,2006;Spicheretal.,2008a,b).Doxorubicin, adrugcommonlyusedinthetreatmentofawiderangeofcan- cers,showedinvivoparasiticidalpropertiesagainstE.multilocularis afterbindingtopolyisohexylcyanoacrylatenanoparticles(Liance etal.,1993).Theinvitroandinvivoeffectsof2-methoxyestradiol, artemisininandartemisininderivativesagainstE.granulosusandE.

multilocularislarvalstageswereevaluated(Spicheretal.,2008a,b).

Furthermore,theisoflavonegenisteinandthegenisteinderivative Rm6423exhibited profoundinvitro activitiesagainstthemen- tionedparasites(Naguleswaranetal.,2006).

Fluoropyrimidineweredevelopedinthe1950sfollowingthe observationthatrathepatomasusedexogenousuracilmorerapidly than normal tissues, indicating that uracil metabolism was a potentialtargetforantimetabolitechemotherapy(Rutmanetal., 1954).Heidelbergerandcolleaguessynthesized5-fluorouracil(5- FU), which differs from uracil by virtue of a flourine atom in placeofhydrogenatthecarbon-5positionofthepyrimidinering (Heidelbergeretal.,1957).Thisdrugiswidelyusedinthetreat- mentofarangeofcancersincludingbreastcancerandcancersof theaerodigestivetract,buthashadthegreatestimpactincolorectal tumors(Longleyetal.,2003).5-FUhasinvitroandinvivoantiprolif- erativeeffectonendometrioticcells(Ngôetal.,2010).Furthermore, anti-malarialactivityof5-FUagainstPlasmodiumfalciparumhas beendemonstrated(Rathodetal.,1989).

Anotheranticanceragentisaditerpeneplantproductderived fromthewesternyew Taxus brevifolia,namelypaclitaxel(PTX) (Wanietal.,1971).PTXhasantineoplasticpropertiesandisused totreatcertainhumanmalignancies(RowinskyandDonerhower, 1995;Huizingetal.,1995).InvitrostudieshaveshownthatPTX inhibitsthegrowthoftheapicomplexanparasitesP.falciparumand Toxoplasmagondii(Pouvelleetal.,1994;Estesetal.,1998).

Theaimofthepresentworkwastodeterminetheinvitroeffect of5-FUandPTXagainstE.granulosusgerminalcells,protoscoleces andcyst.

2. Materialsandmethods

2.1. Drugtreatments

Paclitaxel(Indena,Milan,Italy)and5-FU(RocheLaboratories, Neuilly-sur-Seine, France) were dissolvedin dimethyl sulphox- ide(DMSO) atadrugconcentrationof10mg/ml.5-FUand PTX were added to the medium 199 (Gibco BRL) resulting in final

concentrationsof10,5and1␮g/ml(5-FU:75,37.5,7.5␮M;PTX:12, 6,1.2␮M).Thefinalvolumeofdrugsolutionaddedtothemedium was0.1%.Controlculturecontained0.1%DMSO.

2.2. Parasitematerialandcellculture

Hydatidcystsfromliverandlungsofnaturallyinfectedcattle wereobtainedfromaslaughterhouselocatedinthesoutheastof theBuenosAiresprovince,Argentina.E.granulosuscellculturewas obtainedusingpreviouslyreportedmethods(Albanietal.,2010).

Inbrief,cellswereculturedat37^◦Cinmedium199supplemented with 10% FBS, 10% hydatid fluid, reducing agents (5×10⁻⁵M 2-mercaptoethanol and 100␮M l-cysteine), 2mM l-glutamine (Bio-Rad,USA),40␮g/mlglucose(Sigma,USA),1mMsodiumpyru- vate (Sigma, USA) and antibiotics (penicillin, streptomycin and gentamicin100␮g/ml).E.granulosuscells werecultivatedforat least4weeks.Theculturemediumwaschangedweeklyandcells weresubculturedonceaweekatasplitratio1:2(Albanietal., 2010).

