Antitumor effect of laticifer proteins of Himatanthus drasticus (Mart.) Plumel Apocynaceae

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ContentslistsavailableatScienceDirect

Journal

of

Ethnopharmacology

j o u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m

Antitumor

effect

of

laticifer

proteins

of

Himatanthus

drasticus

(Mart.)

Plumel

– Apocynaceae

Kristiana

C. Mousinho

a

_,

_Cecília

_de

_C.

_Oliveira

a

_,

_José

_Roberto

_de

_O.

_Ferreira

a

_,

_Adriana

_A.

_Carvalho

a

_,

Hemerson

Iury

F. Magalhães

a

_,

_Daniel

_P.

_Bezerra

b

_,

_Ana

_Paula

_N.N.

_Alves

d

_,

_Letícia

_V.

_Costa-Lotufo

a

_,

Claúdia

Pessoa

a

_,

_Mayara

_Patrícia

_V.

_de

_Matos

c

_,

_Márcio

_V.

_Ramos

c

_,

_Manoel

_O.

_Moraes

a,∗ a_Departamento_de_Fisiologia_e_{Farmacologia,}_Faculdade_de_Medicina,_Universidade_Federal_do_Ceará,_Fortaleza,_Ceará,_Brazil

b_Departamento_de_Fisiologia,_Universidade_Federal_de_Sergipe,_São_Cristóvão,_Sergipe,_Brazil

c_Departamento_de_Bioquímica_e_Biologia_Molecular,_Universidade_Federal_do_Ceará,_Fortaleza,_Ceará,_Brazil d_Departamento_de_Clínica_{Odontológica,}_Universidade_Federal_do_Ceará,_Fortaleza,_Ceará,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22December2010 Receivedinrevisedform27April2011 Accepted28April2011

Available online 6 June 2011

Keywords:

Himatanthusdrasticus Apocynaceae Janaguba Laticiferproteins Sarcoma180

Walker256carcinosarcoma

a

b

s

t

r

a

c

t

Ethnopharmacologicalrelevance:Himatanthusdrasticus(Mart.)Plumel–Apocynaceaeisamedicinalplant popularlyknownasJanaguba.Itsbarkandlatexhavebeenusedbythepublicforcancertreatment,among othermedicinaluses.However,thereisalmostnoscientificresearchreportonitsmedicinalproperties.

Aimofthestudy:TheaimofthisstudywastoinvestigatetheantitumoreffectsofHimatanthusdrasticus

latexproteins(HdLP)inexperimentalmodels.

Materialsandmethods:TheinvitrocytotoxicactivityoftheHdLPwasdeterminedonculturedtumorcells. HdLPwasalsotestedforitsabilitytoinducelysisofmouseerythrocytes.Invivoantitumoractivitywas assessedintwoexperimentalmodels,Sarcoma180andWalker256carcinosarcoma.Additionally,its effectsontheimmunologicalsystemwerealsoinvestigated.

Results:HdLPdidnotshowanysignificantinvitrocytotoxiceffectatexperimentalexposurelevels.When intraperitoneallyadministered,HdLPwasactiveagainstbothinvivoexperimentaltumors.However, itwasinactivebyoraladministration.Thehistopathologicalanalysisindicatesthattheliverandkidney wereonlyweaklyaffectedbyHdLPtreatment.ItwasalsodemonstratedthatHdLPactsasan immunomod-ulatoryagent,increasingtheproductionofOVA-specificantibodies.Additionally,itincreasedrelative spleenweightandtheincidenceofmegakaryocytecolonies.

Conclusion:Insummary,HdLPhassomeinterestinganticanceractivitythatcouldbeassociatedwithits immunostimulatingproperties.

1. Introduction

Himatanthusis a small Apocynaceae genus composed of 14

species(Plumel,1991).TheimportanceofthegenusHimatanthus

intraditionalmedicineissupportedbyreportswherepreparations

wereevaluatedformainlyanti-tumor(Bolzanietal.,1999),

anti-inflammatory(Mirandaetal.,2000),anti-ulcerous(Baggioetal.,

Abbreviations: 5-FU, 5-fluorouracil;SBCAL,SociedadeBrasileiradeCiência em AnimaisdeLaboratório; HDLP, Himatanthusdrasticuslatexprotein;MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide;OVA, ovabul-min;PBMC, peripheralbloodmononuclearcells; TGF-␤, transforming growth factor-␤;TNF-␣,tumornuclearfactor-␣.

