Rev. bras. farmacogn. vol.27 número3

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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

A

cytotoxic

Petiveria

alliacea

dry

extract

induces

ATP

depletion

and

decreases

␤-F1-ATPase

expression

in

breast

cancer

cells

and

promotes

survival

in

tumor-bearing

mice

John

F. Hernández

a,b

,

Claudia

P. Urue˜na

a

,

Tito

A. Sandoval

a

,

Maria

C. Cifuentes

a

,

Laura

Formentini

b

,

Jose

M. Cuezva

b

_,

_Susana

_Fiorentino

a,∗

a_Grupo_de_{Inmunobiología}_y_Biología_Celular,_Facultad_de_Ciencias,_Pontificia_Universidad_Javeriana,_Bogotá,_Colombia

b_Departamento_de_Biología_Molecular,_Centro_de_Biología_Molecular_Severo_Ochoa,_Consejo_Superior_de_{Investigaciones}_{Científicas-Universidad}_Autónoma_de_Madrid,_Madrid,_Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17May2016 Accepted20September2016 Availableonline9February2017

Keywords: Petiveriaalliacea

Breastcancer

␤-F1-ATPase Respiration ATPdepletion

a

b

s

t

r

a

c

t

Metabolicplasticityincancercellsassurescellsurvivalandcellproliferationundervariablelevelsof oxy-genandnutrients.Therefore,newanticancertreatmentsendeavortotargetsuchplasticitybymodifying mainmetabolicpathwaysasglycolysisoroxidativephosphorylation.InAmericantraditionalmedicine PetiveriaalliaceaL.,Phytolaccacea,leafextractshavebeenusedforleukemiaandbreastcancertreatments. Herein,westudycytotoxicityandantitumoraleffectsofP.alliaceaextractintumor/non-tumorigeniccell linesandmurinebreastcancermodel.BreastcancercellstreatedwithP.alliaceadryextractshowed reductionin␤-F1-ATPaseexpression,glycolyticfluxtriggeringdiminishedintracellularATPlevels, mito-chondrialbasalrespirationandoxygenconsumption.Consequently,adeclineincellproliferationwas observedinconventionalandthree-dimensionspheresbreastcancercellsculture.Additionally,invivo treatmentofBALB/cmicetransplantedwiththemurinebreastcancerTS/AtumorshowedthatP.alliacea extractviai.p.decreasestheprimarytumorgrowthandincreasessurvivalintheTS/Amodel.

Introduction

Cancer cells may assure survival and proliferation under shiftinglevelsofoxygenand nutrientsthrough metabolic plas-ticitybetweenglycolysisandoxidativephosphorylation(OxPhos) metabolicpathways.Integratingthesepathwaysinglucose oxida-tionprovidesimportantsubstratesasATP,NADHandbiosynthetic precursorsforthecellhousekeepingprocesses(Formentinietal., 2010).MitochondrialactivityandspecificallyOxPhosplaya rele-vantroleinfacilitatingtheexecutionofcelldeath(Cuezvaetal., 2009).InthemitochondrialinnermembraneisfoundtheATPase orcomplexV,amulti-enzymaticcomplexwithtwodomains:a hydrophobicintramembranedomainF0andahydrophilicdomain F1 facing to the matrix leaflet. F1 domain has five sub-units ␣3␤3␥1␦1␧1andthreecatalyticsites(subunit␤and␣/␤ inter-face).Theelectrochemicalgradientgeneratedbythemitochondria REDOXreactions,makespossibletheprotoninfluxintothematrix through F0 domain providing the necessary energy for ADP

∗ Correspondingauthor.

E-mail:susana.fiorentino@javeriana.edu.co(S.Fiorentino).

phosphorylation (Stock et al., 1999; Gledhill et al., 2007).It is wellknownthatcancercells canhavestructuralandfunctional mitochondrial alterations. For instance, it has been shown in human carcinomas that selective repression of ␤-F1-ATPase is inverselycorrelatedwithglyceraldehyde-3-phosphate dehydroge-nase(GAPDH)levelscausingdecreaseinmitochondrialactivityand increaseinglycolyticflux(Cuezvaetal.,2002).Moreover,human breastcancercellsoverexpressedlactatedehydrogenaseisoformA (LDH-A)leadingtoanincreaseinlactatesecretion(Koukourakis et al., 2008). Hence highlactate levels are associated to taxol resistanceand cell proliferation augmentation under a hypoxic microenvironment(Fantinetal.,2006;Zhouetal.,2010).

Recently, dichloroacetate (a pyruvate dehydrogenase kinase inhibitor)hasbeenproposedasanewanticancerdrugspecially forglycolytictumorsposinglimitedsideeffects(Papandreouetal., 2011)andbroadeningthespectrafornewOxPhosregulators.In thisregard,plantscouldbeasourceofcompoundsabletotarget cancermetabolicpathways.

In fact, Petiveria alliacea L., Phytolaccaceae, infusions from leafand roothave beenreportedto haveanti-spasmodic, anti-rheumaticandanti-inflammatoryproperties(Moralesetal.,2001) andparticularlyusedinleukemiaand breastcancertreatments

http://dx.doi.org/10.1016/j.bjp.2016.09.008

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J.F.Hernándezetal./RevistaBrasileiradeFarmacognosia27(2017)306–314

(Garcia-Barriga, 1974; Gupta, 1995). It has been shown that P. alliacea extracts are cytotoxic on leukemia, lymphoma and melanomacelllines(Rossi,1990;Rossietal.,1993;Urue˜naetal., 2008), howeverit poseslow toxicity onhumanfibroblasts and peripheralbloodmononuclearcells(Urue˜naetal.,2008).Recently, wehaveproposedthatantitumoractivityofa dryextractfrom P.alliaceacanbepartlyexplainedbytheglycolyticfluxshifting ofcancercells,asshownon4T1breastcancermodel(Hernandez etal.,2014).

