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epdcx:valueString Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol
http:purl.orgdctermsabstract
Abstract
Background
Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.
Results
Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.
Conclusion
LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
http:purl.orgdcelements1.1creator
Zhou, Ming
Zhao, Yuhua
Ding, Yan
Liu, Hao
Liu, Zixing
Fodstad, Oystein
Riker, Adam I
Kamarajugadda, Sushama
Lu, Jianrong
Owen, Laurie B
Ledoux, Susan P
Tan, Ming
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BioMed Central Ltd
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Ming Zhou et al.; licensee BioMed Central Ltd.
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Molecular Cancer. 2010 Feb 09;9(1):33
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Supplementary Figure S3 Combination of Taxol with o xamate shows synergistic inhibitory effects in Taxol resistant cells by direct cell cou nting



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Supplementary Figure S4 Taxol in combination with o xamate treatment shows better inhibition of MCF7 cells



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Supplementary F tion statues of Cdc2 at Tyrosine igu a re S5 The expression of Bcl-2, Bcl-XL, Cdc2 and phosphoryl 15



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RESEARCHOpenAccess Warburgeffectinchemosensitivity:Targeting lactatedehydrogenase-Are-sensitizes Taxol-resistantcancercellstoTaxol MingZhou 1,3 † ,YuhuaZhao 1 † ,YanDing 1 † ,HaoLiu 1 ,ZixingLiu 1 ,OysteinFodstad 1,4 ,AdamIRiker 1,6 SushamaKamarajugadda 5 ,JianrongLu 5 ,LaurieBOwen 1 ,SusanPLedoux 2 ,MingTan 1,2* Abstract Background: Taxolisoneofthemosteffectivechemotherapeuticagentsforthetreatmentofpatientswithbreast cancer.Despiteimpressiveclinicalresponsesinitially,themajorityofpatientseventuallydevelopresistancetoTaxol. Lactatedehydrogenase-A(LDH-A)isoneofthepredominantisoformsofLDHexpressedinbreasttissue,which controlstheconversionofpyruvatetolactateandplaysanimportantroleinglucosemetabolism.Inthisstudywe investigatedtheroleofLDH-AinmediatingTaxolresistanceinhumanbreastcancercells. Results: Taxol-resistantsubclones,derivedfromthecancercelllineMDA-MB-435,sustainedcontinuousgrowthin highconcentrationsofTaxolwhiletheTaxol-sensitivecellscouldnot.Theincreasedexpressionandactivityof LDH-AweredetectedinTaxol-resistantcellswhencomparedwiththeirparentalcells.ThedownregulationofLDHAbysiRNAsignificantlyincreasedthesensitivityofTaxol-resistantcellstoTaxol.Ahighersensitivitytothespecific LDHinhibitor,oxamate,wasfoundintheTaxol-resistantcells.Furthermore,treatingcellswiththecombinationof TaxolandoxamateshowedasynergisticalinhibitoryeffectonTaxol-resistantbreastcancercellsbypromoting apoptosisinthesecells. Conclusion: LDH-AplaysanimportantroleinTaxolresistanceandinhibitionofLDH-Are-sensitizesTaxol-resistant cellstoTaxol.ThissupportsthatWarburgeffectisapropertyofTaxolresistantcancercellsandmayplayan importantroleinthedevelopmentofTaxolresistance.Toourknowledge,thisisthefirstreportshowingthatthe increasedexpressionofLDH-AplaysanimportantroleinTaxolresistanceofhumanbreastcancercells.Thisstudy providesvaluableinformationforthefuturedevelopmentanduseoftargetedtherapies,suchasoxamate,forthe treatmentofpatientswithTaxol-resistantbreastcancer. Background Taxol(paclitaxel)hasrecentlyemergedasanimportant agentinthetreatmentofhumanbreastcanceraswell asothertumorhistologies,suchasovarian,prostateand non-smallcelllungcancers[1,2].Theprimarycellular targetsofTaxolarethemicrotubulesofcancercells, whichisvitalformitoticactivity,cellularmotilityand proliferativecapacity.Taxolstabilizesthemicrotubule structurebydisruptingthedynamicequilibrium betweensolubletubulindimersandtheirpolymerized form.Itisalsoapotentinhibitorofchromosomal replicationbyblockingcellsinthelateG2ormitotic phasesofthecellcycle[3].Theresistanceofcancer cellstoTaxolandotherchemotherapeuticagentsis knowntoresultinthesubsequentrecurrenceand metastasisofcancer[4,5].Oneknownmechanism involvedwithcancercellresistancetoTaxolandother microtubule-stabilizingagentsisthehigh-expressionof themembraneP-glycoproteinthatfunctionsasadrugeffluxpump[6].Othercellularmechanismsincludethe alterationsoftubulinstructure[7-9],changesinthe drug-bindingaffinityofth emicrotubules[10]andcell cyclederegulation[11,12].However,thedetailedmolecularmechanismsthatmaycontributetoTaxolresistanceofcancercellsarestillnotfullyunderstood. *Correspondence:mtan@usouthal.edu † Contributedequally 1 MitchellCancerInstitute,UniversityofSouthAlabama,Mobile,Alabama, USA Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 2010Zhouetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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Cancercells,unliketheirnormalcounterparts,use aerobicglycolysiswithreduc edmitochondrialoxidative phosphorylationforglucosemetabolism.Thispersistence ofhighlactateproductionbycancercellsinthepresence ofoxygen,knownasaerobicglycolysis,wasfirstnotedby OttoWarburgmorethan75yearsago[13-15].Itwas recognizedthatsincecancercellshaveincreasedcell growthandenergyneedstosustaincellproliferation,elevatedglycolyticactivityinsuresthatadequateATPlevels areavailabletomeetthedemandsofrapidlyproliferating tumorcellswithinahypoxicmicroenvironment[16]. Additionally,Taxol-resistantcancercellsmayescapethe therapeuticeffectsofTaxolvi atheeffluxtransportsystemspresentwithintumorcells.However,drugefflux andmetabolismconsumeslargeamountsofATPthatis generatedviaglycolysis,protectingcellsfromthelethal effectsofTaxolbysustainingtheenergyneededforcellulardrugeffluxandmetabolism.Thus,theenergydistributionconsumedinTaxol-resistantcellsmustbe dramaticallyalteredinordertoaccommodateforboth cellviabilityandlong-termsurvival. Lactatedehydrogenase-A(LDH-A)isoneofthemain isoformsofLDHexpressedinb reasttissue,controlling theconversionofpyruvatetolactateofthecellularglycolyticprocess[17].IthasbeenshownthatLDH-A playsakeyroleinglycolysis,growthpropertiesand tumormaintenanceofbreastcancercells[16,18].To understandthecellularmechanismsinvolvedinthe resistanceofbreastcancer cellstoTaxol,weinvestigatedontheassociationofLDH-AandTaxolresistance inbreastcancercellsandtheroleofLDH-Aintumor therapeuticsanddrugsensitivity.Ourresultsshowthat comparedwiththeirparentalcells,theincreasedexpressionandactivityofLDH-AinTaxol-resistantcells directlycorrelatewiththeirsensitivitytoglycolysisinhibitoroxamate.Furthermore,geneexpressionknockdownexperimentswithsiRNAspecificforLDH-Ashow anincreasedsensitivityofthesecellstoTaxol.Inaddition,treatmentofbreastcancercellswiththecombinationofTaxolwithoxamate,r evealsansynergistically inhibitoryeffectuponcellviability.Takentogether, LDH-AplaysanimportantroleinTaxolresistanceof breastcancercells,servingasapromisingtherapeutic targetforovercomingTaxolresistance.Furthermore, thedataareconsistentwiththeroleofLDH-Aasan essentialtumormaintenancegene,providingfurther insightintothecellularandmolecularmechanisms involvedinTaxol-resistantbreastcancer.MethodsCellsandcellcultureBreastcancercellsMDA-MB -435(MDA-435),MDAMB-231(MDA-231),MCF7andBT474werepurchased fromAmericanTypeCultureCollection(ATCC). 435TR1and435TRPcellsareTaxol-resistantsingle cloneorpooledclones,whichweredevelopedfromparentalMDA-435cellsbytreatedwithgraduallyincreasingconcentrationsofTaxolincellculturemedium. MDA-231celllinewithstableknockdownofLDH-A wasconstructedthroughtransfectionofMSCV-based retroviralvector(MSCV/LTRmiR30-PIG).Allofthese cellswereculturedinDMEM/F-12(MediatechInc.)and supplementedwith10%FBSandPenicillin/ Streptomycin.MorphologicalobservationofTaxol-resistantcellsThecellswereseededin6-wellplatesat3105cells perwellinduplicate.After12hrincubation,cellswere treatedwithorwithout20nMTaxolfor24hrs,with untreatedcellsservingascontrols.Thecellswere washedtwicewithPBSandthenfixedwithmethanol/ acetone(1:1),subsequentlystainedwith4 ’ ,6-diamidino2-phenylindole(DAPI)inordertovisualizethemorphologyofcellnucleus.Themorphologyofcellswas observedwiththefluorescencemicroscope.CellapoptosisassayThecancercellsweretreatedwith20nMTaxolfor48 hrs.Twomethodswereusedtodetectapoptosis.1)The earlystageofapoptosiswasdetectedbyAnnexinV/propidiumiodidestainingwiththeApoptosisDetectionKit (BDPharMingen).Briefly,aliquotsof105Taxol-treated cellswereincubatedwithAn nexinV/propidiumiodide for15minatroomtemperature.Thecellswerethen analyzedbyflowcytometry(BDLSRII).2)Thelate stageofapoptosiswasdetectedbyCellDeathDetection ELISAPLUSkit(Roche)accordingtothemanufacturer ’ sinstruction.WesternblottingCellswereharvestedandlysedinabuffercontaining50 mMTris-HCl,pH7.5,150mMNaCl,2mMEDTA,1% Triton,1mMPMSFandProteaseInhibitorCocktail (Sigma)for20minonice.Lysateswereclearedbycentrifugationat14,000rpmat4Cfor10min.Supernatantswerecollectedandproteinconcentrationswere determinedbytheBradfordassay(Bio-rad).TheproteinswerethenseparatedwithaSDS/polyacrylamidegel andtransferredtoaNitrocellulosemembrane(Bio-rad). AfterblockinginPBSwith5%non-fatdrymilkfor1hr, themembraneswereincubatedovernightat4-8Cwith theprimaryantibodiesinPBSwith5%non-fatdrymilk. Thefollowingantibodieswereutilized:anti-LDHArabbitantibody(1:1000,CellSignaling);anti-PARPrabbit antibody(1:1000,CellSignaling),anti-cleavedPARP Rabbitantibody(1:1000,CellSignaling),anti-Bcl2rabbit antibody(1:1000,CellSignaling),anti-Bcl-XLrabbit monoclonalantibody(1:1000,CellSignaling),anti-Cdc2Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page2of12

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mousemonoclonalantibody( 1:1000,CellSignaling),, anti-p-Cdc2(Y15)rabbitmo noclonalantibody(1:1000, CellSignaling),andantib -actinmonoclonalantibody (1:2000,Sigma).Membraneswereextensivelywashed withPBSandincubatedwithhorseradishperoxidase conjugatedsecondaryanti-mouseantibodyoranti-rabbit antibody(1:2,000,Bio-rad).Afteradditionalwasheswith PBS,antigen-antibodycompl exeswerevisualizedwith theenhancedchemiluminescencekit(Pierce).DetectionofLDHActivityThetotalLDHactivityincelllysateswasexamined accordingtothemanufacturer ’ sinstructionsofthe LDH-cytotoxicityassaykit(BioVision).Briefly,2105cellswereseededina24-wellplateonedaybefore assayingandallsampleswereanalyzedintriplicate. Thencellswerecollected,washedandextractedforproteintomeasureLDHactivity.Resultswerenormalized basedupontotalprotein.siRNAExperimentssiRNAoligonucleotidesforLDH-Awaspurchasedfrom Sigma,withascrambledsiRNA(Sigma)usedasacontrol.Transfectionwasperf ormedusingtheOligofectamineTransfectionreagent(Invitrogen)accordingtothe manufacturer ’ sprotocol.Forty-eighthoursaftertransfection,whole-celllysateswerepreparedforfurtheranalysisbyWesternblot,LDHactivityandTaxol cytotoxicityassay.CellViabilityAssayAtotalof5103~1104cells/wellwereseededin 96-wellplates.Twenty-fourhourslater,themediumwas replacedwithfreshmediumwithorwithoutTaxoland incubatedfor24or48hrs,respectively.Taxolincombinationwithvariousconcentrationsofoxamatewerealso usedtotreatthecellsinordertoinvestigatetheeffect ofdrugcombinations.Cellv iabilitywasdeterminedby twomethods.1)UsingCellTiter96AqueousOneSolutionCellProliferationAssay(Promega)accordingtothe manufacturer ’ sprotocol;2)byTypanBluestainingand directcellcountingusinghematocytometer.StatisticalanalysisTheunpairedStudent ’ s t -testwasusedforthedataanalysis.Alldatawereshownasmeanstandarderror (SE).Astatisticaldifferenceof P <0.05wasconsidered significant.ResultsSelectionandcharacterizationofTaxol-resistantcancer cellsMDA-435cellsweretreatedwithgraduallyincreasing concentrationsofTaxolincellculturemediumfor selectionofTaxol-resistantcells.AftersuccessiveTaxol treatmentsfordurationof3months,severalresistant cellclonesweredevelopedfromtheMDA-435cellline. Taxol-resistantclone1(43 5TR1)andTaxol-resistant pooledclones(435TRP)wereusedforallsubsequent experimentsinthisstudy. TocomparethesurvivalcapacityofbothTaxol-sensitiveandTaxol-resistantcells,MDA-435,435TR1and 435TRPcellsweretreatedwith20nMTaxolfor24hrs. Taxol-sensitiveMDA-435cellsshowedcellrounding andblebbingwithemptyspacesvisualizedwithinthe cells.Thissuggestedthatalargeportionofthesecells werearrestedinG2/Mphase,withsomeofthesecells undergoingapoptosis.However,noobviousmorphologicalchangewasobservedinTaxol-resistant435TR1and 435TRPcells(Fig.1A).Earlystageapoptosiswasexaminedbyflowcytometryanalysisafterstainingwith AnnexinV/propidiumiodide,andlatestageapoptosis wasdetectedbyaCellDeathDetectionELISAPLUS kit,whichexaminestheDNAfragmentationinthe apoptoticcells.BothassaysdetectedasmallerpercentageofapoptoticcellsinTaxol-resistant435TR1and 435TRP,comparedtotheirparentalMDA-435cells aftertreatmentwith20nMTaxolfor48hrs(Fig.1B). TheproteinexpressionofthecleavedPoly(ADP-ribose) polymerase(c-PARP),animportantmarkerofcaspasemediatedapoptosis[19,20],wasalsoexaminedbyWesternblottingafterthecellsweretreatedwith20nM Taxolfor48hrs.Wefoundmuchlowerlevelsof cleavedPARPandcorrespondinglymuchhigherlevels ofun-cleavedPARPinTaxol-resistant435TR1and 435TRPcells,comparedtoparentalMDA-435cells(Fig. 1C).Cellviabilityassayshowedthat435TR1and 435TRPcellscouldtoleratemuchhigherconcentrations ofTaxolcomparedtoMDA-435cells,withtheirIC50 concentrationsfoundtobemorethan30-foldhigher thanthoseofMDA-435cells(Fig.1D).IncreasedexpressionandactivityofLDH-AinTaxolresistantcellsToexaminetheroleofLDH-AinmediatingTaxol resistanceinhumanbreast cancercells,theexpression ofLDH-AwasexaminedinMDA-435,435TR1and 435TRPcells.WefoundthatLDH-Alevelsweremarkedlyincreasedin435TR1and435TRPcells,compared totheirparentalMDA-435cells(Fig.2A).Theactivity ofLDHwasalsoincreasedabout2-foldinTaxol-resistant435TR1and435TRPcells,comparedtoMDA-435 cells(Fig.2B).Theseresultsi ndicatedthatTaxolresistanceiscorrelatedwiththeincreasedLDH-Aexpression andactivity.Interestingly,treatmentwithTaxolresulted intheinductionofLDH-Aexpressioninadose-dependentpatterninMDA-435cells(Fig.2C).WealsoidentifiedthatLDHactivitycouldalsobeinducedbyTaxolinZhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page3of12