2.3. Growthinhibitoryassayonisolatedcells

E.granulosuscellswereseededin24-wellmicroplates(5×10⁵ cells/wellin1mlmedium).Forthisexperimentcellculturesafter 24hofsubculturewereused.5-FUandPTXwereaddedinserial concentrations(10,5and1␮g/ml)andcultureswereincubatedfor 7days.Atdays0,2,5and7viabilitywasassessedbytrypanblue dye(Sigma,USA)exclusionusingahemocytometer.Cultureswere followedmicroscopicallytodeterminetheappearanceofmorpho- logicalalterations.Atdays2,5and7oftreatment,sampleswere takenforscanningelectronmicroscopy(SEM).

2.4. Protoscolecescollectionandinvitroincubationprocedures Protoscoleceswereremovedfromcystsunderasepticcondi- tionsandwashed severaltimeswithphosphate-bufferedsaline (PBS, pH 7.2). Viability was assessed as previously described (Elissondoetal.,2006).Viableandfree protoscoleces(2000per Leightonttube)wereculturedin10mlofmedium199,containing 60␮g/mlpenicillin,100␮g/mlstreptomycin, 50␮g/mlgentami- cinand4mg/mlglucose.Invitroincubationswereperformedat 37^◦C withoutchangesofmedium.5-FUandPTXwereaddedto themediumresultinginfinalconcentrationsof10,5and1␮g/ml.

Culturetubeswerefollowedmicroscopicallyeveryday.Samples ofprotoscoleces(approximately90–100protoscolecesin180␮lof incubationmedium)fromeachtreatmentandthecontrolswere takenevery5–6daysforviabilityassessment.Allexperimentswere performedintriplicateandwererepeatedthreetimes.Addition- ally,ultrastructuralstudieswithSEMandtransmissionelectron microscopy(TEM)wereperformed.

2.5. Ethicstatement

Animalproceduresandmanagementprotocolswerecarriedout in accordance withthe 2011revised formof The Guide for the CareandUseofLaboratoryAnimalspublishedbytheU.S.National Institute of Health. Unnecessary animal suffering was avoided throughoutthestudy.CF-1mice(bodyweight25±5g)wereused.

Theanimalswerehousedinatemperature-controlled(22±1^◦C), light-cycled(12-hlight/darkcycle)room.Foodand waterwere givenadlibitum.

2.6. Determinationofinfectivitytomice

Viableandfreeprotoscoleces(2000perLeightonttube)were culturedin10mlofmedium.5-FUwasaddedtothemediumat

Fig.1.SurvivalofE.granulosuslarvalcellsaftertreatmentwith5-FUandPTX.Eachpointrepresentsthemeanpercentageofvitalcellsfromthreedifferentexperiments (DMSO:dimethylsulphoxide).

afinalconcentrationof10␮g/ml.Protoscolecesincubatedincul- turemediumcontainingDMSOservedascontrols.At24dayspost incubation (p.i.),protoscoleces ofeither 5-FUorcontrol groups wererinsedinmedium199.Aftersedimentation,theywereresus- pendedinmedium199supplemented with60␮g/ml penicillin, 100␮g/mlstreptomycinand50␮g/mlgentamicinandtheconcen- trationwasadjustedto3000protoscoleces/ml.Eachexperiment wasperformedinquadruplicate.

EightCF-1micewereallocatedintotwogroups(n=4):Thecon- troland5-FUgroups wereinfected byintraperitoneal injection with0.5mlofprotoscolecessuspensionfrominvitroDMSOand5- FUtreatments,respectively.At7monthspostinfection,micewere necropsiedandexaminedforlarvalgrowth.

2.7. Mouseinfectionandcollectionofcysts

FemaleCF-1micewereinfectedbyintraperitonealinoculation with1500E.granulosusprotoscoleces/animal,suspendedin0.5ml of medium199(Gibco). At8 monthspost-infection,mice with experimentalsecondaryCEwereeuthanized,andnecropsywas carried out immediatelythereafter. Atnecropsy, theperitoneal cavitywasopenedandthehydatidcystswerecarefullyremoved (Elissondoetal.,2009).