Herein, we demonstrated that a dry extract from P. alli-aceacauseschangesinmitochondrialactivitycharacterizedbya decreasein␤-F1-ATPaseexpressionandATPdepletionleadingto adecreaseinbreastcancercellproliferationinvitroandinvivo.

Materialsandmethods

Plantmaterialandextractionprocedure

Petiveria alliacea L., Phytolaccaceae, leaves and stems (local name“anamu”)werecollectedinCachipay,Cundinamarca, Colom-biaonApril2009andidentifiedbyCarlosParrafromtheColombian National Herbarium; voucher number COL 569765 (Colombian EnvironmentalMinistryagreementnumber 1927relatedtothe use of genetic resources and derivatives products). P. alliacea extractionprocedureand chemicalcharacterization were previ-ouslydescribed (Urue˜naet al.,2008).Briefly,dryground leaves and stemswereextracted with96%ethanol (15±5◦_C),_filtered andconcentratedunderreducedpressure.Ethanolicextractwas trapped on fumed silica, fractionated with ethyl acetate and extractedwithmethanol:wateryieldingadryextract(DER gen-uine:10000–11000:1).Thecompoundsidentifiedin dryextract fromP.alliaceawere:benzaldehyde,leridol,petiveral,myricetin, petiveral4-ethyl, pinitol,dibenzyldisulfideanddibenzyl trisul-phide. HPLC chromatographic fingerprint was acquired in a Jasco®_PU2089plus_equipped_with_a _UV_detector₍₂₅₄_nm)_using a C18 column and water/acetonitrile gradient as mobile phase (Hernandezetal.,2014).TomeetEMAguidelines,theactivemarker selectedwasdibenzyldisulfide,a reportedcytotoxiccompound (Cifuentesetal.,2009)foundata concentrationof2.6mg perg ofextract.

Celllines

4T1,TS/A,3T3andHS578TcelllineswereculturedinDMEM andHCT116celllineinMcCoy’s5Amedium,bothsupplemented withfetal calf serum (FCS) heat-inactivated (10%), l-glutamine

(2mM),penicillin (100U/ml), streptomycin (100␮g/ml), HEPES buffer(0.01M) andsodiumpyruvate(1mM)(EurobioToulouse, FR).MCF12Fcelllinewascultured inDMEM/F-12medium sup-plementedwithfetalhorseserum(5%),epidermalgrowthfactor (20ng/ml),humaninsulin(10␮g/ml),hydrocortisone(500ng/ml), choleratoxin(100mg/ml),penicillin(100U/ml)andstreptomycin (100␮g/ml).Celllineswereincubatedunderhumidified environ-mentat37◦_C_and_5%_CO₂_.

Invitrocytotoxicityassays

Cytotoxic effects were evaluated using methylthiazol tetra-zolium (MTT, Sigma-Aldrich,Saint Louis, MO) and trypan blue dye assays. Cells (5×103 cells/well) were seeded in 96-wells plateswithdifferentconcentrationsofdryextractfromP.alliacea (250–0.95␮g/ml)orethanol(0.02%)asnegativecontrolfor48h. ProliferationwasestimatedbyMTTassayaccordingtoprocedure previouslydescribed(Urue˜naetal.,2008).TheIC50valuewas esti-matedwithnonlinearregressionanalysis(GraphPadPrism5for Windows).

Westernblots

Cellsweresuspendedinlysisbuffercontaining25mMHEPES, 2.5mM EDTA,0.1% TritonX-100,1mM PMSFand 5mg/ml leu-peptin.Extractswerecentrifugedat11,000×gfor15minat4◦_C. Supernatantsprotein concentrationwasdeterminedwith Brad-fordproteinassay.Cellularproteins(7–20␮g)werefractionated bySDS/PAGE (12%)andtransferred ontoPVDFmembranes. Pri-marymonoclonalantibodieswere: anti-␤-F1-ATPase (1:50,000) (Cuezvaetal.,2002),anti-Hsp60(1:10,000)andanti-NADH9 (com-plexI39kDa)(1:1000)(Aceboetal.,2009),anti-ComplexIIIsubunit Core 2 (1:1000) from Abcam; anti-SDH (succinate dehydroge-nase)(1:500)fromLifeTechnologies;anti-␤-actin(1:20,000)and anti-tubulin(1:5000)fromSigma-Aldrich.Peroxidase-conjugated anti-mouseoranti-rabbitIgG(NordicImmunology,1:3000)were usedassecondaryantibodies.Blotsrevealedwithluminol electro-chemiluminescence(ECL)reagent(AmershamPharmaciaBiotech, LittleChalfont,UK).

Oxygenconsumptionestimation

CellularoxygenconsumptionratesweredeterminedinanXF24 ExtracellularFluxAnalyzer(SeahorseBioscience).Cells(4×104) wereseededinXF24-wellcellculturemicroplates(Seahorse Bio-science), treated with dry extract from P. alliacea or ethanol, incubatedat37◦_C_and_5%_CO₂ _for₆_or₂₄_h._After_treatment_the followingsubstanceswereconsecutivelyinjectedtoachieve the indicatedfinalconcentration:oligomycin(OLIGO,6␮M), dinitro-phenol(DNP)0.5mM,rotenone1␮Mandantimycin1␮M.

Glycolysisfluxestimation

Cells(1.5×105)wereseededandallowedtogrowuntil60%of confluence.Todetermineglycolyticrates,cells weretreatedfor 3,6,24or48hwithdryextractfromP.alliaceaorethanolwith orwithoutOLIGO6␮M.Aftertreatmentmediumwasreplacedby freshone(FCS0.5%)andcellswereallowedtorestduring2h. Sam-plemedium(100␮l)wasprecipitatedwithperchloricacid(6%), neutralized(KOH20%),andglycine–hydrazine–EDTAbuffer(1M: 0.4M:1.3mM)containingLDH(RocheDiagnosticsGmbH)and␤ -NADHhydrate (Sigma-Aldrich) wasadded (Govindarajan etal., 2007).Lactatelevelswereestimatedat340nminaShimadzu spec-trophotometer.ProteincontentwasestimatedbyBradfordprotein assay(Sigma-Aldrich).

