Rev. bras. farmacogn. vol.27 número3

(1)

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

Original

Article

Bee

venom

induces

apoptosis

and

suppresses

matrix

metaloprotease-2

expression

in

human

glioblastoma

cells

Mohsen

Sisakht

a

_,

_Baratali

_Mashkani

a

_,

_Ali

_Bazi

b

_,

_Hassan

_Ostadi

a

_,

_Maryam

_Zare

a

_,

Farnaz

Zahedi

Avval

a

_,

_Hamid

_Reza

_Sadeghnia

c,e

,

Majid

Mojarad

d

,

Mohammad

Nadri

a

,

Ahmad

Ghorbani

e

_,

_Mohmmad

_Soukhtanloo

a,e,∗

a_Department_of_Medical_{Biochemistry,}_School_of_Medicine,_Mashhad_University_of_Medical_Sciences,_Mashhad,_Iran b_Faculty_of_Allied_Medical_Sciences,_Zabol_University_of_Medical_Sciences,_Zabol,_Iran

c_Division_of_{Neurocognitive}_Sciences,_Psychiatry_and_Behavioral_Sciences_Research_Center,_Mashhad_University_of_Medical_Sciences,_Mashhad,_Iran d_Department_of_Genetic,_School_of_Medicine,_Mashhad_University_of_Medical_Sciences,_Mashhad,_Iran

e_{Pharmacological}_Research_Center_of_Medicinal_Plants,_Mashhad_University_of_Medical_Sciences,_Mashhad,_Iran

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7August2016 Accepted29November2016 Availableonline6March2017

Keywords:

Beevenom Glioblastoma

Matrixmetalloproteinase-2 Apoptosis

Metastasis Zymography

a

b

s

t

r

a

c

t

Glioblastomaisthemostcommonmalignantbraintumorrepresentingwithpoorprognosis,therapy resistanceandhighmetastasisrate.Increasedexpressionandactivityofmatrixmetalloproteinase-2, amemberofmatrixmetalloproteinasefamilyproteins,hasbeenreportedinmanycancersincluding glioblastoma.Inhibitionofmatrixmetalloproteinase-2expressionhasresultedinreducedaggressionof glioblastomatumorsinseveralreports.Inthepresentstudy,weevaluatedeffectofbeevenomon expres-sionandactivityofmatrixmetalloproteinase-2aswellaspotentialtoxicityandapoptogenicpropertiesof beevenomonglioblastomacells.HumanA172glioblastomacellsweretreatedwithincreasing concen-trationsofbeevenom.Then,cellviability,apoptosis,matrixmetalloproteinase-2expression,andmatrix metalloproteinase-2activityweremeasuredusingMMTassay,propidiumiodidestaining,realtime-PCR, andzymography,respectively.TheIC50valueofbeevenomwas28.5␮g/mlinwhichitleadstodecrease ofcellviabilityandinductionofapoptosis.Incubationwithbeevenomalsodecreasedtheexpression ofmatrixmetalloproteinase-2inthiscellline(p<0.05).Inzymography,therewasareversecorrelation betweenbeevenomconcentrationandtotalmatrixmetalloproteinase-2activity.Inductionofapoptosis aswellasinhibitionofmatrixmetalloproteinase-2activityandexpressioncanbesuggestedasmolecular mechanismsinvolvedincytotoxicandantimetastaticeffectsofbeevenomagainstglioblastomacells.

Introduction

Glioblastoma is recognized as the most common malignant primarybraintumorwithaparticularlypoorprognosis.Despite multipletherapeuticstrategiessuchassurgery,radiotherapy,and chemotherapy, no effective treatment has been identified for glioblastoma (Haar et al., 2012; Naik et al., 2013). Moreover, resistanceofbraintumorstoavailabledrugshasbecomea clin-icalchallenge(Haaretal.,2012).Therefore,developmentofnew natural therapeutic strategies is necessary. In different experi-ments, it was illustrated that the glioma cells show ability to produce and secrete various matrix metalloproteinases (MMP)

∗ Correspondingauthor.

