Contents lists available atScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a
Impact of chemotherapy on metabolic reprogramming:
Characterization of the metabolic profile of breast cancer MDA-MB-231 cells using 1 H HR-MAS NMR spectroscopy
Roberta M. Maria
a, Wanessa F. Altei
b, Heloisa S. Selistre-de-Araujo
b, Luiz A. Colnago
a,∗aEmbrapaInstrumentac¸ão,RuaXVdeNovembro,1452,SãoCarlos,SP,13560-970,Brazil
bLaboratóriodeBioquímicaeBiologiaMolecular,DepartamentodeCiênciasFisiológicas,UniversidadeFederaldeSãoCarlos(UFSCar),Rodovia WashingtonLuís,km235,CaixaPostal676,SãoCarlos,SP,13565-905,Brazil
a r t i c l e i n f o
Articlehistory:
Received19May2017
Receivedinrevisedform21August2017 Accepted28August2017
Keywords:
Breastcancer Anticancertreatment Metabolomics Pathways
Nuclearmagneticresonance
a b s t r a c t
Doxorubicin,cisplatin,andtamoxifenarepartofmanychemotherapeuticregimens.However,studies investigatingtheeffectofchemotherapyonthemetabolismofbreastcancercellsarestilllimited.We used1Hhigh-resolutionmagicanglespinning(HR-MAS)NMRspectroscopytostudythemetabolicprofile ofhumanbreastcancerMDA-MB-231cellseitheruntreated(control)ortreatedwithtamoxifen,cisplatin, anddoxorubicin.1HHR-MASNMRsinglepulsespectraevidencedsignalsfromallmobilecellcompounds, includingfattyacids(membranes),water-solubleproteins,andmetabolites.NMRspectrashowedthat phosphocholine(i.e.,abiomarkerofbreastcancermalignanttransformation)signalswerestrongerin controlthanintreatedcells,butsignificantlydecreasedupontreatmentwithtamoxifen/cisplatin.NMR spectraacquiredwithCarr-Purcell-Meiboom-Gill(CPMG)pulsesequencewereinterpretedonlyqual- itativelybecausesignalareaswereattenuatedaccordingtotheirtransverserelaxationtimes(T2).The CPMGmethodwasusedtoidentifysolublemetabolitessuchasorganicacids,aminoacids,cholineand derivatives,taurine,guanidineacetate,tyrosine,andphenylalanine.Thefattyacidvariationsobserved bysinglepulseaswellasthelactate,acetate,glycine,andphosphocholinevariationsobservedthrough CPMG1HHR-MASNMRhavepotentialtocharacterizebothresponderandnon-respondertumorsina molecularlevel.Additionally,weemphasizedthatcomparabletumors(i.e.,withthesameorigin,inthis casebreastcancer)mayrespondtotallydifferentlytochemotherapy.Ourobservationsreinforcethethe- orythatalterationsincellularmetabolismmaycontributetothedevelopmentofamalignantphenotype andcellresistance.
©2017ElsevierB.V.Allrightsreserved.
1. Introduction
Chemotherapyisthefundamentaltreatmentformostofthedis- seminatedcancers[1].Nevertheless,sometumorcellscansurvive andbecomeresistanttothetherapy[2].Drugresistancehasbeena considerablelimitationfortreatmentstosucceed[1].Fortunately, extensiveeffortshavebeenmadetowardstheunderstandingof resistancemechanisms,suchasoncogenicsignalingpathwaysthat resultfromgenomicinstabilityofcancercells[2].
Therearethousandsofalterationsinpointmutations,translo- cation,amplifications, and deletionsthatmaycontributetothe resistanceof cancercells todrugs, being the mutational range ableto diverge even among histopathologically similar tumors
∗Correspondingauthor.
E-mailaddress:[email protected](L.A.Colnago).
[3].Moreover,thesealterationsarevariedandappeartochange betweendistinctcelltypesanddifferenttimecourses,increasing thecomplexityofthetumorprocess[4].Itisbecomingperceptible, though,thatmanykeyoncogenicsignalingpathwaysconvergeto readjusttumorcellmetabolisminordertosupporttumorgrowth [3].Theseobservationssupportthetheorythatalterationsincellu- larmetabolismmaycontributetothedevelopmentofamalignant phenotype[5].Duetoitshighsensitivityandrapidresponseto changesintheenvironment,metabolismanalysisisoftenclaimed to best reflect the tumor phenotype [6]. The rapid metabolite changescausedbydiseasesortherapeuticagentsmaybeconsid- eredasadvantagesforunderstandingthemechanismsinvolvedin cellresponse[6].Shouldanticancerchemotherapiesbethecase, rapidmetabolicchangescanbeoftendetectedpriortoanyclinical symptoms,allowingthepreventionofsideeffectsoreventoswitch therapies[6].
http://dx.doi.org/10.1016/j.jpba.2017.08.038 0731-7085/©2017ElsevierB.V.Allrightsreserved.