∗Correspondingauthorat:DepartamentodeFisiologiaeFarmacologia,UFC,Rua Cel.NunesdeMelo,1127,60430-270Fortaleza,Ceará,Brazil.Tel.:+558533668255; fax:+558533668333.

E-mailaddress:odorico@ufc.br(M.O.Moraes).

2005),anti-spasmodic(Rattmannetal.,2005),antimicrobial(Souza

etal.,2004),andantileishmanial(Castilloetal.,2007)activities.

Himatanthus drasticus (Mart.) Plumel – Apocynaceae is a

medium-sizedtreegrowingonfirmgroundinSouthAmerica.It

isamedicinalplantpopularlyknownasjanaguba,tiboma,

jasmim-manga, raivosa, pau-de-leite, joanaguba, and sucuúba (Plumel,

1991).Itsbarkandlatexhavebeenusedbythepublicmainlyfor

cancertreatment,asananti-inflammatorymedication,andto

stim-ulatetheimmunesystem(Amaroetal.,2006).Usually,latexplus

water(janagubamilk)ordecoctionsofthebarkorlatexaretakenat

adoseofonecupaboutthreetimesaday.Althoughseveral

medici-naluseshavebeendescribed,therearealmostnoscientificresearch

reportstothisregard.

In a preliminary study of the phytochemical and biological

effects of Himatanthus drasticus, the ethanolic extract of stem

bark was evaluated biologically. This extract was found to be

toxic against brine shrimp, but showedno antimicrobialeffect

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422 137 (2011) 421–426

against the pathogens tested in vitro (Enterobacter,

Streptococ-cusandEscherichiacoli).Additionally, itshowedantinociceptive

(writhing test in rats) affect (Colares et al., 2008). Leite et al.

(2009)demonstratedthelatexfromHimatanthusdrasticustobe

acytoprotectiveagentagainstethanol-inducedulcerformationin

mice.

Theaimofthisstudywastoinvestigatetheantitumoreffectsof

Himatanthusdrasticuslatexproteins(HdLP)inexperimental

mod-els.InordertoevaluatethetoxicologicalaspectsrelatedtoHdLP

treatment,histopathologicalandmorphologicalanalysesoftreated

animalswerealsoperformed.

2. Materialandmethods

2.1. Reagents

5-Fluorouracil (5-FU), O-phenylenidiamine dihydrochloride,

Ficoll-Hypaque, phytohemagglutinin, resazurin, and ovalbumin

werepurchasedfromSigmaChemicalCo.(St.Louis,MO,USA);

rab-bitantimouse totalIg(IgG,A,M) wasfromSerotec(Kidlington,

Oxford,UK).Allotherreagentswereofanalyticalgrade.

2.2. Plantmaterial,latexcollectionandextractionof Himatanthusdrasticuslatexproteins(HdLP)

Latex was obtained in May 2010 from uncultivated plants

locatedinthevicinityofFortaleza,StateofCeará,Brazil.Botanical

materialwasidentifiedattheHerbariumPriscoBezerra

(Departa-mentodeBiologia,UniversidadeFederaldoCeará).Avoucherwas

registeredunderthecode40408.

Thelatexwasobtainedaftercuttingstemandallowingittoflow

intotapwaterinordertogiveanequalmixtureofvolumes.The

sampleswereinitiallycentrifuged(5000×g)at10◦_C_for₂₅_min.

Thepelletwasdiscardedandthesolublephasewasdialyzedagainst

distilledwaterfor60hat25◦_C_with_water_being_renewed_three

timesdaily.Finally,thedialyzedmaterialwascentrifugedas

previ-ouslydone,andcleansupernatantwasrecovered,freezedriedand

usedinallfurtherdeterminations.Thisfraction,comprisingalmost

allsolublelatexproteinwascalledHdLP.

2.3. Animals

Atotalof56Swissmice(female,25–30g)and40Wistarrats

(female,180–220g),obtainedfromthecentral animalhouse of

UniversidadeFederaldoCeará–Brazil,wereused.Animalswere

housedincageswithfree accesstofoodandwater. Allanimals

werekeptundera12:12hlight–darkcycle(lightsonat6:00a.m.).