E-mail:soukhtanloom@mums.ac.ir(M.Soukhtanloo).

enzymes (Rooprai and McCormick, 1996; Forsyth et al., 1999). Ithasbeenproposedthatextracellularmatrixdegradation, trig-geredby MMP-2activationviainteraction withtissue inhibitor of metalloproteinase-2, is essential for invasion of glioma cells (Fillmoreetal.,2001).Additionally,extracellularmatrix degrada-tion,especially byMMP-2, releasesgrowthfactorsandprovides morefreespacestovascularextension.Growthfactorsreleasedby MMPsuchasvascularendothelialgrowthfactor,fibroblastgrowth factor-2andtransforminggrowthfactorbetamayexerta signifi-canteffectsininductionofangiogenesis(EgebladandWerb,2002). Therefore,protocolsaimingtotargetMMP-2activitymaybecome apromisingtherapeuticstrategyfortreatmentofglioma(Abeetal., 1994;Deryuginaetal.,1997;Senotaetal.,1998).

Beevenom(BV,apitoxin)isoneofanaturalbiologicalcomplex compoundwithmanydifferenttherapeuticeffectsincluding neu-roprotective,anti-allergic,andanti-angiogenesisproperties(Huh

http://dx.doi.org/10.1016/j.bjp.2016.11.006

(2)

etal.,2010;Kimetal.,2011;Shinetal.,2014).Thetwomain compo-nentsofBVaremelittinandphospholipaseA2.Ithasbeenreported

thatmelittinhasproapoptoticeffectandshowsanti-tumor activ-ity (Orˇsoli ´c,2012). The BVhas alsobeen utilized in treatment ofvarietyinflammatory conditionssuchasrheumatoid arthritis (Kwon etal., 2001; Park et al., 2004).These effects are shown tobemediatedbyinhibitionofnuclearfactor kappa-light-chain-enhancerofactivatedBcells,mitogen-activatedproteinkinaseand Ca2+_/calmodulin_signaling_pathways₍_Cho_et_al.,_2010;_Park_et_al.,

2010).Studiesonglioblastomacelllinesrevealedthatdisturbance ofCa2+_/calmodulin_signaling_pathway_could_result_in_tumor_cells

apoptosisthroughinhibitionofDNAsynthesis(Tsuruoetal.,1982; Orˇsoli ´c,2009).Also,ithasbeenpreviouslyreportedthatBVinduces cellcyclearrest inhumancervicalcancercells(Ipet al.,2008). In thepresent study, antiproliferative and apoptogenic proper-tiesofvenomofhoneybeeonhumanA172gliomacancercells wereinvestigated.Also,becauseofcriticalroleofMMP(especially MMP-2)ininvasionofglioblastoma(Luetal.,2004),thepossible inhibitoryeffectsofBVonMMP-2expressionandactivitywere evaluated.

Materialandmethods

Materials

Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT),propidiumiodide(PI),TritonX-100 andgelatinwerepurchasedfromSigma(St.Louis,USA).RPMI-1640 mediaandfetal bovineserum(FBS)andpenicillin-streptomycin solutionwerefromGibco(LifeTechnologies,Carlsbad,USA).The venomofhoneybee(persica,workerbees)waspurchasedfrom RoyanZahr(Isfahan,Iran). TotalRNAextractionkit,agarosegel, greenviewerdyeandtheentiresolventsandflaskswereprepared fromParstousco (Iran). HumanA172 glioblastomaand normal murineL929fibroblastcelllineswereobtainedfromPasteur Insti-tute,Iran.

Cellcultureandtreatment:TheA172andL929cellswere cul-turedinRPMI1640medium,supplementedwith10%fetalbovine serumand1%penicillin-streptomycinandincubatedin37◦_C_and 5%CO2.The BVstock solution(15mM) wasprepared in

phos-phatebuffersaline,pH7.2.Forcellviabilityassay,thecellswere seededin96-wellcultureplates(1×104_cells/well)._Then,_the_RPMI

mediawaschanged byfreshonecontainingvarying concentra-tions(5–160␮g/ml)ofBVorreferencedrug(cisplatinat70␮g/ml).

Thecellswereincubatedfor24or48hin37◦_C_and_5%_CO₂_._For apoptosisassay,thecellswereculturedin6wellplates(1×105

cellsperwell) andtreatedwithBVat itsIC50concentrationfor

48h.

