Peganum harmala L.'s anti-growth effect on a breast cancer cell line

Peganum

harmala

L.

’s

anti-growth

effect

on

a

breast

cancer

cell

line

Somayeh

Hashemi

Sheikh

Shabani

a,1

,

Sahar

Seyed

Hasan

Tehrani

a,1

,

Zohreh

Rabiei

a

,

Sattar

Tahmasebi

Enferadi

a,

*

,

Gian

Paolo

Vannozzi

b

a_{NationalInstituteofGeneticEngineeringandBiotechnology,Tehran,Iran} b_{DipartimentidiSienzeAgraria,UniversitadegliStudidiUdine,Udine,Italy}

ARTICLE INFO

Articlehistory: Received1May2015

Receivedinrevisedform24August2015 Accepted25August2015

Availableonline27October2015

Keywords:

Extrinsic/intrinsicapoptosis Harmaline

Harmine

ABSTRACT

ThisresearchwasdonetoevaluatetheinductionofapoptosisinMDA-MB-231breastcancercelllineby Peganumharmala’sextract,inwhichasignificantamountofß-carbolinesisincluded.Theapoptosis incidencewasassessedthroughAnnexin-V-Flouskit.Theexpressionsofgenesthroughwhichintrinsic apoptosispathwayareinvolved,Bax,Bcl-2,Bid,andPuma,overthegenestheexpressionsofwhichare linkedtoextrinsicapoptosispathway,TRAIL,Caspase8,p21,andp53,wereexaminedbyRT-PCRand Real-timePCR.Theresultsdemonstratethattheextractdecreasesthegrowthrateofthecancercellline throughinducingapoptosismechanism.Aslongastheexpressionofanti-apoptosisBcl-2genreduced dramatically,anover-expressioninBaxandPumageneswasmonitoredindicatingactivationofintrinsic apoptosispathway.Anotableover-expressionobservedwithTRAILandCaspase8genesaswellasBid gene.Thelatterisanintermediateforbothintrinsicandextrinsicpathwaysofapoptosis.

ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Apoptosis isa programmedcelldeathwhichis importantin controllingcellnumber and proliferation.Apoptosis inducesby twomajorpathways;extrinsicandintrinsicpathways[8].Extrinsic pathway instigates by death signals that ligate to the death receptorsuchasTRAIL(tumornecrosisfactor-related apoptosis-inducing ligand) or Fas (TNF Receptor Superfamily) genes. The deathreceptorsarethememberofthetumornecrosisfactor(TNF) receptor gene superfamily containing death domain which is importantintransmittingthedeathsignalfromthecell’ssurfaceto intracellularsignalingpathways.Theligationofdeathreceptorand death signal caused receptor trimerization, recruiting adaptor molecules such as FADD (Fas-Associated protein with Death Domain)whichresultsintheactivationoftheinitiator Caspase-8and lead to activatedownstream effectorcaspases (cysteine-asparticproteasesorcysteine-dependentaspartate-directed pro-teases)suchascaspase-3andfinallyDNAfragmentation[9,3].

The other pathway, intrinsic or mitochondrial pathway is triggeredbythereleaseofapoptosisfactorssuchascytochromec. The Bcl-2 (B-cell lymphoma 2) family proteins are crucial for

intrinsicpathwaythatinhibitorinitiateapoptosis.Antiapoptotic Bcl-2familymemberssuchasBcl-2andMcl-1inhibitapoptosis; howeverthedomainofproapoptoticmemberssuchasBax (Bcl-2-associatedXprotein)andBak(Bcl-2homologousantagonist/killer) promote apoptosis and also suppress oncogenesis. Bid (BH3 interacting-domaindeathagonist),a Bcl-2familyproteinwitha BH3 domain only, is a linkage between intrinsic pathwayand extrinsicpathway[9,8].

Evadingapoptosisisoneofthehallmarkofcancercells[13,14]. Theresearcherspostulatedthatinducingapoptosisincancercells whichiscausingnodamagetonormalcellscanbeapracticaldrug fortreatingcancer.Therefore,ongoingcancertherapiesarelooking fortheanti-cancerstriggeringapoptosisincancercells[10,3,9].

