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

Original

Article

Growth

inhibition

of

human

breast

cancer

cells

and

down-regulation

of

ODC1

and

ADA

genes

by

Nepeta

binaloudensis

Akbar

Safipour

Afshar

,

Fatemeh

Saeid

Nematpour,

Mahshid

Meshkani,

Arezosadat

Khafi

DepartmentofBiology,NeyshaburBranch,IslamicAzadUniversity,Neyshabur,Iran

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10February2016 Accepted8July2016

Availableonline15September2016

Keywords:

Adenosinedeaminase Anticancer Cytotoxicity

Ornithinedecarboxylase

a

b

s

t

r

a

c

t

NepetabinaloudensisJamzad,Lamiaceae,isararemedicinalplantendemictoIran.Inspiteofmanystudies

aboutthechemicalconstituentsandantibacterialeffectsofthisspecies,noreporthasbeenprovidedabout

itscytotoxicandanticanceractivities.InthisstudywehaveevaluatedtheeffectsofEtOH70%,hexane

andaqueousextractsofN.binaloudensisonthecellproliferationandn-hexaneextractontheexpression

ofadenosinedeaminaseandornithinedecarboxylase1genesinbreastcancercelllines(MCF-7,

MDA-MB-231)comparedtonon-cancerline(MCF-10A).Thecelllinesweresubjectedtoincreasingdoses

oftheextractsrangingfrom10to320␮g/ml.CellviabilitywasquantifiedbyMTSassay.Expressionof

adenosinedeaminaseandornithinedecarboxylase1geneswasanalyzedbyrealtimePCR.N.binaloudensis

inhibitedthegrowthofmalignantcellsinatimeanddose-dependentmanner.AmongextractsofN.

binaloudensis,thehexaneextractwasfoundtobemoretoxiccomparedtootherextracts.Resultsshowed

amarkeddecreaseintheexpressionofornithinedecarboxylase1andadenosinedeaminasegenesin

cancercelllines.At60␮g/mlconcentrationofN.binaloudensishexaneextractornithinedecarboxylase

1andadenosinedeaminasemRNAexpressionwerereduced4.9foldand3.5foldinMCF-7celllineand

3.6foldand2.6foldinMDA-MB-231celllinecomparedtocontrol,respectively.Theresultofourstudy

highlightsthepotentialinfluencesofN.binaloudensishexaneextractonornithinedecarboxylase1and

adenosinedeaminasegenesexpressioninbreastcancercellsanditsrelationtoinhibitionofcancercell

growth.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Canceristhemostcommoncauseofdeathintheworld.The majorityofdeathsfromcanceroccurinwomenwithbreastcancer. Statisticsindicatethatin2014about29%ofcancerswerebreast cancersamongAmericanwomen(Siegeletal.,2014).InIranalso breastcancerrateshaveincreasedinfemales(Mousavietal.,2009). Manyinvestigationsareperformedtodetectthegeneswhichare involved in breast cancer. One of themost important genes is ornithinedecarboxylase(ODC)which encodesa keyenzyme in polyaminessynthesis(Zhuetal.,2012;Xuetal.,2015).Several studiesindicatethatthereareclosecorrelationbetweenODC activ-ityinthecellsandregulationofcellproliferation(Dengetal.,2008;

Elmetsand Athar,2010; Hayesetal., 2011).Consequently,any

changesinODCactivityandpolyamineslevelhaveeffectsoncell differentiationandproliferation.Inbreastcancercells,estrogens induceODCactivity.So,thelevelofODCandestrogenisincreased

∗ Correspondingauthor.

E-mail:Asafshar@iau-neyshabur.ac.ir(A.S.Afshar).

inthesecells(Zhuetal.,2012).Inaddition,AdenosineDeaminase gene(ADA)encodestheenzymethatcatalyzeshydrolyzationof adenosinetoinosinandparticipatesinpurinesmetabolism,which hasacrucialroleindevelopmentofimmunesystemand matura-tionofmammaliancells(Rietal.,2010;RobertsandRoberts,2012). RecentstudiessuggestthatthelevelofADAenzymeisincreasedin breastcancercellsincontrastwithnormalcells(Aghaeietal.,2010;

Mahajanetal.,2013).Therefore,decreasingofODCandADAgene

expressionandrespectiveenzymeactivitiescanbeagoodstrategy forbreastcancertherapy.