2.8. Proceduresforinvitroincubationofcysts

Groupsof10cysts(2–5mm)wereplacedinLeightontubescon- taining10mlofmedium199with5-FUorPTXatthefollowingfinal concentrationsof10,5and1␮g/ml.Culturesweremaintainedat 37^◦Cwithoutchangesofmediumduringtheentiredrugincubation period(Elissondoetal.,2007).Culturetubeswerefollowedmacro- andmicroscopicallyeveryday.Samplesofcystsfromeachofthe dosinggroupsandthecontrolsweretakenandthenfixedforelec- tronmicroscopy.Thecriteriaforcystsvitalitywasassessedonthe basisofstructuralvesicleintegrityandtheultrastructuralobserva- tionofthegerminallayerasdescribedbyElissondoetal.(2007).All

experimentswereperformedintriplicateandwererepeatedthree times.

2.9. Electronmicroscopy

E.granulosuscellsampleswereprocessedfor SEM(scanning electronmicroscopy)usingtheprotocoldescribedinAlbanietal.

(2010).Samplesofprotoscolecesandcystsculturedinvitrowere processedforSEMandTEMasdescribedbyElissondoetal.(2006, 2007).

2.10. Statisticalanalysis

Log-ranktestwasusedtoassessthesurvivaldifferencesoflarval cells andprotoscolecesafterexposureto5-FUandPTX.Allsta- tisticalanalyseswereperformedusingtheBioEstat5.0software (Ayresetal.,2007).Pvalueslessthan0.05wereconsideredtobe statisticallysignificant.

3. Results

3.1. InvitroE.granulosuscellculture

Theeffectof5-FUandPTXonthegrowthofE.granulosuscells isshowninFig.1.Controltreatmentalwaysshowedanincreasein cellnumberreachinganaverageof1.5×10⁶totalcellsatday7.

Incontrast,5-FUandPTXinhibitedthegrowthofE.granulo- suscellsinatimedependentmanner.At5daysp.i.,thenumber oftreated-cellswas64%lowerthanthatobservedinuntreated- cells. Besides,5-FUand PTXat aconcentrationof 10␮g/mlnot onlyinhibitedthegrowthofcellsbutalsoprovokedaconsiderable reductioninthecellnumberafter7days.

StudiesbySEMrevealedthatstructuraldamagewasproduced indrug-treatedcells(Fig.2).At7daysp.i.,cellsincubatedwith 10␮g/mlof5-FUorPTXshowedmorphologicalalterationaslossof turgidity,cellularcontractionandpresenceofblebsonthesurface.

Fig.2. ScanningelectronmicroscopyofE.granulosusisolatedlarvalcellsincubatedinvitrowith5-FUandPTXduring7days.(a)Control(1400×);(b)detailofcontrolcells (10,000×);(c)cellstreatedwith10␮g/ml5-FU(10,000×);(d)cellstreatedwith10␮g/mlPTX(10,000×).

Fig.3.SurvivalofE.granulosusprotoscolecesafterexposureto5-FUandPTX.Eachpointrepresentsthemeanpercentageofvitalprotoscolecesfromthreedifferent experiments(DMSO:dimethylsulphoxide).

3.2. Invitroprotoscolecesincubation

ThesurvivalofE.granulosusprotoscolecesincubatedwithdif- ferentconcentrationsofof5-FUandPTXisshowninFig.3.Although bothtreatmentshadaprotoscolicidaleffect,5-FUhadaconsider- ablystrongereffectthandidPTX.

Control protoscoleces cultured in medium 199+DMSO remainedviable(94.7±0.2%)after30daysofincubation(Fig.3).No changesinstructureandultrastructurewereobservedthroughout theexperimentalperiod(Figs.4a,5a,5band6a).

Treatment with 5-FU produced dose- and time-dependent effects(Fig.3).Themaximumprotoscolicidaleffectwasfoundwith 10␮g/mlof5-FU.Viabilitywasreducedto28.3±8.4%after18days ofincubationandreached0%after24days.Lossofprotoscolecex viabilityin5␮g/mltreatedculturesbecameclearerafter18days, wherethepercentagevaluewas50.9±13.6%.Atday24,viability

was8.7±2.9%andreached0%after30days.Adelayedprotoscol- icidaleffectwasobservedwith1␮g/mlof5-FU.Inthiscase,the viabilitydiminishedtonear50%after24daysofincubation.Viabil- itywas0%atday48(datanotshown).