PeganumharmalaL.(Nitrariaceae)isalocalremedydistributed inthecentralAsia,Middle EastandNorthAfrica.Thisplanthas been used asfolk medicine mostly because of its antibacterial effect for long time. Studies show the inhibitory effects of P. harmalaonbacteria,parasites,viruses,andcancercells[2].Several reports demonstrated that ß-carboline alkaloids, one of the phytochemicalcompoundofP.harmala,intercalatetoDNAleads toDNAtopoisomerasesinhibition[1].Thatstudyaddressedthat harmalineandharmalolinducemelanogenesisthroughp38MAPK signaling[17].Theresearcherssuggestedtheanti-cancereffectofP. harmalahappensmainlyasaconsequenceofinducingapoptosis

[4,11]. Indeed, the impact of harmine on B16f-10 melanoma revealedthatitactivatesbothintrinsicandextrinsicpathwaysby up-regulating Bax, Bid, p53 and Caspase-8 genes and down-regulatingBcl-2[11].Bycontrast,harmineanditsderivativeshave

*Corresponding author at: National Institute of Genetic Engineering and Biotechnology, Tehran freeway 15km, P.O.BOX 14925/16, Pajouhesh BLV 1417863171,Tehran,Iran.Fax:+982144787399.

E-mailaddress:[email protected](S.TahmasebiEnferadi).

1

Thefirsttwoauthorsareconsideredasthefirstauthor.

http://dx.doi.org/10.1016/j.btre.2015.08.007

2215-017X/ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Biotechnology

Reports

noeffectontheexpressionofp53andBaxwhencellstreatedwith P.harmala.Theover-expressionofFasanddown-regulationofBcl-2 incancercellstreatedwithharmine/itsderivativeswasobserved, too[6].Caoetal.[4],havereportedthatexpressionofBcl-2,Mcl-1 (inducedmyeloidleukemiacelldifferentiationproteinMcl-1)and Bcl-xl(B-celllymphoma-extralarge)geneswerereducedinHepG cancercellswhiletheexpressionofBaxdidn’tchangesuggesting thatitinducesintrinsicpathwayinapoptosis[4].Chenetal.[7]

used9harminederivatives(includingharmine)toinvestigatetheir antitumoreffectsandacutetoxicitiesinmicethroughtheanalyses ofIC50andtheexpressionofBcl-2gene.However,theystatedthat further studies on the effects of harmine derivatives on key regulators for tumor cell apoptosis were needed. Hamsa and Kuttan [12] have demonstrated the anti-angiogenic activity of harmineusinginvivoand invitroassaysystemsandconcluded strong angiogenic inhibitory of harmine with the ability to decrease the proliferation of vascular endothelial cells and reduction in the expression of various pro-angiogenic factors. ZhaoandWink[21]havestudiedharmineactivityontelomerase byanalyzing thetelomericrepeatamplificationprotocol(TRAP) anddemonstratedsignificantinhibitionoftelomeraseactivityand an induction of an accelerated senescence phenotype by over-expressingelementsofthep53/p21pathwayasaresultofharmine treatmentwhich exhibits a pronounced cytotoxicity on cancer cells.Sunetal.[19]andZhangetal.[20]havestudiedtheeffectsof paclitaxeland/orharmineonthecellmigrationandinvasionintwo humangastriccancercelllinesandit waselucidatedthatthose compoundsprovidea synergisticeffectongrowth inhibitionof cancercellsviathedownregulationofCyclooxygenase-2(COX-2) expression.

Despite all the aforementioned evidences, the underlying mechanismofharmine effectoncancercelllinemostofwhich isnotclearyethasnotbeenthoroughlysupported.

