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ORIGINALRESEARCHOpenAccessRadiosensitizationofnoradrenalinetransporter-expressingtumourcellsbyproteasomeinhibitorsandtheroleofreactiveoxygenspeciesColinRae1,MathiasTesson1,JohnWBabich2,MarieBoyd3andRobertJMairs1*AbstractBackground:Theradiopharmaceutical131I-metaiodobenzylguanidine(131I-MIBG)isusedforthetargetedradiotherapyofnoradrenalinetransporter(NAT)-expressingneuroblastoma.
Enhancementof131I-MIBG'sefficacyisachievedbycombinationwiththetopoisomeraseIinhibitortopotecan-currentlybeingevaluatedclinically.
Proteasomeactivityaffordsresistanceoftumourcellstoradiationandtopoisomeraseinhibitors.
Therefore,theproteasomeinhibitorbortezomibwasevaluatedwithrespecttoitscytotoxicpotencyasasingleagentandincombinationwith131I-MIBGandtopotecan.
Sinceelevatedlevelsofreactiveoxygenspecies(ROS)areinducedbybortezomib,theroleofROSintumourcellkillwasdeterminedfollowingtreatmentwithbortezomiborthealternativeproteasomeinhibitor,MG132.
Methods:Clonogenicassayandgrowthoftumourxenograftswereusedtoinvestigatetheeffectsofproteasomeinhibitorsaloneorincombinationwithradiationtreatment.
Synergisticinteractionsinvitrowereevaluatedbycombinationindexanalysis.
Thedependencyofproteasomeinhibitor-inducedclonogenickillonROSgenerationwasassessedusingantioxidants.
Results:Bortezomib,inthedoserange1to30nM,decreasedclonogenicsurvivalofbothSK-N-BE(2c)andUVW/NATcells,andthiswaspreventedbyantioxidants.
ItalsoactedasasensitizerinvitrowhenadministeredwithX-radiation,with131I-MIBG,orwith131I-MIBGandtopotecan.
Moreover,bortezomibenhancedthedelayofthegrowthofhumantumourxenograftsinathymicmicewhenadministeredincombinationwith131I-MIBGandtopotecan.
MG132andbortezomibhadsimilarradiosensitizingpotency,butonlybortezomib-inducedcytotoxicitywasROS-dependent.
Conclusions:Proteasomeinhibitionshowspromiseforthetreatmentofneuroblastomaincombinationwith131I-MIBGandtopotecan.
SincethecytotoxicityofMG132,unlikethatofbortezomib,wasnotROS-dependent,thelatterproteasomeinhibitormayhaveafavourabletoxicityprofileinnormaltissues.
Keywords:Bortezomib;Proteasome;131I-metaiodobenzylguanidine;Neuroblastoma;RadiosensitizerBackgroundNeuroblastoma,themostcommonsolidextra-cranialtumourinchildren,accountsforapproximately15%ofallchildhoodcancerdeaths.
Itisadiseaseofthepost-ganglionicsympatheticnervoussystemwhichcommonlyarisesintheadrenalgland.
Mostneuroblastomacellsex-pressthenoradrenalinetransporter(NAT),acharacteristicthatenablesdiagnosticimagingandtherapyusingtheradiolabellednoradrenalineanaloguemetaiodobenzylgua-nidine-123I-MIBGand131I-MIBG,respectively.
Approxi-mately95%oftumoursshowaffinityforMIBG[1].
Although131I-MIBGisassociatedwiththerapeuticsuccessintheformoflong-termremissionsandpalliation,itislikelythatformaximumefficacy,thereisarequirementforthisradiopharmaceuticaltobeadministeredincom-binationwithotherchemotherapeuticagents[2].
Wehavepreviouslydemonstratedthepotentialuseof131I-MIBGincombinationwiththetopoisomeraseIinhibitortopotecan*Correspondence:Robert.
Mairs@glasgow.
ac.
uk1RadiationOncology,InstituteofCancerSciences,UniversityofGlasgow,GarscubeEstate,GlasgowG611BD,ScotlandFulllistofauthorinformationisavailableattheendofthearticle2013Raeetal.