Animalsweretreatedaccordingtotheethicalprinciplesfor

ani-malexperimentationofSBCAL(SociedadeBrasileiradeCiênciaem

AnimaisdeLaboratório),Brazil.TheAnimalStudiesCommitteeof

UniversidadeFederaldoCearáapprovedtheexperimentalprotocol

(number08/08).

2.4. Cells

ThecytotoxicityofHdLPwastestedagainstHL-60(leukemia),

MDA-MB-435 (melanoma), SF-295 (brain), and HCT-8 (colon)

humancancercelllines,allobtainedfromtheNationalCancer

Insti-tute,Bethesda,MD,USA.CellsweregrowninRPMI-1640medium

supplemented with 10% fetal bovine serum, 2mM glutamine,

100␮g/mlstreptomycinand100U/mlpenicillin,andincubatedat

37◦_C_with_a_5%_CO₂_atmosphere.

Inordertogethealthyhumanperipheralbloodmononuclear

cells(PBMC),heparinizedblood(fromhealthy,non-smokerdonors

whohadnottakenanydrugatleast15dayspriortosampling)was

collected,andPBMCwereisolatedbyastandardmethodof

density-gradientcentrifugationoverFicoll-Hypaque.PBMCwerewashed

andresuspended.Cellsweregrownunderthesameconditionsas

aboveplustheadditionofphytohemagglutinin(4%).

Sarcoma180tumorcellshadbeenmaintainedintheperitoneal

cavityofSwissmiceandWalker256carcinosarcomatumorcells

hadbeenmaintainedbyintramuscularinoculationofthemedial

sideofthethighofWistarratsintheLaboratoryofExperimental

OncologyfromtheUniversidadeFederaldoCearásincethe

mid-1980s.

2.5. InvitrocytotoxicevaluationofHdLP

2.5.1. DeterminationoftheeffectofHdLPonculturedtumorcells

Tumorcellgrowthwasquantifiedbytheabilityoflivingcellsto

reducetheyellowdye

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product

(Mossman, 1983).For allexperiments,cellswereseededin

96-wellplates(105_cells/well_for_adherent_cells_or_0.5

×105_cells/well

forsuspendedcellsin100␮lofmedium).After24h,thetest

sub-stance(0.39–50␮g/ml),dissolvedinsaline,wasaddedtoeachwell

(usingtheHTS—high-throughputscreening—Biomek3000;

Beck-manCoulterInc.,Fullerton,CA,USA)andincubatedfor72h.5-FU

wasusedasthepositivecontrol.Attheendofincubation,theplates

werecentrifugedandthemediumwasreplacedbyfreshmedium

(150␮l)containing0.5mg/mlMTT.Threehourslater,theformazan

productwasdissolvedin150␮lDMSOandtheabsorbancewas

measuredusingamultiplatereader(DTX880Multimode

Detec-tor,BeckmanCoulterInc.,Fullerton,CA,USA).Thedrugeffectwas

quantifiedasthepercentageofcontrolabsorbanceofreduceddye

at595nm.