Nowadays,studiesinvestigatinghowmetabolisminbreastcan- cerisaffectedbychemotherapyarestilllimited[7–10].Wehave previouslystudiedtheinfluence ofchemotherapyonmetabolic profile of the estrogen receptor-positive human breast cancer cell lineMCF-7 using 1H high-resolution magic angle spinning (HR-MAS)NMRspectroscopy[11].Thistechniqueconfirmedthat doxorubicin,cisplatin, and tamoxifen had strongeffects onthe metabolicprofileofMCF-7cells[11].
In this paper,1H HR-MAS NMR spectroscopywas alsoused to investigatethe effect of the anticancer drugs − particularly doxorubicin, cisplatin, and tamoxifen − on intact estrogen- negative human breast cancer MDA-MB-231 cells. These cells lacktheexpressionsof estrogen,progesterone,and humanepi- dermal growth factor receptor-2/neu (TNBC), which implies in totally hormone-independent, poorly-differentiated tumors [4].
The 1H HR-MAS NMR results presented heredemonstrate that themetabolicprofileofintactMDA-MB-231cellsismoreaffected bythese drugs than thatof MCF-7 cells. 1H HR-MAS NMR has beenshowntobeahighlyreproducibletoolinthefieldofcancer cellmetabolomics.Additionally,weemphasizedthatcomparable tumors(i.e.,withthesameorigin,inthiscasebreastcancer)may respondremarkablydifferentlytochemotherapy.Theseobserva- tionsstrengthenthetheorythatalterationsincellularmetabolism maycontributetothedevelopmentamalignantphenotypeandcell resistancetotherapy.
2. Materialsandmethods 2.1. Breastcancercellculture
MDA-MB-231–estrogenreceptor (ER)-negativehumanbreast adenocarcinomacellline− cellswereobtainedfromtheRiode JaneiroCellBank(BCRJ,Rio deJaneiro,RJ, Brazil).MDA-MB-231 tumorcellswerechosenbecausethesearepoorlydifferentiated invivoandleadtotheworstprognosis.MDA-MB-231cellswere growninRoswellParkMemorialInstitute(RPMI)medium(Thermo FisherScientificInc.,Waltham,MA,USA)supplementedwith10%of heat-inactivatedfetalbovineserum(FBS,ThermoFisherScientific Inc.).Cellswereseededatadensityof1.0×106cellsper75cm2of cellcultureflaskandmaintainedat37◦Cinahumidifiedincuba- torcontainingwith5%ofCO2for72h.Thisallowedcellstoattach totheflaskandbecomeconfluent.Themediumwasremovedand subcultureswereobtainedbytreatingcellswithtrypsininphos- phatebufferedsaline(PBS,SigmaAldrich)for2min.Subsequently, 4mLofculturemediumwasaddedandcellswerecentrifugedat 1.200rpmfor5mintoformpellets.Aftercentrifuging,10LofD2O wasaddedtoapproximately40LofMDA-MB-231pelletinPBS.
Thisprocedurewasaccomplishedintriplicate.Thecellviabilitywas testedbyTrypanBlueexclusionpriortothemeasurements.
2.2. Breastcancercelltreatment
MDA-MB-231 cells were cultured as described above. After confluence, cells weretreated withthedrugs (i.e.,doxorubicin, cisplatin,andtamoxifen),individually,for24heach.Theculture mediumwassupplementedwith3,1,and25Mofdoxorubicin, cisplatin, and tamoxifen, respectively. We relied upon previ- ouscytotoxicityassayswithdoxorubicin[12],cisplatin[13],and tamoxifen [12] to choose such drug concentrations. Cisplatin-, tamoxifen-,anddoxorubicin-treatedcellsweredetachedfromthe cultureflaskusingtrypsin.Oncecentrifuged,thepelletswereresus- pendedinD2OtoNMRanalysis.Thisprocedurewascarriedoutin triplicateforeachdrug.ThesamplespHwereapproximately7.2.