;licenseeSpringer.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Raeetal.
EJNMMIResearch2013,3:73http://www.
ejnmmires.
com/content/3/1/73[3,4],thepoly(ADP-ribose)polymerase(PARP)inhibitorPJ34[5]anddisulfiram[6].
Thereisgrowinginterestintargetingtheproteasomeforanti-cancertherapy.
Abnormallyhighproteasomeex-pressionandactivityareobservedinmanycancercellsandarecloselyrelatedtocellularproliferation[7].
Prote-olysisbythe26Sproteasomeisanessentialmetabolicprocesswhichregulatesthedegradationoftumoursup-pressors,transcriptionfactorsandproteinsinvolvedincellcyclecontrolaswellasmutantanddamagedpro-teins.
Inhibitionofproteasomefunctioncausestheab-normalaccumulationofmanyintracellularproteins,resultingincellcyclearrestandapoptosis[8].
Cancercellsaremoresensitivetothemodulationofproteasomeactivitythannormalcellsandproteasomein-hibitionincreasesthesensitivityofcancercellstovariousanti-canceragents[9].
Aswellashavingefficacyassingleagents,proteasomeinhibitorshavebeendemonstratedtoenhancetheanti-tumouractivityofotherdrugs,includinginhibitorsoftopoisomeraseI[10]andhistonedeacetylase[11].
Proteasomeinhibitionalsosensitizescancercellstoradiationbydown-regulationoftheDNAdamagere-sponse[12],bypreventionoftheactivationofradiation-inducednuclearfactor-κB(NF-κB)[9]andthroughcellcyclearrestintheradiosensitiveG2/Mphase[13].
BortezomibisthefirstproteasomeinhibitorapprovedbyUSFDAforthetreatmentofmultiplemyeloma.
Thisdrughasbeendemonstratedtosuppresstumourgrowthandangiogenesisinsolidtumoursincludingbreast,prostate,lung,neuroblastoma,andmesothelioma[14].
Bortezomib'ssensitizationofcancercellstoradiationtreatment[15,16]alsosuggestsitmaybesuitableforcombinationwith131I-MIBGtherapyinneuroblastomapatients.
Indeed,bortezomibhasrecentlybeenusedincombinationwith90Y-ibritumomabor153Sm-lexidronamforthetreatmentofnon-Hodgkinlymphomaormultiplemyeloma,respectively[17,18].
Inexperimentalmodelsofneuroblastoma,bortezomibhasbeenshowntoinhibitcellproliferation;increasesurvivalofhumantumourxe-nograftsinathymicmice;inhibitangiogenesis[19,20];andenhancethecytotoxicityoftopotecan[10],doce-taxel[21],andretinoids[22].
Acquireddrugresistanceisanimportantcauseofneuroblastomatreatmentfailureandrelapse[23].
Encouragingly,bortezomibisnotasubstrateformultidrugresistance-associatedproteins[19],anditinducescelldeathregardlessofp53status[20].
Furthermore,inchildren,bortezomibisassociatedwithminimalsystemictoxicity[24].
Normalcellshaverelativelylowconcentrationsofreact-iveoxygenspecies(ROS)andhighantioxidantcapacity,whereascancercellsgenerateabnormallyhighlevelsofROSduetoaberrantmetabolism[25].
Bortezomib-inducedapoptoticsignallinginculturedhumancancercellsisinitiatedbyROS,andapoptosisispreventedbyadministrationofantioxidants[26].
Bortezomib-inducedROSgenerationmayalsoberesponsibleforsomesideef-fectsassociatedwiththedrugwhichcurrentlylimititsclinicaluse.
Inparticular,peripheralneuropathy,whichcanaffectupto30%ofpatientsreceivingchemotherapy,maybeinducedbyROS[27].
Therefore,inordertomin-imisenormaltissuetoxicity,itisnecessarytocharacterizethemodeofactionofbortezomibandalternativeprote-asomeinhibitors.
Inthisstudy,wedeterminedthecapacityofbortezo-mibtoenhancethesensitivityofNAT-expressingcellstoradiotherapyintheformX-radiationor131I-MIBG.
