ibandronate increases the expression of the pro apoptotic gene fas by epigenetic mechanisms in tumor cells

IbandronateupregulatedFASexpressioninatimeanddose dependentmanner,whichwasresponsiblefordownregulationofcell viability/proliferation In line with the effects observed on caspase activiti

Ibandronate increases the expression of the pro-apoptotic gene FAS by epigenetic

a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Center Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria

b

Ludwig Boltzmann Cluster Oncology and Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria

c

Department of Orthopedics, SMZ-OST, Danube Hospital, Vienna, Austria

1 Introduction

The3rdgenerationofaminobisphosphonateslikealendronate,

risedronate, zoledronicacid, ibandronate and other compounds

affectboneresorptionofosteoclastsbyinhibitingisoprenylationof

thesmallGTP-binding proteinsand are thereforeusedas

anti-resorptivetreatmentofosteoporosis.Additionallytotheireffects

onbone,thereis growingevidenceforananticanceractivityof

thesedrugs [1–8] However, themechanismsinvolved in these

effectsremainpoorlyunderstood

Biochemically,aminobisphosphonatesactprincipallyby

inhi-bitingfarnesylpyrophosphate(FPP)synthase–anenzymeofthe

mevalonatepathway–therebypreventingthepost-translational

modification(prenylation)ofsmallguanosinetriphosphate

(GTP)-binding proteins (small-GTPases) that is essential for their

function Because small GTP-binding proteins modulate nearly

everycellularactivity,itis clearthatfunctionalinhibition from

membersofthisproteinfamilyinfluencesgrowthand differentia-tionofnormalcellsaswellasoftumorcells[3,5,7,9–11] Thefoundingmembersofthelargefamilyofsmall-GTPasesare thethreeRAS-proteins,HRAS,NRASandKRAS.Activationofthe RAS/RAF/MEK/ERK pathway, for instance by mutation of the involvedGTPasesortheirregulatingmembers,isresponsiblefor thedevelopmentofaplethora ofcancers[12–14] andtargeting thispathwayseemstobeapromisingstrategyintumortherapy

DNA-methyla-tion processes [17–19] Epigenetic processes include histone modifications and methylation of CpGs (cytosine-guanosine dinucleotides)ontheDNA,especiallyongenepromoters.Changes

ofthemethylationstateofgenepromotersleadtoalterationin geneexpressionpatterns.Thisinfluences cellulardifferentiation andapoptosisandthustumorformation(forreviewseeRefs.[19– 22]).DNAmethylationdependentinactivationoftumor suppres-sorgeneslikecellcycleinhibitors(e.g.CDKN1A,CDKN1B,CDKN2A, CDKN2B)andLOX(lysyloxidase)aswellofthepro-apoptoticgene FAS(TNFreceptorsuperfamily,member6)isoftenobservedduring developmentofneoplasticdiseases.Promoter CpG-hypermethyla-tion of these genes was found in colon cancers [23], prostate carcinomas [24–26], breast cancers [27–29] or hematologic malignancies[30–33].Consequently,severalDNAdemethylating

A R T I C L E I N F O

Article history:

Received 18 July 2012

Accepted 16 October 2012

Available online 24 October 2012

Keywords:

Bisphosphonates

Ibandronate

Epigenetic DNA-methylation

Small GTPases

FAS apoptosis

A B S T R A C T Thereisgrowingevidencethataminobisphosphonateslikeibandronateshowanticanceractivitybyan unknownmechanism.Biochemically,theypreventposttranslationalisoprenylationofsmallGTPases, thusinhibitingtheiractivity.Intumorcells,activatedRAS-GTPase,thefoundingmemberofthegene family,down-regulatestheexpressionofthepro-apoptoticgeneFASviaepigeneticDNA-methylationby DNMT1.WecomparedibandronatetreatmentinneoplastichumanU-2osteosarcomaandinmouse