2.3. 1HHR-MASNMRanalyses
Each pellet was packed into a zirconium HR-MAS rotor containing10Lofdeuteriumoxideandsodiumtrimethylsilyl- [2,2,3,3-2H4]-1-propionate(TMSP). Thesystemwasadjusted to 0.00ppm whereas the spin was set to the magic angle (i.e., 54.7◦ relative to the magnetic field’s z direction). 1H-HR-MAS spectroscopy was carried out at 14.1T (600MHzfor 1H) and 5kHzofspinningrateusinganAVANCE600BRUKERNMRspec- trometer. The spectrawere acquiredwith a 1.5s presaturation pulse, an acquisition time of 4.63s (32K points), a 4s recycle delay, and an accumulation of 256 transients. Additionally, a Carr-Purcell-Meiboom-Gill(CPMG)spin-echotrainwasemployed before dataacquisition by means of applying120 cyclessepa- ratedby1.2msechoes.Thefreeinductiondecay(FID)signalwas multipliedbya1.0Hz(0.0025ppm)linebroadeningaswellasa two-foldzerofillingforFouriertransform.TheAdvancedChem- istryDevelopment(ACD)Labs softwarewasusedfor automatic phaseadjustmentandbaselinecorrection.Sampleswerealsoana- lyzed using two-dimensional (2D) NMR spectroscopy methods, such as COSY (Correlation spectroscopy), 1H–13C-HSQC (proton- carbonheteronuclearsingle-quantumcorrelationspectroscopy),and
1H–13C-HMBC (proton-carbon heteronuclear multiple bond cor- relation spectroscopy). 1H HR-MAS spectraof cancer cells were identified using correlated spectroscopy (2D homonuclear and heteronuclearNMR experiments,includingCOSY,1H–13C-HSQC, and1H–13C-HMBC).Onlinedatabases−e.g.,HumanMetabolome Database (HMDB) and Chenomx − were alsosuitable for such assignments.One-dimensional(1D)1HHR-MASspectrawerenor- malizedbycorrectingbaselineoffsettozeroaswellasdividingeach datapointbythesumofalldatapoints,beingtheresultingvalues multipliedby1000.
3. Results
Figs.1and2show1HHR-MASNMRspectraofhumanbreast cancerMDA-MB-231control cells(Con) andMDA-MB-231cells treated withtamoxifen (Tamo), cisplatin(cis), and doxorubicin (Doxo).BothsinglepulseandCPMG1HHR-MASNMRspectraof thethree culturesfor each treatment(triplicates)are shownin Supplementaryinformation.
Thespectrapresentedin thesefigureswereacquiredwitha singlepulse(Fig.1)andtheCPMGsequence(Fig.2).Singlepulse spectrashowthesignalsfromallmobilecompoundspresentin thecells,includingfattyacids(membranes)aswellassomewater- solubleproteinsandmetabolites.SpectraacquiredwiththeCPMG sequence(Fig.2)showonlythesharpsignalofthehighlymobile water-solublemetabolites.
TheCPMGsequencehasbeenusedasatransverserelaxation time(T2)filter(T2filter)sinceitattenuatesmoreefficientlybroad (shorter T2) rather than sharp (longer T2) NMR peaks. There- fore,allsignalspresentedinFig.2wereattenuatedaccordingto theirT2 valuesaswellasthetotal echotime usedintheCPMG sequence.Consequently,theCPMGspectra(Fig.2)cannotbeused inquantitativeanalyses, but onlyintheirqualitativeand semi- quantitativecounterparts(i.e.,onlylargedifferencesinpeakareas canbeattributedtovariationsinmetabolitecontents).
Figs.1A–Dand2A–Dpresent1HHR-MASNMRspectra,from0.5 to4.5ppm,ofCon(A),Tamo(B),Cis(C),andDoxo(D).Figs.1A‘–D‘
and2A‘–D‘show1HHR-MASNMRspectraofthesamesamples
−i.e.,Con(A‘),Tamo(B‘),Cis(C‘),andDoxo(D‘)−,butfrom5.0 to10.0ppm.TheverticalaxesofspectraA‘toD‘werefour-fold magnified.BothsinglepulseandCPMG1HHR-MASNMRspectra ofthethreeculturesforeachtreatment(triplicates)areshownin SupplementaryInformation.