PBMCcellgrowthwasdeterminedbytheAlamar blueassay

(Ahmed et al.,1994).For all experiments,cells wereseeded in

96-wellplates(0.3×106_cells/well_for_suspended_cells_in₁₀₀_␮_l_of

medium).After24h,thetestsubstance(0.39–50␮g/ml),dissolved

insaline,wasaddedtoeachwell(usingtheHTS–high-throughput

screening–Biomek3000–BeckmanCoulter,Inc.,Fullerton,CA,

USA) and incubated for 72h. 5-FU was used as the positive

control. Twenty-four hoursbefore theend of incubation, 10␮l

ofstock solution(0.312mg/ml) ofAlamar Blue (Resazurin) was

addedtoeachwell.Theabsorbancewasmeasuredusinga

mul-tiplatereader(DTX880MultimodeDetector,BeckmanCoulter®₎

and the drug effect was quantified as the percentage of

con-trolabsorbanceat570nmand595nm.TheabsorbanceofAlamar

Blueinculturemediumismeasuredatahigherwavelengthand

lowerwavelength. Theabsorbance ofthemedium isalso

mea-sured atthe higher and lowerwavelengths. Theabsorbance of

themediumaloneissubtractedfromtheabsorbanceofmedium

plusAlamar Blueat thehigher wavelength.Thisvalueiscalled

AOHW.The absorbanceofthemediumalone issubtractedfrom

theabsorbanceofmediumplusAlamarBlueatthelower

wave-length.ThisvalueiscalledAOLW.Acorrectionfactor R0 canbe

calculated from AOHW and AOLW, where R0=AOLW/AOHW. The

percent Alamar Blue reduced is then expressed as follows: %

reduced=ALW−(AHW×R0)×100.

2.5.2. DeterminationoftheeffectofHdLPonmouseerythrocytes

Thetest wasperformedin 96-wellplates usinga 2%mouse

erythrocyte suspensionin 0.85% NaCl containing 10mm CaCl2,

following themethoddescribed byJimenez etal. (2003).HdLP

wastestedatconcentrationsrangingfrom8to200␮g/ml.After

incubation at room temperature for 30min and centrifugation,

the supernatant was removed and the hemoglobin released

was measured spectrophotometrically as the absorbance at

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137 (2011) 421–426 423

2.6. InvivoantitumorevaluationofHdLP

2.6.1. DeterminationoftheeffectofHdLPontumorgrowthin miceandrats

2.6.1.1. Sarcoma 180 tumor protocol. Ten-day-old Sarcoma 180

ascitestumorcells(2×106_cells_per₅₀₀_␮_l)_were_implanted

subcu-taneouslyintothelefthindgroinofmice(asdescribedbyBezerra

etal.,2008).Onedayafterinoculation,HdLP(10or20mg/kg,by

intraperitonealadministration;50or100mg/kg,byoral

adminis-tration)wasdissolvedinsalineandadministeredfor7days.

2.6.1.2. Walker256carcinosarcomatumorprotocol.Eight-day-old

Walker256carcinosarcomatumorcells (2×106_cells_per₅₀₀_␮_l)

wereimplantedsubcutaneouslyintothelefthindgroinofrats(as

describedbyMoraesetal.,1997).Onedayafterinoculation,HdLP

(10or20mg/kg,byintraperitonealadministration)wasdissolved

insalineandadministereddailyfor7days.

Onday8,theanimalsweresacrificedbycervicaldislocation.The

tumors,livers,spleens,andkidneys wereexcised,weighed,and

fixedin10%formaldehyde.Percentinhibition(%)wascalculated

bythefollowingformula:percentinhibition(%)=[(A−B)/A]×100,

whereAisthetumorweightaverageofthenegativecontrol,and

Bisthatofthetreatedgroup.Bodyweightsweredeterminedat

thestartandonthelastdayoftreatment.5-FU(25mg/kg/day)was

usedasthepositivecontrol.Negativecontrolwastreatedwiththe

vehicleusedfordilutingthetestsubstance(saline).

2.6.2. Toxicologicalanalyses–histopathologyandmorphological analyses

Afterfixationwithformaldehyde,tumors,livers,spleens,and

kidneysweregrosslyexaminedforsizeorcolorchangesand

hem-orrhage. Next, portions of thetumor, liver, spleen, and kidney

werecutintosmallpieces,followedbystainingofthe

histologi-calsectionswithhematoxylinandeosin.Histologicalanalysiswas

performedbylightmicroscopy.Thepresenceandextentofliver,

kidneyorspleenlesionsattributedtodrugsweredetermined.

2.7. Immunomodulatoryanalyses

2.7.1. Subcutaneousimmunization

Two groups of eight Swiss mice were immunized

subcuta-neouslywithasingledoseofovabulmin(OVA)(2mg/kg)orOVA

(2mg/kg)plusHdLP(40mg/kg).Themicewerebledfromthe

retro-orbitalplexustoobtainserumsamplespriortoimmunizationand

at7,14and21daysafterimmunization.