Furthermore,triplecombinationtherapy,consistingofbortezomibwith131I-MIBG+topotecan,wasevaluatedincomparativeinvestigations.
Wealsocomparedthemechanismsofcytotoxicityofbortezomibwithadiffer-entclassofproteasomeinhibitor,MG132,withrespecttodependenceonROS-inducedcelldeath.
MethodsReagentsBortezomibwasagiftfromMilleniumPharmaceuticals(Cambridge,MA,USA),MG132waspurchasedfromSigma-Aldrich(Dorset,UK)andtopotecanfromAxxoraUKLtd.
(Nottingham,UK).
AllcellculturemediaandsupplementswerepurchasedfromLifeTechnologiesLtd.
(Paisley,UK),andallotherchemicalswerefromSigma-Aldrich(Dorset,UK).
No-carrier-added131I-MIBGwaspreparedusingasolid-phasesystemwhereintheprecursorofMIBGwasattachedtoaninsolublepolymerviathetin-arylbond[28].
CellcultureHumanneuroblastoma-derivedSK-N-BE(2c)cellswerepurchasedfromtheAmericanTypeCultureCollection(Manassas,VA,USA).
TheUVWcelllinewasderivedfromahumanglioblastoma[29].
Celllineswereauthenti-catedin-houseusingtheAmpF/STRIdentifilerkit(Ap-pliedBiosytems,Warrington,UK).
SK-N-BE(2c)cellsweremaintainedinDMEMcontaining15%(v/v)fetalcalfserum(FCS).
UVWcellsweretransfectedtoexpresstheNATgene,facilitatingtheactiveuptakeofMIBG,aspreviouslydescribed[30]andweremaintainedinMEM,containing10%(v/v)FCSand1mg/mlgeneticin.
TransfectantsweredesignatedasUWV/NAT.
ClonogenicsurvivalassayCellswereseededin25-cm2flasksat105cells/flask.
Whencultureswereinexponentialgrowthphase,mediumwasre-movedandreplacedwithfreshmediumcontainingthepro-teasomeinhibitorsbortezomiborMG132,theantioxidantsN-acetyl-L-cysteine(NAC,1mM)ortiron(4,5-Dihydroxy-1,3-benzenedisulfonicaciddisodiumsaltmonohydrate,1mM),orvariouscombinationsoftheseagents.
ThisRaeetal.
EJNMMIResearch2013,3:73Page2of10http://www.
ejnmmires.
com/content/3/1/73enabledacomparisonofNACandtironwithrespecttothecontributionofROSgenerationtothecytotoxicityoftheproteasomeinhibitors.
Cellswereincubatedwithdrugsfor24hat37Cin5%CO2.
Inseparatetreat-ments,cellswereexposedtoX-raysusinganRS225irradiator(Xstrahl,Surrey,UK)atadose-rateof1.
33Gy/min,thenincubatedfor24hat37°Cin5%CO2.
Aftertreatment,cellswereseededforclonogenicassayasprevi-ouslydescribed[3,4].
CombinationtreatmentsThecytotoxicinteractionbetweenbortezomibandradi-ationwasexaminedusingclonogenicassayandcombin-ationindexanalysis,accordingtothemethodofChouandTalalay[31].
Inthisanalysis,thetoxicityinducedbysingledrugsandscheduledcombinationsisinvestigatedusingtheequationCI=(D)1/(Dx)1+(D)2/(Dx)2,where(D)1and(D)2arethedosesofeachagentwhichinhibitx%ofcellgrowthwhenusedincombinationand(Dx)1and(Dx)2arethedosesofeachdrugwhichinhibitx%ofcolonieswhenusedassingleagents.
Initially,exponentiallygrowingcellsweretreatedwitheachagentalonetodetermineeffectivedoses.
Cellsweresubsequentlytreatedwitharangeofdosesofbortezomibandradiation,usingafixeddoseratioofbortezomibtora-diation,sothattheproportionalcontributionofeachagentinthemixtureswouldbethesameatalltreatmentintensities.
Thefixeddoseratiowasequivalentto7.
6nMbortezomib:3.
8GyX-radiation,basedontheirrespectiveIC50values.
