Targeting the Nuclear Import Receptor Kpnb1 as an Anticancer Therapeutic

15 Pages·2016·1.58 MB·English

Published OnlineFirst February 1, 2016; DOI: 10.1158/1535-7163.MCT-15-0052
Molecular
SmallMoleculeTherapeutics
Cancer
Therapeutics
Targeting the Nuclear Import Receptor Kpnb1 as
an Anticancer Therapeutic
Pauline J.van der Watt1, Alicia Chi1,Tamara Stelma1,Catherine Stowell1, Erin Strydom1,
Sarah Carden1, LiselotteAngus1, KateHadley1, Dirk Lang2,WeiWei3, MichaelJ.Birrer3,
John O.Trent4, and VirnaD. Leaner1
Abstract
Karyopherin beta 1 (Kpnb1) is a nuclear transport receptor tissue origins. Minimum effect on the proliferation of non-
thatimportscargoesintothenucleus.Recently,elevatedKpnb1 cancer cells was observed at the concentration of INI-43 that
expression was found in certain cancers and Kpnb1 silencing showed a significant cytotoxic effect on various cervical and
withsiRNAwasshowntoinducecancercelldeath.Thisstudy esophageal cancer cell lines. A rescue experiment confirmed
aimed to identify novel small molecule inhibitors of Kpnb1, that INI-43 exerted its cell killing effects, in part, by targeting
and determine their anticancer activity. An in silico screen Kpnb1. INI-43 treatment elicited a G –M cell-cycle arrest in
2
identifiedmoleculesthatpotentiallybindKpnb1andInhibitor cancercellsandinducedtheintrinsicapoptoticpathway.Intra-
ofNuclearImport-43,INI-43(3-(1H-benzimidazol-2-yl)-1-(3- peritonealadministrationofINI-43significantlyinhibitedthe
dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine)was growthofsubcutaneouslyxenograftedesophagealandcervical
investigatedfurtherasitinterferedwiththenuclearlocalization tumor cells. We propose that Kpnb1 inhibitors could have
of Kpnb1 and known Kpnb1 cargoes NFAT, NFkB, AP-1, and therapeutic potential for the treatment of cancer. Mol Cancer
NFYandinhibitedtheproliferationofcancercellsofdifferent Ther;15(4);560–73.(cid:2)2016AACR.
Introduction nucleoporins(Nups)thatcomprisetheNPC.Onceonthenucle-
oplasmic side of the NPC, the complex is dissociated by the
Karyopherin b1 (Kpnb1) is a member of the Karyopherin b binding of RanGTP to Kpnb1. The binding of RanGTP triggers
superfamily of nuclear transport proteins. There are over 20
thedissociationofthecomplexduetothefactthatRanGTPand
membersoftheKaryopherinbproteinfamily,whichcanfunction KpnashareanoverlappingbindingsiteonKpnb1.RanGTPbinds
aseitherimportorexportreceptors,mediatingeitherthenuclear withhigheraffinitytothissite,displacingKpna,andcausingthe
entryorexitofproteins(1).Kpnb1,alsoknownasImportinb,isa
cargo protein to be released (2). Upon cargo release, RanGTP-
majornuclearimportreceptorinthecellthattransportsproteins Kpnb1 translocates back into the cytoplasm where RanGTP is
containinganuclearlocalizationsignal(NLS)throughthenuclear hydrolyzedtoRanGDPandKpnb1isreleasedforanotherround
porecomplex(NPC)intothenucleus. ofnucleartransport.WhileKpnb1classicallytransportsitscargo
The classical nuclear import pathway is characterized by the
proteinsinconcertwithanadaptor,itcanfunctiononitsown,for
recognitionoftheNLSonthecargoproteinbytheKpnb1adaptor
example,inthetransportofthesterolregulatoryelement-binding
protein,Karyopherina(Kpna),alsoknownasImportina.After
protein2(SREBP-2;ref.3).
cargorecognition,KpnabindsKpnb1andthetrimericcomplex Kpnb1notonlyplaysanimportantroleinthenuclearimportof
translocates into the nucleus, via Kpnb1-interactions with the
cargoes,butalsointheregulationofmitoticprogressionwhenthe
nuclearenvelopebreaksdown.Thisishighlightedbythefactthat
ectopicoverexpressionofKpnb1resultsindistinctmitoticdefects
1DivisionofMedicalBiochemistry,DepartmentofIntegrativeBiomed- (4,5).TherightbalanceofKpnb1isthusnecessaryforcorrectcell
icalSciences,InstituteofInfectiousDiseaseandMolecularMedicine, functioning. Interestingly, recent work has shown that Kpnb1
SAMRC/UCT Gynaecological Cancer Research Centre, Faculty of expression is upregulated in certain cancers. We previously
HealthSciences,UniversityofCapeTown,CapeTown,SouthAfrica.
2Confocal and Light Microscope Imaging Facility, Department of showedthatKpnb1mRNAandproteinareexpressedatelevated
HumanBiology,FacultyofHealthSciences,UniversityofCapeTown, levelsincervicaltumorsandcervicalcancercelllines(6),andthat
CapeTown,SouthAfrica.3CenterforCancerResearch,TheGillette thepromoterismorehighlyactiveincervicalcancercellsduetoits
CenterforGynecologicOncology,MassachusettsGeneralHospital,
HarvardMedicalSchool,Boston,Massachusetts.4DepartmentofMed- activationbythecell-cycleregulator,E2F(7).Smithandcollea-
icine, J.G. Brown Cancer Center, University of Louisville, Louisville, gues (2010) found that Kpnb1 mRNA was elevated in ovarian
Kentucky. cancercelllinesandtransformedovariancells(8)andKuusisto
Corresponding Author: Virna D. Leaner, Division of Medical Biochemistry, andcolleagues(2011)alsodescribedincreasedlevelsofKpnb1in
FacultyofHealthSciences,UniversityofCapeTown,Observatory,CapeTown, severaltransformedcelllines(9).Theselinesofevidencesuggest
7925,SouthAfrica.Phone:[phone];Fax:[phone];E-mail: thattheKpnb1proteinisassociatedwithcellulartransformation
[email protected] and cancer. Indeed, inhibition of Kpnb1 protein expression in
doi:10.1158/1535-7163.MCT-15-0052 cancercellsleadstoapoptosis(6),highlightingtheroleofthis
(cid:2)2016AmericanAssociationforCancerResearch. protein in cancer development. Inhibition of Kpnb1 protein
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Kpnb1asanAnticancerTarget
expressioninnoncancercells,however,hasonlyaminoreffecton pyrrolo[5,4-b]quinoxalin-2-amine)tobeaneffectiveinhibitorof
cell viability (6), pointing to a potential use for Kpnb1 as an Kpnb1-mediatednuclearimportwithanticanceractivity.
anticancertherapeutictarget(10).