Usingmedicinalplantextractsasanticancerdrugsis amore effectivewayforcancertherapy. About50%ofprescribeddrugs intheworldoriginatefromplants(VanSlambroucketal.,2007). NepetabinaloudensisJamzad,Lamiaceae,isanendemicandrare medicinalplantwhichgrowsexclusivelyintheBinaludMountains inKhorasanRazavi provincein NortheastofIran(Tundisetal., 2013).Thisplanthasbeenappliedwidelyintraditionalmedicine forthetreatmentofrespiratorydisease,digestiveproblemsand osteoarthritis(KoochekiandRezvaniMoghaddam,2012).Previous chemicalinvestigationofN.binaloudensishasshownthepresence ofnepetalactoneand1,8cineole(RustaiyanandNadji,1999).Some

http://dx.doi.org/10.1016/j.bjp.2016.07.005

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studiesweredoneaboutantibacterialactivitiesofN.binaloudensis butitscytotoxicityandanticancereffectshavenotbeenstudied yet.Therefore,thepurposeofthestudywastodeterminethe anti-cancereffectsofN.binaloudensisextractontwobreastcancercell lines(MCF-7,MDA-MB-231)andnormalbreastcellline(MCF-10A). Also,thepresentstudyisfocusedonevaluatingthelevelofODC1 andADAgeneexpressioninthenormalandcancerousbreastcells.

Materialandmethods

Plantcollectionandextraction

Shoots ofNepetabinaloudensis Jamzad,Lamiaceae,were col-lected from BinaludMountains in Neyshabur, Khorasan Razavi province,northeastofIran.TheplantwasidentifiedbyMr.M.R. Joharchi,fromFerdowsiUniversityofMashhadHerbarium(FUMH). Voucherspecimen(No.1025) wasdeposited withherbariumof IslamicAzadUniversity, NeyshaburBranch.The extraction pro-cesswasperformedfromdriedshoots.Thedriedshoots (100g) werepercolatedwith100mlofeachsolvent(EtOH70%,hexaneand H2O)for4hseparately.Theextractswerefilteredandthesolvents wereevaporatedundervacuumat45◦Ctoaffordcrudeextracts. Dryextractsweredissolvedindimethylsulfoxide(DMSO)andthen subjectedtocytotoxicandgeneexpressionassays.

Celllinesandculturemedium

Twohumanbreastcancercelllines(MCF-7,MDA-MB-231)and onenormal breast cellline(MCF-10A) wereobtainedfromcell culturelaboratoryoftheFacultyofSciences,FerdowsiUniversity (Mashhad,Iran)andPasteurInstitute(Tehran,Iran).Thecellswere culturedin25cm2flaskswithRPMI-1640(RPMISigma)and sup-plementedwith10%fetalbovineserum(FBS,Gibco)and100U/ml penicillinand100␮g/mlstreptomycinsolutions,at37◦Cwith5% CO2and95%humidity.Themediumwasexchangedeverydayand thecellswerepassagedevery3–4days.

Cytotoxicityassay

ThecytotoxicactivityoftheextractswasdeterminedusingMTT assay(2,3,5dipheniltetrazoliumbromide).Cellswereplatedwith densityof3×104cells/dishin96wellplatesandwereincubated for24hat37◦C.Then,thecellsweretreatedwithdifferent concen-trationsofextracts(0,10,20,40,80,160and320␮g/ml)andwere incubatedfor24,48and72h.A2mMMTTsolutionwasaddedto eachwellandplatewasincubatedfor4h.Themediumwasthen discardedand100mlDMSO(dimethylsulfoxide)wasaddedand theplatewasshakenfor10min.Finally,opticaldensitywas deter-minedin540nmusingELISAmicroplatereader(Awareness,Palm City,FL,USA).Eachexperimentwasperformedintriplicate.Results areexpressedasthepercentagegrowthinhibitionwithrespectto theuntreatedcells.