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Figure1 CharacterizationofTaxol-resistantcells .A,MDA-435,435TR1and435TRPcellsweretreatedwith20nMTaxolfor24hrsandtheir morphologywasobservedunderfluorescencemicroscope.Thephaseimageofthesecellswasshownatthetopandthenucleusstainedby DAPIwasshownatthebottom(200).B,MDA-435,435TR1and435TRPcellsweretreatedwith20nMTaxolfor48hrsandapoptosiswas examinedbyflowcytometryusingAnnexinV/PIstainingandbyCellDeathDetectionELISAPLUSKit.Foldinductionvaluewascalculated followingtheformula:mUofthesample(cellstreatedwithTaxol)/mUofthecorrespondingnegativecontrol(cellswithoutTaxoltreatment).C, Taxol-resistantcellsandtheirparentalcellsweretreatedwithoutorwith20nMTaxolfor48hrs,thenpoly(ADP-ribose)polymerase(PARP)and itscleavedprotein(c-PARP)wereanalyzedbyWesternblottingwithspecificantibodies,respectively. b -actinwasusedasaloadingcontrol.D, CellviabilityanalysiswasperformedtoevaluatecytotoxicityofTaxoltoMDA-435andTaxol-resistant435TR1and435TRPcellsundertreatment withindicatedconcentrationsofTaxolfor48hrs. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page4of12

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theTaxol-resistantcells(datanotshown).Tostudythe mechanismthatmaycontributetotheincreasedexpressionandactivityofLDH-A,MDA-435cellsweretreated withCHXtoblockproteinsynthesisandthecellswere furthertreatedwithorwithoutTaxolfordifferenttimes, theproteinstabilityofLDH-AwasmeasuredbyWesternblot(Fig.2D).TheresultshowedthatLDH-AproteinismorestableinTaxoltreatedcellsthanthatof untreatedcells.Wefurth ercomparedthemRNAlevel ofLDH-AinTaxol-treatedand-untreatedcellsbyqRTPCR(Fig.2E).TheresultshowedthatTaxoltreatment increasedthemRNAexpressionofLDH-A.These resultssuggestthatbothpr oteinstabilityandmRNA inductionbyTaxolcontributetotheup-regulationof LDH-Ainthesecells.ThedownregulationofLDH-Are-sensitizesTaxol-resistant cellstoTaxolTheincreaseofLDH-AexpressionandLDHactivity detectedinTaxol-resistan tcellssuggeststhatLDH-A mayplayacriticalroleinTaxolresistance.Therefore, theeffectofLDH-Adownregulationonthesensitivity ofTaxolwasinvestigated.AfterLDH-AwasdownregulatedefficientlybyspecificsiRNAtoLDH-A(Fig.3A), LDHactivitywasdecreasedabout40%inMDA-435 cellsandabout55%in435TR1cells(Fig.3B).Sincethe expressionandactivityofLDH-Awasupregulatedin Taxol-resistantcancercells(Fig.2),wehypothesized thatthedownregulationofLDH-AbysiRNAmightresensitizeTaxol-resistantcellstoTaxol.Tothisend, LDH-AwasknockeddownwithsiRNAin435TR1and parentalMDA-435cellsrespectively,andthenthecells weretreatedwithdifferentconcentrationsofTaxol.The downregulationofLDH-Aincreasedthesensitivityof thesecellstoTaxol,withTaxol-resistant435TR1cells showingabouta3-10foldincreaseincellgrowthinhibitionunder50-100nMTaxoltreatmentmeasuredby bothMTSassay(Fig.3C)anddirectcellcounting (Additionalfile1,Figure.S1).Interestingly,435TR1cells showedamuchgreateroverallincreasedsensitivityto TaxolcomparedtotheirparentalMDA-435cells(Fig. 3Cand3D).Similarassayswereperformedinanother breastcancercelllineBT474(Fig.4A-C),wherethe knockdownofLDH-AexpressionbysiRNAincreased thesensitivitytoTaxolbyatleast2-fold.Tofurther confirmtheseresults,MDA-231cellswithstableknockdownofLDH-Abyshort-hairpinRNA(shRNA)were used.Comparedtothoseof controlMDA-231cells, LDH-Aexpression(Fig.4D)andLDHactivity(Fig.4E) weredramaticallydecreasedinLDH-AstablyknockdowncellsandthesecellsshowedamuchgreateroverallincreasedsensitivitytoTaxol(Fig.4F).Theseresults demonstratedthatLDH-Aplaysanimportantrolein Taxolresistance.SinceLDHisacriticalenzymeinthe glycolyticpathway,ourresultssuggestthatinhibitionof glycolysismayre-sensitizeTaxol-resistantcellstoTaxol.ThecombinationofTaxolwithoxamateshowssynergistic inhibitoryeffectonbreastcancercellsOxamateisapyruvateanalog thatdirectlyinhibitsthe convertingprocessofpyruvatetolactatebyLDH,therefore,inhibitscellglycolysi s[21].Wefirstexaminedthe effectofoxamateonLDHactivityandcellviabilityof MDA-435and435TR1cells.Oxamatetreatmentledto adecreaseofLDHactivity(Fig.5A)andaninhibitionof cellviability(Fig.5B)inadose-dependantmanner,in bothMDA-435and435TR1cells.ComparedtoMDA435cells,Taxolresistant435TR1cellsshowedagreater sensitivitytooxamate,consistentwiththeresultsof LDH-AknockdownbysiRNA( Fig.3).Sinceglycolysis andmitochondrialoxidativep hosphorylationarelinked processes[16],andwehavepreviouslyshownthat LDH-Aiscriticalinregulat ingglycolysisandgrowthof breastcancercells[18],wereasonedthattheincreased expressionandactivityofLDH-AinTaxol-resistant cellsmayleadtoanincreaseofglycolysisandadecrease ofmitochondrialoxidativephosphorylation.Thus,aspecificinhibitorofthemitochondrialoxidativephosphorylation,oligomycinwasutilizedtotreatthesecells.As expected,Taxol-resistant435TR1cellsweremoreresistanttooligomycin(Additionalfile2,Figure.S2).These resultsfurthersupportthenotionthatincreasedTaxol sensitivitybyoxamateisaconsequenceoftheinhibition ofcellularglycolysis. SincedownregulationofLDH-AbysiRNAoroxamate significantlyinhibitedtheviabilityoftheTaxol-resistant cells,wefurtherinvestigatedtheeffectsofcombining Taxolwithglycolysisinhibit oroxamateonTaxol-resistantbreastcancercells.InbothTaxol-resistant435TRP and435TR1cells(Fig.6Aand6B;Additionalfile3,Figure.S3),andinBT474cells(Fig.6C),Taxolcombined withoxamateweremuchmoreeffectiveininhibiting cellviabilitycomparedwitheitheragentgivenalone. Similartreatmentcombinationswereperformedin anotherbreastcancercellline,MCF7,withsimilar resultsobtained(Additionalfile4,Figure.S4).Taken together,thecombinationofTaxolwithoxamatehasa greatercapacitytoinhibitTaxol-resistantcellscompared toeitheragentgivenalone. TofurtherinvestigatethemechanismofoxamateinducedTaxolre-sensitiza tion,weexaminedcellular apoptosisinthesecells.PARP,anuclearproteinthat canbeeasilycleavedbycaspases,hasbeenwidelyused asanapoptosismarker[19,20].Theexpressionlevelof totalPARPandcleavedPARP(c-PARP)wereexamined in435TR1cellsaftertreatmentwithTaxol,oxamate,or theircombinationfor48hrs,respectively.Wefounda significantincreaseofthelevelsofcleavedPARPafterZhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page5of12

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treatmentwiththecombinationofTaxolandoxamate comparedtotreatmentwithsingleagent(Fig.6D).This indicatesthatcellularapoptosisisamechanisminvolved intheincreasedcellgrowthinhibitoryeffectofthecombinationtreatmentofTaxolwithoxamate.DiscussionInthisstudy,weinvestigatedtheroleofLDH-Ainthe acquiredTaxolresistanceinmultiplehumanbreastcancercelllines.WeidentifiedthatcomparedtoTaxol-sensitivecells,Taxol-resist antcellspossessanincreased expressionandactivityofLDH-A,withits downregulationresultinginanincreasedsensitivityof Taxolresistant-cellstoTaxo l.Inaddition,comparedto Taxol-sensitivecells,Taxol-resistantcellsshowahigher sensitivitytotheLDHinhibitoroxamate.Furthermore, whencomparedtosingleagenttherapy,treatingcells withthecombinationofTaxolandoxamateshowa muchstrongerinhibitoryeffectonTaxol-resistantbreast cancercellsbypromotingcellularapoptosis.These resultsdemonstratethatLDH-Aplaysanimportantrole inTaxolresistanceandpotentiallyitcanserveasatherapeutictargetforovercomingTaxolresistancein patientswithbreastcancer. Figure2 IncreasedLDH-AexpressionandactivityinTaxol-resistantcells .A,Westernblotwasperformedwithananti-LDH-Aantibodyof totalcellextractfromMDA-435,435TR1and435TRPcells.The b -actinproteinwasusedasaloadingcontrol.B,LDHactivityinMDA-435,435TR1 and435TRPwereexamined.C,MDA-435cellsweretreatedwithincreasingconcentrationsofTaxolfor24hrs.Thecelllysateswereprepared andWesternblottingwascarriedoutwithantibodiesagainsttoLDH-Aand b -actin.D,LDHAproteinstabilityassaywasperformedinMDA-435 cellsunderthetreatmentsofTaxolat4nMandCHXat50ug/mlfollowedbyWesternblottingassaytoexamtheproteinexpressionlevelof LDHAat0and8hrs(top).TherelativeintensityofLDHAbandwasnormalizedtoits b -actinloading(bottom).E,LDHAmRNAlevelwas detectedbyreal-timePCRunder2nMTaxolinMDA-435cells.TheLDHAprimersusedforPCRare:forward,5 ’ -tggagtggaatgaatgttgc-3 ’ ; reverse:5 ’ -atagcccaggatgtgtagcc-3 ’ Columns ,meanofthreeindependentexperiments; bars ,SE.***, P <0.001. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page6of12

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Taxolisawidelyusedchemotherapeuticagentforthe treatmentofseveraltypesofcancers,includingbreast cancer.Taxolresistancemayresultinthesubsequent recurrenceandmetastasisofcancer,ultimatelyresulting indeath.Althoughextensiveinvestigationshavebeen doneinregardstotheresistanceofcancercellsto Taxol,thespecificmechanismsinvolvedarestillpoorly understood.Cancercellsaredifferentfromnon-neoplasticcellsintheirmetabolicproperties,withnormalcells relyingprimarilyontheprocessofmitochondrialoxidativephosphorylation,consumingoxygenandglucoseto produceenergy.Incontrast,cancercellsdependmostly uponglycolysis,theanaerobicbreakdownofglucose intotheenergy-storingmoleculeATP,eveninthepresenceofavailableoxygen[13-15,22,23].Recently, researchendeavorshavebeenactivelytriedtomakeuse oftheseuniquebioenergeticpropertiestoenhancethe therapeuticefficacyofkillingcancercells. LDH-AisoneofthemainisoformsofLDHexpressed inbreasttissue,catalyzingtheconversionofpyruvateto lactate[17].Weandothershavepreviouslyshownthat LDH-Aplaysacriticalroleinglycolysis,growth Figure3 KnockdownofLDH-AincreasesthesensitivityofTaxol-resistant435TR1cellstoTaxol .A,MDA-435and435TR1cellswere transfectedwithscramblesiRNA(Ctr)orLDH-AsiRNA.48hrsaftersiRNAtransfection,celllysateswerepreparedandWesternblottingwas performedwithantibodiesagainstLDH-A.The b -actinproteinwasusedasaloadingcontrol.B,LDHactivitywasexaminedfromlysatesofMDA435and435TR148hrsaftersiRNAtransfection.CandD,24hrsaftersiRNAtransfection,MDA-435and435TR1cellswereseededinto96-well platesatthedensityof8103cellsperwell,andtreatedwithTaxol(5nMand10nMforMDA-435,50nMand100nMfor435TR1)for48hrs. ThenthecellviabilitywasdetectedusingaMTSreagent.Dataarepresentedasthepercentageofviabilityinhibitionmeasuredincellstreated withoutTaxol. Columns ,meanofthreeindependentexperiments; bars ,SE.*, P <0.05,**, P <0.01,***, P <0.001. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page7of12

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propertiesandtumormaintenanceofbreastcancercells [16,18].StudieshaveshownthattheLDH-Aexpression incancercellsisassociatedwithradiosensitivity[24]. LDH-AinhibitionresultsinincreasedapoptosisviaROS productionincellwithfumaratehydratasedeficiency andwasviewedasatherapeuticstrategyfortreatment ofhereditaryleiomyomatosisandrenalcellcancer[25]. However,theroleofLDHinTaxolresistanceofcancer cellhasnotbeenexplored.Inthisstudy,weselecteda panelofTaxol-resistantcellsbygraduallyincreasingthe concentrationofTaxolinthecellculturemedium.We usedthese,andotherthreebreastcancercelllines,to studytheexpressionandactivityofLDH-AinthedevelopmentofTaxolresistance.Toourknowledge,thisis thefirstreporttoprovidedirectevidenceinsupportof aroleforLDH-AinacquiredTaxolresistancein humanbreastcancercells. WefoundthatTaxoltreatmentresultedinthe increasedLDH-Aexpressionandactivationincancer cells,whichappearsasaresultoftheinductionof Figure4 KnockdownofLDH-Aincreasesthesensitivityofb reastcancercelllinesBT474andMDA-231toTaxol .A,BT474cellswere transfectedwithscramblesiRNA(Ctr)orsi-LDHA.48hrsaftersiRNAtransfection,celllysateswerepreparedandimmunoblotanalyseswere carriedoutwithantibodiesagainstLDHAand b -actin.B,48hrsaftersiRNAtransfection,celllysateswerepreparedandLDHAactivitywas examined.DataareshowninpercentageofLDHactivityrelativetoCtr-transfectedcells.C,48hrsaftersiRNAtransfection,cellswereseededinto 96-wellplatesatthedensityof8103cellsperwell.12hrsafterincubation,thecellswerethentreatedwithvariousconcentrationsofTaxolfor 48hrs.ThenthecellviabilitywasdetectedusingaMTSreagent.Dataarepresentedasthepercentageofviabilityinhibitionmeasuredincells treatedwithoutTaxol.D,LDHAproteinexpressioninMDA-MB-231(Ctr)andstableLDHAknowdownMDA-MB-231cells(sh-LDHA)were detectedtoevaluatetheefficiencyofLDH-AknockdownbyusinganLDHAantibody. b -actinwasusedasaloadingcontrol.E,LDHactivitywas examinedinMDA-MB-231cellswithandwithoutstablyknockdownofLDH-A.DataareshowninpercentageofLDHactivityrelativetoCtrcells. F,MDA-MB-231cellswithorwithoutstablyknockdownofLDH-Awereseededinto96-wellplatesatthedensityof8103cellsperwell.12hrs afterincubation,thecellsweretreatedwithvariousconcentrationsofTaxolfor48hrs.ThenthecellviabilitywasdetectedusingaMTSreagent. DataarepresentedasthepercentageofviabilityinhibitionmeasuredincellstreatedwithoutTaxol. Columns ,meanofthreeindependent experiments; bars ,SE.*, P <0.05,**, P <0.01,***, P <0.001. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page8of12