Although evidence points to its potential as an anticancer Materials and Methods
target,currentlyCrm1(Exportin1)istheonlynucleartransport
Insilicoscreening
proteinthathasbeeninvestigatedasatherapeutictargetforcancer
A computational screen was performed at the Molecular
treatment.Thewell-knownnaturalCrm1inhibitor,leptomycinB
Modelling Facility of the James Graham Brown Cancer Centre,
(LMB),wasshowntopossessstrongantitumoractivityinvitrobut
UniversityofLouisville(Louisville,KY),usingarationalstructure-
waspoorlytoleratedinvivo.Thiswasproposedtobeduetoitsoff-
basedapproach,toidentifycompoundsthatbindtheoverlapping
target effects; hence several more recent selective inhibitors of Ran-andKpna2-bindingregionofKpnb1,basedontheircrystal
nuclear export (SINE) were developed and have shown great
structures (PDB codes 1ibr, 2bku, and 1qgk). A library of
potentialinpreclinicalstudies.Onesuchcompound,Selinexor,
12,662,570 unique chemical compounds in the 2010 ZINC
iscurrentlybeingevaluatedinclinicaltrials(11).Thesuccessin
drug-likedatabase(30)wasscreenedagainstthetargetsitesusing
targetingCrm1highlightsthepotentialeffectivenessoftargeting
the 1ibr structure, according to the methodology described in
nuclear transport proteins for cancer therapy. Recently, a few
(31). Compounds were ranked according to their predicted
inhibitorsofnuclearimporthavebeendescribedbutnonehave
Kpnb1-bindingaffinity.
been tested for specificity, nor have they been tested for their
anticancereffects.Smallmoleculepeptidomimeticinhibitorsof
Kpna/b-mediatedtransportwereidentifiedinaninvitroscreen; Chemicalcompounds
Forty-seven of the top-scoring compounds were purchased
however, these inhibitors have low potency and are not cell
permeable, hence no inhibition of Kpna/b-mediated nuclear from MolPort, ChemBridge, and Enamine and screened for
import could be observed in vivo (12). Peptide inhibitors that theirabilitytoblocknuclearimport,aswellastheireffecton
bind Kpna with a strong affinity have also been described, yet normalandcancercellviability.Compoundswereobtainedin
thesedonotinhibitKpnb1directly(13).Ivermectinisabroad- apowderformanddissolvedinDMSOtoastockconcentration
spectrumantiparasiticreagentrecentlyfoundtoinhibitKpna/b, of 100 mmol/L. Inhibitor of Nuclear Import-43, INI-43 (3-
but it does not appear to block import mediated by Kpnb1 (1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-
b]quinoxalin-2-amine) was obtained from Chembridge (ZINC
alone (14). Karyostatin 1A (15) and importazole (16) are the
first novel small molecule inhibitors of Kpnb1 to be identi- identification no. 20547783). Cisplatin was obtained from
fied, which inhibit RanGTP binding to Kpnb1; however, their Sigma and dissolved in 150 mmol/L NaCl. Ivermectin and
importazolewereobtainedfromSigmaanddissolvedinDMSO.
off-target effects have not yet been examined, nor have their
anticancer effects.There thus remainsaneed fortheidentifica- leptomycinBwasalsoobtainedfromSigmainmethanol:water
tionofnoveleffectiveKpnb1inhibitorsandforthesetobetested (7:3).AG1478wasobtainedfromCalbiochemanddissolvedin
DMSO.
fortheireffectsoncancercells.
ThevalueofKpnb1inhibitionasananticancerapproachliesin
Celllinesandtissueculture
thefactthatthetargetingofthisproteinresultsinthedisruptionof
AllcelllinesweremaintainedinDMEMcontaining10%FBS,
numerouscellularpathwayscriticalfortumorprogression.Thisis
100 U/mL penicillin, and 100 mg/mL streptomycin, except for
sincehostsofproteins,criticalfortumorigenesis,arerelianton
Kpnb1.DiversecancerprocessesrelyonKpnb1-mediatednuclear MCF12Abenign breastcancercells (obtainedfromtheATCC),
whichweremaintainedin50%HAMSF12and50%DMEM,
import.Forexample,arecentreviewhighlightsitsroleincontrol
supplementedwith5%FBS(Gibco),100U/mLpencillin,100
of the epithelial-to-mesenchymal transition (EMT; ref. 17).
Kpnb1hasbeenshowntobeinvolvedinthenuclearimportof mg/mLstreptomycin20ng/mLEGF(Gibco),100ng/mLCholera
Toxin(Sigma),10mg/mLinsulin(Gibco)and500ng/mLhydro-
a number of EMT-promoting proteins, including Notch (18),
Snail (19), and Smad (20). This role for Kpnb1 in promoting cortisone(Sigma),andtheovariancancercelllines,A2780,CP70,
andOVCAR3,whichweremaintainedinRPMI,containing10%
EMT suggests that it is necessary for cancer cell invasion and
FBS,100U/mLpenicillinand100mg/mLstreptomycin.KYSE30
metastasis.Indeed,thenucleartransportproteins,RanandCrm1,
andKYSE150cellswereobtainedfromDSMZ(32)andWHCO5
havebothrecentlybeenimplicatedaskeyproteinsinthemeta-
static progression of cancer (21–23). Another role for Kpnb1 and WHCO6 oesophageal carcinoma cell lines were acquired
whichhasrecentlybeenhighlightedisitsroleininflammation from Dr R. Veale (33). FG0 and DMB normal skin fibroblasts
(24).Kpnb1hasbeenshowntobeacriticalmediatorofinflam- were obtained from the Department of Medicine, UCT. Unless
mationduetoitsnuclearimportofmanyinflammatory-associ- specifiedabove,allothercelllineswereobtainedfromtheATCC
ated transcription factors, including NFkB (25), STAT family (Rockville).HeLa,SiHa,CaSki,MS751,C33A,WHCO5,SVWI38,
WI38,CCD-1068SK,FG0,WHCO6,Kyse30,andMDA-MB-231
members (26), AP1 (27), and GATA-3(28). Cancer-related
inflammation has been termed the seventh hallmark of cancer celllineswereauthenticatedbyDNAprofilingusingtheCellID
(29),andtheroleforKpnb1inthisprocesshighlightsitsregu- System(Promega).
lation of diverse cellular functions necessary for cancer
progression. Luciferaseassays
The inhibition of Kpnb1 represents a powerful approach to ToassayforNFATluciferaseactivity,30,000HeLacellswere
targetthecommunicationroutesharedbymanycancer-sustain- platedperwellin24-wellplates,andtransfectedwith50ngGFP-
ing pathways. In this study, we performed a structure-based in NFATplasmid(Addgeneplasmid#24219,giftofJerryCrabtree;
silicoscreentoidentifyinhibitorsofKpnb1,andidentifiedINI-43 ref.34),50ngNFAT-luciferase(Addgeneplasmid#10959,giftof
(3-(1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl) TorenFinkel;ref.35),and5ngpRL-TK,using0.4mLGenecellin
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vanderWattetal.