Colonyformationassay

TheeffectofhexaneextractonMCF7andMDA-MB-231cells wasinvestigatedbycolonyformationassay(Frankenetal.,2006). Briefly,thecells(500cells/ml)wereallowedtogrowin60mmPetri dishesfor12h.Subsequently,thecellsweretreatedfor48hwith hexaneextract(10,30and60␮g/ml),or0.1%DMSO.Thecolonies werefixedandstainedwith0.2%crystalvioletandcountedunder stereomicroscope.

Real-timePCR

Theexpression levelsofODC1and ADAgenes,key genesin breastcancercells,wereanalyzedusingRealtimePCRassay.The cells wereculturedinthree groupsand treatedwithincreasing concentrations (0–60␮g/ml) ofthe hexane extract dissolvedin DMSOfor48h.After48htotalcellularRNAwasextractedfrom thetreated and untreated (control) cells using Easy BLUEtotal RNA extractionkit (iNtRON). Thetreatments wererepeatedfor threetimes.ThequalityofRNAwasdeterminedbyabsorptionat 260nmusingT80UV-VisibleSpectrophotometer(PGInstruments). Foreachspecimen2␮gofRNAwasreversetranscribedintocDNA withreversetranscriptionreactionmixtureofPowercDNA Syn-thesisKit(iNtRON).PrimersofODC1,ADAand␤ACTweredesigned usingGeneRunnersoftware(version4).

Primer sequences for ODC1 were: ODC1-FW: 5′ -GTGGGTGATTGGATGCTCTTTG-3′ ODC1-RV: 5-ACCAGGCTAACTA CTCGCTCAA-3′.

Primer sequences for ADA were: ADA-FW: 5′-ACCAG GCTAACTACTCGCTCAA-3′ ADA-RV: 5-TCAGTAAAGCCCATG TCCCGTT-3′.

Primer sequences for ␤ACT were: ACT-FW: 5′ -TCCATCATGAAGTGTGACGT-3′ ACT-RV: 5-GAGCAATGATCTTGAT CTTCAT-3′.

Real-timePCRwascarriedoutusingSYBRGreenmethodonan ABIStep-Oneinstrument(AppliedBiosystems,USA).ThecDNAwas takenasatemplateforPCRamplificationofADAandODC1genes and␤ACTgene(controlgene).Eachreactionwasrepeatedforthree times.APCRreactionmixtureof20␮lcontained10␮lSYBRGreen mastermix,0.4␮lReverseprimerand0.4␮lForwardprimer,6.2␮l dH2O(RNasefree)and3␮lcDNA(4ng).Threepairsofprimerswere usedseparately.Thethermalcyclingconditionswereasfollow:1 denaturationcycleof95◦C for10minand40cyclesof95Cfor 15s,60◦Cfor60s(annealingandextensiontemperature).A neg-ativecontrolwasusedineachruntoassessspecificityofprimers andpossiblecontamination.TherealtimePCRdatawereanalyzed usingtherelativegeneexpression(Ct)method,asdescribed inAppliedBiosystemsUserBulletinNo.2(LivakandSchmittgen, 2001).Briefly,thedataarepresentedasthefoldchangeingene expressionnormalizedtothereferencegene(␤-actin)andwere determinedusingtheequationfoldchange=2−Ct.

Statisticalanalysis

One-wayanalysisofvarianceandFisher’sLSDwereusedfordata analysis.Allresultswereexpressedasmean±SEM,andpvalues lowerthan0.05werejudgedassignificant.