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LDH-AmRNAexpressionbyTaxol.ThedownregulationofLDH-AbyLDH-AsiRNAandinhibitionofLDH byoxamateledtoincreasedsensitivitytoTaxolinall threebreastcancercelllinesexaminedinthisstudy. ThisindicatedthatTaxoltreatmenttriggersafeedforwardcycleinwhichTaxol-inducedactivationofLDH resultsincancercellsbettersurvivalunderTaxoltreatment,likelythroughpromotingcellglycolysis.Arecent studyhasshownthatcancercellsinhibitcytochrome c mediatedapoptosisbyamechanismthroughderegulatedglucosemetabolism[26].Thus,theTaxol-induced highexpressionandactivityofLDH-Adetectedin Taxol-resistantcellscouldbeawayofadaptationof thesecellstoTaxoltreatmentandtomodulateglucose metabolismandglycolysistoavoidapoptosisinducedby Taxol.TargetingLDHbyLDHsiRNAorLDHinhibitor oxamateinterruptsthefeedforwardcycleandrenders there-sensitizationtoTaxol.Theseresultsindicatethat LDHmaypotentiallyserveasanexcellenttargetfor overcomingTaxolresistanceinhumanbreastcancer patients. Up-regulationofantiapopt oticBcl-2familymembers, suchasBcl-2andBcl-XL,wasreportedtocontributeto Taxol-inducedapoptosis[27].Inaddition,wepreviously reportedthatthephosphorylationontyrosine-15of Cdc2byErbB2inbreastcancercellsresultingadelayed MphaseentryandleadingtoanincreasedTaxolresistance[11].Wefoundthatcomparedtotheparental MDA435cells,Taxol-resistantMDA435TR1and MDA435TRPcellsexpresslowerBcl-2andlowerphosphorylationlevelofCdc2attyrosine-15(Additionalfile 5,Figure.S5).BasedontheknownfunctionsofBcl2 andY15-Cdc2inTaxolresistance,theseresultscannot explaintheincreasedresistanceinMDA435TR1and MDA435TRPcells.However,wefoundthatBcl-XLwas upregulatedinTaxol-resista ntcells(Additionalfile5, Figure.S5).Thismightbeanotherreasoninadditionto LDH-AfortheincreasedTaxolresistanceinthesecells. Itwillbeinterestingtoexaminetherelationship betweenLDH-AandBcl-XLinthesecellsinourfuture studies. ThedifferencesincytotoxicityweresomewhatmodestwhentheLDH-AwereknockeddownbysiRNA. OneofthereasonsmightbetherelativelylowsensitivityofMTSassaytodetectcelltoxicityinourexperiments.Anotherpossiblereasonmightbetherelatively lowknockingdownefficiencyofLDH-AbythesiRNA. Inaddition,asfar,thereisnoanysinglemolecule Figure5 Taxol-resistantcellsaremoresensitivetoglycolysisinhibitoroxamate .A,MDA-435and435TR1cellsweretreatedwithvarious concentrationsofoxamatefor48hrs,thenLDHactivitywasdetected.DataareshownasthepercentageofLDHactivityinhibitiondetectedin cellstreatedwithoutoxamate.B,MDA-435and435TR1weretreatedwithvariousconcentrationsofoxamatefor48hrs.Thenthecellviability wasdetectedusingaMTSreagent.Dataarepresentedasthepercentageofviabilityinhibitionmeasuredincellstreatedwithoutoxamate. Columns ,meanofthreeindependentexperiments; bars ,SE.*, P <0.05.**, P <0.01. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page9of12

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reportedthatcanfullyaccountforTaxolresistancein breastcancercells.OurresultsandpreviousstudiessuggestthatmultiplemechanismsmaycontributetoTaxol resistanceandTaxolresistancemaybeasumeffectof multiplemechanisms/pathw ays,whichsuggeststhata strategyofcombinationaltherapyisneededtoovercome theresistancetoTaxol.Toidentifythemoleculesthat maycontributetoTaxolresistanceisimportantforthe managementofTaxolresistantbreastcancer.Nevertheless,ourstudyhasshownthatthecombinationofTaxol andLDH-Ainhibitoroxamate dramaticallyincreased theinhibitoryeffectonthegrowthofTaxol-resistant cancercells.Thispotentiallycanbeaneffectivestrategy toovercomeTaxolresistance. ThecombinationofTaxolwithoxamatewasfoundto bemoreeffectiveinkillingTaxol-resistantcells,comparedtoeitherTaxoloroxamatetreatmentalone.The combinationtherapyrevealsasynergisticinhibitory effectbypromotingbreastcancercellapoptosis(Fig.6). Apoptosisisapredominantmechanismbywhichcancer chemotherapeuticagentskillcells[28].Althoughoxamateiscapableofinhibitingcellcycleprogressionfrom Figure6 CombinationofTaxolwithoxamateshowssynergisticinhibitoryeffectsofTaxol-resistantandBT474cells .AandB,8103perwellof435TRPand435TR1cellswereplatedin96-wellplatesandthentreatedwithTaxol(Tax),Oxamate(Oxa)aloneorTaxolplus Oxamate(Tax+Oxa)withtheindicatedconcentrationsfor48hrs.CellviabilitywasexaminedbyMTSassay.Dataarepresentedasthe percentageofviabilityinhibitionmeasuredincellstreatedwithoutTaxandOxa.C,BT474cellsweretreatedwithTax,OxaaloneorTaxplusOxa withtheindicatedconcentrationsfor48hrs..Cellviabilitywasmeasuredbydirectcellcounting.Dataarepresentedasthepercentageof viabilityinhibitioncountedincellstreatedwithoutTaxandOxa.D,435TR1cellsweretreatedwith100nMTaxolor/and40mMoxamatefor48 hrs,celllysateswerepreparedandWesternblottingwerecarriedoutwithantibodiesagainsttotalPARP(Top)oritscleavedproteinc-PARP (Middle). b -actinwasusedasaloadingcontrol(Bottom). Columns ,meanofthreeindependentexperiments; bars ,SE.***, P <0.001. Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page10of12

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G2toMphase[29],wereporthereanovelfunctionvia inducingapoptoticcelldeath,withimportantimplicationsintheclinicaltreatmentofTaxol-resistantcancers, suchasbreastcancer. TheoriginofMDA-MB-435cellshasrecentlybeen calledintoquestion[30,31].However,alatestliterature indicatedthatcurrentstocksofbothMDA-MB-435 cellsandM14melanomacellsareinfactMDA-MB-435 breastcancercellsinsteadofM14melanomacellline [32].Nevertheless,wealsoexaminedthreemorebreast cancercelllines,ErbB2-overexpressingBT474and ErbB2-low-expressingMDA-231andMCF-7,inorder toconfirmourfindingsfromMDA-MB-435cells. Insummary,thepresentstudyrevealsthatLDH-A playsanimportantroleinTaxol-resistance,withTaxolinducedexpressionandactivityofLDH-Aservingasan importantmechanismfortheacquiredresistanceof humanbreastcancercellstoTaxol.Thisstudyprovides valuableinformationforthedevelopmentoftargeted therapiescapableofinhibitingkeytargets,suchasLDHA.Furtherstudiesareneededtodemonstratewhether thedownregulationofLDH-Amediatedre-sensitization ofbreastcancercellstoTaxolisindeedaconsequence ofinhibitionofglycolysis.Anotherquestionarisesasto whetherthedownregulationofotherkeymoleculesin theglycolyticpathwaymayhavethesameeffectasthe downregulationofLDH-A.Inconclusion,theresultsof ourstudyhighlighttheimportanceofLDH-Ainitsrole inTaxol-resistanceandopenthedoorforpossibletherapeuticinterventionsinpatientsthathavedevelopeda resistancetoTaxol.ConclusionLDH-AplaysanimportantroleinTaxolresistanceand inhibitionofLDH-Are-sensit izesTaxol-resistantcells toTaxol.Thisstudyprovidesvaluableinformationfor thefuturedevelopmentanduseoftargetedtherapies, suchasoxamate,forthetreatmentofpatientswith Taxol-resistantbreastcancer.ListofabbreviationsLDH-A:Lactatedehydrogenase-A;DMEM:Dulbecco ’ s modifiedEaglemedium;c-PARP:thecleavedPoly (ADP-ribose)polymerase;CHX:Cycloheximide.Additionalfile1:FigureS1. KnockdownofLDH-Are-sensitizes435TR1 celltoTaxolbydirectcellcounting.435TR1cellsweretransfectedwith scramblesiRNA(Ctr)orsi-LDHA.24hrsaftersiRNAtransfection,cells weretreatedwith50nMor100nMTaxolfor48hrs.Cellnumberswere directlycountedbyTypanBlueStaining.Dataarepresentedasthe percentageofviabilityinhibitioncountedincellstreatedwithoutTaxol. Columns ,meanofthreeindependentexperiments; bars ,SE.*, P <0.05,**, P <0.01.si-LDHAtransfectionefficiencywasshowedontherightpanel. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1476-4598-9-33S1.PDF] Additionalfile2:FigureS2.Taxol-resistantcellsaremoreresistant tomitochondrialoxidativephosphorylationinhibitoroligomycin MDA-435and435TR1cellswereseededinto96-wellplateatdensityof5 103cellsperwell.12hrsafterincubation;cellsweretreatedwith variousconcentrationsofoligomycinfor24hrs.Thenthecellviability wasdetectedusingaMTSreagent,anddataarepresentedasthe percentageofviabilityinhibitionmeasuredincellstreatedwithout oligomycin. Columns ,meanofthreeindependentexperiments; bars ,SE. ***, P <0.001. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1476-4598-9-33S2.PDF] Additionalfile3:FigureS3. CombinationofTaxolwithoxamateshows synergisticinhibitoryeffectsinTaxol-resistantcellsbydirectcell counting.435TR1and435TRPcellswereseededin24-wellplatesand treatedwithTax,OxaaloneorTaxplusOxawiththeindicated concentrationsfor48hrs.CellnumberswerecountedbyTypanBlue Staining.Dataarepresentedasthepercentageofviabilityinhibition countedincellstreatedwithoutTaxandOxa. Columns ,meanofthree independentexperiments; bars ,SE.*, P <0.05.**, P <0.01.***, P <0.001. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1476-4598-9-33S3.PDF] Additionalfile4:FigureS4. CombinationofTaxolwithoxamateshows betterinhibitionofMCF7cells.A,1104perwellofMCF7cellswere platedinto96-wellplateandtreatedwithTaxol,Oxa,orTaxplusOxa withindicatedconcentrationsfor48hrs.Cellviabilitywasexaminedby MTSassay.Dataarepresentedasthepercentageofviabilityinhibition measuredincellstreatedwithoutTaxandOxa. Columns ,meanofthree independentexperiments; bars ,SE.*, P <0.05,**, P <0.01.B,MCF7cells weretreatedwith10nMTaxolor/and16mMoxamatefor48hrsand celllysateswerepreparedforWesternblottingusingantibodiesagainst totalPARP(Top)oritscleavedproteinc-PARP(Middle). b -actinwasused asaloadingcontrol(Bottom). Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1476-4598-9-33S4.PDF] Additionalfile5:FigureS5. TheexpressionofBcl-2,Bcl-XL,Cdc2and phosphorylationstatusofCdc2atTyrosine15.MDA-435,435TR1andTRP cellswerecollected,lysedandimmunoblotanalyseswerecarriedout withantibodiesagainstBcl-2,Bcl-XL,Cdc2andp-Cdc2-Y15andtubulin. Clickhereforfile [http://www.biomedcentral.com/content/supplementary/1476-4598-9-33S5.PDF] Acknowledgements MingTanisaVincentF.Kilborn,Jr.CancerResearchScholar.WethankDr. EddieReedfromUSAMitchellCancerInstituteforcriticallyreadingthe manuscriptandMs.AmyBrownfortheeditorialassistance.Wearegrateful tothesupportfromTheVincentF.Kilborn,Jr.CancerResearchFoundation (M.Tan)andTheNorwegianRadiumhospitaletLegater(Project334003,M. Tan.andO.Fodstad). Authordetails1MitchellCancerInstitute,UniversityofSouthAlabama,Mobile,Alabama, USA.2DepartmentofCellBiologyandNeuroscience,UniversityofSouth Alabama,Mobile,Alabama,USA.3CancerResearchInstitute,CentralSouth University,Changsha,China.4InstituteforCancerResearch,TheNorwegian RadiumHospital,UniversityofOslo,Norway.5DepartmentofBiochemistry andMolecularBiology,UniversityofFlorida,Gainesville,Florida,USA.6OchsnerCancerInstitute,OchsnerHealthSystem,NewOrleans,Louisiana, USA. Authors ’ contributions MZdesignedandcarriedoutthemajorityoftheexperimentsanddrafted themanuscript.YZ,YD,HL,ZLinvolvedinexperimentaldesignandcarried outsomeexperiments,andhelpedtorevisethemanuscript.SKandJLZhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page11of12

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contributedthekeyreagents.OF,AR,LO,SLhelpedtorevisethe manuscript.MTconceivedthestudyandsupervisedtheoverallexperimental design,executionandrevisedthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:15August2009 Accepted:9February2010Published:9February2010 References 1.HenleyD,Isbill1M,Fernando1R,Foster1JS,Wimalasena1J: Paclitaxel inducedapoptosisinbreastcancercellsrequirescellcycletransitbut notCdc2activity. CancerChemotherPharmacol 2007, 59(2) :235-249. 2.TanM,YuD: MolecularmechanismsoferbB2-mediatedbreastcancer chemoresistance. AdvExpMedBiol 2007, 608 :119-129. 3.FrankelAndrea,BuckmanRobert,KerbelSRobert: AbrogationofTaxolinducedG2-MArrestandApoptosisinHumanOvarianCancerCells GrownasMulticellularTumorSpheroids. CancerRes 1997, 57 :2388-2293. 4.ChenLP,CaiSM,FanJX,LiZT: PEBARegimen(Cisplatin,Etoposide, Bleomycin,andAdriamycin)intheTreatmentofDrug-Resistant Choriocarcinoma. 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JBiolChem 1999, 274(33) :22932-22940. 21.ChangGG,HuangSM,ChiouSH: Kineticmechanismoftheendogenous lactatedehydrogenaseactivityofduckepsilon-crystallin. ArchBiochem Biophys 1991, 284(2) :285-291. 22.DeBerardinisRJ,LumJJ,HatzivassiliouG,ThompsonCB: Thebiologyof cancer:metabolicreprogrammingfuelscellgrowthandproliferation. CellMetab 2008, 7 :11-20. 23.KroemerG,PouyssegurJ: Tumorcellmetabolism:cancer ’ sAchilles ’ heel. CancerCell 2008, 13 :472-482. 24.GorlachA,AckerH: pO2-andpH-gradientsinmulticellularspheroidsand theirrelationshiptocellularmetabolismandradiationsensitivityof malignanthumantumorcells. BiochimBiophysActa 1994, 1227 :105-112. 25.XieH,ValeraVA,MerinoMJ,AmatoAM,SignorettiS,LinehanWM, SukhatmeVP,SethP: LDH-Ainhibition,atherapeuticstrategyfor treatmentofhereditaryleiomyomatosisandrenalcellcancer. Molecular CancerTherapeutics 2009, 8(3) :626-635. 26.Vaughn1EAllyson,DeshmukhMohanish: Glucosemetabolisminhibits apoptosisinneuronsandcancercellsbyredoxinactivationof cytochromec. NatCellBiol 2008, 10(12) :1477-1483. 27.BasuA,DuBoisG,HaldarS: PosttranslationalmodificationsofBcl2family members – apotentialtherapeutictargetforhumanmalignancy. Front Biosci 2006, 11 :1508-1521. 28.FisherDE: Apoptosisincancertherapy:crossingthethreshold. Cell 1994, 78 :539-42. 29.ThornburgJM,NelsonKK,ClemBF,LaneAN,ArumugamS,SimmonsA, EatonJW,TelangS,ChesneyJ: Targetingaspartateaminotransferasein breastcancer. BreastCancerRes 2008, 10(5) :R84. 30.SellappanS,GrijalvaR,ZhouX,YangW,EliMB,MillsGB,YuD: Lineage infidelityofMDA-MB-435cells:expressionofmelanocyteproteinsina breastcancercellline. CancerRes 2004, 64 :3479-3485. 31.WelchDR: Technicalconsiderationsforstudyingcancermetastasisin vivo. ClinExpMetastasis 1997, 15 :272-306. 32.ChambersAF: MDA-MB-435andM14celllines:identicalbutnotM14 melanoma?. CancerRes 2009, 69(13) :5292-5293. doi:10.1186/1476-4598-9-33 Citethisarticleas: Zhou etal .: Warburgeffectinchemosensitivity: Targetinglactatedehydrogenase-Are-sensitizes Taxol-resistantcancercellstoTaxol. MolecularCancer 2010 9 :33. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Zhou etal MolecularCancer 2010, 9 :33 http://www.molecular-cancer.com/content/9/1/33 Page12of12