Fig.1.Normalizedsinglepulse1HHR-MASNMRspectraofhumanbreastcancer MDA-MB-231cells.Controlcells(AandA‘)andcellstreatedwithtamoxifen(Band B‘)[12],cisplatin(CandC‘)[13],anddoxorubicin(DandD‘)[12].Theverticalaxes ofspectraA‘toD‘werefour-foldmagnified.Numbersindicatetheassignmentsto methyl[1]andmethylenegroups[2]offattyacidsandproteins,respectively;to alanine[3],acetate[4],phosphocholine[5],betaine[6],andguanidineacetate[7];
andtoolefinichydrogensofunsaturatedfattyacids[8],tyrosine[9],phenylalanine [10],andUTP/UDP/UMP[11].AtoDindicatespectrafrom0.5to4.5ppm,whereas A‘toD‘indicatespectrafrom5.0to10.0ppm.SeealsoFig.S1.
3.1. Spectraassignments
All spectra displayed in Fig. 1 showed strong, broad peaks overlappedwithsharp, weakones,thelatterbeingassigned to water-solublemetabolitessimilartothoseobservedinMCF-7cells [11]. The broad, strong peaks at 0.9 [1] and 1.3ppm [2] have beenassignedtomethylandmethylenegroupsoffattyacidsand proteins, respectively.These peaksalso comprisedsignals from metabolitesthat were better resolvedin Fig.2. The Con spec- trum(Fig. 1A)alsoexhibited strongpeaksat 3.22[5],3.26[6], and3.78ppm[7],whichhavebeenattributedtophosphocholine, betaine,and guanidinoacetate, respectively. The broad peak at 5.3ppm[8]hasbeenassignedtoolefinichydrogensofunsaturated fattyacids.
Fig.2showsCPMGspectrainwhichsixteenmetaboliteshave been assigned: lactate/threonine (1/2), alanine [3], acetate [4], glutamate/glutamine (5/6), acetone [7], creatine [8], choline [9], phosphocholine [10], taurine [11], glycine [12], guanidine acetate [13], tyrosine [14], phenylalanine [15], and uridine- triphosphate/uridine-diphosphate/uridine-monophosphate (UTP/UDP/UMP) [16]. These assignments have been published elsewhere[14,11].
Fig.2.1HHR-MASNMRspectra,acquiredwithCPMGpulsesequence,ofthefol- lowingbreastcancerMDA-MB-231cells:MDA-MB-231controlcells(AandA‘)and MDA-MB-231cellstreatedwithtamoxifen(BandB‘)[12],cisplatin(CandC‘)[13], anddoxorubicin(DandD‘)[12].TheverticalaxesofspectraA‘toD‘werefour-fold magnified.Numbersindicatepeaksattributedtolactate/threonine(1/2),alanine[3], acetate[4],glutamate/glutamine(5/6),acetone[7],creatine[8],choline[9],phos- phocholine[10],taurine[11],glycine[12],guanidineacetate[13],tyrosine[14], phenylalanine[15],andUTP/UDP/UMP[16].AtoDindicatespectrafrom0.5to 4.5ppm,whereasA‘toD‘indicatespectrafrom5.0to10.0ppm.SeealsoFig.S2.
3.2. ImpactofchemotherapyonMDA-MB-231cellmetabolism
Thespectrum(Fig.1B),whereinvirtuallyonlybroadpeaksare observed,evidencesastrongeffectoftamoxifenonthemetabolic profile.Thephosphocholinepeak[5],consideredasabiomarkerof breastcancermalignanttransformation,wasverysmallinTamo spectrum (see also Fig. S1B, Supplementary Information). The strong,sharppeakobservedat1.91ppm[4]hasbeenassignedto acetate(Fig.2B).
Cisspectrum(Fig.1C)alsoshowsthestrongeffectofchemother- apyonMDA-MB-231metabolicprofile.Phosphocholine[5] and betaine [6] peaks at approximately 3.25ppm were remarkably suppressedby Cis.The expansion of Cis spectrum from 5.0 to 10.0ppm(Fig.1C‘)revealedpeaksat6.8and7.2ppm[9];7.31,7.36, and7.41ppm[10];and7.94ppm[11].Theaforementionedpeaks havebeenassignedtotyrosine,phenylalanine,andUTP/UDP/UMP, respectively.Thestrongersharppeaksobservedat1.30/1.32,1.47, and3.56ppmhavebeenattributedtolactate/threonine(1/2),ala- nine[3],andglycine[12],respectively(Fig.2C).