CCL-51breast cancercellsaswell asin theimmortalizednon-neoplastic MC3T3-E1osteoblastic cells Ibandronateattenuatedcellproliferationinallcelllinestested.Intheneoplasticcellswefound up-regulationofcaspasessuggestingapoptosis.FurtherwefoundstimulationofFAS-expressionasaresult

ofepigeneticDNAdemethylationthatwasduetodown-regulationofDNMT1,whichwasrescuedby re-isoprenylation by both geranylgeranyl-pyrophosphate and farnesylpyrophosphate In contrast, ibandronatedidnotaffectFASandDNMT1expressioninMC3T3-E1non-neoplasticcells.Datasuggest thatbisphosphonatesviamodulationoftheactivityofsmall-GTPasesinduceapoptosisinneoplastic cellsbyDNA-CpG-demethylationandstimulationofFAS-expression.Inconclusiontheshownepigenetic mechanismunderlyingtheanti-neoplasticactivityoffarnesyl-transferase-inhibition,alsoexplainsthe clinicalsuccessofotherdrugs,whichtargetthispathway

ß2012ElsevierInc.Allrightsreserved

* Corresponding author at: Ludwig Boltzmann Institute of Osteology, 1st Medical

Department, Hanusch Hospital, Heinrich Collin-Str 30, A-1140 Vienna, Austria.

Tel.: +43 1 91021 86933; fax: +43 1 91021 86929.

E-mail address: franz.varga@osteologie.at (F Varga).

ContentslistsavailableatSciVerseScienceDirect

j our na l ho me p a ge : w ww e l se v i e r com / l oc a te / b i och e mph a rm

0006-2952/$ – see front matter ß 2012 Elsevier Inc All rights reserved.

to reactivate genes such as FAS which plays a key role in

immortalityofcancerstemcells[34]

IthasrecentlybeenshownthatactivatedRASpreventscellular

apoptosis by epigenetic inhibition of Fas expression through

stimulationoftheRAF/MEK/MAPK1pathwaywithsubsequentFas

promoter methylation via DNMT1

(DNA-(cytosine-5-)-methyl-transferase1),anenzymeresponsibleforCpGmethylationduring

cellreplication[17].Similarly,inosteoblasts,extracellularmatrix

(collagentypeI)preservesCpG-methylationoftheFaspromoter

viaMAPK1andDNMT1,thuspreventingapoptosisofproliferating

osteoblasts [19] Although, utmost efforts have been spent to

clarifytherelevanceandtheregulationofcytosinemethylationfor

physiologicalandpathologicaldevelopment,onlyfewprogresses

havebeenmadeuntilnow.TheinvolvementofRASandothersmall

GTP-binding proteins in bisphosphonates’ activity and the

knowledge of apoptotic effects on bone cells, also of

bispho-sphonatesofthe3rdgeneration[1,4,6,7,35–37],suggestthatthese

drugscouldmodulateCpG-methylationofgenepromoters

Here, we demonstrate that the aminobisphosphonate

iban-dronate modulates the DNA methylation status of the FAS

promoterbyinfluencing theisoprenylatepathwayinhuman

U-2osteosarcoma(OS)cellsandCCL-51cells,amurinemammary

gland tumor cell line, but not in non-neoplastic immortalized

MC3T3-E1 cells Treatment with ibandronate leads to

re-expression of FAS and to increased activity of

apoptosis-associatedcaspasesinthetumorcelllines.KnockdownofFAS

mRNAexpressionbysiRNAtechniquelargelyre-establishescell

viabilityinibandronatetreatedneoplasticU-2OScells.Ourdata

suggest that epigenetic mechanisms play a key role in the

apoptoticactivityofbisphosphonates,andpossiblymanyoftheir

effectsoncellularphysiologyincludingsystemicchangeswithin

anorganism

2 Materialsandmethods

2.1 Cellculture

MC3T3-E1cells,aclonalpre-osteoblasticcelllinederivedfrom

newbornmousecalvaria(kindlydonatedbyDr.Kumegawa,Meikai

University,DepartmentofOralAnatomy,Sakado,Japan)andthe

humanosteosarcoma cell line U-2 OS were cultured in

alpha-minimumessentialmedium(a-MEM;Biochrom,Berlin,Germany)