2.7.2. Measurementofspecificantibody

Specificantibodiesinserumweredetectedbyenzyme-linked

immunosorbentassay(ELISA).Inordertoevaluatetheabilityof

HdLPtoincreasetheresponseelicitedbyOVA,total Ig

antibod-iesagainstOVAweredeterminedusingOVA(50␮g/well)-coated

plates.Theplateswereincubatedat37◦_C_for₁_h_and_washed_three

timeswithPBS-0.05%Tween.Theplateswereblockedwith5%

non-fatmilkin10mMpotassiumphosphatebuffer,pH7.2,with0.9%

NaCl(PBS)for2hat37◦_C_and_washed_once,_after_which₁₀₀_␮_l_of

theappropriateserumdilutedinPBSwasaddedandtheplates

rein-cubatedfor2hat37◦_C._The_plates_were_washed_again_three_times

withPBS-0.05% Tween and treated withperoxidase-conjugated

rabbitantimousetotalimmunoglobulins(100␮l/well,1:1000final

dilution)for2hatroomtemperature.Theplatesweresubsequently

washedthreetimeswithPBS-Tween.Thereactionwasdeveloped

bytheadditionoforthophenylenediaminefollowedbyincubation

for10minat37◦_C._The_intensity_of_the_resulting_color_was_read_at

450nmusingamultiplatereader(DTX880MultimodeDetector,

BeckmanCoulterInc.,Fullerton,CA,USA).

Table1

EffectofHimatanthusdrasticuslatexproteins(HdLP)onhumancelllines.

Cellline Histotype HdLPIC50(␮g/ml) 5-FUIC50(␮g/ml)

HL-60 Leukemia >50 12.599.72–16.29 HCT-8 Colon >50 0.36 0.18–0.71 SF295 Brain >50 0.38 0.23–0.63 MDA-MB-435 Melanoma >50 1.36 0.98–1.88 PBMC Lymphocytes >50 >5

DataarepresentedasIC50valuesand95%confidenceintervalfromthree indepen-dentexperiments,performedinduplicate.5-Fluorouracil(5-FU)wasusedasthe positivecontrol.

2.8. Statisticalanalysis

Dataarepresentedasmean±SEMorIC50valuesandtheir95% confidence intervals (CI 95%)obtainedby nonlinear regression.

Thedifferencesbetweenexperimentalgroupswerecomparedby

ANOVA(analysisofvariance)followedbyStudent–Newman–Keuls

(P<0.05).Allstatisticalanalyseswereperformedusingthe GRAPH-PADprogram(IntuitiveSoftwareforScience,SanDiego,CA,USA).

3. Results

3.1. InvitroevaluationofHdLPcytotoxicity

TheinvitroeffectsofHdLPagainsthumantumorcelllineswere determinedandshowednocytotoxicactivityatthetested con-centrations(Table1).5-FU,usedasthepositivecontrol,showed

IC50valuesrangingfrom0.36to12.59␮g/mlforHCT-8andHL-60,

respectively.

HdLPwasalsotestedfor itsability toinducelysis ofmouse

erythrocytes.HdLPwasnothemolyticevenatthehighest

concen-trationtested(200␮g/ml,datanotshown).

3.2. InvivoantitumorevaluationofHdLP

Theeffects ofHdLP onmicetransplanted withSarcoma180

tumororratstransplantedwithWalker256carcinosarcomaare

presentedinFig.1.HdLP,givenintraperitoneally,wasactiveagainst

bothexperimentaltumorsshowingaverysimilarprofile.However,

itwasinactivebyoraladministration.

InmiceinoculatedwithSarcoma180,theaveragetumorweight

of thecontrol was2.52±0.14gonday 8. Intraperitoneal HdLP

reduced tumor weightto 1.60±0.18 and 1.66±0.18g atdoses

of10and 20mg/kg/day,respectively(Fig.1A).Thesereductions

gaveinhibitionratesof36.46%and34.22%.At25mg/kg/day,5-FU

reducedtumorweightby48.02%withinthesameperiod.OralHdLP

hadnoeffectontumorweight(datanotshown).

InratsinoculatedwithWalker256carcinosarcoma,theaverage

tumorweightofthecontrolwas8.36±0.55gonday8.

Intraperi-tonealHdLPreducedtumorweightto3.52±0.94and3.74±0.95g

atdosesof10and20mg/kg/day,respectively(Fig.1B).These

reduc-tionsgaveinhibitionratesof57.91%and55.23%.At25mg/kg/day,

5-FUreducedtumorweightby56.34%withinthesameperiod.