Results

CytotoxicityofvariousextractsofNepetabinaloudensis

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120

MCF-10A (H2O)

24 h 48 h 72 h

10 20 40 80 160 320 0

100 80 60 40

Cell viability

, %

20 0

120 MCF-10A (EtOH)

10 20 40 80 160 320 0

100 80 60 40

Cell viability

, %

Cell viability

, %

20 0

120

MDA-MB-231 (H2O)

MDA-MB-231 (EtOH)

MDA-MB-231 (n-hexane)

MCF7 (EtOH)

MCF7 (n-hexane) 10 20 40 80 160 320

0 100

80 60 40 20 0

120 100 80 60 40 20 0

120 100 80 60 40 20 0

120 100 80 60 40 20 0 120 100 80 60 40 20 0 120

MCF7 (H2O)

10 20 40 80 160 320 0

10 20 40 80 160 320

0 0 10 20 40 80 160 320

10 20 40 80 160 320

0 0 10 20 40 80 160 320

100 80 60 40 20 0

120 MCF 10A (n-hexane)

Extract concentration (µg/ml) Extract concentration (µg/ml) Extract concentration (µg/ml) 10 20 40 80 160 320

0 100

80 60 40 20 0

24 h 48 h 72 h 24 h 48 h 72 h

Fig.1.CytotoxiceffectsofNepetabinaloudensisvariousextractsonMCF-10A(Normal),MBA-MD-231andMCF-7(cancer)breastcelllines.Cellsweretreatedfor24,48and 72hwithdifferentconcentrationsofextracts.CytotoxicitywasdeterminedbyMTTassay.Resultsarethemean±SEMofthreeindependentexperiments.

Table1

IC50values(␮g/ml)fordifferentextractsofNepetabinaloudensisincelllines.

Plantextract Cellline

MBA-MD-231 MCF-7 MCF-10A

24h 48h 72h 24h 48h 72h 24h 48h 72h

EtOH70% >300 138.4 97.42 249.5 101.9 65.96 >300 >300 >300

Hexane 234.17 92.18 61.29 132 60.89 39.24 >300 >300 147.8

H2O >300 >300 >300 >300 281.7 229.5 >300 >300 >300

sensitivetothecytotoxiceffectsoftheextractscomparedto MBA-MD-231cells.

Fig.2showsthephotomicrographicimagesofMCF-7and MBA-MD-231celllines treated by hexane extract of N.binaloudensis. Cytotoxicity effects were coupled with morphological changes includingdecreaseincellvolumeandshrinkingofthecells.

Paclitaxel(700nM)wasusedasapositivecontrol.Paclitaxelat thisconcentrationdecreasedtheviabilityofMCF-7and MBA-MD-231cellsto6.3%±0.6and12.2±0.3incomparisonwithuntreated controlreceivedanequalvolumeofthesolventrespectively(data notshown).

Invitroclonogenicsurvivalassay

AsgiveninFig.3thereisasignificantdosedependent inhibi-tionincolonyformationinMCF-7andMBA-MD-231afterhexane extracttreatments.Theresultswereshowedadecreaseincolony formationby30–50%inMBA-MD-231whilea45–70%decrease

wasobservedinMCF-7whentreatedwith30␮g/mland60␮g/ml n-hexaneextractrespectively.

ChangesintheexpressionofODC1andADA

Ourresultsshowedthatexposureofcells toincreasing con-centrations of N. binaloudensis hexane extract for 48h caused significantdecreaseinthemRNAlevelsofODC1andADAincancer celllinesbut notnon-cancercells.TheeffectofN.binaloudensis on selected genes expression was shown tobe concentration-dependent.N.binaloudensishexaneextractatconcentrationsof10, 30and60␮g/mlsignificantlydecreasedtheODC1expressionby 1.4,2.3and4.97fold,respectivelyinMCF-7cellline. Moreover, thisextractsignificantlyreducedmRNAlevelsofODC1in MDA-MB-231celllineby1.2,1.7and3.6foldwith10,30and60␮g/ml, respectively(Fig.4).