MMTassay:EffectofBVonA172andL929cellviabilitywas determinedusingMTTassayasdescribedpreviously(Mortazavian etal.,2012;Ghorbanietal.,2015).Briefly,10␮lofMTTreagent

(5mg/ml)wasaddedtoeachwell,andtheplateswereincubated furtherfor4hin37◦_C._At_the_end_of_incubation_time,_media_was removedandformazancrystalsweredissolvedbyadding100␮l

dimethylsulfoxide.Finally,absorbancewasreadat545nmusing ELISAplatereader(Statfax-2100).Theassaywascarriedoutin triplicate.

Apoptosisanalysis:AftertreatmentwithBV,thefloatingand adherentcells were harvested and incubated witha hypotonic buffercontainingpropidiumiodidefor30min(Mortazavianetal., 2013;Sadeghniaetal.,2014).Thesampleswerethensubjectedto theflowcytometryfordeterminationofapoptoticcells.

RNAextractionandcDNAsynthesis:Thecellswerecultured in T25-flasks and treated with different concentrations of BV (0–10␮g/ml).ThentotalRNAwasextractedusingParstousRNA

extractionkit(Iran)accordingtothemanufacturer’sinstruction. QualityofextractedRNAwascheckedbyrunningon1%agarose gelinthepresenceofcybersafeorgreenviewer(Parstous). Syn-thesisofcDNAwasperformedusingParstouskitaccordingtothe manufacturer’sinstruction.

Real time-PCR: MMP-2 primers were designed as follows: forward; 5′_{-AACTACGATGACGACAGCAAGT-3}′ _and _reverse; ₅′ -AGGTGTAAATGGGTCCCATCA-3′_._Quantitative_RT-PCR_was_carried outonStratagene3000instrument.NetvolumeofPCRreaction was20␮lcontaining1␮lcDNA,1␮lmixedprimer,10␮l

Syber-greendye,0.4␮lRoxdyeand7.6␮ldistilledwater.Temperature

profile was designed as an initial denaturation phase at 95◦_C (5min). Following this, reaction continued in 35 cycles with temperatureprofileasdenaturation(94◦_C,₃₀_s),_annealing₍₅₇◦_C, 30s)andextension(72◦_C,₄₅_s)._A_final_period_of₇₂◦_C_for₅_min wasconsideredtoensuremaximumproductionofPCRproducts. TheexpressionofMMP-2genewasnormalizedto glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene.

Gelatinzymography:ToassessenzymaticactivityofMMP-2, gelatinzymographywasperformedtoidentify72kDa (pro-MMP-2)and62kDa(active-MMP-2)isoformsoftheprotein.Thecells wereculturedin96wellplates(1×104_cells/well)_for₁₂_h._Then

theywerewashedwithPBS,andsubjectedtotreatmentwith dif-ferentBVconcentrations(0–10␮g/ml)inserumfreemediumfor

24h. ToevaluatethedirecteffectofBVonMMP-2,differentBV concentrationswereaddedtotheA172cellularsupernatantand incubatedfor24h.Then,themediawereresolvedon8%SDS-PAGE containing1%gelatinasenzymesubstrate.TheSDSwasremoved andtheenzymeactivityregeneratedbywashingthegelsin2.5% triton-X100forthreetimes.Subsequently,gelswereincubatedin developingbuffer(Tris50mMpH7.4,CaCl210mM,NaN30.02%and

steriledH2O)at37◦Cfor42h.Theareaofdigestedgelatinwas

visu-alizedbycounterstainingusingCoomassiebrilliantblueR-250,and quantifiedasrelativenumericalvalueswitharbitraryunitsusing NIHImageJ1.42qsoftware.

Statisticalanalysis

Allresultsarepresentedasmean±standarderrorofthemean (SEM).Thevalueswerecomparedusingtheone-wayanalysisof variancefollowedbyTukey’sposthoctestformultiple compar-isons.Thep-valueslessthan0.05wereconsideredtobestatistically significant.

Results

EffectofBVoncellviability

AsshowninFig.1,BVdecreasedviabilityofglioblastomacells inaconcentration-dependentmannerwiththeIC50valueof28.52

and28.3␮g/mlfor24and48h,respectively.After24hof

incuba-tion,viabilityofcellstreatedwith5,10,20,40,80and160␮g/ml

ofBVwas76±3.5,66±2.5,61±0.5(p<0.05),41±0.5(p<0.001), 27±0.5(p<0.001)and 25±5%(p<0.001) ofcontrol (100±4%), respectively.Atconcentrationsof40–160␮g/ml,the

antiprolifer-ativeeffectofBVwasmorethanthatof70␮g/mlcisplatin.This

effectofBVwasalsomorethancisplatinwhenthecellsincubated for48h.Comparedtountreatedcells,cellviabilitywas70±4.5, 63±2.5,60±3(p<0.05),41±1(p<0.001),25±0.5(p<0.001)and 25±1%(p<0.001),respectively.