ThemetabolicprofileofMDA-MB-231cellswasnotsignificantly changedbyDoxotreatment(Fig.1D).Themaindifferencewasthe phosphocholinepeakat3.22ppm[5],whichdecreasedininten- sity,whilethebetainepeakat3.25ppm[6]remainedpractically unchangedwhencomparedwithuntreatedcells(Fig.1A).Likein
Cistreatment − insmaller intensities,though −, thespectrum expandedfrom5.0to10.0ppm(Fig.1D‘)demonstratedpeaksat 6.8and7.2ppm[9];7.31,7.36,and7.41ppm[10];and7.94ppm [11], whichhave beenassigned to tyrosine,phenylalanine, and UTP/UDP/UMP,respectively. Thestrongersharp peaksobserved at1.30/1.32and3.22ppmwereasattributedtolactate/threonine (1/2)and phosphocholine[10], respectively (Fig.2D). However, other minor sharp peaks were observed, suchas that closeto 2.5ppmthatwasassignedtoglutamate/glutamine(5/6)(Fig.2D).
Creatine,taurine,andguanidineacetate(peaksat3.03,3.45,and 3.78ppm,respectively)alsopresentedreducedintensitiesinthe spectrumrecordedinDoxo-treatedMDA-MB-231cells.
The areas below single pulse 1H HR-MAS NMR spec- tra (Fig. 1) showed the following variations: stronger peak 3 in Cis>Doxo>Tamo∼Con as well as stronger peak 4 in Tamo>Cis∼Doxo∼Con.Tamoand Cisspectrashowedstronger peaks1and2thanthoseofConandDoxo(referalsotoFig.S1B-B‘
andC-C‘). Largerareascorrespondingtomethyl[1]andmethy- lene[2]indicatethatTamoandCiseitherinducedthesynthesis orreducedtheuptakeofsaturatedfattyacids.Therefore,thespec- trumofcontrol(Fig.1A)andDoxo-treatedcells(Fig.1D)showed thehighestofphosphocholine[5]contents.Thispeaksignificantly decreasedinTamoandCisspectra.
Thebetainepeak[6]wassimilarinConandDoxospectra,but substantiallydecreasedinTamoandCisspectra(Fig.1BandC).
Betaineisconsideredtobeamajorosmolyteincellsandplaysan essentialroleincellularprotectionagainstenvironmentalstress, includinghightemperatureandosmoticimbalance[15].Italsoreg- ulatescellvolumeandstabilizesproteins[16].Therefore,regardless ofthetherapeuticintervention,onlydoxorubicininducedastress conditioninMDA-MB-231cells.
Thepeakat3.78ppm,whichwasassignedtoguanidineacetate [7],decreasedafteralltreatments(Fig.1).Theareaattributedto olefinichydrogens[8]waslargerinCis>Tamo>Doxo>Con(see alsoSupplementaryInformation).Thespectraofdrug-treatedcells revealedhigherproteincontents(signalfrom6.5to10ppm)than those ofcontrol cells (Fig.1A‘).However,thespectracisplatin- treatedcells(Fig.1C‘)and,toalowerextent,ofdoxorubicin-treated cells(Fig.1D‘)indicated thepresence oftyrosine,phenylalanine, andUTP/UDP/UMP(peaks9–11,respectively).
TheareasunderCPMG1HHR-MASNMRsignals(Fig.2)assigned tolactate[1],threonine[2],alanine[3],acetate[4],taurine[11],and glycine[12]werefoundtobesmallerinuntreatedcells(Con)than intheirtreatedcounterparts.Phosphocholine[10]waspresentat higherconcentrationsinConandDoxo-treatedcells(Fig.2Aand D),whereasacetate[4]contentwashigherincells treatedwith Tamo(Fig.2B).Alanine[3]andglycine[12]contentswerehigher inCisspectra(Fig.2C).Theareaattributedtoglutamate/glutamine (5/6) was smaller in untreated cells (Con) than in the treated ones,butacetone[7]wasobservedinhighercontentsinuntreated cells(Con).Thespectraofdrug-treatedcellsevidencedhigherpro- tein contents(signal from6.5 to10ppm)than those ofcontrol cells(Fig.2A‘).Tyrosine,phenylalanine,andUTP/UDP/UMPpeaks (14–16,respectively),inparticular,weremoreintenseinCisspec- trum(Fig.2C‘)thaninTamo/Doxospectra.
4. Discussion
Identificationofspecificmetabolitesdisplayingalteredlevels aswellasof theirassociative metabolicpathwayscanimprove theunderstandingofbiologicalandpathologicalaspectsinvolved intheprocessfromnormaltoeventuallycancerousstate.Altered pathwaysincludechangesinketonebodies(acetone),glycolysis (lactate),andenergyandlipidmetabolisms.