Histopathologicalanalysisofthetumorsextirpatedfrom

Sar-coma180controlmiceorWalker256carcinosarcomacontrolrats

showedgroupsoflarge,roundandpolygonalcells,with

pleomor-phicshapes,hyperchromaticnucleiandbinucleation.Inthetumors

extirpated from treated animals,extensiveareas of coagulative

necrosiswereobserved(datanotshown).

3.3. Toxicologicalanalyses

AftertreatmentwithHdLP,nosignificantchangesintheweight

ofliversorkidneys wereseeninmiceinoculated withsarcoma

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424 137 (2011) 421–426

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0 10 20 30 40 50 60

a

a a

A

Saline 5-FU 10 20

HdLP

(mg/kg/day)

Tumor weight (g)

Inhibition (%)

0 2 4 6 8 10

0 20 40 60 80

Inhibition

a a a

Tumor weight

B

Saline 5-FU 10 20

HdLP

(mg/kg/day)

Tumor weight (g)

Inhibition (%)

Fig.1.EffectofHimatanthusdrasticuslatexproteins(HdLP)onmicetransplanted withsarcoma180tumor(A)andratstransplantedwithWalker256carcinosarcoma tumor(B).5-Fluorouracil(5-FU,25mg/kg/day)wasusedasthepositivecontrol. Negativecontrolwastreatedwiththevehicleusedfordilutingthetestedsubstance (saline).Theanimalsweretreatedbyintraperitonealadministration,startingone dayaftertumorimplantation,forsevenconsecutivedays.Dataarepresentedas mean±S.E.M.ofeightanimals.a_P_<_0.05_compared_with_saline_group_by_ANOVA followedbyStudent–Newman–Keuls.

However,spleenweightsweresignificantlyincreasedwhen

com-paredtothecontrolgroup,inbothexperimentaltumors(P<0.05).

Bodyweightgainwassignificantlyreducedaftertreatmentwith

HdLP(20mg/kg/day)or5-FU(25mg/kg/day)inmiceinoculated

withsarcoma180.

Histopathologicalanalysesofkidneysremovedfrom

animals-treatedwithHdLPshowedintenseswellingoftubularepithelial

cells,presenceofhyalinecylinderandfocaltubularnecrosis,but

thestructureoftheglomeruliwasessentiallypreserved(datanot

shown).Intheliver,histopathologicalanalysisshowedthat

ani-malstreatedwithHdLPhadnumerousinflammatoryfoci,intense

ballooningdegenerationofhepatocytes,microvesicularsteatosis,

andnecrosis(datanotshown).Inthespleen,HdLP-treated

ani-malsshowedadiscreetincreaseinmegakaryocytecolonies,which

suggestsanimmunomodulatoryactivity(Fig.2).

3.4. Immunomodulatoryanalyses

ToinvestigatetheeffectofHdLPontheinductionofhumoral

immuneresponseinmiceimmunizedwithasingledoseofOVA

(2mg/kg)orOVA(2mg/kg)plusHdLP(40mg/kg),theOVA-specific

antibodylevelsin serumweremeasuredpriortoimmunization

andat7,14and21daysafter,atadilutionof1:3200byELISA.The

resultsareshowninFig.3.HdLPsignificantlyincreasedtheamount

ofOVA-specifictotalIgintheseraoftreatedanimalsatthedoseof

40mg/kgcomparedtotheOVAcontrol(P<0.05).

4. Discussion

The present work reports the antitumor effects of HdLP on

micetransplantedwithSarcoma180andratstransplantedwith

Walker256carcinosarcoma.Thesemodelsareanimal-originated

tumorsfrequentlyusedininvivocancertherapyresearch(Moraes

etal.,1997;Leeetal.,2003;Bezerraetal.,2008;Linsetal.,2009;

Chenetal.,2010).HdLPinhibitedthegrowthofbothexperimental

tumors,withnoeffectoncellproliferationinvitroatthe

concen-trationstested.Additionally,itsimmunoadjuvantactivitywasalso

demonstrated.Thisisthefirstreportdescribingtheantitumorand

immunomodulatorypropertiesofHdLP.