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Supplementary Figure S1 Knockdown of LDH-A re-sen sitizes 435TR1 cell to Taxol measured by direct cell counting



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Supplementary Figure S2 Taxol-resistant cells are m ore resistant to mitochondrial inhibitor oligomycin


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dochead Research
bibl
title
p Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol
aug
au id A1 ce yes snm Zhoufnm Minginsr iid I1 I3 email mingzhou@usouthal.edu
A2 ZhaoYuhuayzhao@usouthal.edu
A3 DingYanyding@usouthal.edu
A4 LiuHaohliu@usouthal.edu
A5 LiuZixingzixingliu@usouthal.edu
A6 FodstadOysteinI4 ofodstad@usouthal.edu
A7 Rikermi IAdamI6 Ariker@ochsner.org
A8 KamarajugaddaSushamaI5 susha80@ufl.edu
A9 LuJianrongjrlu@ufl.edu
A10 OwenBLaurielowen@usouthal.edu
A11 LedouxPSusanI2 sledoux@usouthal.edu
ca A12 TanMingmtan@usouthal.edu
insg
ins Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA
Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama, USA
Cancer Research Institute, Central South University, Changsha, China
Institute for Cancer Research, The Norwegian Radium Hospital, University of Oslo, Norway
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA
Ochsner Cancer Institute, Ochsner Health System, New Orleans, Louisiana, USA
source Molecular Cancer
issn 1476-4598
pubdate 2010
volume 9
issue 1
fpage 33
url http://www.molecular-cancer.com/content/9/1/33
xrefbib pubidlist pubid idtype pmpid 20144215doi 10.1186/1476-4598-9-33
history rec date day 15month 8year 2009acc 922010pub 922010
cpyrt 2010collab Zhou et al; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec
st
Abstract
Background
Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.
Results
Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.
Conclusion
LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
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Background
Taxol (paclitaxel) has recently emerged as an important agent in the treatment of human breast cancer as well as other tumor histologies, such as ovarian, prostate and non-small cell lung cancers abbrgrp
abbr bid B1 1
B2 2
. The primary cellular targets of Taxol are the microtubules of cancer cells, which is vital for mitotic activity, cellular motility and proliferative capacity. Taxol stabilizes the microtubule structure by disrupting the dynamic equilibrium between soluble tubulin dimers and their polymerized form. It is also a potent inhibitor of chromosomal replication by blocking cells in the late G2 or mitotic phases of the cell cycle
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. The resistance of cancer cells to Taxol and other chemotherapeutic agents is known to result in the subsequent recurrence and metastasis of cancer
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. One known mechanism involved with cancer cell resistance to Taxol and other microtubule-stabilizing agents is the high-expression of the membrane P-glycoprotein that functions as a drug-efflux pump
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. Other cellular mechanisms include the alterations of tubulin structure
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, changes in the drug-binding affinity of the microtubules
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and cell cycle deregulation
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. However, the detailed molecular mechanisms that may contribute to Taxol resistance of cancer cells are still not fully understood.
Cancer cells, unlike their normal counterparts, use aerobic glycolysis with reduced mitochondrial oxidative phosphorylation for glucose metabolism. This persistence of high lactate production by cancer cells in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 years ago
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. It was recognized that since cancer cells have increased cell growth and energy needs to sustain cell proliferation, elevated glycolytic activity insures that adequate ATP levels are available to meet the demands of rapidly proliferating tumor cells within a hypoxic microenvironment
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. Additionally, Taxol-resistant cancer cells may escape the therapeutic effects of Taxol via the efflux transport systems present within tumor cells. However, drug efflux and metabolism consumes large amounts of ATP that is generated via glycolysis, protecting cells from the lethal effects of Taxol by sustaining the energy needed for cellular drug efflux and metabolism. Thus, the energy distribution consumed in Taxol-resistant cells must be dramatically altered in order to accommodate for both cell viability and long-term survival.
Lactate dehydrogenase-A (LDH-A) is one of the main isoforms of LDH expressed in breast tissue, controlling the conversion of pyruvate to lactate of the cellular glycolytic process
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. It has been shown that LDH-A plays a key role in glycolysis, growth properties and tumor maintenance of breast cancer cells
16
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. To understand the cellular mechanisms involved in the resistance of breast cancer cells to Taxol, we investigated on the association of LDH-A and Taxol resistance in breast cancer cells and the role of LDH-A in tumor therapeutics and drug sensitivity. Our results show that compared with their parental cells, the increased expression and activity of LDH-A in Taxol-resistant cells directly correlate with their sensitivity to glycolysis inhibitor oxamate. Furthermore, gene expression knockdown experiments with siRNA specific for LDH-A show an increased sensitivity of these cells to Taxol. In addition, treatment of breast cancer cells with the combination of Taxol with oxamate, reveals an synergistically inhibitory effect upon cell viability. Taken together, LDH-A plays an important role in Taxol resistance of breast cancer cells, serving as a promising therapeutic target for overcoming Taxol resistance. Furthermore, the data are consistent with the role of LDH-A as an essential tumor maintenance gene, providing further insight into the cellular and molecular mechanisms involved in Taxol-resistant breast cancer.
Methods
Cells and cell culture
Breast cancer cells MDA-MB-435 (MDA-435), MDA-MB-231 (MDA-231), MCF7 and BT474 were purchased from American Type Culture Collection (ATCC). 435TR1 and 435TRP cells are Taxol-resistant single clone or pooled clones, which were developed from parental MDA-435 cells by treated with gradually increasing concentrations of Taxol in cell culture medium. MDA-231 cell line with stable knockdown of LDH-A was constructed through transfection of MSCV-based retroviral vector (MSCV/LTRmiR30-PIG). All of these cells were cultured in DMEM/F-12 (Mediatech Inc.) and supplemented with 10% FBS and Penicillin/Streptomycin.
Morphological observation of Taxol-resistant cells
The cells were seeded in 6-well plates at 3 × 10sup 5 cells per well in duplicate. After 12 hr incubation, cells were treated with or without 20 nM Taxol for 24 hrs, with untreated cells serving as controls. The cells were washed twice with PBS and then fixed with methanol/acetone (1:1), subsequently stained with 4',6-diamidino-2-phenylindole (DAPI) in order to visualize the morphology of cell nucleus. The morphology of cells was observed with the fluorescence microscope.
Cell apoptosis assay
The cancer cells were treated with 20 nM Taxol for 48 hrs. Two methods were used to detect apoptosis. 1) The early stage of apoptosis was detected by Annexin V/propidium iodide staining with the Apoptosis Detection Kit (BD PharMingen). Briefly, aliquots of 105 Taxol-treated cells were incubated with Annexin V/propidium iodide for 15 min at room temperature. The cells were then analyzed by flow cytometry (BD LSR II). 2) The late stage of apoptosis was detected by Cell Death Detection ELISA PLUS kit (Roche) according to the manufacturer's instruction.
Western blotting
Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM PMSF and Protease Inhibitor Cocktail (Sigma) for 20 min on ice. Lysates were cleared by centrifugation at 14,000 rpm at 4°C for 10 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-rad). The proteins were then separated with a SDS/polyacrylamide gel and transferred to a Nitrocellulose membrane (Bio-rad). After blocking in PBS with 5% non-fat dry milk for 1 hr, the membranes were incubated overnight at 4-8°C with the primary antibodies in PBS with 5% non-fat dry milk. The following antibodies were utilized: anti-LDHA rabbit antibody (1:1000, Cell Signaling); anti-PARP rabbit antibody (1:1000, Cell Signaling), anti-cleaved PARP Rabbit antibody (1:1000, Cell Signaling), anti-Bcl2 rabbit antibody (1:1000, Cell Signaling), anti-Bcl-XL rabbit monoclonal antibody (1:1000, Cell Signaling), anti-Cdc2 mouse monoclonal antibody (1:1000, Cell Signaling),, anti-p-Cdc2(Y15) rabbit monoclonal antibody (1:1000, Cell Signaling), and anti-β-actin monoclonal antibody (1:2000, Sigma). Membranes were extensively washed with PBS and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (1:2,000, Bio-rad). After additional washes with PBS, antigen-antibody complexes were visualized with the enhanced chemiluminescence kit (Pierce).
Detection of LDH Activity
The total LDH activity in cell lysates was examined according to the manufacturer's instructions of the LDH-cytotoxicity assay kit (BioVision). Briefly, 2 × 105 cells were seeded in a 24-well plate one day before assaying and all samples were analyzed in triplicate. Then cells were collected, washed and extracted for protein to measure LDH activity. Results were normalized based upon total protein.
siRNA Experiments
siRNA oligonucleotides for LDH-A was purchased from Sigma, with a scrambled siRNA (Sigma) used as a control. Transfection was performed using the Oligofectamine Transfection reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after transfection, whole-cell lysates were prepared for further analysis by Western blot, LDH activity and Taxol cytotoxicity assay.
Cell Viability Assay
A total of 5 × 103 ~ 1 × 104 cells/well were seeded in 96-well plates. Twenty-four hours later, the medium was replaced with fresh medium with or without Taxol and incubated for 24 or 48 hrs, respectively. Taxol in combination with various concentrations of oxamate were also used to treat the cells in order to investigate the effect of drug combinations. Cell viability was determined by two methods. 1) Using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's protocol; 2) by Typan Blue staining and direct cell counting using hematocytometer.
Statistical analysis
The unpaired Student's it t-test was used for the data analysis. All data were shown as mean ± standard error (SE). A statistical difference of P < 0.05 was considered significant.
Results
Selection and characterization of Taxol-resistant cancer cells
MDA-435 cells were treated with gradually increasing concentrations of Taxol in cell culture medium for selection of Taxol-resistant cells. After successive Taxol treatments for duration of 3 months, several resistant cell clones were developed from the MDA-435 cell line. Taxol-resistant clone 1 (435TR1) and Taxol-resistant pooled clones (435TRP) were used for all subsequent experiments in this study.
To compare the survival capacity of both Taxol-sensitive and Taxol-resistant cells, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 24 hrs. Taxol-sensitive MDA-435 cells showed cell rounding and blebbing with empty spaces visualized within the cells. This suggested that a large portion of these cells were arrested in G2/M phase, with some of these cells undergoing apoptosis. However, no obvious morphological change was observed in Taxol-resistant 435TR1 and 435TRP cells (Fig. figr fid F1 1A). Early stage apoptosis was examined by flow cytometry analysis after staining with Annexin V/propidium iodide, and late stage apoptosis was detected by a Cell Death Detection ELISA PLUS kit, which examines the DNA fragmentation in the apoptotic cells. Both assays detected a smaller percentage of apoptotic cells in Taxol-resistant 435TR1 and 435TRP, compared to their parental MDA-435 cells after treatment with 20 nM Taxol for 48 hrs (Fig. 1B). The protein expression of the cleaved Poly (ADP-ribose) polymerase (c-PARP), an important marker of caspase-mediated apoptosis
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, was also examined by Western blotting after the cells were treated with 20 nM Taxol for 48 hrs. We found much lower levels of cleaved PARP and correspondingly much higher levels of un-cleaved PARP in Taxol-resistant 435TR1 and 435TRP cells, compared to parental MDA-435 cells (Fig. 1C). Cell viability assay showed that 435TR1 and 435TRP cells could tolerate much higher concentrations of Taxol compared to MDA-435 cells, with their IC50 concentrations found to be more than 30-fold higher than those of MDA-435 cells (Fig. 1D).
fig Figure 1caption Characterization of Taxol-resistant cellstext
b Characterization of Taxol-resistant cells. A, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 24 hrs and their morphology was observed under fluorescence microscope. The phase image of these cells was shown at the top and the nucleus stained by DAPI was shown at the bottom (200 ×). B, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 48 hrs and apoptosis was examined by flow cytometry using Annexin V/PI staining and by Cell Death Detection ELISA PLUS Kit. Fold induction value was calculated following the formula: mU of the sample (cells treated with Taxol)/mU of the corresponding negative control (cells without Taxol treatment). C, Taxol-resistant cells and their parental cells were treated without or with 20 nM Taxol for 48 hrs, then poly (ADP-ribose) polymerase (PARP) and its cleaved protein (c-PARP) were analyzed by Western blotting with specific antibodies, respectively. β-actin was used as a loading control. D, Cell viability analysis was performed to evaluate cytotoxicity of Taxol to MDA-435 and Taxol-resistant 435TR1 and 435TRP cells under treatment with indicated concentrations of Taxol for 48 hrs.
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Increased expression and activity of LDH-A in Taxol-resistant cells
To examine the role of LDH-A in mediating Taxol resistance in human breast cancer cells, the expression of LDH-A was examined in MDA-435, 435TR1 and 435TRP cells. We found that LDH-A levels were markedly increased in 435TR1 and 435TRP cells, compared to their parental MDA-435 cells (Fig. F2 2A). The activity of LDH was also increased about 2-fold in Taxol-resistant 435TR1 and 435TRP cells, compared to MDA-435 cells (Fig. 2B). These results indicated that Taxol resistance is correlated with the increased LDH-A expression and activity. Interestingly, treatment with Taxol resulted in the induction of LDH-A expression in a dose-dependent pattern in MDA-435 cells (Fig. 2C). We also identified that LDH activity could also be induced by Taxol in the Taxol-resistant cells (data not shown). To study the mechanism that may contribute to the increased expression and activity of LDH-A, MDA-435 cells were treated with CHX to block protein synthesis and the cells were further treated with or without Taxol for different times, the protein stability of LDH-A was measured by Western blot (Fig. 2D). The result showed that LDH-A protein is more stable in Taxol treated cells than that of untreated cells. We further compared the mRNA level of LDH-A in Taxol-treated and -untreated cells by qRT-PCR (Fig. 2E). The result showed that Taxol treatment increased the mRNA expression of LDH-A. These results suggest that both protein stability and mRNA induction by Taxol contribute to the up-regulation of LDH-A in these cells.
Figure 2Increased LDH-A expression and activity in Taxol-resistant cells
Increased LDH-A expression and activity in Taxol-resistant cells. A, Western blot was performed with an anti-LDH-A antibody of total cell extract from MDA-435, 435TR1 and 435TRP cells. The β-actin protein was used as a loading control. B, LDH activity in MDA-435, 435TR1 and 435TRP were examined. C, MDA-435 cells were treated with increasing concentrations of Taxol for 24 hrs. The cell lysates were prepared and Western blotting was carried out with antibodies against to LDH-A and β-actin. D, LDHA protein stability assay was performed in MDA-435 cells under the treatments of Taxol at 4 nM and CHX at 50 ug/ml followed by Western blotting assay to exam the protein expression level of LDHA at 0 and 8 hrs (top). The relative intensity of LDHA band was normalized to its β-actin loading (bottom). E, LDHA mRNA level was detected by real-time PCR under 2 nM Taxol in MDA-435 cells. The LDHA primers used for PCR are: forward, 5'-tgg agt gga atg aat gtt gc-3'; reverse: 5'-ata gcc cag gat gtg tag cc-3'. Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.
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The downregulation of LDH-A re-sensitizes Taxol-resistant cells to Taxol
The increase of LDH-A expression and LDH activity detected in Taxol-resistant cells suggests that LDH-A may play a critical role in Taxol resistance. Therefore, the effect of LDH-A downregulation on the sensitivity of Taxol was investigated. After LDH-A was downregulated efficiently by specific siRNA to LDH-A (Fig. F3 3A), LDH activity was decreased about 40% in MDA-435 cells and about 55% in 435TR1 cells (Fig. 3B). Since the expression and activity of LDH-A was upregulated in Taxol-resistant cancer cells (Fig. 2), we hypothesized that the downregulation of LDH-A by siRNA might re-sensitize Taxol-resistant cells to Taxol. To this end, LDH-A was knocked down with siRNA in 435TR1 and parental MDA-435 cells respectively, and then the cells were treated with different concentrations of Taxol. The downregulation of LDH-A increased the sensitivity of these cells to Taxol, with Taxol-resistant 435TR1 cells showing about a 3-10 fold increase in cell growth inhibition under 50-100 nM Taxol treatment measured by both MTS assay (Fig. 3C) and direct cell counting (Additional file supplr sid S1 1, Figure. S1). Interestingly, 435TR1 cells showed a much greater overall increased sensitivity to Taxol compared to their parental MDA-435 cells (Fig. 3C and 3D). Similar assays were performed in another breast cancer cell line BT474 (Fig. F4 4A-C), where the knockdown of LDH-A expression by siRNA increased the sensitivity to Taxol by at least 2-fold. To further confirm these results, MDA-231 cells with stable knockdown of LDH-A by short-hairpin RNA (shRNA) were used. Compared to those of control MDA-231 cells, LDH-A expression (Fig. 4D) and LDH activity (Fig. 4E) were dramatically decreased in LDH-A stably knockdown cells and these cells showed a much greater overall increased sensitivity to Taxol (Fig. 4F). These results demonstrated that LDH-A plays an important role in Taxol resistance. Since LDH is a critical enzyme in the glycolytic pathway, our results suggest that inhibition of glycolysis may re-sensitize Taxol-resistant cells to Taxol.
suppl
Additional file 1
Figure S1. Knockdown of LDH-A re-sensitizes 435TR1 cell to Taxol by direct cell counting. 435TR1 cells were transfected with scramble siRNA (Ctr) or si-LDHA. 24 hrs after siRNA transfection, cells were treated with 50 nM or 100 nM Taxol for 48 hrs. Cell numbers were directly counted by Typan Blue Staining. Data are presented as the percentage of viability inhibition counted in cells treated without Taxol. Columns, mean of three independent experiments; bars, SE.*, P < 0.05, **, P < 0.01. si-LDHA transfection efficiency was showed on the right panel.
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Figure 3Knockdown of LDH-A increases the sensitivity of Taxol-resistant 435TR1 cells to Taxol