IntracellularATPdetermination

Intracellular ATP was measured using ATP Bioluminescence AssaykitHS II(RocheDiagnostics). Briefly,1.5×105 cells were platedin6-wellplates,treatedwithdryextractfromP.alliacea, DOG/metforminHCl (Sigma-Aldrich)orethanolduring6h, har-vested, counted, and lysedwithlysis buffer(50␮l) at 20◦_C _for 5min.Adilution(1:100)fromsampleorstandard(50␮l)was trans-ferredtoa96-wellplate,andluciferasereagent(50␮l)wasadded. Theemittedlightwasmeasuredimmediatelyandintegratedusing aPlateChameleonVModel425-156(Hidex)for10s.Blankvalue (noATP)wassubtractedfromeachsample’srawdataandATP con-centrationswerecalculatedfromthestandardcurveandexpressed as␮molper1×105cells.

Spheresnumberandareaestimation

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streptomycin (100␮g/ml). Cells were daily treated during 6 dayswithethanol(negativecontrol),dryextractfromP.alliacea (11␮g/ml),deoxyglucose(DOG,0.24mM)anddoxorubicin(DOX, 0.08␮M).After7days,sphereswerecountedbytwoindependent observersusinganopticalmicroscopeOlympus(10×).Cellculture mediumwas recovered,centrifuged(100×g)during 3min and spheres pelletsuspended in phosphate buffer saline(PBS) and placed onmicroscope slide. Spheres’ areawas measuredusing Axiovision®_software_(Carl_Zeiss).

Animals

Female BALB/c mice,6–12 weeks old were purchased from CharlesRiversLaboratoriesInternational,Inc.(Boston,MA), and housedinouranimalresearchfacilityfollowingtheestablished protocols of the Ethics Committee of the Science Faculty and National and International Legislation for Live Animal Experi-mentation(Colombia Republic, Resolution 8430/1993; National

AcademyofSciences, 2010).Micewerehousedinpolyethylene cageswithfoodandwateradlibitum,controlledtemperature,anda 12-hlight/darkcycle.Beforetreatments,themicewereacclimated foroneweekunderstandardconditions.Thisprojectwasapproved bytheEthicsCommitteeoftheScienceFacultyon29/04/2009.

Tumormodel

TS/Acells(1×104)suspendedin100␮lofPBSwereinjected intotherightmammaryfatpad(subcutaneously[SC])onday0 and then randomly assigned to PBS control group (n=9), DOX group (3mg/kg Al Pharma®_, _n₌₉₎ _or _P. _alliacea _extract _group (250mg/kg, n=8).After5 days ofinoculation, treatmentswere injectedintraperitoneally(i.p.)onceaweekforDOXandtwicea weekforP.alliaceaextractuntil56dayspost-inoculation.Tumors weremeasuredwithVerniercalipersthreetimesaweek,andtumor volumewascalculatedusingthefollowingformula:tumorvolume (mm3₎₌_[(width)2

×length]/2Gallotannin-richCaesalpiniaspinosa

100

80

60

40

20

0

0 25 50 75 100 125 150

P. alliacea dry extract (µg/ml)

Control

125 µg/ml 62.5 µg/ml P. allieacea

Control Doxorubicin

5 µM 2.5 µM

Cell line

4T1 HS578T TS/A HCT116 MCF12F 3T3

29.3 30.3 76.7 88.0

≅ 250

≅ 250 IC50 (µg/ml)

Cell death, %

175 200 225 250

A

B

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20

A

C

D

E

B

16

12

8

4

0

20

16

12

8

4

0

Control Oligo

∗∗ ∗∗

∗∗

∗∗∗

3h 6h 24h

P. alliacea extract 3 µg/ml

48h Control

nmol lactate/

µ

g protein/h

±

SEM

nmol lactate/

µ

g protein/h

±

SEM

nmol lactate/

µ

g protein/h

±

SEM

nmol lactate/

µ

g protein/h

±

SEM

nmol lactate/

µ

g protein/h

±

SEM

Oligo 3h 6h 24h

P. alliacea extract 3 µg/ml 48h

20

16

ns

12

8

4

0

20

16

12

8

4

0

20

16

12

8

4

0

Control Control Oligo Control Oligo P. alliacea

3 µg/ml P. alliacea extract 10 µg/ml

After wash 24h

After wash 48h

3h 6h 24h

Oligo 3h

P. alliacea 12 µg/ml

Fig.2. Petiveriaalliaceaextractdecreasesglycolyticfluxinbreastcancercelllines.(A)4T1,(B)HS578T,(C)MCF12F,(D)HCT116,(E)HS578T.Celllinesweretreatedwitha

P.alliaceadryextract(IC50/10)during3,6,24or48h.Aftertreatmentlactateconcentrationwasevaluatedbyenzymaticassay.Datarepresentthemean±S.E.M.ofatleast

threeindependentexperiments*p<0.05,**p<0.001,***p<0.0001comparedtocontrolusingStudent’sttest.

fractiondecreasestheprimarytumorandfactorsassociatedwith poorprognosisina murinebreast cancermodel (Urue˜naetal., 2013).Astudyofsurvivaldefiningtheendpointofeach individ-ualaccordingtothecriteriaof toxicityand animalwelfarewas conducted.TheanimalswereeuthanizedinaCO₂chamberwhen achieveoneormoreendpointcriteria.

Statisticalanalysis

Resultsareexpressedasmean±S.D.Foroxygenconsumption estimations and mammosphere analyses two-way ANOVAwas usedandunpairedttestforremaininganalyses.Survivalcurves obtainedbytheKaplan–Meiermethodwerestatisticallyanalyzed usingtheLog-ranktest.StatisticalanalysesweredoneusingGraph PadPrism5withap<0.05significance.