TransfectionReagent(CelticMolecularDiagnostics).TheNFAT- IC determinations
50
luciferasereporterplasmidcontainsthreetandemrepeatsofa30- Cells were plated in 96-well plates and treated with varying
bp fragment of the IL2 promoter known to bind NFAT. The concentrationsofdrugfor48hours,afterwhichMTT(Sigma)was
followingdaycellsweretreatedwiththevariouscompoundsand added,andcrystalssolubilized4hourslaterusingSolubilisation
stimulated with 100 nmol/L PMA (Sigma) and 1.3 mmol/L Reagent (10 % SLS in 0.01 mol/L HCl). Absorbencies were
ionomycin (Santa CruzBiotechnology) for 1.5or 3hours, fol- measuredat595nmthefollowingdayusingaBioTekmicroplate
lowingwhichluciferaseactivitywasmeasured. spectrophotometerandIC curvesweregeneratedusingGraph-
50
ToassayforAP-1andp65luciferaseactivities,HeLacellswere PadPrism.
plated in 24-well plates, and transfected with 50 ng of an AP1
luciferasereporter(containingfourcopiesoftheAP-1–binding GenerationofstableKpnb1-GFP–expressingcells
site;ref.36)or50ngofap65luciferasereporter(containingfive TheplasmidforoverexpressionofKpnb1(pEFIRES-Kpnb1-
copies of the p65-binding site; Promega) and 5 ng pRL-TK GFP) was generated by inserting SacI- and NotI-digested
(encodingRenillaluciferase,Promega),using0.16mLTransFectin human Kpnb1-GFP, released from pEGFP-Kpnb1 (kind gift
lipidreagent(Bio-Rad).Thefollowingdaycellsweretreatedwith fromPatriziaLavia,InstituteofMolecularBiologyandPathol-
0.5 or 1(cid:1) the IC50 concentration of INI-43, importazole, or ogy,CNRNationalResearchCouncil,Rome,Italy;ref.4),into
ivermectin for 24 hours. Three hours before reading luciferase the pEFIRES plasmid (kind gift from Yosef Shaul, Weizmann
activity,cellswerestimulatedusing500nmol/LPMA.Luciferase InstituteofScience,Israel;refs.37,38).pEFIRES-expressingGFP
activity was assayed using the Dual-Luciferase Reporter assay only was used as a control. HeLa cells were transfected with
system(Promega),accordingtothemanufacturer'sinstructions, GenecellinTransfectionReagent(CelticMolecularDiagnostics)
andluciferasereadingsweremeasuredusingtheVeritasmicro- and transfected cells were selected using Puromycin (Calbio-
plateluminometer(Promega)andnormalizedtoRenillalucifer- chem,Merck).Poolsofstablytransfectedcellsweremaintained
aseinthesameextract. in 1.5 mg/mL Puromycin.
Immunofluorescence Cellproliferationassays
HeLacellsweregrownonglasscoverslipsandtreatedwith10 ToassayforcellproliferationafterINI-43treatment,cellswere
mmol/L INI-43 for 1.5 or 3 hours. For controls, equivalent platedin96-wellplatesandtreatedwith5or10mmol/LINI-43,
volumes of DMSO were used. For p65 experiments, cells were afterwhichcellproliferationwasmonitoredevery24hoursfor4
treatedwith100nmol/LPMAfor1hourandsubsequentlyfixed days,usingMTT.
with4%paraformaldehyde.Immunofluorescenceanalysiswas
performed using 1:200 p65 antibody (sc-7151X, Santa Cruz
Cell-cycleanalysis
Biotechnology) and 1:300Cy3goatanti-rabbitsecondaryanti-
Cells were plated in 60 mm dishes and treated with the
body(JacksonImmunoResearch).ForKpnb1immunofluorence,
appropriateconcentrationofINI-43for3,6,or24hours.Cells
a1:100Kpnb1antibody(sc-11367,SantaCruzBiotechnology) wereharvestedandfixedin100%ethanol,afterwhichtheywere
wasused.Cellnucleiwerestainedwith0.5mg/mLDAPI.Images stainedwithpropidiumiodideandthecell-cycleprofilesanalyzed
were captured using a Zeiss inverted fluorescence microscope using a BD Accuri C6 flow cytometer (BD Biosciences). Data
under100(cid:1)oilimmersion.
analysiswasperformedusingModFit3.3software.
Kpnb1-GFPfluorescencemicroscopy PARPcleavageanalysis
HeLa-GFP and HeLa-Kpnb1-GFP cells were grown on glass HeLa cells were treated with either the IC or 1.5 (cid:1) IC
50 50
coverslips and treated with 10 mmol/L INI-43 for 45 minutes, concentrationofINI-43,importazole,ivermectin,orleptomycin
afterwhichcellswerefixedusing4%paraformaldehyde.Because Bforvaryingtimepoints,followingwhichcellsandcellfloaters
oftheoverlappingemissionspectraofINI-43andGFP,theINI-43 wereharvestedusingRIPAbuffer(10mmol/LTris-Cl,pH7.4,150
andGFPsignalswereseparatedusingspectralunmixing,usinga mmol/LNaCl,1%sodiumdeoxycholate,0.1%SDS,1%Triton
ZeissLSM510Metaconfocalmicroscope. X-100, 1(cid:1) complete protease inhibitor cocktail (Roche) and
Western blot analysis performed, using a rabbit anti-PARP1/2
Nuclearandcytoplasmicproteinextraction (H-250)antibody(sc-7150).
Nuclearandcytoplasmicproteinfractionationwasperformed
usingtheNE-PERNuclearProteinExtractionKit(ThermoScienti- Mitochondrialandcytoplasmicproteinextractions
fic),accordingtothemanufacturer'sinstructions.HeLaandCaSki Cellsweregrownin10cmplatesandtreatedwith10mmol/L
cellswereeithertreatedwithdifferentconcentrationsofINI-43for INI-43 for various time points. Cells were lysed in subcellular
3hours,ortransfectedwith20nmol/LcontrolsiRNA(sc-37007, fractionationbuffer(250mmol/Lsucrose,20mmol/LHEPES,10
SantaCruzBiotechnology)orKpnb1siRNA(sc-35736,SantaCruz mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L
Biotechnology)for 96hours,beforeprotein extraction. Western EGTA,1mmol/LDTTand1(cid:1)completeproteaseinhibitorcock-
blot analysis was performed using a rabbit anti-NFYA (H-209) tail;Roche),passedthrougha25-Gneedleandincubatedonice
antibody(sc-10779,SantaCruzBiotechnology).Ananti-b-tubulin for20minutes.Thenuclearpelletwasobtainedaftercentrifuga-
(H-235)antibody(sc-9104,SantaCruzBiotechnology)wasused tionat720gfor5minutes.Thesupernatantwasfurthercentri-
tocontrolforcytoplasmicproteinloadingandarabbitanti-TATA- fuged at 10,000 g and the resulting supernatant used as the
box–binding protein (TBP; N-12) antibody (sc-204, Santa Cruz cytoplasmic fraction. The pellet containing the mitochondrial
Biotechnology)tocontrolfornuclearproteinloading.Densitom- fractionwaswashedinsubcellularfractionationbufferandpassed
etrywasperformedusingImageJ. througha25-Gneedle.Themitochondrialfractionwaspelletedat
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Kpnb1asanAnticancerTarget
10,000gandresuspendedinRIPAbuffer.Westernblotanalysis Kpnb1 siRNA and cell viability monitored 5 days after
wasperformedusingamouseanti-CytochromeCantibody(BD transfection. Figure 1 shows that Kpnb1 inhibition results in a
Pharmingen) and an anti-Pereroxiredoxin-3 (PRDX-3; P1247, significantreductionincellnumberincervicalcancer,esophageal
Sigma) antibody was used as mitochondrial protein loading cancer, and transformed cell lines, while noncancer cells were
control. relativelyunaffected(Fig.1A).Kpnb1knockdowninthecancer
andnoncancercellswasconfirmedbyWesternblotanalysis(Fig.