UntreatedMDA-MB-231cellsshowedbroadpeaksrelatedto fattyacids,aspreviouslyobservedinMCF-7cells[11].Thehigher concentrationsoffattyacidsinuntreatedtumorcellsareinline withthehighactivityoffattyacidsynthaseintumortissue,which inturnisassociatedwithbothproliferationandmalignanttrans- formation[17].Essentially,fattyacidsynthaseactivitysupports cancergrowthbyincreasingthebuildingblocksforcellmembranes andlipid-containingmoleculesinvolvedincellsignaling[18].Asa result,thedenovosynthesisoffattyacidsintumorcellswould bepossible[19].Thebroadpeakassignedtoolefinichydrogensof unsaturatedfattyacidsbecamestrongerin treatedcells.Conse- quently,chemotherapiesarebelievedtosignificantlyinducefatty acidsynthesis.
Thetreatments,especiallythoseinvolvingcisplatinandtamox- ifen, significantly reduced phosphocholine content, compound which is consideredasa biomarkerof breast cancermalignant transformation[20].Thisindicatesthatdoxorubicinand,inparticu- lar,tamoxifenandcisplatinreduceditssynthesisinMDA-MB-231 cellsand,asaconsequence,cellinvasiveness.Chemotherapeutic interventionalsodecreasedphosphocholineintensitiesinMCF-7 cells[11],butthedecreasewassmallerthanthatinMDA-MB-231 cells.
Ketone bodies − including -hydroxybutyrate, acetoacetate, and acetone − are formed via -oxidation of fatty acids in mitochondria. Acetoacetate and hydroxybutyrate can originate acetyl-CoAandjointhetricarboxylicacid(TCA)cycle,providing therequiredenergyforcellularactivity[21].Thisprobablyexplains thedeprivationofthesemetabolitesintheacquiredspectra,since onlyacetonewasobservedinuntreatedMDA-MB-231cells.Thus, thehigheracetonecontentcouldbeanindicationofketogenesis inuntreatedcells.AcetonepeakwasimperceptibleinMCF-7cells [11].SinceMDA-MB-231cellshaveaninvasivebehavior,arepoorly differentiatedinvivo,andhaveaworseprognosisthanMCF-7cells, theformersareexpectedtorequiremoreenergyforcellularactivity thanMCF-7cells.
SimilarlytoMCF-7cells[11],lactatelevelsinuntreatedMDA- MB-231 cells were lower than in treated cells. This suggests enhancedglycolyticactivityafterchemotherapy.Becauselactateis theendproductofglycolysis,itsaccumulationimpliesanincreased anaerobic glycolysis. However, lactate area increased similarly upontreatmentsinMCF-7cells[11].Conversely,lactatecontent increased significantly only aftertreatments withcisplatin and doxorubicininMDA-MB-231cells.Tamoxifentreatmentdidnot changesuchpeaksignificantly.Astamoxifenisaselectiveestrogen receptor (ER) modulator and MDA-MB-231 cells were demon- stratedtobeER-negative,thetreatmentwithtamoxifenprobably didnotperturbtheanaerobicglycolysisinMDA-MB-231cellsasit didinMCF-7cells,whichareER-positive.
Theareasassignedtoglutamineandglutamatewereslightly largerinMDA-MB-231treatedcellsthaninuntreatedcells.Glu- tamineplaysanimportantroleinnucleotideandproteinsyntheses aswellasinmitochondrialenergymetabolism[22].Glutamateand glutaminedenoteagroupofglucogenicaminoacidsthatfeatures manybiologicalfunctions.Theseareconsideredtobeveryimpor- tantforcellfunctionmaintenanceandpromotion,beingglutamine animportantmetabolicprecursorinrapidly-dividingcells[23].Cell demandsforglutamineandglutamateareenormousduringsevere illness.Theirlevelsmayeitherdecreaseorincrease,suggestingthat thesemaybecomeconditionallyessentialaminoacidsincancer [24].
ThelargestcreatineareawasobservedinMDA-MB-231cells treated withdoxorubicin. Provided that creatine is widely rec- ognized asa keyintermediate in energymetabolism, increased creatineareaupondoxorubicintreatmentcanbeassociatedwith highenergydemand.