TheeffectofPTXwasalsodoseand time-dependent(Fig.3).

At30daysp.i.with10and5␮g/mlPTX,viabilityofprotoscoleces decreasedtoapproximately60%andreached0%after72days(data notshown).Treatmentwith1␮g/mlPTXalsoshowedprotoscolici- daleffect,with75.3%ofparasitesremainingviableincultureafter 30daysofincubation.Asitwasmentioned,5-FUhadastronger effectthandidPTX.Forthisreason,experimentsofinfectivityto miceweredevelopedonlywith5-FU.

Theresultsofviabilitytestcoincidewiththetegumentalalter- ationsobserveddailybyopticalmicroscope(Fig.4).Between2and 4daysp.i.,thepresenceofnumerousblebsinthetegumentofpro- toscolecestreatedwith10,5and1␮g/mlof5-FUwasobserved.

Fig.4.LightmicroscopyofE.granulosusprotoscolecesincubatedinvitrowith5-FUandPTX.(a)Controlprotoscoleces(cc,calcareuscorpuscles;h,hooks;4daysp.i.;300×);

(b)protoscolecesincubatedwith5␮g/ml5-FU(3daysp.i.;400×).Notethelossofhooks(arrow).Vesiculatedprotoscolex(arrowhead);(c)alteredprotoscolexafter3days ofincubationwith10␮g/ml5-FU.Notethepresenceofnumerousblebs(arrow)andthelostofhooks(300×),(d)vesiculatedprotoscolecesafter5dayswith10␮g/mlPTX (300×).

Otheralterationssuchasrostellardisorganizationandcontraction ofposteriorregioncouldbeseenafter3days(Fig.4bandc).After4 daysofincubationwithdifferentconcentrationsofPTX,vesiculated protoscoleceswereobserved(Fig.4d).Tegumentalalterationsin PTX-treatedprotoscolecescouldbeobservedlater,between5and 7daysp.i.

Theseresultswereconfirmedontheultrastructural levelby SEMandTEM(Figs.5and6).Controlculturesexhibitednoultra- structuralalterationsinparasitetissueduringthewholeincubation period(Figs.5aandband6a).Incontrast,morphologicalandultra- structuraldamagesweredetectedin treated protoscoleces.The primarysiteofdamagewasthetegumentoftheparasite.

Theultrastructuraleffectsobservedafter5-FUincubationwere greater than those caused by PTX (Figs. 5c–f and 6b and c).

At 12 days p.i., SEM analysis of protoscoleces incubated with 5-FU showed the presence of tegumental alterations, rostellar

disorganization,lossofhooks,andsheddingofmicrotrichesofthe scolexregion(Fig.5candd).Lossofmorphologywasevidentwith 5-FUatconcentrationof10␮g/ml(Fig.5e).PTXproducedrostellar disorganisationandlossofmicrotrichesoftherostellarregionafter 12days(Fig.5f).

At 12 days p.i.,observations by TEMof 5-FUtreated proto- scolecesrevealedtheinternaltissuealteredwiththepresenceof numerousvacuoles andlipiddroplets (Fig.6b).Upontreatment with PTX for 12 days, the internal tissue of protoscoleces was slightlyvacuolated(Fig.6c).

3.3. Determinationofinfectivitytomice

Controlprotoscolecesdevelopedanaverageof6±2.3gofcysts, similartotheinoculationoffreshly isolatedprotoscoleces. This resultshowedthattheinfectivityofcontrolprotoscoleceswasnot

Fig.5. ScanningelectronmicroscopyofE.granulosusprotoscolecesincubatedinvitrowith5-FUandPTX.(a)Invaginatedcontrolprotoscolex(18daysp.i.;600×);(b) evaginatedcontrolprotoscolex(rr,rostelarregion;s,suckers;sr,somaregion;18daysp.i.;600×);(c)alteredevaginatedprotoscolexincubatedwith1␮g/ml5-FUduring12 days.Thesheddingofmicrotrichescanbeobservedinthescolexregion(700×);(d)Lossofhooksandmicrotrichesandpresenceofnumerousblebs(arrow)(5␮g/ml5-FU;

12daysp.i.;600×);(e)completelossofmorphology.Notethepresenceofnumerousblebs(arrow)(10␮g/ml5-FU;12daysp.i.;850×);(f)alteredprotoscolexafter18days with10␮g/mlPTX(500×).