Caspase-3/7assay 1B).TheseresultssuggestthatKpnb1expressionandactivityare
HeLacells were plated in96-wellplates and treated with10 necessaryforthesurvivalofcancercells.
mmol/L INI-43. Caspase-3/7 activity was monitored at various
timepointsusingthePromegaCaspase-Glo3/7assay,according IdentificationofpotentialKpnb1inhibitorsusingahigh-
tothemanufacturer'sinstructions.Luminescencewasmeasured throughputinsilicoscreen
using the Veritas microplate luminometer (Promega) and nor- ThekillingofcancercellsafterKpnb1inhibitionwithsiRNA
malized to OD595 readings of MTT experiments performed in presentedauniqueopportunitytoidentifyKpnb1smallmole-
parallel. cule inhibitors with potential anticancer activity. To identify
Kpnb1smallmoleculeinhibitors,astructure-basedvirtualscreen
Xenografttumormodels was performed. Virtual screening has yielded many new lead
AtoxicologystudywasperformedandrevealedthatAthymic compoundsfordifferentproteintargetsandisaprocesswhereby
nudemice(n¼6mice/group)toleratedvariousdosesofINI-43(1 librariesofsmallmoleculesarescreenedtoidentifythosestruc-
mg/kg,10mg/kg,and50mg/kg).Nosideeffectswereexperience tureswhicharemostlikelytobindadrugtarget.Thisscreenaimed
atallthedosesexaminedand50mg/kgwaschosenasthedosefor toidentifycompoundsthatcanbindtheoverlappingRan-and
furtherexperiments.Themiceliverswereexaminedattheendof Kpna2-bindingregionofKpnb1(correspondingtoaminoacids
thetreatmentandnohistologicpathologywasreported.Thisdose 331–363).ThisregionofKpnb1waspreviouslyidentifiedtobe
isalsoinlinewiththatusedforotherchemotherapeuticdrugs criticalforitsfunction,asitsdeletionresultedinaninabilityof
according totheliterature. Thetreatmentschedule isshownin Kpnb1 to transport an NLS-HSA cargo into the nucleus (2).
SupplementaryFig.S3CandS3D.WHCO6andCaSkicellswere Seventy-four potential "hit" compounds were identified and
harvested and resuspended in PBS. For tumor inoculation, numbered according to their predicted Kpnb1-binding affinity,
WHCO6 or CaSki cancer cells (5(cid:1)106 per mouse) were s.c. and47oftheavailabletop-scoringcompoundswerepurchased
implantedintothehindflanksoffemalenudemice.Oncetumors forfurtheranalysis.Astheintentionwastofindaninhibitorof
hadreachedapalpablesize(forCaSkithemicewereseparated nuclearimportwithanticanceractivity,thecompoundswerefirst
into two groups as the tumors took varying lengths of time to tested for their effect on cancer cell viability, using the cervical
reachtheappropriatesize),drugtreatmentwasinitiated,where cancer cell line CaSki as a model cell line. Cell viability as
tumor-bearingmicewererandomizedanddosedi.p.witheither measured using the MTT assay revealed that 16 compounds
vehicle(DMSO)orINI-43(50mg/kg),every2to3daysfor3(for displayed antiproliferative IC50 values of less than 50 mmol/L
WHCO6)or4(forCaSki)weeks.CaSkitumorsgrewslowerhence (SupplementaryTableS1),whichiswithintherangeoftheIC50
the longer treatment schedule. Of note, 50 mg/kg had been obtained using current chemotherapeutic drugs, for example,
chosenastheMTDinatoxicologystudy.Tumorsweremeasured cisplatin(SupplementaryTableS1).
onday0(firsttreatment)andevery3to4daysthereafter,using
calipers, and tumor volume estimated using the following for- InhibitorofNuclearImport-43(INI-43)isidentifiedasa
mula:volume¼(length(cid:1)width(cid:1)width)/2.Miceweresacrificed potentialinhibitorofKpnb1-mediatednuclearimport
3(forWHCO6)or4(forCaSki)weeksafterthecommencement The 16 compounds with cancer cell-killing ability were next
ofdrugtreatment.Day21measurementswereexcludedfromthe tested for the ability to block nuclear import using a lumines-
WHCO6mousestudyduetoonemousehavingtobesacrificed cence-basedscreeningassay.Thisassaywasbasedonthepremise
earlyonday20.Animalethicsapprovalwasobtainedfromthe thatthetranscriptionfactorNFATshuttlesbetweenthenucleus
FacultyofHealthSciencesAnimalEthicsCommittee,University andthecytoplasminaKpnb1-dependent(39)andCrm1-depen-
ofCapeTown,CapeTown,SouthAfrica(referencenumber012/ dentmanner(40).Itsnuclear-cytoplasmictransportisregulated
009). viacalciumexposure(41),whereNFATispredominantlycyto-
plasmic;however,anincreaseinintracellularcalciumlevelsleads
to its nuclear accumulation. Soderholm and colleagues (2011)
Statisticalanalysis
Foralldatacomparisons,theStudentttestwasperformedusing showed by fluorescence microscopy that inhibiting nuclear
MicrosoftExcel.APvalueof<0.05wasconsideredstatistically import via Kpnb1 prevented the nuclear accumulation of
significant. NFAT-GFP in response to stimulation with the calcium iono-
phore,ionomycin(16).WeassayedfornuclearNFATfollowing
cotreatmentwithionomycinandthecompoundsidentifiedinthe
Results
in silico screen. This was performed using an NFAT-luciferase
Kpnb1inhibitionresultsintheselectivekillingofcancercells reporterthatisactivatedbynuclearNFAT.ThephorbolesterPMA
Earlier studies performed in our laboratory showed that the wasusedtocostimulateNFAT,asithasbeenshownpreviously
inhibitionofKpnb1expressionincervicalcancercelllinesusing thatPMAandionomycinelicitsynergisticstimulationofNFAT
siRNAresultedincelldeathviaapoptosis(6).Thiseffectappeared luciferaseactivity(42).ResultsshowedthatstimulationofNFAT-
morespecifictothecervicalcancercellscomparedwithnoncancer transfected HeLa cells with PMA and ionomycin resulted in a
cells. To expand these findings, cancer, transformed and non- significantincreaseinactivationoftheNFATreporter,duetothe
cancercelllinesofdifferenttissueoriginsweretransfectedwith nuclear import of NFAT (Fig. 2B). After screening the 16
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vanderWattetal.