HdLPdidnotshowanysignificantinvitrocytotoxiceffectatthe

experimentalexposurelevels,butitdidshowaninvivoantitumor

effect.Inthepreclinicalanticancerdrug-screeningprogramusedin

thisstudy,anextractthatdisplayedIC50valuesbelow30␮g/mlwas

consideredpromising(SuffnessandPezzuto,1990).HdLPshowed

IC50values greaterthan50␮g/mlforalltumorcelllinestested,

suggestingthattheinvivoantitumoractivitywasnotrelatedto

directantiproliferativeeffects.

As previously cited, the ethanolic extract of stem bark of

Himatanthus drasticus was active in the brine shrimp lethality

test.Itsextractshowedahighlysignificantactivityinthisassay

(LC50=257ppm).Theauthorsattributedtheseeffectstothe

triter-penelupeolcinammate(Colaresetal.,2008).Ontheotherhand,the

effectofitslatexproteinshasneverbeenevaluatedagainsttumor

cellslines.

InthegenusHimatanthus,thelatexofHimatanthussucuubais

usedinitsrawformasanantitumoragent(VanderBerg,1984).

Chemicalstudieshave indicatedthepresence oflupeolacetate,

Table2

EffectofHimatanthusdrasticuslatexproteins(HdLP)onorganweights.

Drug Dose(mg/kg/day) Liver(g/100gbodyweight) Kidney(g/100gbodyweight) Spleen(g/100gbodyweight)

Healthymice

Saline – 4.61±0.19 1.07±0.03 0.26±0.02

MicetransplantedwithS180

Saline – 4.66±0.06 1.08±0.02 0,73±0.02

5-FU 25 4.38±0.09 1.08±0.04 0.35±0.05a

HdLP 10 4.88±0.14 1.17±0.03 1.09±0.06a

HdLP 20 4.97±0.13 1.19±0.04 1.10±0.05a

Healthyrats

Saline – 4.54±0.11 1.01±0.06 0.50±0.01

RatstransplantedwithWalker256

Saline – 4.83±0.11 0.89±0.02 0.43±0.01

5-FU 25 4.95±0.06 1.04±0.04 0.34±0.03a

HdLP 10 4.84±0.30 0.88±0.06 0.50±0.06

HdLP 20 5.24±0.20 0.94±0.17 0.65±0.04a

Dataarepresentedasmean±SEMofeightanimals.5-Fluorouracil(5-FU)wasusedasthepositivecontrol.Negativecontrolwastreatedwiththevehicleusedfordiluting thetestedsubstance(saline).Theanimalsweretreatedbyintraperitonealadministration,startingonedayaftertumorimplantation,forsevenconsecutivedays.

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137 (2011) 421–426 425

Fig.2. EffectofHimatanthusdrasticuslatexproteins(HdLP)onspleensofmicetransplantedwithsarcoma180tumor.Theanimalsweretreatedbyintraperitoneal admin-istration,startingonedayaftertumorimplantation,forsevenconsecutivedays.Photomicrographsshowthehistopathologyofthespleensfromsaline-treated(panelA), 5-FU-treated(25mg/kg/day,panelB),HdLP-treated(10mg/kg/day,panelC),andHdLP-treated(20mg/kg/day,panelD)animals,analyzedbylightmicroscopy(400×).White arrowshowsmegakaryocyte.

alpha-amyrin,andlupeolcinnamates(Mirandaetal.,2000).The

ethanolicextractofleavesofHimatanthusobovatusshowedstrong

cytotoxicactivityincancercelllines(Mesquitaetal.,2009).

Addi-tionally,theethanolicextractofleavesofHimatanthusattenuatus

showedsignificanttoxicityagainstArtemiasalina(Jiménezetal.,

2001).InthefamilyApocynaceae,thelaticiferproteinsofCalotropis

procera(Ait)wasshowntoexhibitselectivecytotoxiceffects on

humancancercelllines.Itwasshown toinhibitDNAsynthesis,

probablyaffectingtopoisomeraseIactivity,leadingtoapoptosisin

tumorcells(Oliveiraetal.,2007).Recently,itsinvivoanticancer

potentialwasalsoevaluated,reinforcingthepotentialoflaticifer

proteinsintreatingneoplasia(Oliveiraetal.,2010).