TheareaassignedtoglycineandalaninewashigherinMDA- MB-231cellstreatedwithcisplatin.Highglycineandalaninelevels hadalsobeenobservedpreviouslyinbreastcancertissues[22].The correlationinalanine/glycinelevelsisbecausealanineisinvolved inglycinesynthesisfrompyruvateandserine[22].Thisbiochemi- calpathwayisimportantsinceglycineisanimportantaminoacid involvedinthesynthesisofproteinsandnucleotides,elementsthat aredecisiveincancerdevelopment.
AcetatepeakwassmallerorabsentinallspectraofMCF-7cells [11].Similarly,suchwasnotpresentinuntreatedMDA-MB-231 cells.However,this peak becamestronger intamoxifen-treated MDA-MB-231 cells. As acetate is the end product of lipid metabolism,theseresultssupportthehypothesisthattamoxifen hasahigherinfluenceoverlipidmetabolismdisordersinMDA- MB-231cellsthaninMCF-7cells.
In summary, the NMR spectra acquired with CPMG pulse sequencewereusedonlyinaqualitativefashionbecausetheareas underthesignals wereattenuated by thetransverserelaxation time(T2).TheCPMGmethodwasusedtoidentifysolublemetabo- lites(e.g.,organicacids,aminoacids,phosphocholine,taurine,and guanidinoacetate). 1H HR-MAS NMR spectroscopy wasremark- ablyefficientindemonstratingtheeffectsoftamoxifen,cisplatin, anddoxorubicinonthemetabolicprofilesofMDA-MB-231cells, whichpresenteddifferencesfromthoseobservedintreatedMCF- 7cells.Wedemonstratedherethatvariationsinlactate,acetate, andphosphocholinecontents,indicatedbysinglepulseHR-MAS NMR spectroscopy,have potential tocharacterize both respon- derandnon-respondertumorsinamolecularlevel.Additionally, weemphasizedthatcomparabletumors−i.e.,withthesameori- gin,inthiscasebreastcancer−mayrespondtotallydifferentlyto chemotherapy.Theseobservationsreinforcethetheorythatalter- ationsincellularmetabolismmaycontributetothedevelopment ofamalignantphenotypeaswellascellresistance.
Conflictofinterest
Theauthorsdeclarethatthereisnoconflictofinterest.
Disclosureofpotentialconflictsofinterest Nopotentialconflictsofinterestweredisclosed.
Acknowledgements
We would like to acknowledge the financial support of the Brazilian agencies FAPESP (grants # 2013/05128-5and
#2013/00798-2)andCNPq(grant#303837/2013-6).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.jpba.2017.08.038.
References
[1]J.M.L.Roodhart,L.G.M.Daenen,E.C.A.Stigter,H.J.Prins,J.Gerrits,J.M.
Houthuijzen,M.G.Gerritsen,H.S.Schipper,M.J.G.Backer,M.vanAmersfoort, J.S.P.Vermaat,P.Moerer,K.Ishihara,E.Kalkhoven,J.H.Beijnen,P.W.B.
Derksen,R.H.Medema,A.C.Martens,A.B.Brenkman,E.E.Voest,Mesenchymal stemcellsinduceresistancetochemotherapythroughthereleaseof platinum-inducedfattyacids,CancerCell20(2011)370–383.
[2]K.Glunde,L.Jiang,S.A.Moestue,I.S.Gribbestad,MRSandMRSIguidancein molecularmedicine:targetingandmonitoringofcholineandglucose metabolismincancer,NMRBiomed.24(2011)673–690.
[3]R.Cairns,I.Harris,T.Mak,Regulationofcancercellmetabolism,Nat.Rev.
Cancer11(2011)85–95.
[4]H.Bando,M.Toi,K.Kitada,M.Koike,Genescommonlyupregulatedby hypoxiainhumanbreastcancercellsMCF-7andMDA-MB-231,Biomed.
Pharmacother.57(2003)333–340.
[5]R.G.Jones,C.B.Thompson,Tumorsuppressorsandcellmetabolism:arecipe forcancergrowth,GenesDev.23(2009)537–548.
[6]M.Palmnas,H.Vogel,ThefutureofNMRmetabolomicsincancertherapy:
towardspersonalizingtreatmentanddevelopingtargeteddrugs?Metabolites 3(2013)373–396.
[7]E.Furman,E.Rushkin,R.Margalit,P.Bendel,Tamoxifeninducedchangesin MCF7humanbreastcancer:invitroandinvivostudiesusingnuclear magneticresonancespectroscopyandimaging,J.Steroid43(1992)189–195.
[8]Z.Huang,Y.Tong,J.Wang,Y.Huang,Lipidsandthecisplatin-resistanceof A549/DDPcells,CancerCellInt.8(2003)1–8.