A 1.4
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Kpnb1 siRNA
1.2
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(cid:3) 1
a
er
olif 0.8
pr
ell 0.6
c
e
v
a(cid:3) 0.4
el
R
0.2
0
HeLa SiHa CaSki Ms751 C33A WHCO5 SVWI38 WI38 CCD-1068SK FG0
B
HeLa SiHa CaSki Ms751 C33A WHCO5 SVWI38 WI38 CCD-1068SK FG0
A A A A A A A A A A
N N N N N N N N N N
RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR RNA 1 siR
Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ Ctl si Kpnβ
Kpnβ1
β-Tubulin
Figure1.
Kpnb1siRNAresultsinsignificantlyreducedcancercellproliferation.A,MTTcellproliferationassayshowingdifferentialsensitivityofcancerandnon-cancercelllines
toKpnb1inhibitionwithsiRNA.Cervicalcancer(HeLa,SiHa,CaSki,Ms751andC33A),esophagealcancer(WHCO5),andtransformedfibroblast(SVWI38)
celllinesalldisplayedsignificantlyreducedcellproliferation5daysaftertransfectionwith20nMKpnb1siRNA((cid:3)(cid:3),P<0.01).ProliferationofWI38,CCD-1068SK,
andFG0noncancercelllines,ontheotherhand,wasunchanged.Resultsshownarethemean(cid:4)SEMofexperimentsperformedinquadruplicate(B).
WesternblotanalysisshowingeffectiveKpnb1knockdowninthecelllinesusedinA.b-tubulinwasusedasaproteinloadingcontrol.
compoundsthatshowedcellkillingeffects,3-(1H-benzimidazol- interfere with the nuclear translocation of p65 in response to
2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2- PMA treatment, as p65 has previously been shown to rely on
amine(ZINCidentificationno.20547783)emergedasapotential Kpnb1foritsnuclearimport(25).HeLacellsweretreatedwith
inhibitorofnuclearimport,hereafterreferredtoasInhibitorof INI-43 and stimulated with PMA, following which p65 local-
NuclearImport-43(INI-43).ThemolecularstructureofINI-43is ization was determined by immunofluorescence. In untreated
showninFig.2A.TreatmentwithINI-43significantlydiminished cells, p65 showed a predominantly cytoplasmic localization
NFATactivationinadose-dependentmanner(Fig.2B).INI-43 andstimulationwithPMAresultedinaclearinfluxofp65into
wastakenupwithinminutesintothecellsandinhibitionofNFAT thenucleus(Fig.3A).TreatmentwithINI-43inthepresenceof
activitywasachievedafter1.5hourataconcentrationof10and15 PMAnoticeablyblockedthenuclearentryofp65at1.5and3
mmol/L(approximately1(cid:1)and1.5(cid:1)itsIC concentration).In hourtimepoints(Fig.3A).Thecellswerecategorizedashaving
50
comparison, recently identified inhibitors of Karyopherin "predominantlycytoplasmic"or"predominantlynuclear"p65
a/b-mediated nuclear transport, ivermectin and importazole andtheratioofnucleartocytoplasmicp65plotted.INI-43was
(14,43),wereunabletoinhibitNFATactivityatthesametime shown to markedly influence p65 nuclear translocation (Fig.
pointandconcentrations(Fig.2B),implicatingINI-43asamore 3B),supportingitspotentialasaninhibitorofnuclearimport
potentinhibitorofnuclearimport.Aftertreatmentfor3hours, via Kpnb1.
however,importazoleandivermectin,likeINI-43,wereableto Luciferase assays were next performed to assay for p65
significantlyinhibitNFATactivityattheirrespectiveIC or1.5(cid:1) nuclear activity after PMA stimulation and INI-43 treatment,
50
IC50concentrations(IC valuesforimportazoleandivermectin andPMA-inducedp65nuclearactivitywasmarkedlyreduced
50
in HeLa cells were calculated to be 25.3 and 17.8 mmol/L, in response to overnight INI-43 treatment at 0.5X IC50 and
respectively; higher than the IC for INI-43 in HeLa cells of IX IC50 concentrations, as indicated by significantly reduced
50
9.3mmol/L; Fig. 2C–E).As a negative control, NFAT activation luciferaseactivity(Fig.3C).Theinhibitionachievedwassim-
wasmeasuredaftera3-hourtreatmentwiththeEGFRinhibitor, ilar to that obtained using importazole and ivermectin
AG1478,atitsIC and1.5(cid:1)IC concentrations,andnochange atconcentrationsrelativetotheirrespectiveIC50s.AP-1lucif-
50 50
inNFATactivationwasobserved(Fig.2F). eraseactivitywasalsomeasuredinresponsetoPMAstimula-
tion and INI-43 treatment, since this transcription factor
INI-43preventstranscriptionfactorandKpnb1accesstothe has also been shown to rely on Kpnb1 for nuclear import
nucleus (25,27). AP-1 luciferase activity was also found to be signif-
As an additional indicator that INI-43 was able to inhibit icantly decreased after INI-43, importazole and ivermectin
nuclearimport,itwasnextinvestigatedwhetherINI-43could treatment (Fig. 3D).