Knockdown of LDH-A increases the sensitivity of Taxol-resistant 435TR1 cells to Taxol. A, MDA-435 and 435TR1 cells were transfected with scramble siRNA (Ctr) or LDH-A siRNA. 48 hrs after siRNA transfection, cell lysates were prepared and Western blotting was performed with antibodies against LDH-A. The β-actin protein was used as a loading control. B, LDH activity was examined from lysates of MDA-435 and 435TR1 48 hrs after siRNA transfection. C and D, 24 hrs after siRNA transfection, MDA-435 and 435TR1 cells were seeded into 96-well plates at the density of 8 × 103 cells per well, and treated with Taxol (5 nM and 10 nM for MDA-435, 50 nM and 100 nM for 435TR1) for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. Columns, mean of three independent experiments; bars, SE. *, P < 0.05, **, P < 0.01, ***, P < 0.001.
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Figure 4Knockdown of LDH-A increases the sensitivity of breast cancer cell lines BT474 and MDA-231 to Taxol
Knockdown of LDH-A increases the sensitivity of breast cancer cell lines BT474 and MDA-231 to Taxol. A, BT474 cells were transfected with scramble siRNA (Ctr) or si-LDHA. 48 hrs after siRNA transfection, cell lysates were prepared and immunoblot analyses were carried out with antibodies against LDHA and β-actin. B, 48 hrs after siRNA transfection, cell lysates were prepared and LDHA activity was examined. Data are shown in percentage of LDH activity relative to Ctr-transfected cells. C, 48 hrs after siRNA transfection, cells were seeded into 96-well plates at the density of 8 × 103 cells per well. 12 hrs after incubation, the cells were then treated with various concentrations of Taxol for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. D, LDHA protein expression in MDA-MB-231 (Ctr) and stable LDHA knowdown MDA-MB-231 cells (sh-LDHA) were detected to evaluate the efficiency of LDH-A knockdown by using an LDHA antibody. β-actin was used as a loading control. E, LDH activity was examined in MDA-MB-231 cells with and without stably knockdown of LDH-A. Data are shown in percentage of LDH activity relative to Ctr cells. F, MDA-MB-231 cells with or without stably knockdown of LDH-A were seeded into 96-well plates at the density of 8 × 103 cells per well. 12 hrs after incubation, the cells were treated with various concentrations of Taxol for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. Columns, mean of three independent experiments; bars, SE.*, P < 0.05, **, P < 0.01, ***, P < 0.001.
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The combination of Taxol with oxamate shows synergistic inhibitory effect on breast cancer cells
Oxamate is a pyruvate analog that directly inhibits the converting process of pyruvate to lactate by LDH, therefore, inhibits cell glycolysis
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. We first examined the effect of oxamate on LDH activity and cell viability of MDA-435 and 435TR1 cells. Oxamate treatment led to a decrease of LDH activity (Fig. F5 5A) and an inhibition of cell viability (Fig. 5B) in a dose-dependant manner, in both MDA-435 and 435TR1 cells. Compared to MDA-435 cells, Taxol resistant 435TR1 cells showed a greater sensitivity to oxamate, consistent with the results of LDH-A knockdown by siRNA (Fig. 3). Since glycolysis and mitochondrial oxidative phosphorylation are linked processes
16
, and we have previously shown that LDH-A is critical in regulating glycolysis and growth of breast cancer cells
18
, we reasoned that the increased expression and activity of LDH-A in Taxol-resistant cells may lead to an increase of glycolysis and a decrease of mitochondrial oxidative phosphorylation. Thus, a specific inhibitor of the mitochondrial oxidative phosphorylation, oligomycin was utilized to treat these cells. As expected, Taxol-resistant 435TR1 cells were more resistant to oligomycin (Additional file S2 2, Figure. S2). These results further support the notion that increased Taxol sensitivity by oxamate is a consequence of the inhibition of cellular glycolysis.
Additional file 2
Figure S2. Taxol-resistant cells are more resistant to mitochondrial oxidative phosphorylation inhibitor oligomycin. MDA-435 and 435TR1 cells were seeded into 96-well plate at density of 5 × 103 cells per well. 12 hrs after incubation; cells were treated with various concentrations of oligomycin for 24 hrs. Then the cell viability was detected using a MTS reagent, and data are presented as the percentage of viability inhibition measured in cells treated without oligomycin. Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.
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Figure 5Taxol-resistant cells are more sensitive to glycolysis inhibitor oxamate
Taxol-resistant cells are more sensitive to glycolysis inhibitor oxamate. A, MDA-435 and 435TR1 cells were treated with various concentrations of oxamate for 48 hrs, then LDH activity was detected. Data are shown as the percentage of LDH activity inhibition detected in cells treated without oxamate. B, MDA-435 and 435TR1 were treated with various concentrations of oxamate for 48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without oxamate. Columns, mean of three independent experiments; bars, SE. *, P < 0.05. **, P < 0.01.
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Since downregulation of LDH-A by siRNA or oxamate significantly inhibited the viability of the Taxol-resistant cells, we further investigated the effects of combining Taxol with glycolysis inhibitor oxamate on Taxol-resistant breast cancer cells. In both Taxol-resistant 435TRP and 435TR1 cells (Fig. F6 6A and 6B; Additional file S3 3, Figure. S3), and in BT474 cells (Fig. 6C), Taxol combined with oxamate were much more effective in inhibiting cell viability compared with either agent given alone. Similar treatment combinations were performed in another breast cancer cell line, MCF7, with similar results obtained (Additional file S4 4, Figure. S4). Taken together, the combination of Taxol with oxamate has a greater capacity to inhibit Taxol-resistant cells compared to either agent given alone.
Additional file 3
Figure S3. Combination of Taxol with oxamate shows synergistic inhibitory effects in Taxol-resistant cells by direct cell counting. 435TR1 and 435TRP cells were seeded in 24-well plates and treated with Tax, Oxa alone or Tax plus Oxa with the indicated concentrations for 48 hrs. Cell numbers were counted by Typan Blue Staining. Data are presented as the percentage of viability inhibition counted in cells treated without Tax and Oxa. Columns, mean of three independent experiments; bars, SE. *, P < 0.05. **, P < 0.01. ***, P < 0.001.
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Additional file 4
Figure S4. Combination of Taxol with oxamate shows better inhibition of MCF7 cells. A, 1 × 104 per well of MCF7 cells were plated into 96-well plate and treated with Taxol, Oxa, or Tax plus Oxa with indicated concentrations for 48 hrs. Cell viability was examined by MTS assay. Data are presented as the percentage of viability inhibition measured in cells treated without Tax and Oxa. Columns, mean of three independent experiments; bars, SE.*, P < 0.05, **, P < 0.01. B, MCF7 cells were treated with 10 nM Taxol or/and 16 mM oxamate for 48 hrs and cell lysates were prepared for Western blotting using antibodies against total PARP (Top) or its cleaved protein c-PARP (Middle). β-actin was used as a loading control (Bottom).
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Figure 6Combination of Taxol with oxamate shows synergistic inhibitory effects of Taxol-resistant and BT474 cells
Combination of Taxol with oxamate shows synergistic inhibitory effects of Taxol-resistant and BT474 cells. A and B, 8 × 103 per well of 435TRP and 435TR1 cells were plated in 96-well plates and then treated with Taxol (Tax), Oxamate (Oxa) alone or Taxol plus Oxamate (Tax+ Oxa) with the indicated concentrations for 48 hrs. Cell viability was examined by MTS assay. Data are presented as the percentage of viability inhibition measured in cells treated without Tax and Oxa. C, BT474 cells were treated with Tax, Oxa alone or Tax plus Oxa with the indicated concentrations for 48 hrs.. Cell viability was measured by direct cell counting. Data are presented as the percentage of viability inhibition counted in cells treated without Tax and Oxa. D, 435TR1 cells were treated with 100 nM Taxol or/and 40 mM oxamate for 48 hrs, cell lysates were prepared and Western blotting were carried out with antibodies against total PARP (Top) or its cleaved protein c-PARP (Middle). β-actin was used as a loading control (Bottom). Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.
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To further investigate the mechanism of oxamate-induced Taxol re-sensitization, we examined cellular apoptosis in these cells. PARP, a nuclear protein that can be easily cleaved by caspases, has been widely used as an apoptosis marker
19
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. The expression level of total PARP and cleaved PARP (c-PARP) were examined in 435TR1 cells after treatment with Taxol, oxamate, or their combination for 48 hrs, respectively. We found a significant increase of the levels of cleaved PARP after treatment with the combination of Taxol and oxamate compared to treatment with single agent (Fig. 6D). This indicates that cellular apoptosis is a mechanism involved in the increased cell growth inhibitory effect of the combination treatment of Taxol with oxamate.
Discussion
In this study, we investigated the role of LDH-A in the acquired Taxol resistance in multiple human breast cancer cell lines. We identified that compared to Taxol-sensitive cells, Taxol-resistant cells possess an increased expression and activity of LDH-A, with its downregulation resulting in an increased sensitivity of Taxol resistant-cells to Taxol. In addition, compared to Taxol-sensitive cells, Taxol-resistant cells show a higher sensitivity to the LDH inhibitor oxamate. Furthermore, when compared to single agent therapy, treating cells with the combination of Taxol and oxamate show a much stronger inhibitory effect on Taxol-resistant breast cancer cells by promoting cellular apoptosis. These results demonstrate that LDH-A plays an important role in Taxol resistance and potentially it can serve as a therapeutic target for overcoming Taxol resistance in patients with breast cancer.
Taxol is a widely used chemotherapeutic agent for the treatment of several types of cancers, including breast cancer. Taxol resistance may result in the subsequent recurrence and metastasis of cancer, ultimately resulting in death. Although extensive investigations have been done in regards to the resistance of cancer cells to Taxol, the specific mechanisms involved are still poorly understood. Cancer cells are different from non-neoplastic cells in their metabolic properties, with normal cells relying primarily on the process of mitochondrial oxidative phosphorylation, consuming oxygen and glucose to produce energy. In contrast, cancer cells depend mostly upon glycolysis, the anaerobic breakdown of glucose into the energy-storing molecule ATP, even in the presence of available oxygen
13
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15
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. Recently, research endeavors have been actively tried to make use of these unique bioenergetic properties to enhance the therapeutic efficacy of killing cancer cells.
LDH-A is one of the main isoforms of LDH expressed in breast tissue, catalyzing the conversion of pyruvate to lactate
17
. We and others have previously shown that LDH-A plays a critical role in glycolysis, growth properties and tumor maintenance of breast cancer cells
16
18
. Studies have shown that the LDH-A expression in cancer cells is associated with radiosensitivity
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. LDH-A inhibition results in increased apoptosis via ROS production in cell with fumarate hydratase deficiency and was viewed as a therapeutic strategy for treatment of hereditary leiomyomatosis and renal cell cancer
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. However, the role of LDH in Taxol resistance of cancer cell has not been explored. In this study, we selected a panel of Taxol-resistant cells by gradually increasing the concentration of Taxol in the cell culture medium. We used these, and other three breast cancer cell lines, to study the expression and activity of LDH-A in the development of Taxol resistance. To our knowledge, this is the first report to provide direct evidence in support of a role for LDH-A in acquired Taxol resistance in human breast cancer cells.
We found that Taxol treatment resulted in the increased LDH-A expression and activation in cancer cells, which appears as a result of the induction of LDH-A mRNA expression by Taxol. The downregulation of LDH-A by LDH-A siRNA and inhibition of LDH by oxamate led to increased sensitivity to Taxol in all three breast cancer cell lines examined in this study. This indicated that Taxol treatment triggers a feedforward cycle in which Taxol-induced activation of LDH results in cancer cells better survival under Taxol treatment, likely through promoting cell glycolysis. A recent study has shown that cancer cells inhibit cytochrome c-mediated apoptosis by a mechanism through deregulated glucose metabolism
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. Thus, the Taxol-induced high expression and activity of LDH-A detected in Taxol-resistant cells could be a way of adaptation of these cells to Taxol treatment and to modulate glucose metabolism and glycolysis to avoid apoptosis induced by Taxol. Targeting LDH by LDH siRNA or LDH inhibitor oxamate interrupts the feedforward cycle and renders the re-sensitization to Taxol. These results indicate that LDH may potentially serve as an excellent target for overcoming Taxol resistance in human breast cancer patients.
Up-regulation of antiapoptotic Bcl-2 family members, such as Bcl-2 and Bcl-XL, was reported to contribute to Taxol-induced apoptosis
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. In addition, we previously reported that the phosphorylation on tyrosine-15 of Cdc2 by ErbB2 in breast cancer cells resulting a delayed M phase entry and leading to an increased Taxol resistance
11
. We found that compared to the parental MDA435 cells, Taxol-resistant MDA435TR1 and MDA435TRP cells express lower Bcl-2 and lower phosphorylation level of Cdc2 at tyrosine-15 (Additional file S5 5, Figure. S5). Based on the known functions of Bcl2 and Y15-Cdc2 in Taxol resistance, these results can not explain the increased resistance in MDA435TR1 and MDA435TRP cells. However, we found that Bcl-XL was upregulated in Taxol-resistant cells (Additional file 5, Figure. S5). This might be another reason in addition to LDH-A for the increased Taxol resistance in these cells. It will be interesting to examine the relationship between LDH-A and Bcl-XL in these cells in our future studies.
Additional file 5
Figure S5. The expression of Bcl-2, Bcl-XL, Cdc2 and phosphorylation status of Cdc2 at Tyrosine 15. MDA-435, 435TR1 and TRP cells were collected, lysed and immunoblot analyses were carried out with antibodies against Bcl-2, Bcl-XL, Cdc2 and p-Cdc2-Y15 and tubulin.
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The differences in cytotoxicity were some what modest when the LDH-A were knocked down by siRNA. One of the reasons might be the relatively low sensitivity of MTS assay to detect cell toxicity in our experiments. Another possible reason might be the relatively low knocking down efficiency of LDH-A by the siRNA. In addition, as far, there is no any single molecule reported that can fully account for Taxol resistance in breast cancer cells. Our results and previous studies suggest that multiple mechanisms may contribute to Taxol resistance and Taxol resistance may be a sum effect of multiple mechanisms/pathways, which suggests that a strategy of combinational therapy is needed to overcome the resistance to Taxol. To identify the molecules that may contribute to Taxol resistance is important for the management of Taxol resistant breast cancer. Nevertheless, our study has shown that the combination of Taxol and LDH-A inhibitor oxamate dramatically increased the inhibitory effect on the growth of Taxol-resistant cancer cells. This potentially can be an effective strategy to overcome Taxol resistance.
The combination of Taxol with oxamate was found to be more effective in killing Taxol-resistant cells, compared to either Taxol or oxamate treatment alone. The combination therapy reveals a synergistic inhibitory effect by promoting breast cancer cell apoptosis (Fig. 6). Apoptosis is a predominant mechanism by which cancer chemotherapeutic agents kill cells
B28 28
. Although oxamate is capable of inhibiting cell cycle progression from G2 to M phase
B29 29
, we report here a novel function via inducing apoptotic cell death, with important implications in the clinical treatment of Taxol-resistant cancers, such as breast cancer.
The origin of MDA-MB-435 cells has recently been called into question
B30 30
B31 31
. However, a latest literature indicated that current stocks of both MDA-MB-435 cells and M14 melanoma cells are in fact MDA-MB-435 breast cancer cells instead of M14 melanoma cell line
B32 32
. Nevertheless, we also examined three more breast cancer cell lines, ErbB2-overexpressing BT474 and ErbB2-low-expressing MDA-231 and MCF-7, in order to confirm our findings from MDA-MB-435 cells.
In summary, the present study reveals that LDH-A plays an important role in Taxol-resistance, with Taxol-induced expression and activity of LDH-A serving as an important mechanism for the acquired resistance of human breast cancer cells to Taxol. This study provides valuable information for the development of targeted therapies capable of inhibiting key targets, such as LDH-A. Further studies are needed to demonstrate whether the downregulation of LDH-A mediated re-sensitization of breast cancer cells to Taxol is indeed a consequence of inhibition of glycolysis. Another question arises as to whether the downregulation of other key molecules in the glycolytic pathway may have the same effect as the downregulation of LDH-A. In conclusion, the results of our study highlight the importance of LDH-A in its role in Taxol-resistance and open the door for possible therapeutic interventions in patients that have developed a resistance to Taxol.
Conclusion
LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
List of abbreviations
LDH-A: Lactate dehydrogenase-A; DMEM: Dulbecco's modified Eagle medium; c-PARP: the cleaved Poly (ADP-ribose) polymerase; CHX: Cycloheximide.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MZ designed and carried out the majority of the experiments and drafted the manuscript. YZ, YD, HL, ZL involved in experimental design and carried out some experiments, and helped to revise the manuscript. SK and JL contributed the key reagents. OF, AR, LO, SL helped to revise the manuscript. MT conceived the study and supervised the overall experimental design, execution and revised the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
Ming Tan is a Vincent F. Kilborn, Jr. Cancer Research Scholar. We thank Dr. Eddie Reed from USA Mitchell Cancer Institute for critically reading the manuscript and Ms. Amy Brown for the editorial assistance. We are grateful to the support from The Vincent F. Kilborn, Jr. Cancer Research Foundation (M. Tan) and The Norwegian Radiumhospitalet Legater (Project 334003, M. Tan. and O. Fodstad).
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 Material Information
Title: Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol
Series Title: Molecular Cancer 2010, 9:33
Physical Description: Mixed Material
Creator: Zhou M
Zhao Y
Ding Y
Liu H
Liu Z
Fodstad O
Riker AI
Kamarajugadda S
Lu J
Owen LB
Ledoux SP
Tan M
Publication Date: 40218
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Full Text