Furthermore,HdLPactsasanimmunomodulatoryagent,

rais-ingtheproductionofOVA-specificantibodies.Additionally,itwas

demonstrated that HdLP increased relative spleen weight, and

increasedmegakaryocyticnests,corroboratingthehypothesisthat

itactsthroughimmunestimulation.Thus,thesedatasuggestthat

themechanismofbeneficialtherapeuticeffectselicitedbyHdLP

seemedtobeattributedtothepotentiationofhost-defensethrough

theenhancementofimmunity,inagreementwithitstraditional

use.

Interestingly, the immunostimulant potential of the genus

Himatanthushasbeenevaluated.ThelatexofHimatanthus

sucu-ubaincreasednitricoxide(NO)andtumornuclearfactor-␣(TNF-␣)

anddecreasedtransforminggrowthfactor-␤(TGF-␤)productionin

macrophages,suggestinganimmunomodulatoryactivity(Soares

etal.,2010).InthefamilyApocynaceae,experimentalstudieswith

proteinsof thelatex of C. procerasuggested that theactive LP

fraction(PII)displaysinvivoinhibitionoftumorgrowth,

proba-blybymediatinganincreasedimmunopharmacologicalresponse

(Oliveiraetal.,2010).

Hepatotoxicityandnephrotoxicityareverycommonsideeffects

ofcancerchemotherapeuticdrugs.Hepaticdysfunctioninducedby

vincristineandetoposide(KingandPerry,2001)andrenal

toxic-ityinducedbycisplatin(Pinzanietal.,1994)aresuchexamples.

Inthepresentstudy,theintegrityofliverandkidneysinanimals

subjectedtoHdLPtreatmentwereevaluatedbyhistopathological

analyses.

TheliverofHdLP-treatedanimalsshowednumerous

inflam-matory foci, intense ballooning degeneration of hepatocytes,

microvesicularsteatosis,andnecrosis,suggestingintrinsic

hepato-toxicity.However,regenerationofhepatictissuesoccursinmany

diseases.Thisoccursevenwhenhepatocellularnecrosisispresent.

Preimmune 7 14 21

0.0 0.4 0.8 1.2 1.6

Days after immunization

*

Absorbance

(6)

426 137 (2011) 421–426

Inhepaticdegeneration,regenerationiscompletewhenthe

con-nectivetissueispreserved(ScheuerandLefkowitch,2000;Kummar

etal.,2004).ThehepaticalterationsobservedinHdLP-treated

ani-malscouldbeconsideredreversible(McGeeetal.,1992;Scheuer

andLefkowitch,2000;Kummaretal.,2004).

Thekidneysremovedfromanimals-treatedwithHdLPshowed

intense swelling of tubular epithelium cell, presence of

hya-linecylinderandfocaltubularnecrosis,butthestructureofthe

glomeruliwasessentiallypreserved.Necrosisoftherenaltubule

epitheliummayoftenoccurasaconsequenceofthe

administra-tionofvariouscancerchemotherapeuticdrugs(OlsenandSolez,

1994).Itisworthmentioningthatthehistopathologicalanalyses

ofHdLP-treatedanimalsdemonstratedthattheinterstitialtissues

werepreserved,suggestingthatregenerationispossible(Curran,

1990;OlsenandSolez,1994).

Insummary,thehistopathologicalanalysisindicatesthatthe

liverandkidneywereonlyweaklyaffectedbyHdLPtreatment.

Anyway,the HdLP-inducedsystemic toxicity effectneedstobe

moreextensivelyevaluatedtoassessthesafety.Then,further

stud-iesmustbedonetowell-definedandunderstandtheunderline

mechanisminvolvedinHdLPtoxiceffect.

5. Conclusion

Briefly,thisworkshowedthatHdLPexhibitedantitumoreffects

againstexperimentaltumorswithoutsubstantialtoxicity.In

addi-tion, this activity seems to be related to its immunostimulant

properties.

Acknowledgments

TheauthorsaregratefultotheBrazilianagenciesFINEP,CNPq,

BNB/FUNDECI,PRONEX,andCAPESforfellowshipsandfinancial

support.SilvanaFranc¸a dos Santosprovidedexcellent technical

assistanceand Dr. A. Leyvahelped withEnglish editing of the

manuscript.

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