564 MolCancerTher;15(4)April2016 MolecularCancerTherapeutics
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Kpnb1asanAnticancerTarget
A B
2.5
Uns(cid:3)mulated
DMSO
N 2
iFInNihgI-iub4ri3etoi2sr.oidfeKnptnifibe1d.Aa,sstarupcottuernetoiaflINI- N N NH2 AT ac(cid:2)vity 1.5 * 1105 μμmmooll//LL +PMA/ionomycin
43(3-(1H-benzimidazol-2-yl)-1-(3- N NF *
dimethylaminopropyl)pyrrolo[5,4- N ve 1
b]quinoxalin-2-amine).B,effectof HN a(cid:2)
INI-43andotherKpnb1inhibitorson Rel
0.5
NFATpromoteractivation.HeLacells
weretransfectedwithNFATreporter
andexpressionplasmidsand 0
incubatedinthepresenceofPMA INI-43 Importazole Ivermec(cid:3)n
andionomycinfor1.5hours,withor
without10or15mmol/LINI-43, C D
importazoleorivermectin.Results 2.5 2.5
* *
showasignificantinductionofNFAT
* *
pPrinerMcdorAumec/aotisoitoiennnrogmainIcNytaIci-vci4nitti3yvaacnitnodentdacheeNsniFgptArnraeTifitsiecowanninctshteof AT ac(cid:2)vity 1.25 AT ac(cid:2)vity 1.25
((cid:3),P<0.05),butnotimportazole NF NF
osfotrirmiv3uehlraeotmuedrescwitniintth.hCeP–MpEAr,ecsaeenllndscweioenororemycin Rela(cid:2)ve 0.15 Rela(cid:2)ve 0.15
absenceofINI-43,importazole,or
ivermectinattheirrespectiveIC50or
1.5(cid:1)IC50concentrations.Allthree 0 0
inhibitorswereabletosignificantly 0 0 10 15 μmol/L INI-43 0 0 25 40μmol/L importazole
decreaseNFATtranscriptional +PMA/ionomycin +PMA/ionomycin
activation((cid:3),P<0.05).F,as E F
anegativecontrol,NFAT 4.5 4
transcriptionalactivitywas *
4 3.5
measuredinthepresenceof
PMA,ionomycin,andEGFR vity 3.5 vity 3
inhibitorAG1478for3hours, (cid:2) 3 (cid:2)
panrodmnootesrigancifiticvaantitoenffweactsoonbsNeFrvAeTd. FAT ac 2.5 FAT ac 2.25
Resultsshownarerepresentative ve N 2 ve N 1.5
ofexperimentsperformedin (cid:2) 1.5 (cid:2)
a a
quadruplicateandrepeatedatleast Rel 1 Rel 1
twoindependenttimes.
0.5 0.5
0 0
0 0 20 30 μmol/L Ivermec(cid:3)n 0 0 40 60 μmol/L AG1478
+PMA/ionomycin +PMA/ionomycin
TheabilityofINI-43toblockthenuclearimportfunctionof proteinfractionwasnotconcentratedenoughtodetectNFY-A
Kpnb1 was tested further by investigating the localization of protein.
anotherKpnb1cargo,NFY-A(44),bysubcellularfractionation To address the question of whether the inhibitory effect of
and Western blot analysis. Cells were treated with INI-43, INI-43 on nuclear import associated with Kpnb1, we investi-
followingwhichnuclearandcytoplasmicproteinfractionswere gated the localization of Kpnb1 itself in control and INI-43–
isolated. Western blot analysis was then performed to deter- treatedcells.WefoundthattreatmentofHeLacellswithINI-43
minethelocalizationofNFY-A,andNFY-Alevelswerefoundto resulted in altered subcellular localization of endogenous
decrease in the nuclear fraction after 3-hour INI-43 treatment Kpnb1. In untreated cells, endogenous Kpnb1 predominantly
(Fig.3E).ToverifythatthebandingpatternobservedafterINI- localized to the nucleus (Fig. 4A). Treatment with INI-43
43treatmentwasasexpectedafterinhibitionofKpnb1,Kpnb1 resulted in a change in Kpnb1 subcellular localization to
wassilencedusingsiRNA,andnuclearandcytoplasmicprotein cytoplasmicandperinuclearwithin45minutesofexposureto
fractions isolated for Western blot analysis. Similar results as the compound, suggesting that the compound acts via inter-
those obtained with INI-43 treatment were found where the fering with Kpnb1. To further support this result, a HeLa cell
amounts ofNFY-AweredecreasedinthenucleusafterKpnb1 line stably overexpressing Kpnb1-GFP was generated, termed
inhibition using siRNA (Fig. 3F). In both cases of INI-43 HeLa-Kpnb1-GFP,aswellasacontrolcelllineoverexpressing
treatment and Kpnb1 siRNA treatment, NFY-A could not be EGFP alone, termed HeLa-GFP. The HeLa-Kpnb1-GFP cells
observed in the cytoplasm. It is possible that the cytoplasmic expressed the Kpnb1-GFP at equivalent levels to that of
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vanderWattetal.
A p65-Cy3 DAPI Merged B
10 μmol/L
INI-43 PMA
Cytoplasmic
120
− − Nuclear
100
%) 80
5 (
− + yt p6 60
C
/
uc 40
N
1.5 hr + 20
0
− + + + PMA
1.5 hour 3 hour
3 hr + INI-43 INI-43
C D
Uns(cid:3)mulated
Uns(cid:3)mulated
c(cid:2)vity 6700 D01 .MX5 ISXCO I5C050 +PMA ac(cid:2)vity 220500 D01 .MX5 ISXCO I5C050 +PMA
p65 luciferase a 23450000 AP-1 luciferase 110500 *
ve ve 50
a(cid:2) 10 a(cid:2)
el el
R 0 R 0
INI-43 importazole Ivermec(cid:3)n INI-43 importazole Ivermec(cid:3)n
E F
Whole cell Cytoplasm Nucleus
Whole cell Cytoplasmic Nuclear
0 10 15 0 10 15 0 10 15 μmol/L INI-43 A A A
N N N
NFY Ctl siRNA Kpnβ1 siR Ctl siRNA Kpnβ1 siR Ctl siRNA Kpnβ1 siR
TBP
NFY
β-tubulin
TBP
1.2
ar protein (cid:2)ve to TBP 00..681 Kpnβ1
NFY nucleevels rela 00..240 β-Tubulin
l 0 10 15
Conc. INI-43 (μmol/L)
Figure3.
INI-43interfereswiththenuclearentryofendogenousKpnb1cargoes.A,immunofluorescenceanalysisshowingpredominantlycytoplasmicp65inunstimulatedcells
andnuclearp65inPMA-stimulatedHeLacells.Treatmentwith10mmol/LINI-43for1.5and3hourspreventsp65fromenteringthenucleusinresponse
toPMA.B,quantitationofnuclearandcytoplasmicp65fluorescenceinapproximately200cellspercondition.Cellswerescoredashavingpredominantly
cytoplasmicorpredominantlynuclearp65fluorescence.CandD,p65(C)andAP-1(D)promoter-drivenluciferaseactivityincreasesinresponsetoPMA
treatmentandthisincreasecanbesignificantlydampenedinthepresenceofovernighttreatmentofHeLacellswithKpnb1inhibitorsINI-43,importazole,and
ivermectinat0.5(cid:1)and1(cid:1)theirrespectiveIC50concentrations((cid:3),P<0.05).Resultsshownarethemean(cid:4)SEMofexperimentsperformedinquadruplicate
(E)WesternblotanalysesshowingreducedNFYnuclearlocalizationinfractionatedproteinlysatesofCaSkicellstreatedwith10mmol/LINI-43for3hours.b-tubulin
wasusedasacytoplasmicloadingcontrolandTBPasanuclearloadingcontrol.QuantificationofNFYnuclearproteinlevelsinuntreatedandINI-43–treated
cellsisshownrelativetoTBPinthesameextract.F,WesternblotanalysesshowinginhibitionofKpnb1expressionusingsiRNAandaresultantsimilardecrease
inNFYnuclearlocalizationasthatobservedusingINI-43.