supplemented with50mg/mL ascorbic acid (Sigma–Aldrich, St

Louis, MO), 5% fetal calf serum (Biochrom), and 10mg/mL

gentamycin (Sigma–Aldrich) CCL-51 cells, a murine mammary

glandtumorcellline,wereculturedineagleminimumessential

medium(EMEM,Sigma–Aldrich)supplementedwith292mg/mL

L-glutamine,10%fetalcalfserumand10mg/mLgentamycin.Allcells

were cultured in humidified air under 5% CO2 at 378C For

propagation,cells weresubculturedtwice a weekusing 0.001%

pronaseE(Roche,Mannheim,Germany)and0.02%EDTAin

Ca2+-andMg2+-freephosphate-bufferedsaline(PBS)beforeachieving

confluence To prevent a potential phenotypic drift during repeated sub-cultures,thecellswerenot usedfor morethan4 weeksafterthawing.Forexperiments,cellswereseededinculture dishesatadensityof20,000/cm2asuntreatedcontrolsortreated with the indicated compounds at times and concentrations specified Ibandronate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate(FPP)were purchasedfrom Sigma-Aldrich.Ibandronate wasdissolvedin water and aliquots were frozenat208C

2.2 Cellviability/proliferation

Toassesscellmetabolicactivity,acommerciallyavailable,MTT similarassay(EZ4U;Biomedica,Vienna,Austria)wasused.Forthis purpose,thecelllineswereincubatedwithincreasing concentra-tionsofibandronate(1–50mMforMC3T3-E1andU-2OScellsor 1–200mMforCCL-51cells).Afteracomparabledoublingtimefor allthreecelllinestheassaywasperformedfollowingtheprotocol

ofthesupplier

2.3 Cellcounting Cellswereseededin24multi-wellculturedishesatadensityof 20,000/cm2 and wereeitherleft untreated(controls)or treated withibandronate,GGPPandFPPattheindicatedconcentrations for72h.Thereafter,cellsweredetachedwith0.001%pronaseEand thenumber ofviable cells wasassessed withCasycell counter (SchaerfeSystems,Germany).Eachexperimentwasperformedin quadruplicateandexperimentswerecarriedouttwice

2.4 Measurementofcaspaseactivity Caspase3/7andcaspase8activitiesweremeasuredbyusing theCaspase-Glo3/7andCaspase-Glo8assayKit(Promega,Corp., Madison, WI)following manufacturesinstructions.Briefly, after treatments,cellswerelysedandsubstratecleavagebycaspases was measured by the generated luminescent signal witha 96 multi-wellluminometer(Glomax,Promega).Eachexperimentwas performed in quintuplicate and experiments were carried out twice

2.5 IsolationofnucleicacidsandexpressionanalysisbyqRT-PCR DNAandRNAwereextractedusingaDNA/RNAIsolationKit (Qiagen,Hilden,Germany)followingmanufacturersinstructions cDNAwassynthesizedfrom0.5mgRNAusingthe1stStrandcDNA SynthesisKit(Roche)asdescribedbythesupplier.Theobtained cDNAwassubjectedtoPCRamplificationwithareal-timecycler usingFastStartSYBR-GreenMasterMix(Roche)forthegenesFas and Dnmt1 (primers are shown in Table 1 The qRT-PCR was performedwith45cyclescomposedof30sdenaturationat958C,

30s annealing atthe indicated temperature(Table1) and 30s extensionat728Cafter10minofinitialdenaturationat958C.For

Table 1

Primer sequences.

Primer for gene expression (mouse and human)