Inthepreviousstudy,theanti-cancerimpactofP.harmalaon cancercelllineswasconfirmedbyMTTtest[18].Inthepresent study,theapoptosisassaywasusedtoelucidate theanticancer effectofP.harmala’sseedextract.TheexpressionofBax,Bcl-2,Bid andPuma(p53upregulatedmodulatorofapoptosis)genesbeing involvedinintrinsic pathwayhasbeenstudiedassomerelated mechanisms of P. harmala’s extract. Furthermore, the gene expressionof TRAILandCaspase-8 ascandidatesgenesinvolved in extrinsic apoptosis pathway was monitored. For further confirmation, p53 and p21 were traced using quantitative procedures,real-timePCR.

2.Materialsandmethods

2.1.Cellcultureandpreparation

The breastcancercelllineMDA-MB-231,wasobtainedfrom National Institute of Genetic Engineering and Biotechnology, NIGEB.ThecellswereculturedinDMEMmediumsupplemented with 10% heat-inactivated FBS, penicillin (100

m

mL
1), and streptomycin (100

m

mL
1)at 37_{C ina humidifiedatmosphere}

containing5%CO2maintained.

2.2.Chemicals

P. harmala seeds were collected from the mountains of Shahrekord, Chaharmahalo Bakhtiari province, Iran. The P. harmala’salkaloidextracthasbeenpreparedaccordingtoSeyed HasanTehranietal.[18].

Annexin-v-fluosstainingKit(Roche AppliedScience,France), RNAextractkit(RocheAppliedScience,France),RNeasyMiniKit Qiagen(Cat.No.74104),MasterMixPCR(Iintron),QuantiFastSYBR Green (Qiagen, Cat. No. 204054), Harmine (286044-1G, Sigma, USA),Harmaline(51330-1G,Sigma,USA)andDMEM,Trypsin,and FBS(bioidea,Italy)wereusedforourexperiments.

2.3.Apoptosisassay

Annexin-v-fluosstainingKitusedfordifferentiatingapoptotic cellsfromnecroticcellsintheearlystageofapoptosisbybindingto thecellsincludingphosphatidylserine.Cancercellline MDA-MB-231wereseeded(1000cells/well)in24wellplateandincubated for24hat37Cin5%CO2atmosphere.Theconcentrationof30and

100

m

g.ml
1ofP.harmala’sseedextractwasaddedandincubated forfurther24and48h.Afterincubationthemediumdepleted,for stainingwithannexin-V,each wellwas incubatedfor15minat 25Cin 100

m

lofasolutioncontaining20

m

lof Annexin-Vand 20

m

lpropidiumiodide(30

m

gml
1)in1mlHepesbuffer(10mM Hepes/NaOH pH 7.4; 140mM NaCl; 5mM CaCl2). Cells were

washedtwicewithphosphate-bufferedsalineandanalyzedunder a fluorescence microscopy using an excitation wavelength of 480nmanddetectionintherangeof515–565nm.

2.4.RNAextractionandcDNAsynthesis

TotalRNAwasextractedfromMDA-MB-231cellstreatedwith 30

m

g.ml
1 concentration of alkaloid extract for 24h (as the

Table1

Listofprimerpairsusedcorrelatedwithapoptosispathwayinhumancells. Accessionno.a

minimumlethaldosethatkillsapproximately50%ofcellsoccurred at24h(IC50)obtained byMTT test, this combination of time/ concentrationhasbeenchosenfortherestofanalysis)usingthe RNA extraction Kit (RNeasy Mini Kit Qiagen (Cat. No. 74104)) accordingtothemanufacturer’sprotocol.ThequalityoftheRNA samples and concentration were precisely determined using NanoDrop spectrophotometer. RNA reverse transcription was performedin a final volume of 20

m

l using PrimeScriptTM 1st strandcDNASynthesisKit(Takara,Cat.No.6110,Iran).

2.5.Primerdesign

UponasurveyonNCBIGenBank,therelatedsequencesforBax, Bcl-2,Bidand Puma, TRAILand Caspase-8 involved in apoptosis pathwayand also p53gene were selectedand the appropriate primerpairsweredesignedwithlengthsrangingfrom70to186bp

withtheprogramsOligo6.0.BLASTNsearchesconductedagainst nr(thenonredundantsetofGenBank,EMBL,andDDBJdatabase sequences)toconfirmthetotalgenespecificityofthenucleotide sequences chosen for the primers. To avoid amplification of contaminatinggenomicDNA,oneofthetwoprimersortheprobe wasplaced atthejunctionbetweentwoexonsorin adifferent exon. Designed primers were purchased from Gene fanavaran (Iran).TheprimersdetailsarereportedinTable1.