566 MolCancerTher;15(4)April2016 MolecularCancerTherapeutics
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Kpnb1asanAnticancerTarget
P P
F F
G G
A B 1- C 1-
β β
INI-43 Kpnβ1-Cy3 DAPI Merged FP pn FP pn
G K G K
a- a- a- a-
L L L L
e e e e
H H H H
−
Kpnβ1-GFP
Kpnβ1 GFP
GAPDH
+
GAPDH
D INI-43 EGFP Kpnb1-GFP E p65-Cy3 DAPI Merged
INI-43 PMA
− HeLa − −
HeLa- − −
GFP
+
HeLa-
KpnB1- − −
GFP
F p65-Cy3 DAPI Merged G p65-Cy3 DAPI Merged
INI-43 PMA INI-43 PMA
HeLa − + + +
HGeFPLa - − + + +
HeLa-
KpnB1- − + + +
GFP
HeLa-GFP HeLa-Kpnβ1-GFP
H 120 Cytoplasmic I 120 Cytoplasmic
Nuclear Nuclear
100 100
Nuc/Cy p65 (%) 468000 Nuc/Cy p65 (%) 468000
20 20
0 0
− + + PMA − + + PMA
3 hour INI-43 3 hour INI-43
Figure4.
A,INI-43interfereswiththenuclearentryofendogenousKpnb1.ImmunofluorescenceanalysisshowingthatINI-43treatmentaltersKpnb1subcellularlocalization.
HeLacellsweretreatedwith5mmol/LINI-43for45minutesandfluorescentimagescapturedusingaconfocalmicroscope.Kpnb1localizationaltered
frompredominantlynucleartocytoplasmicandperinuclear.BandC,WesternblotanalysisofKpnb1(B)andGFP(C)expressioninHeLacellsconfirmingthe
establishmentofcellsstablyexpressingGFP(HeLa-GFP)orKpnb1-GFP(HeLa-Kpnb1-GFP).GAPDHwasusedtocontrolforproteinloading.D,fluorescence
microscopyshowingGFPandKpnb1-GFPlocalizationafter45minutesINI-43treatment.E,immunofluorescentanalysisofp65expressioninHeLa,Hela-EGFP,
andHeLa-Kpnb1-GFPuntreatedcellsshowspredominantlycytoplasmicp65localization.F,treatmentofHeLa,Hela-EGFP,andHeLa-Kpnb1-GFPwith
0.5mmol/LPMAresultsinnucleartranslocationofp65.G,treatmentofHeLaandHela-EGFPcellswith10mmol/LINI-43for3hourspreventsp65from
enteringthenucleusinresponsetoPMA,whereasnuclearp65isobservedinKpnb1-overexpressingHeLa-Kpnb1-GFPcells.HandI,quantitationofnuclearand
cytoplasmicp65fluorescenceinapproximately100cellspercondition.Cellswerescoredashavingpredominantlycytoplasmicorpredominantlynuclear
p65fluorescence.
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vanderWattetal.
endogenous Kpnb1. Therefore, approximately twice as much Anchorage-independent proliferation of cancer cells was
Kpnb1 was expressed in the HeLa-Kpnb1-GFP cells than the next measured in response to INI-43 treatment. Cells were
controlcells(HeLaandHeLa-GFP;Fig.4B).AGFPWesternblot grownonpolyheme-coatedplatesandcellproliferationmon-
analysis confirmed the overexpression of EGFP in the control itored 9 days after treatment. Both cervical and esophageal
cellline(Fig.4C).ThelocalizationofGFPinthesecells(HeLa- cancercellcolonyformationwasinhibitedinthepresenceof
GFP and HeLa-Kpnb1-GFP) was analyzed after INI-43 treat- 10 mmol/L INI-43 (Fig. 5G), confirming that INI-43 inhibits
mentfor45minutes.First,theINI-43fluorescencesignalhadto bothanchorage-dependentand-independentproliferationof
be excluded from the GFP signal, due to their overlapping cancer cells.
emissionspectra.Thereafter,ashiftinKpnb1-GFPlocalization Toinvestigatewhetherthecelldeath observedafterINI-43
was noted upon INI-43 treatment, where reduced nuclear treatmentwasasaresultofKpnb1inhibition,weperformeda
Kpnb1-GFP fluorescence was observed in response to INI-43, rescue experiment using the HeLa-Kpnb1-GFP cell line (Fig.
further supporting the fact that INI-43 interferes with Kpnb1 4B).AlthoughKpnb1-GFPoverexpressionresultedinamodest
nuclearentry(Fig.4D).EGFPlocalizationdidnotchangeupon but significant increase in the IC50 for INI-43 (from 8.33 in
INI-43 treatment, as expected. HeLa-GFP cells to 9.6 in HeLa- Kpnb1-GFP cells), cells over-
Wethenpostulatedthatifthechangeinsubcellularlocalization expressingKpnb1wereconsiderablymore resistant to INI-43
ofp65wasasaresultofperturbingKpnb1function,overexpres- treatment at concentrations of 5 and 10 mmol/L INI-43, and
sionofKpnb1shouldrescuetheinhibitoryeffectsofINI-43.We showedsignificantlymoreviablecellscomparedwithcontrols
therefore next performed a rescue experiment using the HeLa- (Fig. 5H). The rescue experiment therefore confirmed that
Kpnb1-GFPcells.ImmunofluorescentanalysisoftheKpnb1cargo INI-43, in part, exerted its cell killing effects by targeting
proteinp65showedapredominantcytoplasmiclocalizationin Kpnb1, as overexpression of Kpnb1 significantly rescued cell
untreatedHeLa,HeLa-GFP,andHeLa-Kpnb1-GFPcells(Fig.4E). viability.
Although stimulation of these cells with PMA resulted in the
nucleartranslocationofp65(Fig.4F),apretreatmentwithINI-43 INI-43treatmentresultsintheinductionofapoptosis
blockedthenuclearentryofp65inHeLaandHeLa-GFPcontrol The mechanism of INI-43–induced cell death was next
cells(Fig.4G).However,inHeLa-Kpnb1-GFPcellsnuclearentry investigated. Cell-cycle analysis showed that INI-43 treatment
of p65 wasrestored (Fig. 4G, bottom). Quantificationof these resultedinanincreaseinthepopulationofcellsintheG2–M
resultsisshowninFig.4Hand4I.Theseresultssuggestthatthe stageofthecellcycleat6hoursaftertreatment(Fig.6A).This
inhibitoryeffectsofINI-43onnuclearimportcanberescuedby correlates with the important role played by Kpnb1 in the
Kpnb1 overexpression, providing evidence that INI-43 acts via regulation of mitosis. The sub-G1 population of cells was
Kpnb1. significantly increased after 10 mmol/L INI-43 treatment for
24 hours and as little as 6 hours with 15 mmol/L INI-43
INI-43treatmentresultsincancercelldeathviaaG –Mcell- (Supplementary Fig. S2). The induction of a cell-cycle block
2
cyclearrestandcelldeathbyINI-43canberescuedviaKpnb1 and accumulation of a sub-G cell population suggested that
1
overexpression apoptosismightbeactivated;hencethecleavageofPARP-1was
As the focus of this study was to identify nuclear import measuredbyWesternblotanalysisasanindicatorofapoptosis.