Primer for Fas promoter methylation assessment

Primer for DNMT1 chromatin immune-precipitation of the Fas promoter

(4319413E,AppliedBiosystems,ForsterCity,CA)intherespective

mastermixaccordingtothesupplierssuggestedconditions(Applied

Biosystems).AllPCRswereperformedintriplicateandexpression

wasevaluatedusingthecomparativequantitationmethod[38].For

each of the three time independent biological replicates, the

triplicate results of the qRT-PCR were averaged and this mean

valuewastreatedasasinglestatisticalunit

2.6 FASsiRNAtransfection

For FAS depletion by siRNA, U-2 OS cells were seeded at

20,000cells/cm2 in a 48-multi-well plate and transfected with

100nM of a mixture of two FASsiRNAs(SASI_Hs01 00079050,

SASI_Hs01_00079052, Sigma–Aldrich) by electroporation using

the Neon Transfection System (Invitrogen) following supplier’s

protocol.Next daycells weretreated with7.2mMibandronate

(EC50) After 72h of incubation, cell metabolic activity was

measuredwiththeEZ4Uassayasdescribedabove.Furthermoreto

controlFASmRNAandproteinknockdown,cellswereseededina

six-multi wellplate or 5cm petri dish at 20,000cells/cm2 and

transfected with the FAS siRNA as described above 96h after

transfectionmRNAorproteinwasisolatedasdescribedaboveand

belowandFASmRNAandproteinexpressionwasmeasuredby

qRT-PCRandimmune-blot,respectively

2.7 Proteinisolationandimmuneblotting

Forwholecellproteinextraction,cellswerewashedtwicewith

PBS,scrapedinSDSsamplebuffer(2%SDS,100mMb

-mercap-toethanol,125mMTris–HCl,pH6.8)heatedat958Cfor5minand

sonicatedforfurther5min.Todividecellproteinsintocytosolic

andmembranefractions,cellsweredetachedfromculturevessels

washedwithPBSandincubatedinhypotonicbuffer(10mMHEPES

pH7.8,1.5mMMgCl2,10mMKCl,0.5mMDTT,0.2mMPMSF)for

10minonice.Then,cellswerebrokenby20strokesinaDounce

homogenizatorandsuspensionswerecentrifugedat3300gfor

20minat48C.Supernatantswerecentrifugedforanother30min

at 15,000g at 48Cand the supernatants thus obtained were

saved as cytosolic fraction The pellets were dissolved in SDS

samplebufferconstitutingthemembranefraction.Proteincontent

was measured with Coomassie protein assay (Thermo Fisher

Scientific,Waltham,MA)orwithbicinchoninicacidassay(Sigma–

Aldrich)fortheSDSextracts.Forimmune-blotting30mgofprotein

extractsforRASandFASor50mgofproteinextractsforDNMT1

were fractionated on 12%, 10% or 8% SDS-PAGE, respectively

FollowingSDS-gelelectrophoresis,theproteinsweretransferredto

nitrocellulosefilters(Millipore)andblockedovernightwith10%

blockingreagent(RocheAppliedScience,Mannheim,Germany)in

TNBuffer (50mMTris, 125mM NaCl,pH8).Subsequently, the

filters were incubated for 1h at room temperature with an

antibodyagainstDNMT1(K-18,SantaCruzBiotechnology,Santa

Cruz,CA),againstFASandagainstRAS(610197and610002,BD

TransductionLaboratoriesTM)alldiluted1:200inblockingbuffer

Afterwards, filters werewashedthree times withimmune blot

washbuffer(TNbuffercontaining0.01%Tween)andincubatedfor

anadditionalhourwithananti-goatIgGHRP-labeledsecondary

antibody(Sigma–Aldrich)diluted1:160,000inblockingbufferor

withan anti-mouse IgG/anti-rabbitIgG horseradish peroxidase

(HRP)-labeledsecondaryantibody(Roche)diluted1:10,000inthe

blockingbuffer,respectively.Finally,theblotswerewashedagain

threetimeswithimmune-blotwashingbufferbeforedetectionof

lightemissionwiththeBMchemiluminescenceimmuneblotting

kit (Roche Applied Science) as described by the supplier

Chemiluminescence was measured with an image acquisition

system(Vilber-Lourmat,France)

Cell fractionation was controlled by immunoblotting using antibodiesagainstintegrin-b5(ITGB5,H-96,SantaCruz Biotech-nology,SantaCruz,CA)forthemembraneandmitogen-activated proteinkinase4(MAPK4,TransductionLaboratories,BD,Franklin Lakes,NY)forthecytosolicfraction(Suppl.Fig.3)

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.bcp.2012.10.016 2.8 Specificpromotermethylation