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Control

MCF-7

MD

A-MB-231

20 µg/ml 80 µg/ml 320 µg/ml

Fig.2.MorphologicalchangesofMCF-7andMDA-MB-231cellstreatedwithdifferentconcentrationsofthehexaneextractofNepetabinaloudensisfor48hviewedunder invertedphase-contrastmicroscope(200×magnification).Reduceincellpopulationwasnotedwiththeincreaseintheconcentrationofthetreatmentascomparedtothe control(untreatedcells).

MCF7 MDA-MB-231

Number of colonies

, %

120

100

80

60

40

20

0

0 10

Extract concentration (µg/ml)

∗∗

∗∗

30 60

120

100

80

60

40

20

0

0 10

Extract concentration (µg/ml)

∗∗

∗∗

30 60

Fig.3.ColonyformationassayshowsasignificantreductionincolonyformationinMCF-7andMDA-MB-231celllinestreatedwithdifferentconcentrationofhexaneextract ofNepetabinaloudensis.Theresultswerepresentedasmean±SEM,n=3(**p<0.01).

dose-dependentmannerby1.32,1.83and3.49foldwith10,30and 60␮g/ml,respectively.AsshowninFig.5,at10,30and60␮g/ml concentrationofN.binaloudensisextractADA mRNAexpression wasreduced1.21, 1.56and 2.56fold in MDA-MB-231cellline, respectively(Fig.5).Incancercelllines,mRNAexpressionofMCF-7 wasreducedmorethanthatofMDA-MB-231.

Discussion

Several studies suggest an increased polyamine concentra-tion in breast cancertissue compared to normal breast tissue and polyamines biosynthesis inhibitors decrease thegrowth of canceroustumors (Brownet al., 2009; Arisanet al.,2012; Zhu et al.,2012).Ornithine decarboxylase (ODC1),is a key enzyme in polyamine biosynthesis which decarboxylates Ornithine to putrescine(WangandJiang,2014).Furthermore,adenosine deami-nase(ADA)enzyme,whichcatalyzesthehydrolyticdeaminationof adenosinetoinosine,playsanimportantroleinpurinemetabolism andAdenosinehomeostasis(Garcia-Giletal.,2015).Ithasbeen proved to be a direct correlation between breast cancer and

increasedADAenzymeactivityincancertissueofpatients(Gocmen

etal.,2009;Rietal.,2010;Ogataetal.,2011).Therefore,ODC1and

ADAgenesaretheappropriatetargetinanticancerresearch. Accordingtopreviousstudiesandtheimportanceof recogniz-inganti-cancermechanismsofplantextracts,inpresentstudythe effectsofvariousextractsofN.binaloudensisonthecell prolifer-ationandtheexpressionofADAandODCgenesinbreastcancer celllines(MCF-7,MDA-MB-231)withrespecttonon-cancerlines (MCF-10A)wereevaluated.

Theresultsshowedthataftertreatmentofcells(at24,48and 72h)withextractsofhexaneandEtOH70%,excludingH2Oextract, alinearrelationshipbetweenthedoseswiththepercentageofcell viabilitywasobserved.Accordingtotheresultsofthisstudyhexane andEtOHextractofthisplanthasanti-cancereffects.

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1.2

0

0 10 30 60 0 10 30 60

0 10 30 60

0.2 0.4 0.6

ODC1 mRNA (f

old)

MCF-10A

MCF-7

Extract concentration (µg/ml)

∗∗

∗∗

∗∗

∗∗ ∗∗

MDA-MB-231

0.8 1

1.2

0 0.2 0.4 0.6

ODC1 mRNA (f

old) 0.8

1

1.2

0 0.2 0.4 0.6 0.8 1

Fig.4.Quantitativereal-timePCRanalysisofODC1mRNAexpressioninonenormalbreastcellline(MCF-10A)andtwocancerousbreastcelllines(MDA-MB-231and MCF-7).ThecellswereincubatedwiththeindicatedconcentrationsofthehexaneextractofNepetabinaloudensisfor48h.ODC1mRNAlevelsdecreaseincancercelllinesin dose-dependentmanner.RelativeabundanceofmRNAisobtainedbynormalizationto␤-actinexpression.Resultsarethemean±SEMofthreeindependentexperiments. **p<0.01comparedtocontrol.