Results

Petiveriaalliaceadryextractismorecytotoxictobreastcancer celllineswhilesparingtofibroblastsandepithelialbreastcells

Dryextract from P.alliacea is cytotoxicto breast and colon tumorcell lines in a dose-dependent mannerwhile sparingto fibroblasts(3T3)and non-tumorigenicepithelial breastcell line (MCF12F)(Fig.1A).ThecorrespondingIC₅₀is30␮g/mlforhuman HS578Tandmurine4T1breastcancercells,77␮g/mlformurine TS/Abreastcancercelllineand88␮g/mlforcolontumorcellline HCT116(Fig.1A).Cytotoxicityobservedinbreastcancercelllines wasassociatedtomorphologicalchangeslikeincreaseofcellular volume,theappearanceofrefringentvesiclesanddetachmentfrom theculturesurface(Fig.1B).

Reductionintheglycolyticfluxwasobservedaftertreating4T1 andHS578Tcelllines(3and6h)withP.alliaceaextract,at sub-cytotoxicconcentrations–IC50/10th–(Fig.2,panelAandB).The extracteffectisearlyandtransientdisappearingafter24h, regard-lesswhethertheextractremainsornotinthecellculture(Fig.2, panelE).Noeffectisobserved onMCF12F orHCT116celllines (Fig.2,panelCandD,respectively)after24htreatment,neither intheexpressionofglycolyticenzymesasGAPDH,pyruvatekinase (PK)andLDH(datanotshown).Thelattersuggeststhattheextract compoundsmaybindanyglycolyticenzymeinareversibleway promotingthetransientdecreaseintheglycolyticflux.

Petiveriaalliaceadryextracttreatmentcausesdecreasein mitochondrialrespiration,ATPsynthaseexpressionand intracellularATPconcentrationonbreastcancercelllines

MitochondrialOxPhosproteinexpressionofNADH9(complex I), succinatedehydrogenase (complexII30kDairon–sulfur sub-unit),cytochromeb-c1subunit2(complexIIICoreIIsubunit)and ␤-F1-ATPase (complexV)weredetermined toassess P.alliacea extractactivity.Wefoundthattheexpressionof␤-F1-ATPase pro-teinwasaffectedbythetreatmentinbothbreastcancercelllines (Fig.3).

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J.F.Hernándezetal./RevistaBrasileiradeFarmacognosia27(2017)306–314 Control 1

A

B

30 48 52 52 42 30 39 48 52 52 42 SDH 3.5 3 2.5 2 1.5 1 0.5

Control 24 h

0

2 ₇ 20 12

10 8 6 4 2 0 15 10 5 0 6 5 4 3 2 1 0 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 3.5 3 2 1 0 3 2.5 2 1.5 1 0.5 Control Core II/α-tubulin

SDH/α-tubulin β-F1/β-actin

∗

24 h Control 24 h

Control 24 h Control 24 h

Control 24 h Control

NADH9/α-tubulin SDH/α-tubulin Core II/α-tubulin β-F1/β-actin

24 h 0 Core II β-F1-ATPse α-tubulin β-actin SDH NADH9 Core II β-F1-ATPse α-tubulin β-actin

2 3 1 2 3

P. alliacea extract (3 µg/ml)

Control

1 2 3 1 2 3

P. alliacea extract (3 µg/ml)

Fig.3. Petiveriaalliaceaextractdecreases␤-F1-ATPaseexpressioninbreastcancercelllines.(A)4T1and(B)HS578TcelllinestreatedwithaP.alliaceadryextractfor24h. NADH9,SDH,andCOREIIproteinexpressionwerealsodeterminedshowingnoeffectaftertreatment.RepresentativeWesternblotsandtheircorrespondinghistogramsare shownofthreeindependentpreparations(lanes1–3).Theleftsideofblotshowsproteinmolecularmassin(kDa).Blackbarsrepresentnormalizedbandswith␣-tubulinor

␤-actininarbitraryunits.Resultsarethemean±S.E.M.ofthreeindependentexperiments.*p<0.05comparedtocontrolusingStudent’sttest.

100 80 60 40 20 0 0 50 100 150

0 10 20

Rotenone Rotenone Antimycin Antimycin Control P. alliacea Control ∗ ∗∗ ∗∗ P. alliacea Oligomycin Oligomycin DNP DNP 30 Time (min) OCR (pmol/min/ µ g protein) ± SEM OCR (pmol/min/ µ g protein) ± SEM OCR (pmol/min/ µ g protein) ± SEM OCR (pmol/min/ µ g protein) ± SEM

40 50 60

0 10 20 30

Time (min)

40 50 60 BR MR OSR

BR MR OSR

150 0 20 40 60 80 100 100 50 0

Fig.4.Petiveriaalliaceaextracttreatmentreducesbasalrespiration,maximalrespirationandoxygenconsumptionrate(OCR)associatedtoATPproductionin4T1cells. After6h(upperpanel)and24htreatment(lowerpanel)theOCRwasmeasuredfollowingtheadditionoftheindicatedagents(left).Histogramsshowthecomparisonin basalrespiration(BR),maximalrespiration(MR)andoligomycinsensitiverespiration(OSR)betweencontrolandP.alliaceaextractcellstreated.Resultsareexpressedasthe mean±SEMoftwoindependentexperiments.*p<0.05,**p<0.001comparedtocontrolusingatwo-wayANOVA.