(3)

120

100

80

60

40

20

0

Control Cisplatin 5 10 20

Bee venom (µg/ml)

After 24 h After 48 h

Cell viability

, %

40 80 160

∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

∗

Fig.1.EffectofbeevenomonviabilityofhumanA172glioblastomacells.Thecells weretreatedwithdifferentconcentrationsofbeevenomorcisplatin(70␮g/ml)

asreferencedrugfor24or48h.*p<0.05versuscontrolcells;***p<0.001versus controlcells.

EffectofBVonapoptosis

EffectofBVonapoptosisofglioblastomacellsisshowninFig.3. Flowcytometry analysisrevealedthat incontrolconditiononly 14±5%ofglioblastomacellswereinapoptosisstage.However,in

140

120

100

80

60

40

20

0

0 5 10 20

Bee venom (µg/ml)

Cell viability

, %

40 80 160

Fig.2. EffectofbeevenomonviabilityofmurineL929fibroblastcells.Thecellswere treatedwithdifferentconcentrationsofbeevenomfor48h.

thepresenceof28.52␮g/ml(IC50value)ofBV,percentageofthe

apoptoticcellswas64±5%whichissignificantlyhigherthanthose inuntreatedcellpopulation(p<0.01).

EffectofBVonMMP-2expression

RT-PCRanalysisofMMP-2expressionshowedthatincubation withBVsignificantlyreducedthelevelofMMP-2mRNAinA172

128

0

M1

100 ₁₀1 ₁₀2

FL2-H

103

70

60

50

40

30

20

10

0

0 28.52

∗∗

Bee venom (µg/ml)

Apoptotic cells

, %

104

Ev

ents

128

0

M1

Bee venom (28.52 µg/ml) -treated A172 cells Control A172 cells

100 ₁₀1 ₁₀2

FL2-H

103 ₁₀4

Ev

ents

(4)

2.00

1.00

∗ ∗

0.00

–1.00

–2.00

–3.00

Control 0.1

Bee venom concentation (µg/ml)

MMP-2 f

old changes

1

Fig.4. EffectofbeevenomonMMP-2geneexpression.MMP-2mRNAlevelwas measuredinuntreatedcells(control)andthoseexposedto0.1␮g/mland1␮g/ml

BV.*p<0.05.GAPDHhousekeepinggeneservedasinternalcontrol.

cells.Comparedtocontroluntreatedcells,levelofMMP-2mRNA foldchangeswere−0.1and−2.1at0.1and1␮g/mlofBV

concen-trations,respectively(p<0.05,Fig.4).

EffectofBVonMMP-2activity

Treatment with BV significantly reduced the quantity of detectableMMP-2enzymeincellularsupernatant(p<0.001). Inter-estingly,ratioofactive(62kDa)isoformofMMP-2waselevatedby increasingBVconcentration(Fig.5).

Discussion

IthasbeenrecentlysuggestedthatBVasacytotoxicagentmay haspotentialtherapeuticeffectsincancer(Ipetal.,2008;Orˇsoli ´c, 2009,2012).Inpresentwork,theeffectsofBVoncellviability, apoptosisandMMP-2expressionandactivitywereinvestigatedon humanA172glioblastomacells.OurdatashowedthatBVtreatment decreasedcellularviabilityinaconcentration-dependentmanner throughitsproapoptoticaction.ThisantiproliferativeeffectofBV atIC50valueof28.52␮g/mlwasapproximatelycomparablewith

theeffectofcisplatinat70␮g/ml.Furthermore,BVtreatmentdid

notaffectviabilityofnormalfibroblasticcellsindicatingadegree ofspecificityformalignantcells.Therefore,itseemsthatBV com-ponentscanbegoodcandidateforfutureclinicaltrialsforcancer therapy.BVconstitutesanenormoussourceofenzymesand bioac-tivepeptides(e.g.melittinandphospholipaseA2),anditsbeneficial

actionsontumorcellsmaybeduetheeffectsofasingleconstituent orbytheeffectsofseveralofitsconstituentsonthetumorcells (Orˇsoli ´c,2012).