1.2

0 0.2 0.4 0.6

ODC1 mRNA (f

old) 0.8

1.2

0 0.2 0.4 0.6

AD

A mRNA (f

old) 0.8 1

1.2

0 0.2 0.4 0.6 0.8 1

MCF-10A

0 10 30 60

0 10 30 60

0 10 30 60

MDA-MB-231

∗∗

∗∗

∗∗

∗∗

MCF-7

Extract concentration (µg/ml)

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components(Mohammadpouretal.,2013).Thecytotoxiceffects ofthesemonoterpeneshavebeenpreviouslyshownagainst dif-ferentcelllines.1,8-cineole,themaincompoundpresentinthe N.binaloudensisoil, hasbeen reportedtoinhibit thegrowthof liver-derived(HepG2)andextrahepatic(A549)celllines(Rodenak

Kladniew et al., 2014). The results of another study indicated

1,8-cineolesuppressedhumancolorectalcancerproliferationby inducingapoptosis(Murataetal.,2013).Alphaterpineolanother keycomponentoftheoil,hasalsobeenreportedtoinhibitsthe growthoftumorcellsthroughamechanismthatinvolves inhibi-tionoftheNF-␬Bpathway(Hassanetal.,2010).␦-Terpineolinhibits cellgrowthandinducesapoptosisinhumanlivercancerBEL-7402 cells(Wuetal.,2014).Anti-tumoreffectof␣-pineneonhuman hep-atomacelllinesthroughinducingG2/Mcellcyclearresthasbeen reported(Chenetal.,2015).It wasdemonstratedthat␣-pinene inhibitedBEL-7402cellsbyarrestingcellgrowthintheG2/Mphase ofthecellcycle,downregulatingCdc25CmRNAandprotein expres-sion,and reducingcycledependenceonkinase1(CDK1)activity (Chenetal.,2014).

Amongtheextracts,hexaneextractexhibitedthelargest cyto-toxic effects on cancercells. This can bedescribed by the low polarityofhexane,whichextractlow-polarcompoundsthatare eitherfavorablyabsorbedthroughthecellorhavecytotoxic activ-ity.Therefore,itisassumedthatmostofthecytotoxiccompounds wereconcentratedinthehexaneextract(Tayarani-Najaranetal.,

2013).

The results of our study revealed that the cytotoxicity of thehexane extractofN.binaloudensisontheestrogen receptor-positivecellline(MCF7)(ER+/PR+/HER2−)wasstrongerthanthat ofER/PR/HER2triplenegativebreastcancercellline(MDA-MB231). SuchfindingsaresimilartothosefoundintheworkofSrisawatetal.

(2015)whofoundahigherapoptosisandgrowthinhibitionratein

MCF-7cellsthaninMDA-MB-231cells.Thehighercytotoxiceffect ofN.binaloudensisextractonMCF-7wasprobablyduetolackof estrogenstimulationinthisexperiment.

Thedecreaseofthecellsviabilitycouldbebecauseofanincrease ofapoptosisand/ordecreasedcellreproduction(Islametal.,2013). Inourstudy,acolonyformationassaywasperformedtoevaluate thehexaneextractcapabilityofarrestingproliferationof cancer-ouscellsbyloweringreproductiveability.Ourresultsrevealedthat cytotoxiceffectsofhexaneextractcouldbeduetoadecreaseofcell proliferation.