However,itmustbetakenintoaccountthattreatmentsignificantly lowers basal respiration explaining the no change in respira-tion.DNP,isaprotonionophorethatinduceselectrontransport chaintofunctionatitsmaximumratebycollapsingmitochondrial

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2.0

1.5

1.0

0.5

0.0

ETOH P. alliacea

(30 µg/ml)

P. alliacea

(3 µg/ml)

DOG (1.2mM)+ MET (0.5mM)

Treatment

∗∗∗ ∗∗ ∗∗

A

T

P [

µ

M]/1

×

10

5 cel

Fig.5. PetiveriaalliaceaextracttreatmentdecreasesintracellularATP.4T1cellswere treatedwithP.alliaceadryextractordeoxyglucoseplusmetformin(DOG+MET) during6h.ATPwasmeasuredbyabioluminescenceassay.ATPconcentrationis expressedas␮molper1×105cells.Thefigurerepresentsonefromthree

indepen-dentexperiments.Resultsareexpressedasthemean±S.D.**p<0.001,***p<0.0001 comparedtocontrolusingStudent’sttest.

expressionlevelorfunctionalityof␤-F1-ATPaseisaltered(Fig.4, upperpanel).Similarresultswereobtainedafter24htreatment (Fig.4,lowerpanel).

IntracellularATPlevelin4T1 cellswasmeasuredafterusing DOG/metformin (1.2mM/0.5mM) as a severe ATP synthesis inhibitor,whichcausesa5-foldreductioninintracellularATP.Our treatmentshoweda 2.5-folddecrease(Fig.5)in 4T1cells, con-firmingthatmitochondrialATPsynthesisisdecreasebyP.alliacea extracttreatment.

AdryextractfromPetiveriaalliaceadecreases4T1spheresand conventionalcellcultureproliferation

Anoutstandingmodelfordrugscreeningarethespheressince theyaremidwaybetweenconventionalculturesandinvivotumors (Pampaloniet al., 2007).Aftersix days of P.alliacea treatment atsub-cytotoxicconcentrations,asignificantdecreaseinspheres’ numbernearly74%and75%inareawasobserved(Fig.6AandB).A comparablebehaviorwasobservedwithDOGandDOXtreatments. Decreaseinviablecellsnumber(60%)wasobservedin4T1 conven-tionalcellcultureafter6daystreatmentwithDOGorP.alliacea extractatsub-cytotoxicconcentrations(Fig.7).

Petiveriaalliaceadryextractdecreasestheprimarytumorand promotessurvivalinaTS/Amurinebreastcancermodel

ToassesstheantitumoreffectofP.alliaceaextract,a murine modelofmetastaticbreastcancerwasused.Previously,an esti-matedlethaldose50 (LD50) of1545mg/kg for theextract was reported(Hernandezetal.,2014),thustherapeuticdoseevaluated was6-foldlowertoassurelowtoxicity.FemaleBALB/cmicewere inoculatedwithSCinjectionof1×104TS/Acells.Afterfivedays, thetumorswerepalpable,andthemiceweretreatedwithP. alli-aceaextract(250mg/kg,twiceaweek),vehicle(PBS)orpositive control(DOX, 3mg/kgonce a week).Fig.8A shows thatP. alli-aceaextractsignificantlyreducedtumorgrowthcompared with thecontrol,becomingincreasinglymarkedfromday32,atday42 thetumorachievesavolumeof881mm3_in_control_group com-pareto343mm3_in_P._alliacea_treatment._Likewise,_DOX_treatment reducestumorvolumeinasignificantlymannershowingavolume of87mm3_at_day₄₂_with_a_67%_of_animals_free_of_tumor._Due_to_a

15

A

B

C

800 000 ∗∗∗

∗∗∗ ∗∗∗ ∗∗∗

∗∗∗

600 000

400 000

200 000

0 10

5

0

Control DOG

Treatment

Spheres n

umber

±

SD

Area (

µ

m

2)±

SD

DOX

P. alliacea

Control P. alliacea (6 µg/ml) DOG (0.24 mM) DOX (0.08 µM)

Control DOG

Treatment

DOX

P. alliacea

Fig.6. Petiveriaalliaceaextracttreatmentdecreasesthespheresinnumberandarea.4T1singlecellscultureonultra-lowattachmentplatesweretreatedwithaP.alliacea

dryextract(IC50/5),deoxyglucose(DOG,0.24mM))ordoxorubicin(DOX,0.08␮M)duringsixdays.(A)Sphereswerecountedbytwoindependentobserversonday7thusing

anopticalmicroscopyOlympus(10×).(B)SpheresareawasdeterminedusinganopticalmicroscopyOlympus(10×)andimagesanalyzedwithsoftwareAxiovision®_._(C)

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500 000

400 000

300 000

200 000

100 000

0

ETOH ∗∗∗

∗∗∗ ∗∗∗

DOG Treatment

Number of viab

le cells

±

SD

DOX P. alliacea

Fig.7.ContinuoustreatmentwithPetiveriaalliaceadryextractusingsub-cytotoxic concentrationsdecreases4T1cellviability.4T1cellsweretreatedwithP.alliacea

dryextract(IC50/5),deoxyglucose(DOG,0.24mM))ordoxorubicin(DOX,0.08␮M)

duringsixdays.Atday7thviablecellswerecountedusingtrypanbluedye.Results areexpressedasmean±S.D.fromthreeindependentexperiments.***p<0.0001 comparedtocontrolusingatwo-wayANOVA.

markeddecreaseinthecontrolgrouppopulationbeforeday50th (77%),weevaluatethesurvivaloftreatmentsgroupsfollowingthe endpointcriteriaoftoxicityandanimalwelfareforeach individ-ual.AsshowninFig.8BP.alliaceaextractincreasessurvivalto48.5 dayscomparedto38daysin controlgroup(p=0.0055Log-rank test)whileDOXtreatmentimprovessurvivalto70dayscompared tocontrol(p=0.0004Log-ranktest).