Zhou et al. Molecular Cancer 2010, 9:33
http://www.molecular-cancer.com/content/9/1/33


e MOLECULAR
CANCER


Warburg effect in chemosensitivity: Targeting

lactate dehydrogenase-A re-sensitizes

Taxol-resistant cancer cells to Taxol

Ming Zhou'3t, Yuhua Zhaolt, Yan Dingit, Hao Liu', Zixing Liu', Oystein Fodstad'4, Adam I Riker'6,
Sushama Kamarajugadda5, Jianrong Lus, Laurie B Owen', Susan P Ledoux2, Ming TanI'2*


Abstract
Background: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast
cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol.
Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which
controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we
investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.
Results: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in
high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of
LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH
A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific
LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of
Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting
apoptosis in these cells.
Conclusion: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant
cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an
important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the
increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study
provides valuable information for the future development and use of targeted therapies, such as oxamate, for the
treatment of patients with Taxol-resistant breast cancer.


Background
Taxol (paclitaxel) has recently emerged as an important
agent in the treatment of human breast cancer as well
as other tumor histologies, such as ovarian, prostate and
non-small cell lung cancers [1,2]. The primary cellular
targets of Taxol are the microtubules of cancer cells,
which is vital for mitotic activity, cellular motility and
proliferative capacity. Taxol stabilizes the microtubule
structure by disrupting the dynamic equilibrium
between soluble tubulin dimers and their polymerized
form. It is also a potent inhibitor of chromosomal

* Correspondence mtan@usouthal edu
t Contributed equally
Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama,
USA


0 BioMed Central


replication by blocking cells in the late G2 or mitotic
phases of the cell cycle [3]. The resistance of cancer
cells to Taxol and other chemotherapeutic agents is
known to result in the subsequent recurrence and
metastasis of cancer [4,5]. One known mechanism
involved with cancer cell resistance to Taxol and other
microtubule-stabilizing agents is the high-expression of
the membrane P-glycoprotein that functions as a drug-
efflux pump [6]. Other cellular mechanisms include the
alterations of tubulin structure [7-9], changes in the
drug-binding affinity of the microtubules [10] and cell
cycle deregulation [11,12]. However, the detailed mole-
cular mechanisms that may contribute to Taxol resis-
tance of cancer cells are still not fully understood.


2010 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commonn
Attribution Licene (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.






Zhou et al. Molecular Cancer 2010, 9:33
http://www.molecular-cancer.com/content/9/1/33


Cancer cells, unlike their normal counterparts, use
aerobic glycolysis with reduced mitochondrial oxidative
phosphorylation for glucose metabolism. This persistence
of high lactate production by cancer cells in the presence
of oxygen, known as aerobic glycolysis, was first noted by
Otto Warburg more than 75 years ago [13-15]. It was
recognized that since cancer cells have increased cell
growth and energy needs to sustain cell proliferation, ele-
vated glycolytic activity insures that adequate ATP levels
are available to meet the demands of rapidly proliferating
tumor cells within a hypoxic microenvironment [16].
Additionally, Taxol-resistant cancer cells may escape the
therapeutic effects of Taxol via the efflux transport sys-
tems present within tumor cells. However, drug efflux
and metabolism consumes large amounts of ATP that is
generated via glycolysis, protecting cells from the lethal
effects of Taxol by sustaining the energy needed for cellu-
lar drug efflux and metabolism. Thus, the energy distri-
bution consumed in Taxol-resistant cells must be
dramatically altered in order to accommodate for both
cell viability and long-term survival.
Lactate dehydrogenase-A (LDH-A) is one of the main
isoforms of LDH expressed in breast tissue, controlling
the conversion of pyruvate to lactate of the cellular gly-
colytic process [17]. It has been shown that LDH-A
plays a key role in glycolysis, growth properties and
tumor maintenance of breast cancer cells [16,18]. To
understand the cellular mechanisms involved in the
resistance of breast cancer cells to Taxol, we investi-
gated on the association of LDH-A and Taxol resistance
in breast cancer cells and the role of LDH-A in tumor
therapeutics and drug sensitivity. Our results show that
compared with their parental cells, the increased expres-
sion and activity of LDH-A in Taxol-resistant cells
directly correlate with their sensitivity to glycolysis inhi-
bitor oxamate. Furthermore, gene expression knock-
down experiments with siRNA specific for LDH-A show
an increased sensitivity of these cells to Taxol. In addi-
tion, treatment of breast cancer cells with the combina-
tion of Taxol with oxamate, reveals an synergistically
inhibitory effect upon cell viability. Taken together,
LDH-A plays an important role in Taxol resistance of
breast cancer cells, serving as a promising therapeutic
target for overcoming Taxol resistance. Furthermore,
the data are consistent with the role of LDH-A as an
essential tumor maintenance gene, providing further
insight into the cellular and molecular mechanisms
involved in Taxol-resistant breast cancer.

Methods
Cells and cell culture
Breast cancer cells MDA-MB-435 (MDA-435), MDA-
MB-231 (MDA-231), MCF7 and BT474 were purchased
from American Type Culture Collection (ATCC).


435TR1 and 435TRP cells are Taxol-resistant single
clone or pooled clones, which were developed from par-
ental MDA-435 cells by treated with gradually increas-
ing concentrations of Taxol in cell culture medium.
MDA-231 cell line with stable knockdown of LDH-A
was constructed through transfection of MSCV-based
retroviral vector (MSCV/LTRmiR30-PIG). All of these
cells were cultured in DMEM/F-12 (Mediatech Inc.) and
supplemented with 10% FBS and Penicillin/
Streptomycin.

Morphological observation of Taxol-resistant cells
The cells were seeded in 6-well plates at 3 x 105 cells
per well in duplicate. After 12 hr incubation, cells were
treated with or without 20 nM Taxol for 24 hrs, with
untreated cells serving as controls. The cells were
washed twice with PBS and then fixed with methanol/
acetone (1:1), subsequently stained with 4',6-diamidino-
2-phenylindole (DAPI) in order to visualize the mor-
phology of cell nucleus. The morphology of cells was
observed with the fluorescence microscope.

Cell apoptosis assay
The cancer cells were treated with 20 nM Taxol for 48
hrs. Two methods were used to detect apoptosis. 1) The
early stage of apoptosis was detected by Annexin V/pro-
pidium iodide staining with the Apoptosis Detection Kit
(BD PharMingen). Briefly, aliquots of 105 Taxol-treated
cells were incubated with Annexin V/propidium iodide
for 15 min at room temperature. The cells were then
analyzed by flow cytometry (BD LSR II). 2) The late
stage of apoptosis was detected by Cell Death Detection
ELISA PLUS kit (Roche) according to the manufac-
turer's instruction.

Western blotting
Cells were harvested and lysed in a buffer containing 50
mM Tris-HC1, pH 7.5, 150 mM NaC1, 2 mM EDTA, 1%
Triton, 1 mM PMSF and Protease Inhibitor Cocktail
(Sigma) for 20 min on ice. Lysates were cleared by cen-
trifugation at 14,000 rpm at 4C for 10 min. Superna-
tants were collected and protein concentrations were
determined by the Bradford assay (Bio-rad). The pro-
teins were then separated with a SDS/polyacrylamide gel
and transferred to a Nitrocellulose membrane (Bio-rad).
After blocking in PBS with 5% non-fat dry milk for 1 hr,
the membranes were incubated overnight at 4-8C with
the primary antibodies in PBS with 5% non-fat dry milk.
The following antibodies were utilized: anti-LDHA rab-
bit antibody (1:1000, Cell Signaling); anti-PARP rabbit
antibody (1:1000, Cell Signaling), anti-cleaved PARP
Rabbit antibody (1:1000, Cell Signaling), anti-Bcl2 rabbit
antibody (1:1000, Cell Signaling), anti-Bcl-XL rabbit
monoclonal antibody (1:1000, Cell Signaling), anti-Cdc2


Page 2 of 12






Zhou et al. Molecular Cancer 2010, 9:33
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mouse monoclonal antibody (1:1000, Cell Signaling),,
anti-p-Cdc2(Y15) rabbit monoclonal antibody (1:1000,
Cell Signaling), and anti-p-actin monoclonal antibody
(1:2000, Sigma). Membranes were extensively washed
with PBS and incubated with horseradish peroxidase
conjugated secondary anti-mouse antibody or anti-rabbit
antibody (1:2,000, Bio-rad). After additional washes with
PBS, antigen-antibody complexes were visualized with
the enhanced chemiluminescence kit (Pierce).

Detection of LDH Activity
The total LDH activity in cell lysates was examined
according to the manufacturer's instructions of the
LDH-cytotoxicity assay kit (BioVision). Briefly, 2 x 105
cells were seeded in a 24-well plate one day before
assaying and all samples were analyzed in triplicate.
Then cells were collected, washed and extracted for pro-
tein to measure LDH activity. Results were normalized
based upon total protein.

siRNA Experiments
siRNA oligonucleotides for LDH-A was purchased from
Sigma, with a scrambled siRNA (Sigma) used as a con-
trol. Transfection was performed using the Oligofecta-
mine Transfection reagent (Invitrogen) according to the
manufacturer's protocol. Forty-eight hours after trans-
fection, whole-cell lysates were prepared for further ana-
lysis by Western blot, LDH activity and Taxol
cytotoxicity assay.

Cell Viability Assay
A total of 5 x 103 ~ 1 x 104 cells/well were seeded in
96-well plates. Twenty-four hours later, the medium was
replaced with fresh medium with or without Taxol and
incubated for 24 or 48 hrs, respectively. Taxol in combi-
nation with various concentrations of oxamate were also
used to treat the cells in order to investigate the effect
of drug combinations. Cell viability was determined by
two methods. 1) Using CellTiter 96 Aqueous One Solu-
tion Cell Proliferation Assay (Promega) according to the
manufacturer's protocol; 2) by Typan Blue staining and
direct cell counting using hematocytometer.

Statistical analysis
The unpaired Student's t-test was used for the data ana-
lysis. All data were shown as mean + standard error
(SE). A statistical difference of P < 0.05 was considered
significant.