inhibitor(s) with anticancer activity, the cell killing effect of INI-43treatment(at10mmol/L)resultedinatime-dependent
INI-43 was tested using a panel of cell lines of various tissue increaseincleavedPARP-1,fromasearlyas3hours,butmore
origins. INI-43 was found to kill cancer cell lines of cervical, visibly at 6 hours, after treatment (Fig. 6B). Treatment for 24
esophageal,ovarian,andbreastoriginwithanIC orapproxi- hourswith15mmol/Lresultedinalmostcompletecelldeath,
50
mately10mmol/L(SupplementaryTableS2).Alldose–response hencedegradationoftheloadingcontrol,GAPDH.Treatment
curves had sharp slopes, quantified by the Hill slope (Supple- of HeLa cells with the IC and 1.5 (cid:1) IC concentrations of
50 50
mentary Table S2 and Supplementary Fig. S1). INI-43 also importazole,ivermectin,andtheCrm1inhibitorleptomycinB,
showed elevated toxicity inthe cancer celllines incomparison similarly resulted in the accumulation of cleaved PARP-1,
withthenoncancerlines.Specifically,itshowedanapproximate although this was only visible at later time points (Fig. 6C).
2- to 3-fold selectivity toward cancer cell lines over noncancer This reveals that INI-43 works like other nuclear transport
mesenchymalcells. inhibitors, whereby it results in the induction of cancer cell
ToconfirmthatINI-43wasmoreeffectiveatkillingcancercells apoptosis.ToinvestigatewhetherINI-43treatmentresultedin
compared with noncancer cells, representative cancer (cervical the activation of apoptosis associated with the intrinsic mito-
and oesophageal) and noncancer cell lines, were grown in the chondrialpathway,CytochromeCsubcellularlocalizationwas
presenceofINI-43,andcellproliferationmeasuredoveraperiod investigated,asCytochromeCreleasefromthemitochondriais
of5days.ProliferationassaysshowedthatINI-43hadadramatic a characteristic feature of apoptosis mediated by the intrinsic
impactoncancercellviabilityinlessthan24hours,andwithin48 mitochondrialpathway.OurresultsshowedthatINI-43treat-
to72hourscompletecelldeathwasobservedataconcentrationof ment resulted in a decrease in mitochondrial Cytochrome C
10mmol/LINI-43(Fig.5A–D).Thiseffectwassustainedoverthe levels,inatime-dependentmannerfrom6hoursposttreatment
assay time period and there was no population of cells that (Fig.6D),suggestingthattheintrinsicmitochondrialpathway
recovered from the treatment. Noncancer fibroblast cells, how- isactivateduponINI-43treatmentandresponsibleforcancer
ever,proliferatedrelativelynormallyinthepresenceof10mmol/L celldeath.Finally,caspase-3/7activitywasmeasuredafterINI-
INI-43(Fig.5EandF).Thisresultsuggestsadifferentialsensitivity 43treatmentofHeLacells.Asignificantincreaseincaspase-3/7
ofcancerandnoncancercellstoINI-43,whereat10mmol/LINI- activitywasobservedboth6and24hoursafter10mmol/LINI-
43treatment,thecancercellsunderwent100%celldeathbutthe 43 treatment (Fig. 6E), correlating with the release of Cyto-
noncancercellswerelargelyunaffected. chrome C and cleavage of PARP-1 at these time points, and
568 MolCancerTher;15(4)April2016 MolecularCancerTherapeutics
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Kpnb1asanAnticancerTarget
A B
CaSki 1.5 HeLa
0.8
Cell prolifera(cid:3)on (OD595) 0000....4062 U510 nμ tμmrmeoaolt/elL/d LIN INI-I4-343 Cell prolifera(cid:3)on (OD595) 010...500 51U 0nμ tμmrmeoaolt/elL/d Lc 4IN3I-43
1 2 3 4 5 0 1 2 3 4 5
Time (days)
Time (days)
C KYSE30 D WHCO6
2.0 2.0
Untreated
595) 5 μmol/L INI-43 95) 5U nμtmreoalt/eLd INI-43
OD 1.5 10 μmol/L INI-43 D5 1.5 10 μmol/L INI-43
on ( n (O
a(cid:3) 1.0 (cid:3)o 1.0
Cell prolifer 0.5 ell prolifera 0.5
0.0 C 0.0
1 2 3 4 5 1 2 3 4 5
Time (days) Time (days)
DMB FG0
E F
Untreated 0.20
595) 0.3 51 0μ μmmool/lL/ LIN INI-I4-343 D595) 0.15 U5 nμtmreoalt/eLd INI-43
D O 10 μmol/L INI-43
n (O 0.2 on (
o (cid:3) 0.10
(cid:3) a
ell prolifera 0.1 Cell prolifer 0.05
C 0.0 0.00
1 2 3 4 5 0 1 2 3 4 5
Time (days) Time (days)
G H
HeLa KYSE30
1.2 HeLa-GFP
HeLa-Kpnβ1-GFP
d 1
e
at
e y
Untr abilit 0.8
vi
ell 0.6
c
NI-43 a(cid:3)ve 0.4
L I Rel
ol/ 0.2
m
μ
0 0
1 0 5 10
Concentra(cid:3)on INI-43 (μmol/L)
Figure5.
INI-43inhibitsbothanchorage-dependentand-independentcancercellproliferation.A–F,effectof5and10mmol/LconcentrationsofINI-43ontheproliferation
ofCaSki(A),HeLa(B),Kyse30(C),WHCO6(D),DMB(E),andFG0(F)cells.ProliferationwasmonitoredusingtheMTTassay.Resultsshowthatcancer
cells,CaSki,HeLa,Kyse30,andWHCO6aremoresensitivetoINI-43treatmentcomparedwithnoncancerDMBandFG0cells.Resultsshownarethemean(cid:4)SEMof
experimentsperformedinquadruplicateandrepeatedatleasttwoindependenttimes.G,anchorage-independentcolonyformationofHeLaandKYSE30
cellsgrowninthepresenceorabsenceof10mmol/LINI-43.INI-43treatmentresultsintheinabilityofHeLaandKYSE30cellstoformcolonies.H,Kpnb1
overexpressionaffectsHeLacellviabilityinresponsetoINI-43treatment.MTTassayshowingreducedviabilityofGFP-expressingcellsinresponseto48-hourINI-43
treatment,andsignificantlylesscelldeathinKpnb1-GFP–overexpressingcells((cid:3),P<0.05).Resultsshownarethemean(cid:4)SEMofexperimentsperformedin
quadruplicateandrepeatedatleastthreeindependenttimes.
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