[9]F.Chiaradonna,R.M.Moresco,C.Airoldi,D.Gaglio,R.Palorini,F.Nicotra,C.
Messa,L.Alberghina,Fromcancermetabolismtonewbiomarkersanddrug targets,Biotechnol.Adv.30(2012)30–51.
[10]A.Shajahan-Haq,M.Cheema,R.Clarke,Applicationofmetabolomicsindrug resistantBreastcancerresearch,Metabolites5(2015)100–118.
[11]R.M.Maria,W.F.Altei,H.S.Selistre-de-Araújo,L.A.Colnago,Effectsof doxorubicin,cisplatin,andtamoxifenonthemetabolicprofileofhuman breastcancerMCF-7cellsasdeterminedby1HHR-MASNMR,Biochemistry (2017),http://dx.doi.org/10.1021/acs.biochem.7b00015.
[12]N.Abu,M.N.Akhtar,W.Y.Ho,S.K.Yeap,N.B.Alitheen,
3-Bromo-1-hydroxy-9,10-anthraquinone(BHAQ)inhibitsgrowthand migrationofthehumanbreastcancercelllinesMCF-7andMDA-MB231, Molecules18(2013)10367–10377.
[13]P.Tassone,P.Tagliaferri,A.Perricelli,S.Blotta,B.Quaresima,M.L.Martelli,A.
Goel,V.Barbieri,F.Costanzo,C.R.Boland,S.Venuta,BRCA1expression modulateschemosensitivityofBRCA1-defectiveHCC1937humanbreast cancercells,Br.J.Cancer88(2003)1285––1291.
[14]R.M.Maria,W.F.Altei,A.D.Andricopulo,A.B.Becceneri,M.R.Cominetti,T.
Venâncio,L.A.Colnago,Characterizationofmetabolicprofileofintact non-tumorandtumorbreastcellsbyhigh-resolutionmagicanglespinning nuclearmagneticresonancespectroscopy,Anal.Biochem.488(2015)14–18.
[15]I.Lee,Betaineisapositiveregulatorofmitochondrialrespiration,Biochem.
Biophys.Res.Commun.456(2015)621–625.
[16]R.Obeid,Themetabolicburdenofmethyldonordeficiencywithfocusonthe betainehomocysteinemethyltransferasepathway,Nutrients5(2013) 3481–3495.
[17]L.Z.Milgraum,L.A.Witters,G.R.Pasternack,F.P.Kuhadja,Enzymesofthefatty acidsynthesispathwayarehighlyexpressedinSitubreastcarcinoma,Clin.
CancerRes.3(1997)2115–2120.
[18]M.Buzzai,D.E.Bauer,R.G.Jones,R.J.DeBerardinis,G.Hatzivassiliou,R.L.
Elstrom,C.B.Thompson,TheglucosedependenceofAkt-transformedcells canbereversedbypharmacologicactivationoffattyacid-oxidation, Oncogene24(2005)4165–4173.
[19]O.Beckonert,J.Monnerjahn,U.Bonk,D.Leibfritz,Visualizingmetabolic changesinbreast-cancertissueusing1H-NMRspectroscopyand self-organizingmaps,NMRBiomed.16(2003)1–11.
[20]G.Eliyahu,T.Kreizman,H.Degani,Phosphocholineasabiomarkerofbreast cancer:molecularandbiochemicalstudies,Int.J.Cancer120(2007) 1721–1730.
[21]J.C.Newman,E.Verdin,Ketonebodiesassignalingmetabolites,Trends Endocrinol.Metab.25(2014)42–52.
[22]M.D.Cao,S.Lamichhane,S.Lundgren,A.Bofin,H.Fjøsne,G.F.Giskeødegård, T.F.Bathen,Metaboliccharacterizationoftriplenegativebreastcancer,BMC Cancer14(2014)941.
[23]A.L.Peterson,A.Walker,E.K.Sloan,D.J.Creek,Optimizedmethodfor untargetedmetabolomicsanalysisofMDA-MB−231,BreastCancerCells (2016).
[24]X.Zhang,L.Xu,J.Shen,B.Cao,T.Cheng,T.Zhao,X.Liu,H.Zhang,Metabolic signaturesofesophagealcancer:NMR-basedmetabolomicsandUHPLC-based focusedmetabolomicsofbloodserum,Biochim.Biophys.Acta—Mol.BasisDis.
1832(2013)1207–1216.