2.6.RT-PCR

Reverse transcriptase reactions contained 20ng of RNA samples,50nMRTrandomhexanucleotideprimers (Amersham PharmaciaBiotech,Piscataway,NJ),1RTbuffer(10mMTris–HCl, 0.1MKCl,0.05%[wt/vol]Tween20,0.75mMEGTA[pH8.3],2mM deoxyribonucleoside triphosphates, 2.5mM MgCl2, 3.3U.

m

l
1

reversetranscriptaseand0.5U.

m

l
1RNasein(allpurchasedfrom cDNAArchivekitofAppliedBiosystems).Thereactionmixturewas sequentiallyincubatedinan Applied Biosystems9800 Thermo-Cycler for 30minat 16C, 30min at 42C, 5minat 85C. The reversetranscriptionreactionwasstoppedbyheatingto95Cfor 5min. Next,0.5U of RNase H (Invitrogen) was added, and the reactionmixturewasincubatedat37Cforanadditional30min. Amplified PCR (15ml) products were electrophoresed on a standard1.8%agarosegelhavingstainedwithethidiumbromide andphotographunderUVlight.

2.7.Real-timequantitativePCR(qPCR)

ThesynthesizedcDNAwasquantifiedbytheQuantiFastSYBR Green(Qiagen,Cat.No.204054)accordingtothemanufacturer’s protocol.ForeachPCRrun,1

m

lprimer(10

m

M)and300ng.

m

l
1 cDNAwereaddedtothePCRmastermixandreachedtothefinal volumeof20

m

l.Real-timePCRwasperformedwithanABIPrism 5700orABIPrism7700machine(AppliedBiosystems,USA)and universal cycling conditions (2min at 50C, 10min at 95C, 40cyclesof15sat95C,and1minat60C).Experimentswere performedwithduplicatesforeachdatapoint.Notemplatewas addedtonegativecontrolreactions.Inordertoquantifyspecific geneexpression,themRNAlevelineachsamplewascalculated relativetobeta-actin.TheCtdatawasdeterminateusingdefault threshold settings. The threshold cycle (Ct) is defined as the fractionalcyclenumberatwhichthefluorescencepassesthefixed threshold. The relative quantification of gene expression was analyzedbythe

DD

Ctmethod.TheexpectedsizeofPCRproducts andnothavingprimerdimerwereconfirmedbygel electrophore-sisonastandard1.2%agarosegelstainedwithethidiumbromide andvisualizedunderUVlight.

2.8.Statisticalanalysis

ThereactionefficiencieswereestimatedusingtheLinRegtool for each sample, the expressionratios weretested by random pairwisereallocationusingRESTsoftware.

3.Results

3.1.Apoptosisanalysis

Apoptosis, a normal phenomenon, through which a suicide program is activated within the cell occurs frequently in multicellularorganisms, however is associated with DNA frag-mentation,cytoplasm shrinkage,membrane changesand finally cell death without releasing harmful substances into the surroundingarea.

UpontreatingMDA-MB-231cancercelllinewithP.harmalaL.’s seed extract several changes were observed depending the concentration and time of exposure of which 30

m

gml
1 concentration of the extract lead to a significant growth rate reductionandtheminimumlethaldosethatkillsapproximately 50%ofcellsat24h(IC50).Providingmoredeleterioussignforthe laterconcentrationwasresponsibleforsmallercellsizeandmore shrunkcytoplasm(dataarenotshown).

3.2.Annexin-v-fluosassay

To validate apoptosis pathway induction and cell death occurrenceasitsconsequence,Annexin-v-fluosassaywasused. Cancer cell line MDA-MB-231 were treated with 30

m

g.ml
1 concentrationof P.harmala L.’sseedextractand wereobserved underfluorescencelightafter24and48h.The100%percentageof

treated cells was fluorescentgreen while untreatedcells didn’t emitanygreenlight(Fig.1).