Accordingtothecytotoxicityresults,hexaneextractexhibited thelargest cytotoxiceffects oncancercells. Therefore, in gene expressionstudieshexaneextractwasusedinsafeconcentrations (0–60␮g/ml).SignificantreductioninexpressionofODC1genewas observedinbreastcancercelllines(MCF-7andMDA-MB-231)after 48htreatmentwithN.binaloudensishexaneextract.Other stud-ieshave showntheinhibitoryeffectsofplantmaterialsonODC activityand expressionin cancerouscelllines andtissues. Liao

etal.(2008)foundthatenzymeactivityandproteinexpression

ofODCwerereducedincurcumintreatedleukemiaHL-60cells. Theysuggestedthatcurcumin-inducedapoptosisoccursthrougha down-regulatingmechanismofODC.Inanotherstudy,Hakimuddin

etal.(2008)reportedmorethan3-foldreductioninODC1mRNA

levelintumorsofwinepolyphenol-treatedmice.

However, the mechanism by which N. binaloudensis extract reducesODC1geneexpressionisunknown.IntheODC1promoter, therearemultiplebindingsitesforAp-1,Ap-2andc-Myc(Liaoetal., 2008).ThemechanismsoftheinhibitionofODC1expressionby N.binaloudensisextractlikelyinvolvetranscriptionalinhibitionof thesefactors.

ODCenzymeactivityisrelatedtotheproductionofpolyamines such as spermine that function as a free radical scavenger

(Smirnovaetal.,2012).InhibitionofODCgeneexpressionbyN.

binaloudensisextractdecreasestheconcentrationofpolyamines.

Thus,reductionofpolyaminescouldincreaseintracellularROSand causeapoptosisincancercells(Wangetal.,2011).

ADA gene expression decreased in the cancerous human breastcelllinestreatedbyN.binaloudensishexaneextract.Many researchersreportedthatplantextractshaveinhibitoryeffecton ADA expression and activity. For example, Durak et al. (2005)

showedthatblackgrapeextractssignificantlyinhibitedtheADA enzymeactivityincancerouscolontissues.Inanotherstudy,ithas beenfoundthatexpressionoftheadenosinedeaminasegenewas consistentlydecreasedbyalltheberriesinintestinaladenoma

tis-sueofrats(Mutanenetal.,2008).Tomatojuicehasbeenreportedto

causessignificantinhibitiononADAactivityintheprostatecancer tissues(Duraketal.,2003).

Our datademonstratethatdecreaseofadenosine deaminase expressionabolishesadvantageofcancercellsinobtainingmore nucleotides inDNA synthesis and proliferation(Namuslu etal., 2014).Also,inhibitionofADAexpressioncausesincreased avail-ability of adenosine which is a protective molecule in case of tumorouscondition(Mahajanetal.,2013).

Inconclusion,thestudydescribedheredemonstrates thatN. binaloudensishexaneextracthasinhibitoryeffectsonODC1and ADAgeneexpressioninbreastcancercellline.Therefore,a possi-blemechanismforobservedcytotoxicityeffectofthisplantextract couldbeexpressioninhibitionofthesegenes.Thisisthefirstreport aboutthecytotoxicityofN.binaloudensishexaneextractby inhibi-tionofODC1andADAinmRNAlevel,hencefurtherstudieswillbe necessarytosupplementourfindings.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authorcontributions

ASAdesigned and supervised theexperiments,analyzed the dataanddraftedthepaper;FSNperformedexperiments,analyzed dataanddraftedthepaper;MMperformedexperimentsand ana-lyzeddata;AKdesignedandanalyzedthedata.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

This work was financially supported by Neyshabur Branch, IslamicAzadUniversityNeyshabur,Iran.

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Imagem

Fig. 1. Cytotoxic effects of Nepeta binaloudensis various extracts on MCF-10A (Normal), MBA-MD-231 and MCF-7 (cancer) breast cell lines
Fig. 2. Morphological changes of MCF-7 and MDA-MB-231 cells treated with different concentrations of the hexane extract of Nepeta binaloudensis for 48 h viewed under inverted phase-contrast microscope (200× magnification)
Fig. 5. Quantitative real-time PCR analysis of ADA mRNA expression in one normal breast cell line (MCF-10A) and two cancerous breast cell lines (MDA-MB-231and MCF-7).

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