Fig.6.TEMofE.granulosusprotoscolecesincubatedinvitrowith5-FUandPTX.(a) Somaregionofacontrolprotoscolex(12daysp.i.;g,glycocalix;dc,distalcytoplasm;

12,000×);(b)Treatedprotoscolex(12daysp.i.,10␮g/ml5-FU).Internaltissuewas alteredwiththepresenceofvacuoles(arrows)andlipiddroplets(l)(12,000×);(c) protoscolexincubatedwith10␮g/mlPTX(12daysp.i.).Notethevacuolationofde distalcitoplasmandinternaltissue(arrows)(15,000×).

affectedduringinvitroincubationfor24days.Lossofinfectivity inprotoscolecesincubatedwith5-FUafter24dayswasobserved, sincealloftheprotoscolecesfailedtodevelopintocystsfollowing theirinoculationintomice.

3.4. Invitrocystsincubation

Survivalof cystsincubatedwiththedifferentconcentrations of5-FUandPTXareshowninFig.7.Theeffectwasmorerapidly detectedincyststreatedwith5-FUthanwhenPTXwereused.

AscanbeseeninFig.8,incubationofE.granulosuscystsinthe presenceof5-FUandPTXresultedindramaticalterationswithin 3to5daysafterinitiationoftreatment.Atlowerconcentrations, similarmorphologicalchangesoccurred,althoughatalowerrate (datanotshown).Alreadyvisiblemacroscopically,theparasitetis- suedetachedfromtheinteriorliningofthelaminatedlayerand formedadenselypackedaggregateinsidethevesicles.Thelami- natedlayerbecametranslucent,mostlybecauselargepartsofthe parasitetissuehadbeenredistributedtoanothersite.

InspectionofcontrolcystsbySEMandTEMrevealedthatthey exhibitedtypicalfeaturesofE.granulosusmetacestodes,withadis- tinctacellularouterlaminatedlayerandanintactgerminallayer comprisedofamultitudeofdifferent,morphologicallyintactcell types(Fig.9aandb).

Theultrastructuraleffectobservedafter5-FU-incubationwas greaterthanthatcausedbyPTX.Clearly,after3dayswith5-FU (10␮g/ml)thegerminallayerlostthemulticellularstructurefea- ture(Fig.9c).At10 daysp.i.,studiesbyTEMrevealedcomplete destructionoftheparasitetissue withthepresence oflamellar bodies(Fig.9d).Ontheotherhand,lesscellswereobservedinthe germinallayerofcystsincubatedwith10␮g/mlofPTX(Fig.9e).

After10 days,thepresenceofnumerous vacuoleswasdetected (Fig.9f).

4. Discussion

Many similarities exist between cancer cells and parasites.

Therefore,a promising startingpoint forthediscoveryof novel drugs to combat parasites is to examine available compounds developedagainstcancerforantiparasiticproperties(Klinkert&

Heussler,2006).Severaldrugs inhibiting proliferationof cancer cellshave beenassayedonEchinococcus metacestodesandpro- toscoleces. This work describes for the first time the in vitro anthelminticeffectoftheanti-cancerdrugs5-FUandPTXonE.

granulosuslarvalcells,protoscolecesandcyst.

Thereare numerous routes and schedules of administration available for 5-FU. The weekly bolus administration of 5-FU at 15mg/kg in clinic produces initial peak plasma levels of approximately50to100␮g/mlwhichrapidlydeclinebyseveral ordersofmagnitude(Calabro-Jonesetal.,1982).Arecommended starting doseof single-agent5-FUgiven by protractedinfusion is 300mg/m²/day (Harris et al., 1990). Continuous infusion of 2300mg/m²/dayproduces5-FUserumconcentrationsontheorder of1␮g/ml(Gremetal.,1993).Inaddition,theexposureto1.3␮g/ml 5-FUdoesnotproducecytotoxiceffectonprimaryendometrialcell cultures(epithelialandstromalcells)(Ngôetal.,2010).