3.3.Geneexpressionanalysis

Thequantitativeandqualitativeanalysesofgeneexpressionby which a reliablecalculation of thegene functionsin a cell get evidencedneedtobetraced.Thus,theexpressionsofthegenes involvedinintrinsicapoptosispathwaynamely,Bax,Bcl-2,Bid,and Pumaaswellasthegenesinvolvedinextrinsicapoptosispathway suchasTRAILandCaspase-8 andtwo moregenes,p21andp53, were evaluated qualitatively and quantitatively by RT-PCR and Real-timePCR,respectively.

InordertoinvestigatemRNAdifferentialexpressionin MDA-MB-231cancercellline,theimpactofP.harmalaL.’sseedextract ([30

m

gml
1]/24h)wereconductedaswell.

Fig.2. ImpactofP.harmalaL.’sseedextractontheexpressionofdifferentgenes. MDA-MB-231cellsweretreatedwith30mgml
1_{oftheextractfor24h.Expression}

ofbeta-actinwasusedtonormalizetheconditions.

Fig.2showschangesintheexpressionofeightgenesinvolving apoptosispathwaycomparedtoahousekeepinggene(beta-actin) inMDA-MB-231celllineundertwoconditions,beingtreatedwith P.harmalaL.’sseedextractandkeepingthecellsuntreated.RT-PCR resultshowed thattheexpressionofpro-apoptoticgenesPuma, Bax,andBidinvolvinginintrinsicpathwayincreasedsigni_ficantly incomparisonwithcontrol,while theanti-apoptoticgeneBcl-2 diminished. Meanwhile the extrinsic gene TRAIL and Caspase8wereup-regulated,alsotheexpressionofthe interme-diategeneinbothintrinsicandextrinsicpathways,Bid,increased meaningfully.Thefigurealsorevealstheoverexpressioninp53and p21genescomparedtotheircontrol(Fig.2).

Accordingly,inreal-timePCRanalysis,theexpressionof pro-apoptotic genes, Bax, Puma, and Bid, increased 4.926, 4.045, 1.57times,respectively,despitetheexpressionofBcl-2decreased (1.307times)whencomparedwithitsrespectedcontrol(Fig.3). Furtherstudiesontheextrinsicgeneexpressiondemonstratedthat thedeathreceptorgenes,TRAILandCaspase-8wereup-regulated rising to 1.571 and 1.867 times, respectively (Figs. 4 and 5). However,the highestover-expression is dedicated top53 gene (3.938).Additionally,theexpressionofp21generoseto1.64time inthetreatedcells(Fig.4).

4.Discussion

Theanti-cancereffectofP.harmala’sseedextractondecreasing thegrowthrateofcancercellshasbeenapprovedaccordingtoour previousstudy.Inthepresentstudy,wescrutinizedthisimpactat molecular level through following up the expression of some relatedgenes.Themorphologicalchangesincludingdamagedcell membrane and cell shrinkage are the features of apoptosis as

suggested by [16] on cancer cell death caused by apoptosis pathway.ThiswasconfirmedbyAnnexin-v-fluosassayaswell.The treatedcellsallofwhichweregreenunderfluorescencelight(and obviouslynotred)weredamagedintotheirnucleolus.Thismeans that apoptosistriggersdeathin cancercells and leavesout the necrosisasan option.Thisphenomenawould beintensifiedby increasingthe exposuretime (from 24hto48h) and themost deleteriouscelldeathwasobservedat48hasshowninFig.1(the mirrorsof1Aand1Cmicrographs).

It is noteworthy that apoptosis is mainly induced by two pathways; extrinsic and intrinsic. Admittedly, in this study we examinedtheeffectofP.harmalaL.’sseedextractthroughwhich apoptosispathwayareinduced.Asaresultofthefactthatextrinsic pathwaygetstriggeredbyligationofdeathsignalandreceptors, Caspase-8 gene is activated as a consequence of intracellular signaling.TheactiveCaspase-8initiateCaspasecascadetheroleof whichonmorphologyofapoptosisisnotclear[8].