Results
Selection and characterization of Taxol-resistant cancer
cells
MDA-435 cells were treated with gradually increasing
concentrations of Taxol in cell culture medium for


selection of Taxol-resistant cells. After successive Taxol
treatments for duration of 3 months, several resistant
cell clones were developed from the MDA-435 cell line.
Taxol-resistant clone 1 (435TR1) and Taxol-resistant
pooled clones (435TRP) were used for all subsequent
experiments in this study.
To compare the survival capacity of both Taxol-sensi-
tive and Taxol-resistant cells, MDA-435, 435TR1 and
435TRP cells were treated with 20 nM Taxol for 24 hrs.
Taxol-sensitive MDA-435 cells showed cell rounding
and blebbing with empty spaces visualized within the
cells. This suggested that a large portion of these cells
were arrested in G2/M phase, with some of these cells
undergoing apoptosis. However, no obvious morphologi-
cal change was observed in Taxol-resistant 435TR1 and
435TRP cells (Fig. 1A). Early stage apoptosis was exam-
ined by flow cytometry analysis after staining with
Annexin V/propidium iodide, and late stage apoptosis
was detected by a Cell Death Detection ELISA PLUS
kit, which examines the DNA fragmentation in the
apoptotic cells. Both assays detected a smaller percen-
tage of apoptotic cells in Taxol-resistant 435TR1 and
435TRP, compared to their parental MDA-435 cells
after treatment with 20 nM Taxol for 48 hrs (Fig. 1B).
The protein expression of the cleaved Poly (ADP-ribose)
polymerase (c-PARP), an important marker of caspase-
mediated apoptosis [19,20], was also examined by Wes-
tern blotting after the cells were treated with 20 nM
Taxol for 48 hrs. We found much lower levels of
cleaved PARP and correspondingly much higher levels
of un-cleaved PARP in Taxol-resistant 435TR1 and
435TRP cells, compared to parental MDA-435 cells (Fig.
1C). Cell viability assay showed that 435TR1 and
435TRP cells could tolerate much higher concentrations
of Taxol compared to MDA-435 cells, with their IC50
concentrations found to be more than 30-fold higher
than those of MDA-435 cells (Fig. 1D).

Increased expression and activity of LDH-A in Taxol-
resistant cells
To examine the role of LDH-A in mediating Taxol
resistance in human breast cancer cells, the expression
of LDH-A was examined in MDA-435, 435TR1 and
435TRP cells. We found that LDH-A levels were mark-
edly increased in 435TR1 and 435TRP cells, compared
to their parental MDA-435 cells (Fig. 2A). The activity
of LDH was also increased about 2-fold in Taxol-resis-
tant 435TR1 and 435TRP cells, compared to MDA-435
cells (Fig. 2B). These results indicated that Taxol resis-
tance is correlated with the increased LDH-A expression
and activity. Interestingly, treatment with Taxol resulted
in the induction of LDH-A expression in a dose-depen-
dent pattern in MDA-435 cells (Fig. 2C). We also identi-
fied that LDH activity could also be induced by Taxol in


Page 3 of 12




















MDA-435


435TR1


Phase








DAPI


MDA-435 435TR1


50,

o



0 -2'
CL*

0 01,'
U-


it It 1io' lI',


Non Taxol

--OT-------- O--.------ '

It 7 2
in ti I2




PARPl -

c-PARP m -

p-actin -----


MDA-435 435TR1 435TRP


o -- -




o k
T ao ram5 \ i(niM


SMDA435 435TR1! 135TRIP
IC50 IC50 T IC50
1 2 4 9 16 32 64 128 265 512
Taxal Treatment (nM)


Figure 1 Characterization of Taxol-resistant cells. A, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 24 hrs and their
morphology was observed under fluorescence microscope. The phase image of these cells was shown at the top and the nucleus stained by
DAPI was shown at the bottom (200 x). B, MDA-435, 435TR1 and 435TRP cells were treated with 20 nM Taxol for 48 hrs and apoptosis was
examined by flow cytometry using Annexin V/PI staining and by Cell Death Detection ELISA PLUS Kit. Fold induction value was calculated
following the formula: mU of the sample (cells treated with Taxol)/mU of the corresponding negative control (cells without Taxol treatment). C,
Taxol-resistant cells and their parental cells were treated without or with 20 nM Taxol for 48 hrs, then poly (ADP-ribose) polymerase (PARP) and
its cleaved protein (c-PARP) were analyzed by Western blotting with specific antibodies, respectively. p-actin was used as a loading control. D,
Cell viability analysis was performed to evaluate cytotoxicity of Taxol to MDA-435 and Taxol-resistant 435TR1 and 435TRP cells under treatment
with indicated concentrations of Taxol for 48 hrs


Zhou et al. Molecular Cancer 2010, 9:33
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Page 4 of 12


435TRP


10 10 10' 10t






Zhou et al. Molecular Cancer 2010, 9:33
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the Taxol-resistant cells (data not shown). To study the
mechanism that may contribute to the increased expres-
sion and activity of LDH-A, MDA-435 cells were treated
with CHX to block protein synthesis and the cells were
further treated with or without Taxol for different times,
the protein stability of LDH-A was measured by Wes-
tern blot (Fig. 2D). The result showed that LDH-A pro-
tein is more stable in Taxol treated cells than that of
untreated cells. We further compared the mRNA level
of LDH-A in Taxol-treated and -untreated cells by qRT-
PCR (Fig. 2E). The result showed that Taxol treatment
increased the mRNA expression of LDH-A. These
results suggest that both protein stability and mRNA
induction by Taxol contribute to the up-regulation of
LDH-A in these cells.

The downregulation of LDH-A re-sensitizes Taxol-resistant
cells to Taxol
The increase of LDH-A expression and LDH activity
detected in Taxol-resistant cells suggests that LDH-A
may play a critical role in Taxol resistance. Therefore,
the effect of LDH-A downregulation on the sensitivity
of Taxol was investigated. After LDH-A was downregu-
lated efficiently by specific siRNA to LDH-A (Fig. 3A),
LDH activity was decreased about 40% in MDA-435
cells and about 55% in 435TR1 cells (Fig. 3B). Since the
expression and activity of LDH-A was upregulated in
Taxol-resistant cancer cells (Fig. 2), we hypothesized
that the downregulation of LDH-A by siRNA might re-
sensitize Taxol-resistant cells to Taxol. To this end,
LDH-A was knocked down with siRNA in 435TR1 and
parental MDA-435 cells respectively, and then the cells
were treated with different concentrations of Taxol. The
downregulation of LDH-A increased the sensitivity of
these cells to Taxol, with Taxol-resistant 435TR1 cells
showing about a 3-10 fold increase in cell growth inhibi-
tion under 50-100 nM Taxol treatment measured by
both MTS assay (Fig. 3C) and direct cell counting
(Additional file 1, Figure. Sl). Interestingly, 435TR1 cells
showed a much greater overall increased sensitivity to
Taxol compared to their parental MDA-435 cells (Fig.
3C and 3D). Similar assays were performed in another
breast cancer cell line BT474 (Fig. 4A-C), where the
knockdown of LDH-A expression by siRNA increased
the sensitivity to Taxol by at least 2-fold. To further
confirm these results, MDA-231 cells with stable knock-
down of LDH-A by short-hairpin RNA (shRNA) were
used. Compared to those of control MDA-231 cells,
LDH-A expression (Fig. 4D) and LDH activity (Fig. 4E)
were dramatically decreased in LDH-A stably knock-
down cells and these cells showed a much greater over-
all increased sensitivity to Taxol (Fig. 4F). These results
demonstrated that LDH-A plays an important role in
Taxol resistance. Since LDH is a critical enzyme in the


glycolytic pathway, our results suggest that inhibition of
glycolysis may re-sensitize Taxol-resistant cells to Taxol.

The combination of Taxol with oxamate shows synergistic
inhibitory effect on breast cancer cells
Oxamate is a pyruvate analog that directly inhibits the
converting process of pyruvate to lactate by LDH, there-
fore, inhibits cell glycolysis [21]. We first examined the
effect of oxamate on LDH activity and cell viability of
MDA-435 and 435TR1 cells. Oxamate treatment led to
a decrease of LDH activity (Fig. 5A) and an inhibition of
cell viability (Fig. 5B) in a dose-dependant manner, in
both MDA-435 and 435TR1 cells. Compared to MDA-
435 cells, Taxol resistant 435TR1 cells showed a greater
sensitivity to oxamate, consistent with the results of
LDH-A knockdown by siRNA (Fig. 3). Since glycolysis
and mitochondrial oxidative phosphorylation are linked
processes [16], and we have previously shown that
LDH-A is critical in regulating glycolysis and growth of
breast cancer cells [18], we reasoned that the increased
expression and activity of LDH-A in Taxol-resistant
cells may lead to an increase of glycolysis and a decrease
of mitochondrial oxidative phosphorylation. Thus, a spe-
cific inhibitor of the mitochondrial oxidative phosphory-
lation, oligomycin was utilized to treat these cells. As
expected, Taxol-resistant 435TR1 cells were more resis-
tant to oligomycin (Additional file 2, Figure. S2). These
results further support the notion that increased Taxol
sensitivity by oxamate is a consequence of the inhibition
of cellular glycolysis.
Since downregulation of LDH-A by siRNA or oxamate
significantly inhibited the viability of the Taxol-resistant
cells, we further investigated the effects of combining
Taxol with glycolysis inhibitor oxamate on Taxol-resis-
tant breast cancer cells. In both Taxol-resistant 435TRP
and 435TR1 cells (Fig. 6A and 6B; Additional file 3, Fig-
ure. S3), and in BT474 cells (Fig. 6C), Taxol combined
with oxamate were much more effective in inhibiting
cell viability compared with either agent given alone.
Similar treatment combinations were performed in
another breast cancer cell line, MCF7, with similar
results obtained (Additional file 4, Figure. S4). Taken
together, the combination of Taxol with oxamate has a
greater capacity to inhibit Taxol-resistant cells compared
to either agent given alone.
To further investigate the mechanism of oxamate-
induced Taxol re-sensitization, we examined cellular
apoptosis in these cells. PARP, a nuclear protein that
can be easily cleaved by caspases, has been widely used
as an apoptosis marker [19,20]. The expression level of
total PARP and cleaved PARP (c-PARP) were examined
in 435TR1 cells after treatment with Taxol, oxamate, or
their combination for 48 hrs, respectively. We found a
significant increase of the levels of cleaved PARP after


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LDHA -
-111--- - -11 ........
3-actin s- 1



D
MDA 435


1-i


I-


U


LDHA


s Taxol treatment
0 1 2 4 (nM)
LDHA ---
100% 120% 140% 240%
p-actin [- '' D-43
MDA-435


UOAA"SI"STVRI -35OVRP


E


0 8 hrs 0 8 hrs
CHX CHX+Taxol
Figure 2 Increased LDH-A expression and activity in Taxol-resistant cells. A, Western blot was performed with an anti-LDH-A antibody of
total cell extract from MDA-435, 435TR1 and 435TRP cells. The p-actin protein was used as a loading control. B, LDH activity in MDA-435, 435TR1
and 435TRP were examined. C, MDA-435 cells were treated with increasing concentrations of Taxo for 24 hrs. The cell lysates were prepared
and Western blotting was carried out with antibodies against to LDH-A and p-actin. D, LDHA protein stability assay was performed in MDA-435
cells under the treatments of Taxol at 4 nM and CHX at 50 ug/ml followed by Western blotting assay to exam the protein expression level of
LDHA at 0 and 8 hrs (top). The relative intensity of LDHA band was normalized to its p-actin loading (bottom). E, LDHA mRNA level was
detected by real-time PCR under 2 nM Taxol in MDA-435 cells. The LDHA primers used for PCR are: forward, 5'-tgg agt gga atg aat gtt gc-3';
reverse: 5'-ata gcc cag gat gtg tag cc-3'. Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.


treatment with the combination of Taxol and oxamate
compared to treatment with single agent (Fig. 6D). This
indicates that cellular apoptosis is a mechanism involved
in the increased cell growth inhibitory effect of the com-
bination treatment of Taxol with oxamate.

Discussion
In this study, we investigated the role of LDH-A in the
acquired Taxol resistance in multiple human breast can-
cer cell lines. We identified that compared to Taxol-sen-
sitive cells, Taxol-resistant cells possess an increased
expression and activity of LDH-A, with its


downregulation resulting in an increased sensitivity of
Taxol resistant-cells to Taxol. In addition, compared to
Taxol-sensitive cells, Taxol-resistant cells show a higher
sensitivity to the LDH inhibitor oxamate. Furthermore,
when compared to single agent therapy, treating cells
with the combination of Taxol and oxamate show a
much stronger inhibitory effect on Taxol-resistant breast
cancer cells by promoting cellular apoptosis. These
results demonstrate that LDH-A plays an important role
in Taxol resistance and potentially it can serve as a ther-
apeutic target for overcoming Taxol resistance in
patients with breast cancer.


Page 6 of 12


8 0 8 Time (hrs)
. + Taxol: 4 nM
+ + CHX: 50 ug/ml

LDHA E
3
,6-actin -


MDA435 treated with
2 nM Taxol


0 16
Time (hrs)















MDA-435 435TR1


Q Q
L.. L
an O I,


SO15

> 125-

m 100-

S75



a 25.


35.

W 30-

S25

C 20-
O
-15.
,m


OCtr
Msi-LDHA










MDA-435 435TR1


Page 7 of 12


ECtr
Msi-LDHA


0 0
04 JI "e 0-t I
Taxol-SOnM Taxol-100nM Taxol-5nM Taxol-10nM
435TR1 MDA-435
Figure 3 Knockdown of LDH-A increases the sensitivity of Taxol-resistant 435TR1 cells to Taxol. A, MDA-435 and 435TR1 cells were
transfected with scramble siRNA (Ctr) or LDH-A siRNA. 48 hrs after siRNA transfection, cell lysates were prepared and Western blotting was
performed with antibodies against LDH-A. The p-actin protein was used as a loading control. B, LDH activity was examined from lysates of MDA-
435 and 435TR1 48 hrs after siRNA transfection. C and D, 24 hrs after siRNA transfection, MDA-435 and 435TR1 cells were seeded into 96-well
plates at the density of 8 x 10 cells per well, and treated with Taxol (5 nM and 10 nM for MDA-435, 50 nM and 100 nM for 435TR1) for 48 hrs.
Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated
without Taxol. Columns, mean of three independent experiments; bars, SE. *, P < 0.05, **, P < 0.01, ***, P < 0.001.