ToanalyzeFaspromoter5-methylcytosinelevels,appropriate fragments of thetargetedpromoter regions weregenerated by digestion of 1mg of genomic DNA with 20U of the CpG methylationinsensitiverestrictionenzymesMboII(Promega) or PstI(Fermentas,St.Leon-Rot,Germany)(forthemurineorforthe humanFasgene,respectively)for1hat378Cfromcellscultured for 24, 48 and 72h with 50mM ibandronate in the medium Subsequently,theenzymewasheatinactivatedat658Cfor20min The Faspromoterregion wasselectedasshown before [39].In brief,inthemurinecelllinestwoCpGrichregionswereanalyzed, thefirstat2.6kbandthesecondstartingat30bpupstreamfrom the Fas transcriptional start site (TSS) [17,19] In the human osteosarcoma cell line, a CpG rich region of the proximal FAS promoterregionwasanalyzed[19].Thisfragmentspannedfrom

79bpupstreamfromtheTSSinto 359bpof thefirstexon and showedanoverallCpGcontentabove50%.PstIdigestiongenerated

asuitablefragmentof942bpcoveringtheCpGrichregion.After digestion,DNAwaspurifiedusingacommerciallyavailablePCR clean-upkitfollowingthesupplier’sinstructions.Inthenextstep methylatedDNAfragmentswerecapturedusingthe‘‘MethylMiner MethylatedDNAEnrichmentKit’’(Invitrogen).Inbrief,methylated DNA was captured by methyl-CpG binding domain protein 2 (MBD2)coupledtomagneticbeadstoseparatethemodifiedfrom the non-modified DNA fractions DNA was eluted from the magnetic beads with 200mL of 2M NaCl solution as single fraction independent from the CpG methylation density and concentratedbyethanolprecipitation.Finally,themean methyla-tionstatus of thefragmentswasdeterminedbyamplifyingthe fragmentsbyquantitativereal-time PCR.Amplificationratios of thebound(methylated)DNAfractiontounbound(unmethylated) DNAfractionwerecalculated(forprimerdesignseeTable1

2.9 DNMT1chromatinimmuneprecipitation(ChIP)onFaspromoter Forthispurpose,cellsweretreatedwith50mMibandronatefor

72h.Subsequently,chromatincross-linking,celllysis,chromatin sharing, DNMT1 immuneprecipitation and DNA clean up were performed with the ChampionChip one-day kit (SABiosciences, Hilden,Germany)followingmanufacturer’sinstructions.Thereby, chromatinfromuntreatedaswellasfromibandronatetreatedcells wasincubatedovernightonarotorat48Cwith4mgofanti-DNMT1 antibody(K-18,SantaCruzBiotechnology,SantaCruz,CA)orwith

4mgofnon-immune serumasnegativecontrol Beforeimmune precipitation,1%(10mL)ofthechromatinwassavedandstoredat

48Cforfurtheruseasreference.DNMT1bindingonappropriateFAS promoterregionswasmeasuredbyqRT-PCR(forChIP-FASpromoter primersseeTable1 ForquantitationoftheqRT-PCRvalues,foreach sample, DNA signal of the DNMT1-precipitated chromatin was normalizedtotheunprecipitatedchromatin

2.10 Statisticalanalysis StatisticalanalyseswereperformedeitherwithANOVAorwith Student’st-testusingPrism4.03(GraphPadSoftware,SanDiego, CA), P0.05 was considered as significant The data of the experimentsarepresentedasmeansstandarddeviation(SD)

3 Results

3.1 Effectsofibandronateoncellmultiplicationandcaspases

activities

After72hoftreatment,ibandronateattenuatedcell

multipli-cationinallcelllinestested,althoughwithdifferenthalfmaximal

effectiveconcentrations(EC50,Fig.1A–C).UsingaMTT-likeassay

to assess cell viability/proliferation, in the bone related cells

MC3T3-E1(Fig.1A)andU-2OS(Fig.1B),ibandronateshoweda

comparable half maximal effect (EC50) with 6.8mM (95% c.i.:

6.00–7.67mM)and7.21mM(95%c.i.:5.90–8.79mM),

respective-ly,whiletheCCL-51cells(Fig.1C)werelesssensitivewithanEC50

of 14.4mM (95% c.i.: 14.0–14.9mM) Using cell counting only

approximatesimilaritieswerefoundforthethreecell-lines.This

maybeexplainedbythestrongchangesincellmorphologyafter

ibandronatetreatment,whichmakesitdifficulttodetermine,ifa

cellisstillviable(Suppl.Fig.1A–C)

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.bcp.2012.10.016

In MC3T3-E1 cells, caspase 8 and particularly caspase 3/7

activitieswerereducedafter72hofibandronate treatmentina

concentrationdependentmannershowingsignificanceat20mM

forcaspase3/7andat100mMforcaspase8(Fig.1D).Anopposite

effectwasseeninthetwotumorcelllinesanalyzed:caspase8as

wellascaspase 3/7 activitiesweresignificantly increasedafter

72hofibandronatetreatmentinhumanU-2OSosteosarcomacells

(Fig.1E)aswellasinmurineCCL-51breasttumorcells(Fig.1F)

3.2 IbandronateupregulatedFASexpressioninatimeanddose

dependentmanner,whichwasresponsiblefordownregulationofcell

viability/proliferation

In line with the effects observed on caspase activities,

ibandronatefailedtosignificantlyregulatethemRNAexpression

of the pro-apoptotic gene Fas in MC3T3-E1 cells at any time

(Fig.2A)andafter72hatanyconcentrationtested(Fig.2B).In

contrast,alreadyafter24hoftreatment,FASmRNAexpressionwas stronglyinducedinU-2OScells(Fig.2C)andafter48hinCCL-51 cells(Fig.2E).Thisstimulationwasdosedependentbutshowed significance only at concentrations of 100mM in U-2 OS cells (Fig.2D)orhigherthan20mMinCCL-51cells(Fig.2F)

To demonstrate the role of FAS in ibandronate induced apoptosis, U-2 OS cells were transfected with FAS siRNA by electroporationandcellviability/proliferationwasmeasuredwith the MTT-like assay (Fig 3A) In ibandronate treated cells, a differenceof19.2%indownregulationofcellviability/proliferation was measured between electroporation of unexposed and electroporationofexposedcells,whichsuggestthat electropora-tionmakescellsmoresensitivetoibandronate.Thisassumptionis supportedbyasimilareffectfoundbytransfectionofthecontrol siRNA.TransfectionofFASsiRNArecoveredcell viability/prolifera-tion to a large extent (87%), suggesting that a second, minor processisinvolvedindown-regulationofcell viability/prolifera-tiontoo.FASknockdownbysiRNAwascontrolledbyFASmRNA expression(Suppl.Fig.2)

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.bcp.2012.10.016 TranslationoftheFASmRNAexpressiontotheproteinlevelis demonstratedinFig.3B.ProteinextractsisolatedfromFASsiRNA transfectedcells,whichwereeitherleftuntreatedortreatedwith ibandronate for 72h, were subjected to immune-blotting and probedwithananti-FASantibody.SimilarlytomRNAregulation, whencomparedtocontrols,ibandronateupregulatedFASprotein expression FAS siRNA transfected cells, however, showed no significantdifferencetocontrols,neitheruntreatednor ibandro-natetreated

3.3 DifferentialregulationofDnmt1expressionbyibandronate SimilartoFasexpression,ibandronate failedtoaffectDnmt1 expressioninMC3T3-E1cellsatanytime(Fig.4A)andafter72hat any concentration (Fig.4B) tested However, Dnmt1 expression wasclearlydownregulatedinbothtumorcelllines.InU-2OScells