Recently and in agreement with our findings, Gajski et al. reportedthatpre-incubationwithBVinducescellsensitizationto cisplatin,andthereforecanimprovethekillingeffectofthisdrug againsthumanglioblastomaA1235cells(Gajskietal.,2016).Also, Ipetal.demonstratedthatBVinducescellcyclearrestand apopto-sisinhumancervicalepidermoidcarcinomacells(Ipetal.,2008). Thepro-apoptoticeffectofBVcanbemediatedthroughintrinsic or extrinsicpathways, two generalways for activation of apo-ptosis.Intrinsicpathwayisinitiatedbymitochondrialreleaseof cytochromecandsubsequentactivationofcaspase-3.Ontheother hand,theextrinsicpathwayisstimulatedwithacelldeathreceptor whichactivatescaspase-8andfinallycaspase-3.Activated caspase-3targetssubstratesthat promote DNAfragmentation (Kirkland

A

B

_{40 000}

62 kDa

85 kDa 70 kDa 60 kDa MW

10 µg/ml 1

0.1 0

72 kDa

30 000

20 000

10 000

0

0 0.1

Bee venom (µg/ml)

MMP-2 Pro-MMP-2

∗∗∗ ##

##

Optical density (arbitr

ar

y units)

1 10

Fig.5.EffectofbeevenomontheactivityofMMP-2enzymeinhumanA172 glioblastomacells.(A)Evaluationofactivitiesofpro-MMP-2(72kDa)andactive MMP-2(62kDa)bygelatinzymography;(B)QuantitativepresentationofMMP-2 activity(verticalaxisrepresentsgelatinolyticactivityasarbitraryunits.***p<0.001 versuscontrolcells(0␮g/ml)regardingpro-MMP-2;##p<0.01versuscontrolcells (0␮g/ml)regardingactiveMMP-2.

et al., 2002; Ghorbani et al., 2015). It has been reported that proapoptotic effect of BV is mediated via a Fas receptor path-wayinvolvingmitochondrialdependentpathwayswhichincreased activationofcaspase-3andthenleadtoapoptosis(Ipetal.,2008). Themaincharacteristic featureofglioblastomaislocal inva-siveness. This feature makes it much difficult to resect tumor completely,and therefore patientshave usually poorprognosis (Luetal., 2004).Thisenhanced invasivenessis associated with increasedproductionandsecretionofMMPsenzymes(Roopraiand McCormick,1996;Forsythetal.,1999;Fillmoreetal.,2001;Luet al.,2004).Luetal.(2004)showedan80%increaseinMMP-2 acti-vationduringinvasionofhumanglioblastomacells.TheMMP-2 triggersextracellularmatrixdegradation,releasesgrowthfactors andprovidesmorefreespacesforvascularextensionandcancer progression(Fillmoreetal.,2001;EgebladandWerb,2002).Our datafromQ-PCRandzymographyshowedaninverserelationship betweenBV concentrationand MMP-2expression and activity. BecausetheinhibitoryeffectofBVonMMP-2activitywasnotseen incell-freesupernatantmedium,thiseffectismostprobably medi-atedbycellularsignaling.IthasbeendemonstratedthatMMP-2 expressionisunderregulationofanintracellularsignalingpathway knownasextracellular-signal-regulatedkinase/mitogen-activated proteinkinase(ERK/MAPK)(StoicaandLungu,2014).However, fur-therstudiesareneededtorevealtheexactmechanismsinvolved inalteringMMP-2expressionandactivitybyBV.

(5)

In conclusion our results showed that BV inhibits viability of glioblastoma cells through induction of apoptosis. BV also decreasedMMP-2expressionsuggestingapotentialrolein inhibi-tionofglioblastomametastasis.Therefore,itcanbegoodcandidate forfutureclinicaltrialsinglioblastomatumors.

Authorship

Studyconceptionanddesign:MS,BM,FZA,MMandMS. Acqui-sitionofdata:MS,ABandMZ.Analysisandinterpretationofdata: MS,HO,MN,HRSandAG.Draftingofmanuscript:AB,FZA,BM,and MajidMojarad.Criticalrevision:MS,HRSandAG.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisworkwassupportedbyagrant(911311)fromMashhad UniversityofMedicalSciences(MUMS),andextractedfromM.Sc. thesispresentedbyMohsenSisakht.