Forcombinationsof131I-MIBGandbortezo-mib,thefixeddoseratiowas7.
6nMbortezomib:1.
5MBq131I-MIBG.
Forthepurposesofcombinationindexana-lysis,simultaneoustreatmentwith{131I-MIBG+topote-can}wasconsideredasoneagent,andthefixeddoseratiowas7.
6nM:0.
5MBq:4.
9nM(bortezomib:131I-MIBG:topo-tecan),asthisdosekilled50%ofclonogenswhenadminis-teredsimultaneouslyincombination.
Threedifferenttreatmentscheduleswereassessed:bortezomibgiven24hbefore,afterorsimultaneouslywithradiation.
Theeffect-ivenessofcombinationsofbortezomibandradiationwasquantifiedbydeterminingacombinationindex(CI)atvariouslevelsofcytotoxicity.
CI1indi-catesynergism,additivityandantagonism,respectively.
TumourxenograftsSix-week-oldfemale,congenitallyathymicmiceofstrainCD1nu/nuwereobtainedfromCharlesRiverplc(Kent,UK).
InvivoexperimentswerecarriedoutinaccordancewiththeAnimals(ScientificProcedures)Act1986.
Tu-moursinathymicmiceformedfromSK-N-BE(2c)andUVW/NATcellsexpresstheNATenablingactiveuptakeof131I-MIBG.
Subcutaneoustumourgrowthwasestab-lishedaspreviouslydescribed[3].
Micewereusedforexperimentaltherapywhenthetumourvolumeshadreachedapproximately100mm3.
Tomonitorpotentialtoxicity,experimentalanimalswereexamineddailyforsignsofdistressandweighedweekly.
Micewereran-domizedintotreatmentgroups,eachconsistingofsixanimalsthatreceived:PBSsolution;0.
8mg/kgbortezo-mibsolution;simultaneousadministrationof131I-MIBG(18or5MBqforSK-N-BE(2c)orUVW/NAT,respect-ively)andtopotecan(1.
75or0.
875mg/kgforSK-N-BE(2c)orUVW/NAT,respectively);oradministrationofbortezomib24hafter{131I-MIBG+topotecan}-allbyi.
pinjection.
Theindicatedactivitiesof131I-MIBGadminis-teredtothemicewereshownpreviouslybyustoinducesignificantdelayofgrowthbutincompletesterilizationofSK-N-BE(2c)andUVW/NATxenografts,andthesimultan-eousadministrationof131I-MIBG+topotecanwasdemon-stratedtobethemosteffectiveschedule[3].
Bortezomibdoses(0.
5to1mg/kg)weresimilartothoseusedinprevi-ouspreclinicalstudies[9].
Tumoursweremeasuredwithcallipersimmediatelybeforetreatmentandtwiceweeklythereafter.
Ontheassumptionofellipsoidalgeometry,diametermeasurementswereconvertedtoanapproximatetumourvolumebymultiplyinghalfthelongestdiameterbythesquareofthemeanofthetwoshorterdiameters.
StatisticsDataarepresentedasmeans±standarderrorofthemean(SEM),unlessotherwisestated,withthenumberofindependentrepetitionsprovidedinthelegendtoeachfigure.
StatisticalsignificancewasdeterminedusingStudent'sttest.
APvalue1indicatepartialantagonism.
Administrationschedulesweresimultaneous,bortezomib24hbeforeX-radiation(BZbefore)andbortezomib24hafterX-radiation(BZafter).
Itali-cisedvaluesindicatesynergy.
Raeetal.
EJNMMIResearch2013,3:73Page4of10http://www.
ejnmmires.
com/content/3/1/73131I-MIBGhadanantagonisticeffect(CI>1)uponthetoxicitytobothcelllinesatallcombinationdoses.
Three-waycombinationtreatmentconsistedofbortezo-miband{131I-MIBG+topotecan}-thelattertwoagentsbeinggivensimultaneously[3].
Supra-additiveclonogeniccellkillwasobservedonlywhenbortezomibwasadminis-tered24hafter{131I-MIBG+topotecan}(Table3).