Taxol is a widely used chemotherapeutic agent for the
treatment of several types of cancers, including breast
cancer. Taxol resistance may result in the subsequent
recurrence and metastasis of cancer, ultimately resulting
in death. Although extensive investigations have been
done in regards to the resistance of cancer cells to
Taxol, the specific mechanisms involved are still poorly
understood. Cancer cells are different from non-neoplas-
tic cells in their metabolic properties, with normal cells
relying primarily on the process of mitochondrial oxida-
tive phosphorylation, consuming oxygen and glucose to


produce energy. In contrast, cancer cells depend mostly
upon glycolysis, the anaerobic breakdown of glucose
into the energy-storing molecule ATP, even in the pre-
sence of available oxygen [13-15,22,23]. Recently,
research endeavors have been actively tried to make use
of these unique bioenergetic properties to enhance the
therapeutic efficacy of killing cancer cells.
LDH-A is one of the main isoforms of LDH expressed
in breast tissue, catalyzing the conversion of pyruvate to
lactate [17]. We and others have previously shown that
LDH-A plays a critical role in glycolysis, growth


Zhou et al. Molecular Cancer 2010, 9:33
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LDHA -- ----

1-actin I S .... _-- ,_


EJCtr
msi-HA


10-
0*

ioi 10






Zhou et al. Molecular Cancer 2010, 9:33
http://www.molecular-cancer.com/connten/9/1/33


BT474


X




LDHA -


3-actin _- -




MDA-MB-231

D

-4,



LDHA -


P-actin w e


2100



a m-


S0-


Ctr si-LDHA


Ctr sh4


1o a2 80
Taxol treatment (nM)


It 20 40
Taxol treatment (nM)


Figure 4 Knockdown of LDH-A increases the sensitivity of breast cancer cell lines BT474 and MDA-231 to Taxol. A, BT474 cells were
transfected with scramble siRNA (Ctr) or si-LDHA. 48 hrs after siRNA transfection, cell lysates were prepared and immunoblot analyses were
carried out with antibodies against LDHA and p-actin. B, 48 hrs after siRNA transfection, cell lysates were prepared and LDHA activity was
examined. Data are shown in percentage of LDH activity relative to Ctr-transfected cells. C, 48 hrs after siRNA transfection, cells were seeded into
96-well plates at the density of 8 x 103 cells per well. 12 hrs after incubation, the cells were then treated with various concentrations of Taxol for
48 hrs. Then the cell viability was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells
treated without Taxol. D, LDHA protein expression in MDA-MB-231 (Ctr) and stable LDHA knowdown MDA-MB-231 cells (sh-LDHA) were
detected to evaluate the efficiency of LDH-A knockdown by using an LDHA antibody. p-actin was used as a loading control. E, LDH activity was
examined in MDA-MB-231 cells with and without stably knockdown of LDH-A. Data are shown in percentage of LDH activity relative to Ctr cells.
F, MDA-MB-231 cells with or without stably knockdown of LDH-A were seeded into 96-well plates at the density of 8 x 103 cels per well. 12 hrs
after incubation, the cells were treated with various concentrations of Taxol for 48 hrs. Then the cell viability was detected using a MTS reagent.
Data are presented as the percentage of viability inhibition measured in cells treated without Taxol. Columns, mean of three independent
experiments; bars, SE., P 0.05, P 0.01, ***, P < 0.001.


properties and tumor maintenance of breast cancer cells
[16,18]. Studies have shown that the LDH-A expression
in cancer cells is associated with radiosensitivity [24].
LDH-A inhibition results in increased apoptosis via ROS
production in cell with fumarate hydratase deficiency
and was viewed as a therapeutic strategy for treatment
of hereditary leiomyomatosis and renal cell cancer [25].
However, the role of LDH in Taxol resistance of cancer
cell has not been explored. In this study, we selected a
panel of Taxol-resistant cells by gradually increasing the


concentration of Taxol in the cell culture medium. We
used these, and other three breast cancer cell lines, to
study the expression and activity of LDH-A in the devel-
opment of Taxol resistance. To our knowledge, this is
the first report to provide direct evidence in support of
a role for LDH-A in acquired Taxol resistance in
human breast cancer cells.
We found that Taxol treatment resulted in the
increased LDH-A expression and activation in cancer
cells, which appears as a result of the induction of


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A


O MDA435
m435TR1
















0 8 24 32 64

Oxamate Treatment (mM)


] MDA435
m435TR1


0 8 24 32

Oxamate Treatment (mM)


Figure 5 Taxol-resistant cells are more sensitive to glycolysis inhibitor oxamate. A, MDA-435 and 435TRI cells were treated with various
concentrations of oxamate for 48 hrs, then LDH activity was detected. Data are shown as the percentage of LDH activity inhibition detected in
cells treated without oxamate. B, MDA-435 and 435TR1 were treated with various concentrations of oxamate for 48 hrs. Then the cell viability
was detected using a MTS reagent. Data are presented as the percentage of viability inhibition measured in cells treated without oxamate.
Columns, mean of three independent experiments; bars, SE. *, P < 0.05. **, P < 0.01.


LDH-A mRNA expression by Taxol. The downregula-
tion of LDH-A by LDH-A siRNA and inhibition of LDH
by oxamate led to increased sensitivity to Taxol in all
three breast cancer cell lines examined in this study.
This indicated that Taxol treatment triggers a feedfor-
ward cycle in which Taxol-induced activation of LDH
results in cancer cells better survival under Taxol treat-
ment, likely through promoting cell glycolysis. A recent
study has shown that cancer cells inhibit cytochrome c-
mediated apoptosis by a mechanism through deregu-
lated glucose metabolism [26]. Thus, the Taxol-induced
high expression and activity of LDH-A detected in
Taxol-resistant cells could be a way of adaptation of
these cells to Taxol treatment and to modulate glucose
metabolism and glycolysis to avoid apoptosis induced by
Taxol. Targeting LDH by LDH siRNA or LDH inhibitor
oxamate interrupts the feedforward cycle and renders
the re-sensitization to Taxol. These results indicate that
LDH may potentially serve as an excellent target for
overcoming Taxol resistance in human breast cancer
patients.
Up-regulation of antiapoptotic Bcl-2 family members,
such as Bcl-2 and Bcl-XL, was reported to contribute to
Taxol-induced apoptosis [27]. In addition, we previously


reported that the phosphorylation on tyrosine-15 of
Cdc2 by ErbB2 in breast cancer cells resulting a delayed
M phase entry and leading to an increased Taxol resis-
tance [11]. We found that compared to the parental
MDA435 cells, Taxol-resistant MDA435TR1 and
MDA435TRP cells express lower Bcl-2 and lower phos-
phorylation level of Cdc2 at tyrosine-15 (Additional file
5, Figure. S5). Based on the known functions of Bcl2
and Y15-Cdc2 in Taxol resistance, these results can not
explain the increased resistance in MDA435TR1 and
MDA435TRP cells. However, we found that Bcl-XL was
upregulated in Taxol-resistant cells (Additional file 5,
Figure. S5). This might be another reason in addition to
LDH-A for the increased Taxol resistance in these cells.
It will be interesting to examine the relationship
between LDH-A and Bcl-XL in these cells in our future
studies.
The differences in cytotoxicity were some what mod-
est when the LDH-A were knocked down by siRNA.
One of the reasons might be the relatively low sensitiv-
ity of MTS assay to detect cell toxicity in our experi-
ments. Another possible reason might be the relatively
low knocking down efficiency of LDH-A by the siRNA.
In addition, as far, there is no any single molecule


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A


Tax-100 Oxa-20Tax+Oxa Tax-100 Oxa-40 Tax+Oxa



BT474 Taxol plus Oxamate


Tax-200 Oxa-40 Tax+EOa Tax-200 Oxa-80 Tax+Oxa


Tax100 Oxa-20 Tax+Oxa Tax-100 Oxa-40 Tax+Oxa


PARP

c-PARP -- -


p-actin ---

435TR1


Figure 6 Combination of Taxol with oxamate shows synergistic inhibitory effects of Taxol-resistant and BT474 cells. A and B, 8 x 10
per well of 435TRP and 435TR1 cells were plated in 96-well plates and then treated with Taxol (Tax), Oxamate (Oxa) alone or Taxol plus
Oxamate (Tax+ Oxa) with the indicated concentrations for 48 hrs. Cell viability was examined by MTS assay. Data are presented as the
percentage of viability inhibition measured in cells treated without Tax and Oxa. C, BT474 cells were treated with Tax, Oxa alone or Tax plus Oxa
with the indicated concentrations for 48 hrs.. Cell viability was measured by direct cell counting. Data are presented as the percentage of
viability inhibition counted in cells treated without Tax and Oxa. D, 435TR1 cells were treated with 100 nM Taxol or/and 40 mM oxamate for 48
hrs, cell lysates were prepared and Western blotting were carried out with antibodies against total PARP (Top) or its cleaved protein c-PARP
(Middle). B-actin was used as a loading control (Bottom). Columns, mean of three independent experiments; bars, SE. ***, P < 0.001.


reported that can fully account for Taxol resistance in
breast cancer cells. Our results and previous studies sug-
gest that multiple mechanisms may contribute to Taxol
resistance and Taxol resistance may be a sum effect of
multiple mechanisms/pathways, which suggests that a
strategy of combinational therapy is needed to overcome
the resistance to Taxol. To identify the molecules that
may contribute to Taxol resistance is important for the
management of Taxol resistant breast cancer. Neverthe-
less, our study has shown that the combination of Taxol
and LDH-A inhibitor oxamate dramatically increased


the inhibitory effect on the growth of Taxol-resistant
cancer cells. This potentially can be an effective strategy
to overcome Taxol resistance.
The combination of Taxol with oxamate was found to
be more effective in killing Taxol-resistant cells, com-
pared to either Taxol or oxamate treatment alone. The
combination therapy reveals a synergistic inhibitory
effect by promoting breast cancer cell apoptosis (Fig. 6).
Apoptosis is a predominant mechanism by which cancer
chemotherapeutic agents kill cells [28]. Although oxa-
mate is capable of inhibiting cell cycle progression from


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C







O
15

0


r-
5-







Zhou et al. Molecular Cancer 2010, 9:33
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G2 to M phase [29], we report here a novel function via
inducing apoptotic cell death, with important implica-
tions in the clinical treatment of Taxol-resistant cancers,
such as breast cancer.
The origin of MDA-MB-435 cells has recently been
called into question [30,31]. However, a latest literature
indicated that current stocks of both MDA-MB-435
cells and M14 melanoma cells are in fact MDA-MB-435
breast cancer cells instead of M14 melanoma cell line
[32]. Nevertheless, we also examined three more breast
cancer cell lines, ErbB2-overexpressing BT474 and
ErbB2-low-expressing MDA-231 and MCF-7, in order
to confirm our findings from MDA-MB-435 cells.
In summary, the present study reveals that LDH-A
plays an important role in Taxol-resistance, with Taxol-
induced expression and activity of LDH-A serving as an
important mechanism for the acquired resistance of
human breast cancer cells to Taxol. This study provides
valuable information for the development of targeted
therapies capable of inhibiting key targets, such as LDH-
A. Further studies are needed to demonstrate whether
the downregulation of LDH-A mediated re-sensitization
of breast cancer cells to Taxol is indeed a consequence
of inhibition of glycolysis. Another question arises as to
whether the downregulation of other key molecules in
the glycolytic pathway may have the same effect as the
downregulation of LDH-A. In conclusion, the results of
our study highlight the importance of LDH-A in its role
in Taxol-resistance and open the door for possible ther-
apeutic interventions in patients that have developed a
resistance to Taxol.


Conclusion
LDH-A plays an important role in Taxol resistance and
inhibition of LDH-A re-sensitizes Taxol-resistant cells
to Taxol. This study provides valuable information for
the future development and use of targeted therapies,
such as oxamate, for the treatment of patients with
Taxol-resistant breast cancer.

List of abbreviations
LDH-A: Lactate dehydrogenase-A; DMEM: Dulbecco's
modified Eagle medium; c-PARP: the cleaved Poly
(ADP-ribose) polymerase; CHX: Cycloheximide.


Additional file 1: Figure S1. Knockdown of LDH-A re-sensitizes 435TR1
cell to Taxol by direct cell counting 435TR1 cells were transfected with
scramble siRNA (Ctr) or si-LDHA 24 hrs after siRNA transfection, cells
were treated with 50 nM or 100 nM Taxol for 48 hrs Cell numbers were
directly counted by Typan Blue Staining Data are presented as the
percentage of viability inhibition counted in cells treated without Taxo
Columns, mean of three independent experiments; bars, SE*, P < 005, **
P < 001 si-LDHA transfection efficiency was showed on the right panel
Click here for file
[ http'//wwwbiomedcentra com/content/supplementary/ 476-4598-9-33
51 PDF]


Additional file 2: Figure S2. Taxol-resistant cells are more resistant
to mitochondrial oxidative phosphorylation inhibitor oligomycin
MDA-435 and 435TR1 cells were seeded into 96-well plate at density of 5
x 103 cells per well 12 hrs after incubation; cells were treated with
various concentrations of oligomycin for 24 hrs Then the cell viability
was detected using a MTS reagent, and data are presented as the
percentage of viability inhibition measured in cells treated without
oligomycin Columns, mean of three independent experiments; bars, SE
""", P< 0001
Click here for file
[ http'//wwwbiomedcentra com/content/supplementary/1476-4598-9-33
S2PDF]
Additional file 3: Figure 3. Combination of Taxol with oxamate shows
synergistic inhibitory effects in Taxol-resistant cells by direct cel
counting 435TR1 and 435TRP cells were seeded in 24-well plates and
treated with Tax, Oxa alone or Tax plus Oxa with the indicated
concentrations for 48 hrs Cell numbers were mounted by Typan Blue
Staining Data are presented as the percentage of viability inhibition
counted in cells treated without Tax and Oxa Columns, mean of three
independent experiments; bars, SE ", P < 005 "*, P < 001 ""*, P < 0001
Click here for file
Shttp//www biomedcentra com/content/supplementary/1476-4598-9-33
S3PDF]
Additional file 4: Figure 54. Combination of Taxol with oxamate shows
better inhibition of MCF7 cells A, 1 x 104 per well of MCF7 cells were
plated into 96wel plate and treated with Taxol, Oxa, or Tax plus Oxa
with indicated concentrations for 48 hrs Cell viability was examined by
MTS assay Data are presented as the percentage of viability inhibition
measured in cells treated without Tax and Oxa Columns, mean of three
independent experiments; bars, SE, P < 005, **, P < 001 B, MCF7 cells
were treated with 10 nM Taxol or/and 16 mM oxamate for 48 hrs and
cell lysates were prepared for Western blotting using antibodies against
total PARP (Top) or its cleaved protein c-PARP (Middle) p-actin was used
as a loading control (Bottom)
Click here for file
[ http'//wwwbiomedcentra com/content/supplementary/1476-4598-9-33
S4PDF]
Additional file 5: Figure 55. The expression of Bcl-2, Bc-XL, Cdc2 and
phosphorylation status of Cdc2 at Tyrosine 15 MDA-435, 435TR1 and TRP
cells were collected, lysed and immunoblot analyses were carried out
with antibodies against Bc 2, Bc-XL, Cdc2 and p-Cdc2-Y15 and tubulin
Click here for file
[ http//wwwbiomedcentra com/content/supplementary/1476-4598-9-33
S5 PDF]



Acknowledgements
Ming Tan is a Vincent F Kilborn, Jr Cancer Research Scholar We thank Dr
Eddie Reed from USA Mitchell Cancer Institute for critically reading the
manuscript and Ms Amy Brown for the editorial assistance We are grateful
to the support from The Vincent F Kilborn, Jr Cancer Research Foundation
(M Tan) and The Norwegian Radiumhospitalet Legater (Project 334003, M
Tan and 0 Fodstad)

Author details
Mithell Cancer Institute, University of South Alabama, Mobile, Alabama,
USA Department of Cell Biology and Neuroscience, University of South
Alabama, Mobile, Alabama, USA Cancer Research Institute, Central South
University, Changsha, China 4 institute for Cancer Research, The Norwegian
Radium Hospital, University of Oslo, Norway Department of Biochemistry
and Molecular Biology, University of Florida, Gainesville, Florida, USA
"Ochsner Cancer Institute, Ochsner Health System, New Orleans, Louisiana,
USA

Authors' contributions
MZ designed and carried out the majority of the experiments and drafted
the manuscript YZ, YD, HL, ZL involved in experimental design and carried
out some experiments, and helped to revise the manuscript SK and JL


Page 11 of 12








Zhou et al. Molecular Cancer 2010, 9:33
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contributed the key reagents OF, AR, LO, SL helped to revise the
manuscript MT conceived the study and supervised the overall experiment
design, execution and revised the manuscript All authors read and
approved the final manuscript

Competing interests
The authors declare that they have no competing interests

Received: 15 August 2009
Accepted: 9 February 2010 Published: 9 February 2010

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doi:1 0.1186/1476-4598-9-33
Cite this article as: Zhou e ao Warburg effect in chemosensitivity:
Targeting lactate dehydrogenase-A re-sensitizes
Taxol-resistant cancer cells to Taxol. Molecular Cancer 2010 9'33


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