1200

A

B

800

400

0

100

150

0

0 15 30 45 60 75 90

0 2 4 7 9 11 14 16 18 21

Time (days)

PBS

∗ ∗ ∗

∗∗∗

∗

∗ DOX (3 mg/kg) (n=9) P. alliacea (250 mg/kg) (n=8)

PBS (n=9) DOX (3 mg/kg) (n=9)

P. alliacea (250 mg/kg) (n=8)

Sur

viv

al, %

T

u

mor v

olume (mm

3)

23 25 28 30 32 35 37 39 42

Fig.8.TumorgrowthinhibitionandincreaseinsurvivalinBALB/cmicebyPetiveria alliaceaextract.BALB/cmiceimplantedSCwith1×104TS/Acellsforfivedaysand

randomlydividedintothreegroups.Group1wastreatedwithPBS(vehicle),group 2wastreatedwith3mg/kgofdoxorubicin,andgroup3wastreatedwith250mg/kg ofP.alliaceaextract.(A)Meantumorvolume.Thegraphrepresentsthemeantumor volume(mm3₎

±S.D.fromeachgroupwith8–9animalspergroup.***p<0.0001 comparedtocontrolusingStudent’sttest.(B)Kaplan–Meiersurvivalcurves rep-resenting%survivalofmicebearingsubcutaneousTS/Abreasttumors.Statistical analysiswasdoneusinglogranktest.**p<0.001,***p<0.0001comparedtocontrol.

Discussion

OurgroupaimstovalidateP.alliaceatraditionaluseforcancer treatment(Chirinos,1992;Gupta,1995;CorreaandBernal,1998). Previously,P.alliaceaextracthasbeenproventoinduce apopto-sisandtodecreasecolonycellgrowthin4T1cells(Urue˜naetal., 2008).Inaddition,hereinweshowedthatthedryextractfromP. alliaceaiscytotoxicinadose-dependentmannertohumanbreast (IC5030␮g/ml)andcolon(IC5088␮g/ml)cancercelllines. How-ever,theplantextractislesscytotoxictomurinefibroblastsand non-tumorigenicepithelialbreastcellline,whenculturedathigh xenobioticconcentration(Fig.1).Thecytotoxicityshowninbreast butnotcoloncancercellsisassociatedwiththedecreasein gly-colyticrate,giventhatthelactateproductionisreduced(Fig.2A). Although,wedidnotfindchangesinglycolyticenzymes expres-sion,thefluctuationsinratewereacuteandreversible.

Suolinnaetal.(1975)havedemonstratedthatflavonoidshaving hydroxylgroupsat3′_,₄′_,₇_either₃_or₅_positions,_like_fisetin, lute-olinorquercetindecreaseglycolyticrateinErhlichascitestumor cells. Such behaviorhasbeenexplained by thelossofNa+_–K+ -ATPaseactivitythatlowersintracellularADPandPi,requiredfor glycolyticratemaintenance(Suolinnaetal.,1975).Previouslywe haveshown,thatP.alliaceaextractcontainsflavonoidsasleridol, petiveraland4-ethylpetiveralwhichhavehydroxylgroupsat5,7 and6positionsrespectively,althoughmethoxylsubstituentsmay bepresentat5or7positions(Urue˜naetal.,2008).Thisunique flavonoidcombinationcouldberesponsibleforthelactate secre-tiondecreaseinbreastcancercelllines.

Herein, we have demonstrated a decrease in ␤-F1-ATPase expressionandmitochondrialrespiration(basalandafterOLIGO addition)inhumanand murinebreast cancercelllinesafter P. alliaceaextracttreatment(Figs.3and4).Also,changesin maxi-mumrespirationcanbeaccountedbytheeffectofP.alliaceaextract onrespiratorycomplexesexpression(Fig.3).Inmammaliancells ␤-F1-ATPaseexpressionisprimarilyregulatedbymechanisms con-trolledattheleveloftranslation(WillersandCuezva,2011).Hence, wesuggestthatP.alliaceaextractmightpartiallyinhibittranslation of␤-F1-ATPasemRNA.

Moreover, flavonoids like quercetin, kaempferol and morin affectmitochondrial ATPaseactivity(ZhengandRamirez,2000). Specifically,quercetinbindstothehydrophobicpocketbetween ␥-and␤TP-subunitbymeansofvanderWaalsforcesandH-bonds preventingtherotationofF1catalyticdomain(Gledhilletal.,2007). Asdiscussedabove,P.alliaceaextractcontainsseveralflavonoids havingaplanarconformationlikequercetin.Wehypothesizedthat P.alliaceaextractflavonoids behavein aquercetin-like manner suchastheonereportedbyZhengandRamirez(2000),bybinding toF1hydrophobicpocketsanddecreasingtherespirationrate.

Celldeathisaddressedtonecrosisorapoptosisdependingupon ATPlevels.StrongATPdepletion(>50%)isacommitmentto necro-siswhilehigherATPlevelsfavorsapoptosis(Leistetal.,1997).Also, wehaveestablisheda2.5-folddecreaseinATPlevelsassociatedto adeclineinglycolyticfluxandOCRin4T1cells(Fig.5).Previously, ourgrouphasreportedthatP.alliaceaextractinducesapoptotic celldeath(Hernandezetal.,2014)whereATPdepletioncouldbe implied.

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Glucose

Glucose 6P

PEP

Pyruvate

P. alliacea

extract

ADP ATP

TCA

OxPhos ADP

Breast cancer cell

Intracellular ATP

cell proliferation

ATP

Basal respiration

O₂ consumption

F1-ATP synthase Acetyl-CoA

Lactate Lactate

Fig.9. AmodelfortheproposedmetabolicmechanismofactionofthecytotoxicextractofPetiveriaalliacea.