BortezomibenhancestumourgrowthdelayInvitroexperimentalresultsindicatednoenhancementoftumourcellkillbyschedulingbortezomib24hbefore{131I-MIBG+topotecan}orsimultaneousadministrationofthecomponentsofthetriplecombination.
Therefore,inordertoreducethenumberofexperimentalanimals,thelatterscheduleswerenotadministeredtoathymicmicebearingxenografts.
Noneoftheanimalsinthisstudyshowedsignofdistress.
TheeffectsofagentsadministeredaloneorincombinationonthegrowthinathymicmiceofxenograftsderivedfromSK-N-BE(2c)andUVW/NATcellsareshowninFigure2.
BortezomibaloneinducedaslightdelayinthegrowthofSK-N-BE(2c)tumours.
Thetimetakentoincreasetumourvolumefivefold(τ5)was14.
7days,comparedtountreatedcontrolτ5of12.
3days.
Simultan-eousadministrationof{131I-MIBG+topotecan}inducedasimilardelayinthegrowthofSK-N-BE(2c)xenografts(τ5=15.
8days).
However,bortezomibadministered24hafter{131I-MIBG+topotecan}resultedinenhancedtumourgrowthdelay,manifestbyaτ5valueof28.
5days.
InxenograftsderivedfromUVW/NATcells,bortezomibalonehadnoeffectongrowthrate,exemplifiedbyτ5valuesof16.
0and15.
9daysforuntreatedcontrolgroupsandbortezomib,respectively.
Incontrast,simultaneousadministrationof131I-MIBGandtopotecaninducedanen-hancementofgrowthdelay(τ5=18.
2days)comparedwithPBS-treatedcontrols.
Bortezomibadministered24hafter{131I-MIBG+topotecan}resultedinafailurebytumourstoachieveafivefoldincreaseinvolumeover42days.
Therefore,inxenograftsderivedfromeitherSK-N-BE(2c)orUVW/NATcells,thetriplecombination,consistingofbortezomibadministered24hafter{131I-MIBG+topote-can},inducedsignificantlygreatergrowthdelaythanborte-zomibaloneorthe131I-MIBG+topotecancombination.
Bortezomib-inducedclonogenickillisROS-dependentThemechanismofbortezomib-inducedclonogeniccellkillwasinvestigatedbydeterminingtheprotectionaffordedbytreatmentwithantioxidants.
TheresultsareshowninFigure3.
InbothSK-N-BE(2c)andUVW/NATcells,themagnitudeofbortezomib-inducedclonogeniccellkillwasdiminishedbyNAC.
Forexample,exposureto10nMbor-tezomibreducedclonogenicsurvivalto20%(SK-N-BE(2c)cells)or49%(UVW/NATcells)ofcontrolvalues,whereasinthepresenceof1mMNAC,thecorrespondingvalueswere83%and93%,respectively.
Thissuggeststhatasig-nificantproportionofthecytotoxicityinducedbybortezo-mibasasingleagentwasduetoROS.
AnalternativeROSscavenger,tiron,completelyblockedbortezomib-inducedcytotoxicityatallconcentrations(Figure3),suggestingadifferentmechanismofactionofNACandtironoradif-ferentdegreeofnullificationofROS.
RadiosensitizationbyMG132isnotROS-dependentMG132,analternativeinhibitorofproteasomeactivity,mayhaveapharmacologicprofiledifferentfromthatofTable2Synergismanalysisofvariousschedulesofadministrationofbortezomiband131I-MIBGEffectlevelCombinationindex*Combinationindex*SK-N-BE(2c)cellsUVW/NATcellsSimultaneousBZbeforeBZafterSimultaneousBZbeforeBZafterED250.
821.
340.
730.
561.
140.
40ED500.
651.
300.
630.
701.
110.
49ED750.
942.
260.
961.
481.
861.
08Asterisk'*'indicatesthatthecombinationindexvaluesaremeansoffourexperiments.
Administrationschedulesweresimultaneous,bortezomib24hbefore131I-MIBG(BZbefore)andbortezomib24hafter131I-MIBG(BZafter).
Italicisedvaluesindicatesynergy.