TheexpressionoftwogenesTRAIL,deathreceptor,and Caspase-8,associatedwithextrinsicpathway,whichwerefollowedby RT-PCRandReal-timePCRdemonstratedup-regulationofbothgenes upontreatingwithP.harmalaL.’sseedextractsuggestingthatthe extractinducesmainlyonapoptosisextrinsicpathway.

On the other hand, to identify the cooperation of intrinsic pathwaysinanti-cancereffectofP.harmalaL.’sseedextract,Bcl-2 genefamilywasmainlyfollowedup.Thepro-apoptoticgenes,BH3 multidomain, Bax (Bcl-2-associated X protein) and Bak (Bcl-2 homologousantagonist/killer)causedpermeabilityin mitochon-drialmembranewhichwasinhibitedbyanti-apoptoticproteins, Bcl-2,Bcl-xl.AsaconsequenceofbindingBH3-onlyproteins(BID, BIM(Bcl-2-likeprotein11),BAD(Bcl-2-associateddeathpromoter), and PUMA to anti-apoptotic proteins, their activities were inhibited. However, in this research Bcl-2 as an anti-apoptotic protein,BaxasaBH3multidomainpro-apoptoticproteinandPUMA as BH3-only domaim pro-apoptotic protein were selected for evaluationofrelatedgenesaffectingtheintrinsicpathway.Bidalso studiednot onlyas a pro-apoptoticproteinbut also aslinkage betweenintrinsicpathwayandextrinsicpathway[8].

Expression analysis of mRNA by RT-PCR and Real-timePCR revealedthatexpressionofantiapoptoticgene,Bcl-2decreasedin treatedcellincomparetocontrolwhilethepro-apoptoticgenes suchasBax,Puma,Bidincreased.Alltheresultssupporteachother and proposethattheP.harmala’s seedextractinducesintrinsic pathwaywhenexposedtocelllines.

Theexpressionchangesofp53andp21inMDA-MB-231cancer celllinetreatedwithP.harmala’sseedextractwerealsostudied. Bothp53andp21wereup-regulatedintreatedcellsincompared with their respected control. Accordingly, it approved that P. harmal’sseedextractdecreasesthecellgrowthrate,hencep53as transcriptionregulatorup-regulatesBax,PumaandTRAIL[15,5].

Fig.5. ApoptosisandgrowthrelatedgeneexpressionincellstreatedbyP.harmala L.’sseedextract.Thep53,p21genesexpressionweresignificantlyelevated. p53:phosphoproteinp53ortumorsuppressorp53,p21:cyclin-dependentkinase inhibitor1 orCDK-interactingprotein1.

Fig.4.Extrinsicapoptosis-relatedgeneexpressionincellstreatedbyP.harmalaL.’s seedextract.TRAILandCaspase-8genesexpressionincreased.

Fig.3.Intrinsicapoptosis-relatedgeneexpressionincellstreatedbyP.harmalaL.’s seedextract.Pro-apoptoticgenes,Bax,Puma,andBid,wereup-regulatedwhilethe anti-apoptoticgene,Bcl-2showedlessexpressioncomparetocontrol.

5.Conclusion

Cancer cells grow and divide further fromnormalcy due to losingthegeneralcontrolsoverthegrowth.Traditionaltreatments involvediminishingthegrowthrateofcancercellsbymeansof exposingitagainstamedicinalherbextract.Inthepresentstudy,P. harmalaL.’sseedextractexposedonaMDA-MB-231cancercell line and its growth inhibition was followed through both morphologicalchangesobservationandfollowinggenesinvolved programmecelldeath.

P.harmalaL.’sseedextractinducedcelldeathanddecreasedthe cellgrowthinthebreastcancercellline.Thecelldeathwascaused byapoptosiswhichwastriggeredbybothintrinsicandextrinsic pathwayswhichsuggestthatherbmightbeusefulforpreventing thedevelopmentoftumors.

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