Invivo assay showedthat treatmentwithP. alliaceaextract twiceaweekviai.p.withadoseequivalenttotheLD_50/6decreases the primary tumor growth and increases survival in the TS/A murinebreastcancermodel.TS/Aisahighlyheterogeneous mam-maryadenocarcinomaoriginatedspontaneouslyinaBALB/cmice thatdevelopsspontaneouslungmetastases andgenerates100% oftumordevelopmentafterinoculationof105_cells_via_SC₍_Nanni etal.,1983).OurresultsshowedthatP.alliaceaextractincreases significantlythemediansurvivalofmicewithaninteresting50%of populationfreeoftumoruntil42days,suggestingthatATP deple-tioncausedbyP.alliaceatreatmentcouldarrestthetumorgrowth atthebeginningphase,astagecharacterizedbyahighlyglycolytic andmitochondrialactivity.Overallwehypothesizethatcontinuous treatmentof cancercells withP.alliaceaextract decrease␤ -F1-ATPaseexpressionandglycolyticfluxtriggeringdiminishedATP levelsandfinallydecreasingcellproliferation.SoATPdepletionin breastcancercelllinescouldpartiallyexplainP.alliaceadryextract cytotoxicandantitumoralactivity.Aproposedmodeldisplaying themetabolismmechanismof actionoftheextract isshownin Fig.9.

Anticancerdrugsthatblockenergyproductionormimic low energyconditionrepresentanewclassofcancerdrugtherapy(Jose andRossignol,2013).Particularly,drugsaffectingmitochondrial complexes(I,IIandIV)likeVLX600anindol-1,2,4-triazinehave shownantitumoractivityincolontumorxenografts(Zhangetal., 2014).Similarly,acytotoxicaqueousextractfromScutellaria bar-batacontainingflavonoids,terpenesand alkaloidsreducesbasal respirationandglycolyticfluxinbreastcancercelllines(Chenetal., 2012).

Conclusions

HerewehavedemonstratedthatATPdepletionanddecreasein mitochondrialexpressionof␤-F1-ATPasecouldpartlyexplainthe P.alliaceadryextractcytotoxicactivity.Suchmechanismseems specifictoepithelialbreastcancercelllineshavingnoeffecton non-tumorigeniccounterparts.Currently,wearestudyingifATP depletionisonlyduetomitochondrialeffectsorNa+_–K+_-ATPase

changesareinvolvedinamechanismsimilartothedescribedby Suolinnaforhydroxylsubstitutedflavonoids.

Authorcontributions

SF,JMC, LFand JFH participate inthe studyconception and experimentsdesign.JFHandTASperformtheacquisitionofdata. JFH,CPU,MCCandTASparticipateindraftingofmanuscript.SF, MCCandJMCaccomplishthecriticalrevisionofmanuscript.All theauthorscontributetoanalysisandinterpretationofdata.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirwork centeronthe publicationof patientdata.

Right to privacy and informed consent.The authors have

obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Acknowledgments

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AdministrativodeCiencia,TecnologíaeInnovación“COLCIENCIAS” (120348925341)andPUJ(ID00004753)fortheirfinancialsupport. SomeexperimentsweresupportedbyMinisteriodeEconomíay Competitividad(SAF2013-41945-R),Spain.LFwasfinancially sup-portedbyAsociaciónEspa˜nolaContraelCáncer(AECC),Spain.PhD studentsJFHandTASwerefinanciallysupportedbyCOLCIENCIAS.

References

Acebo,P.,Giner,D.,Calvo,P.,Blanco-Rivero,A.,Ortega,Á.D.,Fernández,P.L., Ron-cador,G.,Fernández-Malavé, E., Chamorro,M., Cuezva,J.M., 2009. Cancer abolishesthetissuetype-specificdifferencesinthephenotypeofenergetic metabolism.Transl.Oncol.2,138–145.

Chen,V.,Staub,R.E.,Fong,S.,Tagliaferri,M.,Cohen,I.,Shtivelman,E.,2012.Bezielle selectivelytargetsmitochondriaofcancercellstoinhibitglycolysisandOXPHOS. PLoSONE7,e30300.

Chirinos,D.N.,1992.Elmilagrodelosvegetales:Petiveriaalliacea,3rd_ed._Bienes Lacónica,Caracas,Venezuela.

Ciavardelli,D.,Rossi,C.,Barcaroli,D.,Volpe,S.,Consalvo,A.,Zucchelli,M.,DeCola, A.,Scavo,E.,Carollo,R.,D’Agostino,D.,Forli,F.,D’Aguanno,S.,Todaro,M.,Stassi, G.,DiIlio,C.,DeLaurenzi,V.,Urbani,A.,2014.Breastcancerstemcellsrely onfermentativeglycolysisandaresensitiveto2-deoxyglucosetreatment.Cell DeathDis.5,e1336.

Cifuentes,C.,Casta˜neda,D.,Urue˜na,C.,Fiorentino,S.,2009.AfractionfromPetiveria alliaceainducesapoptosisviaamitochondriadependentpathwayandregulates Hsp70expression.UniversitasSci.14,125–134.

Correa,J.,Bernal,H.,1998.EspeciesVegetalesPromisoriasdePaisesdelconvenio AndrésBello,Bogotá,Colombia.

Cuezva,J.,Krajewska,M.,deHeredia,M.,Krajewski,S.,Santamaria,G.,Kim,H., Zapata,J.,Marusawa,H.,Chamorro,M.,Reed,J.,2002.Thebioenergeticsignature ofcancer:amarkeroftumorprogression.CancerRes.62,6674–6681.

Cuezva,J.M.,Ortega,A.,Willers,I.,Sanchez-Cenizo,L.,Aldea,M.,Sanchez-Arago, M.,2009.Thetumorsuppressorfunctionofmitochondria:translationintothe clinics.Biochim.Biophys.Acta1792,1145–1158.

Fantin,V.,St-Pierre,J.,Leder,P.,2006.AttenuationofLDH-Aexpressionuncovers alinkbetweenglycolysis,mitochondrialphysiology,andtumormaintenance. CancerCell9,425–434.

Formentini,L.,Martínez-Reyes,I.,Cuezva,J.,2010.Themitochondrialbioenergetic capacityofcarcinomas.IUBMBLife62,554–560.

Garcia-Barriga,H.,1974.FloraMedicinaldeColombia.InstitutodeCiencias Natu-rales.UniversidadNacional,Bogotá,Colombia.