Table3Synergismanalysisofvariousschedulesofadministrationofbortezomib{131I-MIBG+topotecan}EffectlevelCombinationindex*Combinationindex*SK-N-BE(2c)cellsUVW/NATcellsSimultaneousBZbeforeBZafterSimultaneousBZbeforeBZafterED251.
461.
420.
641.
301.
710.
77ED501.
541.
700.
631.
442.
020.
77ED751.
632.
040.
631.
602.
410.
79Asterisk'*'indicatesthatthecombinationindexvaluesaremeansoffourexperiments.
Administrationschedulesweresimultaneous,bortezomib24hbefore{131I-MIBG+topotecan}(BZbefore)andbortezomib24hafter{131I-MIBG+topotecan}(BZafter).
Italicisedvaluesindicatesynergy.
Raeetal.
EJNMMIResearch2013,3:73Page5of10http://www.
ejnmmires.
com/content/3/1/73bortezomib.
SingleagenttreatmentwithMG132causedaconcentration-dependentreductioninthesurvivalofSK-N-BE(2c)andUVW/NATclonogens(Figure4A).
MG132alsosensitizedbothcelllinestoradiationtreatment(Figure4B,C).
TheIC50valuesobtainedforSK-N-BE(2c)cellsfollowingX-irradiationaloneorwithsimultaneousad-ministrationof150or200nMMG132were3.
72±0.
17,2.
70±0.
18and2.
15±0.
09Gy,respectively.
Forirradi-atedUVW/NATcells,thecorrespondingIC50valueswere4.
23±0.
16,2.
36±0.
09and2.
00±0.
20Gy.
Theseresultsindicatedoseenhancementratiosatthe50%levelofcellkill(DER50),inSK-N-BE(2c)cellsandUVW/NATcells,respectively,of1.
38and1.
79for150nMMG132,and1.
73and2.
12for200nMMG132.
TheseDER50valuesarecomparabletothoseobtainedfollowingtreatmentwithbortezomib,whichrangedfrom1.
32to1.
55.
Thissuggeststhatalthoughthetwoagentshadasimilareffect,theconcentrationsrequireddifferedbyapproximately40-fold.
ThisdifferenceinpotencybetweenbortezomibandMG132haspreviouslybeenreported[13,32,33],thoughnotfullyexplained.
Thedose-dependentkillofSK-N-BE(2c)orUVW/NATclonogensobservedfollowingtreatmentwithMG132wasnotsignificantlyalteredbysimultaneoustreatmentwiththeantioxidantsNACortiron(Figure5).
Therefore,incontrasttotheclonogeniccellkillresultingfrombortezomibtreatment,MG132-inducedkillwasnotmediatedbyROS.
Thissuggeststhatalthoughborte-zomibandMG132bothtargettheproteasome,bothin-duceclonogenickillassingleagentsandbothsensitize020040060080005101520253035Averagetumorvolume(mm3)Averagetumorvolume(mm3)Timeaftertreatment(days)PBSBZM/TBZ+M/TASK-N-BE(2c)B051015202530354045Timeaftertreatment(days)0200400600800PBSBZM/TBZ+M/TUVW/NATFigure2Effectofbortezomibaloneorcombinedwith131I-MIBG/topotecanonthegrowthdelayofexperimentaltumours.
Growthofhumantumourxenograftsderivedfrom(A)SK-N-BE(2c)cellsor(B)UVW/NATcellsinathymicmiceexposedtoPBS,bortezomibalone(0.
8mg/kg),131I-MIBG(18or5MBqforSK-N-BE(2c)orUVW/NAT,respectively)+topotecan(1.
75or0.
875mg/kgforSK-N-BE(2c)orUVW/NAT,respectively)orthecombinationofbortezomibwith{131I-MIBG+topotecan}.
Dataareexpressedasmeantumourvolumeateverytimepoint(mm3)±SD.
Ab-breviations:BZ,bortezomib;M/T,131I-MIBGandtopotecanadministeredsimultaneously.
AControl1mMNAC1mMTiron00.
20.
40.
60.
811.
2Survivingfraction110100********Bortezomibconc.