References

Abe,T.,Mori,T.,Kohno,K.,Seiki,M.,Hayakawa,T.,Welgus,H.G.,Hori,S.,Kuwano,M., 1994.Expressionof72kDatypeIVcollagenaseandinvasionactivityofhuman gliomacells.Clin.Exp.Metastas.12,296–304.

Cho,H.J.,Jeong,Y.J.,Park,K.K.,Park,Y.Y.,Chung,I.K.,Lee,K.G.,Yeo,J.H.,Han,S.M.,Bae, Y.S.,Chang,Y.C.,2010.BeevenomsuppressesPMA-mediatedMMP-9gene acti-vationviaJNK/p38andNF-kappaB-dependentmechanisms.J.Ethnopharmacol. 127,662–668.

Deryugina,E.I.,Bourdon,M.A.,Luo,G.X.,Reisfeld,R.A.,Strongin,A.,1997.Matrix metalloproteinase-2activationmodulatesgliomacellmigration.J.Cell.Sci.110 (Pt19),2473–2482.

Egeblad,M.,Werb,Z.,2002.Newfunctionsforthematrixmetalloproteinasesin cancerprogression.Nat.Rev.Cancer2,161–174.

Fillmore,H.L.,VanMeter,T.E.,Broaddus,W.C.,2001.Membrane-typematrix met-alloproteinases(MT-MMP):expressionandfunctionduringgliomainvasion.J. Neurooncol.53,187–202.

Forsyth,P.A.,Wong,H.,Laing,T.D.,Rewcastle,N.B.,Morris,D.G.,Muzik,H.,Leco,K.J., Johnston,R.N.,Brasher,P.M.,Sutherland,G.,Edwards,D.R.,1999.Gelatinase-A (MMP-2),gelatinase-B(MMP-9)andmembranetypematrix metalloproteinase-1(MT1-MMP) areinvolvedindifferentaspectsofthepathophysiology of malignantgliomas.Br.J.Cancer79,1828–1835.

Gajski,G., ˇCimbora-Zovko,T.,Rak,S.,Osmak,M.,Garaj-Vrhovac,V.,2016.Antitumour actiononhumanglioblastomaA1235cellsthroughcooperationofbeevenom andcisplatin.Cytotechnology68,1197–1205.

Ghorbani,A.,Sadeghnia,H.R.,Asadpour,E.,2015.Mechanismofprotectiveeffect oflettuceagainstglucose/serumdeprivation-inducedneurotoxicity.Nutr. Neu-rosci.18,103–109.

Haar,C.P.,Hebbar,P.,Wallace,G.C.t.,Das,A.,Vandergrift3rd,W.A.,Smith,J.A.,Giglio, P.,Patel,S.J.,Ray,S.K.,Banik,N.L.,2012.Drugresistanceinglioblastoma:amini review.Neurochem.Res.37,1192–1200.

Huh,J.E.,Baek,Y.H.,Lee,M.H.,Choi,D.Y.,Park,D.S.,Lee,J.D.,2010.Beevenominhibits tumorangiogenesisandmetastasisbyinhibitingtyrosinephosphorylationof VEGFR-2inLLC-tumor-bearingmice.CancerLett.292,98–110.

Ip,S.W.,Wei,H.C.,Lin,J.P.,Kuo,H.M.,Liu,K.C.,Hsu,S.C.,Yang,J.S.,Mei,D.,Chiu, T.H.,Han,S.M.,Chung,J.G.,2008.Beevenominducedcellcyclearrestand apo-ptosisinhumancervicalepidermoidcarcinomaCaSkicells.AnticancerRes.28, 833–842.

Kim,J.I.,Yang,E.J.,Lee,M.S.,Kim,Y.S.,Huh,Y.,Cho,I.H.,Kang,S.,Koh,H.K.,2011.Bee venomreducesneuroinflammationintheMPTP-inducedmodelofParkinson’s disease.Int.J.Neurosci.121,209–217.

Kirkland,R.A.,Windelborn,J.A.,Kasprzak,J.M.,Franklin,J.L.,2002.Abax-induced pro-oxidantstateiscriticalforcytochromecreleaseduringprogrammed neu-ronaldeath.J.Neurosci.22,6480–6490.