AtthemaximumtoleratedandrecommendedPhaseIIdosesfor PTXadministeredasa6-hinfusion,plasmalevelshaveapproached 10␮Mfor2–4h,andhaveexceeded1␮Mfor24h,and0.1␮Mfor 48h(Rowinskyetal.,1988).Besides,PTXdidnotalterthegrowth offibroblastsataconcentrationof8.54␮g/ml(Estesetal.,1998).

Ontheotherhand,cleareffectsonPlasmodiumfalciparumwere demonstratedusing5-FUat1.6␮g/mlconcentration(Rathodetal., 1989).Theactivityagainstapicomplexanparasiteswasdetermined usingPTXatconcentrationsbetween0.25to10␮g/ml(Estesetal.,

Fig.7.SurvivalofE.granulosuscystsaftertreatmentwith5-FUandPTX.Viabilitywasmeasuredonthebasisofvesicleintegrity(DMSO:dimethylsulphoxide).

1998).Itwasthereforedecidedtotest5-FUandPTXatconcentra- tions1,5and10␮g/ml.

Nodifferencesbetweentheeffectof5-FUandPTXontheviabil- ityofgerminalcellswerefound.5-FUorPTXinhibitedthegrowth ofE.granulosuscellsinatimedependentmanner.Moreover,5-FU orPTXataconcentrationof10␮g/mlnotonlyinhibitedthegrowth ofcellsbutalsoprovokedaconsiderablereductioninthecellnum- ber.Cellsexposedto5-FUorPTXshowedmorphologicalalteration aslossofturgidity,cellularcontractionandpresenceofblebson cellsurface.

Interesting,larvalcellsweremoresensitivetothedrugsthan protoscolecesandcysts.Isolatedcellsofgerminallayeraremore exposedtotheactionofthedrugsduetotheyaredetachedfrom theirnormalenvironmentandcouldthusbemoresensitive.Pro- toscolecesandcystsare multicellularorganismssurroundedby tegumentthatactsasbarrieragainstthedrugs(Galindoetal.,2008;

Urrea-Parisetal.,1999).Anotherpossibleexplanationcouldbethat asdrugsprimarilytargetcellsinmitosis,post-mitoticcellsofentire larvaecouldsurvivelonger.

Theefficacyof 5-FUandPTX wasalsodemonstratedinvitro onE.granulosusprotoscoleces,andmarkedly differencesonthe effectivenessofbothdrugsweredetected.5-FUhadaconsiderably strongerprotoscolicidaleffectthandidPTX.Besides,itseffectwas dose-andtime-dependent,provokingthecompletelossofviabil- ityafter24daysofincubation.Incontrast,thelossofviabilitywas slowerinPTXtreatedprotoscoleces,reachingtoapproximately60%

after30days.

Moreover,5-FUwasabletokillE.granulosusprotoscoleces,since followinginoculationoftreatedparasitesinmicenocystscouldbe recoveredafter4monthspostinfectioninallmiceinfectedwith 5-FU-treatedparasites.

Themorphologicalandultrastructuralalterationsincludedcon- tractionofthesomaregion,formationofblebsonthetegument, rostellardisorganization,lossofhooks,sheddingofmicrotriches, andthepresenceofvacuolesandlipiddroplets.Thesamealter- ationshavealsobeenreportedbyotherauthorsworkingwithother

drugslikebenzimidazoles(Pérez-Serranoetal.,1994;Elissondo et al., 2006), praziquantel(Urrea-Paris et al.,2000), ivermectin (Casado et al., 2002; Elissondo et al., 2009), and nitasoxanide (Walkeretal.,2004).

Likewise,5-FUproducedagreatereffectonE:granulosusmurine cystthanthatobservedwithPTX.Acorrelationbetweentheinten- sityofdamageandtheconcentrationofthedrugwasfound.The ultrastructural changesobservedbySEM andTEMincludedthe lossofthecharacteristicmulticellularappearanceofthegermi- nalmembrane.Furthermore,thesameultrastucturalalterationsin cellculturesaftertreatmentwith5-FUandPTXcouldbeobserved.