Gledhill,J.,Montgomery,M.,Leslie, A.,Walker,J.,2007.Mechanismof inhibi-tionofbovineF1-ATPasebyresveratrolandrelatedpolyphenols.PNAS104, 13632–13637.

Gong,C.,Bauvy,C.,Tonelli,G.,Yue,W.,Delomenie,C.,Nicolas,V.,Zhu,Y.,Domergue, V.,Marin-Esteban,V.,Tharinger,H.,Delbos,L.,Gary-Gouy,H.,Morel,A.,Ghavami, S.,Song,E.,Codogno,P.,Mehrpour,M.,2013.Beclin1andautophagyarerequired forthetumorigenicityofbreastcancerstem-like/progenitorcells.Oncogene32, 2261–2272.

Govindarajan,B.,Sligh,J.,Vincent,B.,Li,M.,Canter,J.,Nickoloff,B.,Rodenburg,R., Smeitink,J.,Oberley,L.,Zhang,Y.,Slingerland,J.,Arnold,R.,Lambeth,J.,Cohen,C., Hilenski,L.,Griendling,K.,Martinez-Diez,M.,Cuezva,J.,Arbiser,J.,2007. Overex-pressionofAktconvertsradialgrowthmelanomatoverticalgrowthmelanoma. J.Clin.Invest.117,719–729.

Gupta,M.,1995.270PlantasmedicinalesIberoamericanas.ConvenioAndrésBello. ConvenioAndresBelloySubprogramaXdelCYTED,Bogotá,Colombia.

Hernandez,J.,Urue˜na,C.,Cifuentes,C.,Sandoval,T.,Pombo,L.,Castaneda,D.,Asea, A.,Fiorentino,S.,2014.APetiveriaalliaceastandardizedfractioninducesbreast adenocarcinomacelldeathbymodulatingglycolyticmetabolism.J. Ethnophar-macol.153,641–649.

Jose,C.,Rossignol,R.,2013.Rationaleformitochondria-targetingstrategiesincancer bioenergetictherapies.Int.J.Biochem.CellBiol.45,123–129.

Koukourakis,M.,Kontomanolis,E.,Giatromanolaki,A.,Sivridis,E.,Liberis,V.,2008.

SerumandtissueLDHlevelsinpatientswithbreast/gynaecologicalcancerand benigndiseases.Gynecol.Obstet.Invest.67,162–168.

Leist,M., Single, B.,Castoldi, A.S., Kuhnle,S., Nicotera, P., 1997. Intracellular adenosinetriphosphate(ATP)concentration:aswitchinthedecisionbetween apoptosisandnecrosis.J.Exp.Med.185,1481–1486.

Morales,C.,Gomez-Serranillos,M.,Iglesias,I.,Villar,A.,Caceres,A.,2001.Preliminary screeningoffiveethnomedicinalplantsofGuatemala.Farmaco56,523–526.

Nanni,P.,deGiovanni,C.,Lollini,P.,Nicoletti,G.,Prodi,G.,1983.TS/A:anew metas-tasizingcelllinefromaBALB/cspontaneousmammaryadenocarcinoma.Clin. Exp.Metastasis1,373–380.

Pampaloni,F.,Reynaud,E.,Stelzer,E.,2007.Thethirddimensionbridgesthegap betweencellcultureandlivetissue.Nat.Rev.Mol.CellBiol.8,839–845.

Papandreou, I., Goliasova, T., Denko, N., 2011. Anticancer drugs that tar-getmetabolism: isdichloroacetate thenewparadigm? Int.J. Cancer 128, 1001–1008.

Rossi,V.,1990.AntiproliferativeeffectsofPetiveriaalliaceaonseveraltumorallines. Pharmacol.Res.22,434.

Rossi,V.,Marini,S.,Jovicevic,L.,D’Atri,S.,Turri,M.,Giardina,B.,1993.Effects ofPetiveriaalliaceaL.oncellimmunity.Pharmacol.Res.27(Supplement1), 111–112.

Stock,D.,Leslie,A.,Walker,J.,1999.Moleculararchitectureoftherotarymotorin ATPsynthase.Science286,1700–1705.

Suolinna,E.,Buchsbaum,R.,Racker,E.,1975.Theeffectofflavonoidsonaerobic glycolysisandgrowthoftumorcells.CancerRes.35,1865–1872.

Urue˜na,C.,Cifuentes,C.,Castaneda,D.,Arango,A.,Kaur,P.,Asea,A.,Fiorentino, S., 2008. Petiveria alliacea extracts uses multiple mechanisms to inhibit growthofhumanandmousetumoralcells.BMCComplement.Altern.Med.8,

http://dx.doi.org/10.1186/1472-6882-8-60.

Urue˜na, C., Mancipe, J., Hernandez, J., Castaneda, D., Pombo, L., Gomez, Asea,A., Fiorentino,S., 2013. Gallotannin-richCaesalpinia spinosa fraction decreases the primary tumor and factors associated with poor progno-sisin amurinebreast cancermodel. BMC Complement.Altern.Med.13,

http://dx.doi.org/10.1186/1472-6882-13-74.

Willers,I.,Cuezva,J.,2011.Post-transcriptionalregulationofthemitochondrial H+_ATP_synthase:_a_key_regulator_of_the_metabolic_phenotype_in_cancer._Biochim. Biophys.Acta1807,543–551.

Zhang,X.,Fryknäs,M.,Hernlund,E.,Fayad,W.,DeMilito,A.,Olofsson,M.,Gogvadze, V.,Dang,L.,Påhlman,S.,Schughart,L.,2014.Inductionofmitochondrial dys-functionasastrategyfortargetingtumourcellsinmetabolicallycompromised microenvironments.Nat.Commun.5,http://dx.doi.org/10.1038/ncomms4295. Zheng, J., Ramirez, V., 2000. Inhibition of mitochondrial proton F0F1-ATPase/ATPsynthasebypolyphenolicphytochemicals.Br.J.Pharmacol.130, 1115–1123.