Kwon,Y.B.,Lee,J.D.,Lee,H.J.,Han,H.J.,Mar,W.C.,Kang,S.K.,Beitz,A.J.,Lee,J.H., 2001.Beevenominjectionintoanacupuncturepointreducesarthritis associ-atededemaandnociceptiveresponses.Pain90,271–280.

Lu,K.V.,Jong,K.A.,Rajasekaran,A.K.,Cloughesy,T.F.,Mischel,P.S.,2004. Upre-gulationoftissueinhibitorofmetalloproteinases(TIMP)-2promotesmatrix metalloproteinase(MMP)-2activationandcellinvasioninahuman glioblas-tomacellline.LabInvest.84,8–20.

Mortazavian,S.,Ghorbani,A.,Hesari,T.G.,2012.Effectofhydro-alcoholicextractof

Violatricoloranditsfractionsonproliferationofuterinecervixcarcinomacells.

Iran.J.Obst.Gynecol.Infertil.15,9–16.

Mortazavian,S.M.,Parsaee,H.,Mousavi,S.H.,Tayarani-Najaran,Z.,Ghorbani,A., Sadeghnia,H.R.,2013.Acetylcholinesteraseinhibitorspromoteangiogenesisin chickchorioallantoicmembraneandinhibitapoptosisofendothelialcells.Int. J.AlzheimersDis.2013,http://dx.doi.org/10.1155/2013/121068.

Naik,P.P.,Somani,R.R.,Shirodkar,P.Y.,Wagulde,S.,Juvatkar,P.,Kale,M.K.,2013.A reviewon-glioblastomamultiform.Int.J.Pharm.Tech.Res.5,873–878.

Orˇsoli ´c,N.,2009.PotentiationofBleomycinlethalityinHeLaandV79cellsbybee venom.Arch.Ind.Hyg.Toxicol.60,317–326.

Orˇsoli ´c,N.,2012.Beevenomincancertherapy.CancerMetast.Rev.31,173–194.

Park,H.J.,Lee,S.H.,Son,D.J.,Oh,K.W.,Kim,K.H.,Song,H.S.,Kim,G.J.,Oh,G.T.,Yoon, D.Y.,Hong,J.T.,2004.Antiarthriticeffectofbeevenom:Inhibitionof inflamma-tionmediatorgenerationbysuppressionofNF␬Bthroughinteractionwiththe p50subunit.ArthritisRheum.50,3504–3515.

Park,J.H.,Jeong,Y.-J.,Park,K.-K.,Cho,H.-J.,Chung,I.-K.,Min,K.-S.,Kim,M.,Lee, K.-G.,Yeo,J.-H.,Park,K.-K.,2010.MelittinsuppressesPMA-inducedtumorcell invasionbyinhibitingNF-␬BandAP-1-dependentMMP-9expression.Mol.Cells 29,209–215.

Rooprai,H.,McCormick,D.,1996.Proteasesandtheirinhibitorsinhumanbrain tumours:areview.AnticancerRes.17,4151–4162.

Sadeghnia,H.R.,GhorbaniHesari,T.,Mortazavian,S.M.,Mousavi,S.H., Tayarani-Najaran,Z.,Ghorbani,A.,2014.Violatricolorinducesapoptosisincancercells andexhibitsantiangiogenic activityonchickenchorioallantoicmembrane. Biomed.Res.Int.2014,http://dx.doi.org/10.1155/2014/625792.

Senota,A.,Itoh,F.,Yamamoto,H.,Adachi,Y.,Hinoda,Y.,Imai,K.,1998.Relationof matrilysinmessengerRNAexpressionwithinvasiveactivityinhumangastric cancer.Clin.Exp.Metastasis16,313–321.

Shin,S.H.,Kim,Y.H.,Kim,J.K.,Park,K.K.,2014.Anti-allergiceffectofbeevenomin anallergicrhinitismousemodel.Biol.Pharm.Bull.37,1295–1300.

Stoica,G.,Lungu,G.,2014.RoleofMMP2inbrainmetastasis.In:Hayat,M.A.(Ed.), TumorsoftheCentralNervousSystem.SpringerNetherlands,Dordrecht,pp. 195–205.