These changes have been also observed in E. granulosus cysts followinginvitrotreatmentwithothersanticancerdrugssuchas2- methoxyestradiol,genesteinandotherisoflavones(Naguleswaran etal.,2006;Spicheretal.,2008a).Theseultrastructuralchanges weresimilartothosereportedbyothersauthors(Elissondoetal., 2007;Walkeretal.,2004).

Thedifferentmechanismofactioncouldexplainthedisparate efficacyofthesedrugsagainstE.granulosus.5-FUisananalogue of uracil with a fluorine atom at the C-5 position instead of hydrogen.Itrapidlyentersthecellusingthesamefacilitatedtrans- portmechanismasuracil.Thedrugisconvertedintracellularlyto severalactive metabolites:fluoro-deoxyuridinemonophosphate (FdUMP),fluorodeoxyuridinetriphosphate(FdUTP),andfluorouri- dinetriphosphate(FUTP).Theactivemetabolitesof5-FUdisrupt RNAsynthesis(FUTP),inhibittheactionofthymidylatesynthase (TS)–anucleotidesyntheticenzyme(FdUMP)–andcanalsobe directlymisincorporatedintoDNA(FdUTP)(Longleyetal.,2003).

E.granulosusgenomeencodesaTS(EgrG000260100)(Zhengetal., 2013)withconsiderablehomologywithdehumanenzyme.

Bytheotherhand,PTXpromotestheassemblyofmicrotubules andstabilizestubulinpolymersbypreventingtheirdepolymeriza- tion.Thisunusualstabilityresultsintheinhibitionofthenormal dynamicreorganizationofthemicrotubulenetwork(Blagosklonny and Fojo, 1999). Since microtubules are important for mitosis andinterphasefunctionssuchasmaintenanceofcellshape,cell

Fig.8.Morphologicaleffectsof5-FUandPTXtreatmentsonE.granulosuscysts.(a)Controlcyst(3daysp.i.);(b)cystincubatedwith10␮g/mlof5-FU(arrow,germinallayer;

3daysp.i.);(c)cystincubatedwith10␮g/mlofPTX(arrow,germinallayer;5daysp.i.).Bar=300␮m.

Fig.9.RepresentativeimagesofSEMandTEMofmurinecystsincubatedinvitrowith5-FUandPTX.((a)and(b))Controlcysts(a)controlcystwithanintactgerminallayer (gl,germinallayer;ll,laminarlayer;3daysp.i.;SEM500×);(b)Controlcyst(ll,laminarlayer;mt,microtriches;dc,distalcitoplasm;10daysp.i.;TEM12,000×);((c)and(d)) cystincubatedwith10␮g/ml5-FU.(c)Notetheextensivedamageofthegerminallayer(3daysp.i.;SEM300×)(d)Theinternaltissuewasseverelyaffected((b)residual lamillarbodies;10daysp.i.;TEM15,000×);((d)and(e))cystsincubatedwith10␮g/mlPTX.(d)Thegerminallayerisaltered(3daysp.i.;SEM250×);(e)notethepresence ofvacuoles(arrows)(10daysp.i.;TEM12,000×).

motility,and intracellular transport, PTX might beexpected to affectcells duringbothinterphaseandmitoticcellcyclephases (Rowinskyetal.,1988).

In thesearch foran effectivedrug againstE. granulosus, we showedthat5-FUandPTX,atclinicallyachievableconcentrations, inhibitedthesurvivaloflarvalcells,protoscolecesandmetaces- todes.Invivostudiestotesttheantiparasiticactivitiesof5-FUand PTXarecurrentlybeingundertakenonthemurinemodelofcystic echinococcosis.

Acknowledgements

Thisworkwassupportedby thePICT12 no.1164(ANPCyT, Argentina), PIP 0029 (CONICET, Argentina) and Universidad Nacional de Mar del Plata (Grant EXA 581/12 and Grant EXA 672/14),Argentina.

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