(nM)SK-N-BE(2c)BControl1mMNAC1mMTiron00.
20.
40.
60.
811.
2Bortezomibconc.
(nM)Survivingfraction110100******UVW/NATFigure3Effectofantioxidantsonbortezomib-inducedclonogeniccellkill.
Clonogenicassayof(A)SK-N-BE(2c)and(B)UVW/NATcellsex-posedtobortezomibfor24hinthepresenceorabsenceoftheantioxidantsNAC(1mM)ortiron(1mM).
Dataaremeans±SEM,n=4;signifi-canceofdifferences:**p10nM)inSK-N-BE(2c)cells,whereinthetoxicitywasonlypartiallyreversedbyNAC.
Mechanistically,MG132-inducedcytotoxicitymaybecausedbyactivationofthemitochondria-dependentcas-pasecascade,accumulationofpro-apoptoticproteins,suppressionofNF-κBactivationandcellcyclearrestinG2/M[33,36,42].
Therefore,althoughMG132wastoxictocancercellsandenhancedradiation-inducedcellkillinamannersimilartobortezomib,MG132mayhaveare-ducedlikelihoodofROS-relatedsideeffects.
00.
20.
40.
60.
811.
2101001000MG132conc.
(nM)SurvivingfractionA****************************Control1mMNAC1mMTironSK-N-BE(2c)BControl1mMNAC1mMTiron00.
20.
40.
60.
811.
2101001000MG132conc.
(nM)Survivingfraction*************************UVW/NATFigure5EffectofantioxidantsonMG132-inducedclonogeniccellkill.
Clonogenicassayof(A)SK-N-BE(2c)and(B)UVW/NATcellsexposedtoMG132for24hinthepresenceorabsenceofNAC(1mM)ortiron(1mM).
Dataaremeans±SEM,n=4;significanceofdifferences:*p<0.
05,**p<0.
01fromuntreatedcontrol.
Raeetal.
EJNMMIResearch2013,3:73Page8of10http://www.
ejnmmires.
com/content/3/1/73ConclusionTheexperimentalcombinationtherapystudiesreportedhereindicatethepotentialuseofproteasomeinhibitionasameansofenhancingradiation-inducedcancercellkill,especiallywhengivenincombinationwiththeradiophar-maceutical131I-MIBGforthetargetedradiotherapyofneuroblastoma.
Bortezomibisroutinelyusedinthetreat-mentofhaematologicalmalignanciesandhasbeenre-portedtobewelltoleratedinchildren.
Althoughithasbeenshowntobeeffectiveasasingleagentinpre-clinicalmodelsofneuroblastoma,bortezomibisnotonlyminim-allyeffectiveinthetreatmentofvariousothersolidtu-moursbutalsohasbeenassociatedwithtoxicsideeffectswhenadministeredasasingleagent.
Therefore,consider-ationshouldbegiventocombinationtherapiesincludingalternativeproteasomeinhibitorswhichareexpectedtohavepreferableclinicaltoxicityprofiles.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsCRdesignedandcarriedouttheinvitroexperiments,performedtheanalysisoftheresultsanddraftedthemanuscript.
MTcarriedoutthexenograftexperimentsandperformedtheanalysisoftheresults.
MBandJWBmadeasubstantialcontributiontotheconception,designandorganisationoftheconductofthestudy.
RJMcontributedtothedesign,supervision,andpreparationofthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisworkwassupportedbygrantsfromtheNeuroblastomaSociety,MolecularInsightPharmaceuticalsInc,SportAidingMedicalResearchforKids(SPARKS),ChildrenwithCancerUKandGreatOrmondStreetHospitalChildren'sCharityandtheScottishGovernmentalChiefScientistOffice.
WethankDr.
SallyPimlottforradiopharmaceuticalsynthesis.
Authordetails1RadiationOncology,InstituteofCancerSciences,UniversityofGlasgow,GarscubeEstate,GlasgowG611BD,Scotland.
2DepartmentofRadiology,CornellUniversity,Ithaca,NY14850,USA.
3InstituteofPharmacyandBiomedicalSciences,StrathclydeUniversity,GlasgowG40RE,UK.
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