Fig 1 Ibandronate attenuated cell viability/proliferation and regulated caspases 3/7 and 8 After 3 days treatment ibandronate attenuated cell multiplication with a half maximal effect (EC50) at 6.8mM (95% c.i.: 6.00–7.67mM) in MC3T3-E1 cells (A) and 7.21mM (95% c.i.: 5.90–8.79mM) U-2 OS cells (B), while in CCL-51 a slightly higher concentration of 14.4mM (95% c.i.: 14.0–14.9mM) (C) was necessary to show the same effect After the same treatment time, ibandronate down regulated caspase 3/7 in MC3T3-E1 cells significantly already at a concentration of 20mM, while a significant down-regulation of 8 was found not until a concentration of 100mM (D) In U-2 OS (E) and CCL-51 (F), however, ibandronate up regulated both caspases at all concentrations tested Bars represent mean  SD; **P  0.01; ***P  0.001; n = 4.

cellssignificancewasreachedafter72hoftreatmentwith50mM

ibandronate (Fig 4E) At this time, in U-2 OS cells, DNMT1

expression was already strongly down regulated at 20mM

ibandronateinculturemedium(Fig.4D)wherebyacomparable

effect was reached at 100mM ibandronate for CCL-51 cells

(Fig.4F).Fig.6GandHcomparestheeffectof72hoftreatment

with 50mM ibandronate on DNMT1 protein expression by

immuneblotanalysisinthethreecelllinesstudied

3.4 Faspromotermethylationwasalteredintumorcellsafter

ibandronatetreatment

72h after seeding, in MC3T3-E1 cells basal Fas promoter

methylationwasconsiderablylowerasinthetwoneoplasticcell

lines(Table2 Furthermore,in MC3T3-E1 cells,72hof50mM

ibandronatetreatmentfailedtosignificantlyaffectDNMT1binding

onFaspromoter(Fig.5A).After72h,inU-2OScells(Fig.5C)aswell

asinCCL-51cells(Fig.5E)treatmentwithibandronate

significant-lyreducedbindingofDNMT1totheFASpromoter

Reduced binding of DNMT1 to the Fas promoter suggested changesinDNAmethylation,whichwasevaluatedbycomparing theconcentrationofmethyl-cytosinesvs.unmethylatedcytosines

inthepromoter.Whileasexpected,nochangeinthemethylation statusoftheFaspromoterinMC3T3-E1cellswasfound(Fig.5B), ibandronatesignificantlyreducedthemethyl-cytosine concentra-tionoftheFaspromoterinU-2OSalreadyafter24h(Fig.5D)and after48hinCCL-51cells(Fig.5F)

3.5 IbandronatealteredRASlocalizationintheanalyzedtumor cell-lines

Bisphosphonatesinhibittheactivitiesoftheenzymes farnesyl-andgeranylgeranyl-diphosphatesynthase,whichcausedisruption

Fig 2 Ibandronate up regulated FAS expression in U-2 OS and CCL-51 but not in MC3T3-E1 cells (A and B) At 100mM ibandronate up regulated FAS expression in U-2 OS already after 24 h (C) while in CCL-51 not until 48 h (E) In both cell lines the regulation was dose dependent, which reached significance at 100mM in U-2 OS (D) and at 50mM

in CCL-51 (F) Bars represent mean  SD; *P  0.05; **P  0.01; n = 3.

of small-GTPases prenylation thus altering their sub-cellular

distribution Cells were either left untreated or treated with

ibandronate.Thereafter,proteinsofthemembraneaswellasof

cytoplasmatic fractionwere isolated and subjected to immune

blotting.AsshowninFig.6A,inMC3T3-E1cellsRASwasprimarily

localizedin the cytoplasm and, therefore, ibandronate did not

affectRASlocalizationinthiscell-line.InU-2OScells(Fig.6B)as

wellasinCCL-51cells(Fig.6C)RASwasprimarilylocalizedincell

membranes.Afterexposureto50mMibandronatefor72h,inboth

celllinesaclearshiftinRASlocalizationfromthecellmembranes

tocytoplasmwasseen(Fig.6BandC)

Cell fractionation was controlled by immune blotting of

integrin-b5 for the membrane and mitogen-activated protein

kinase4forthecytosolicfraction(Suppl.Fig.3)

3.6 Bisphosphonateinducedapoptosisoftumorigeniccellswas

differentiallyrescindedbyGGPPandFPP

Inhibitionofprenylationofsmall-GTPasesbybisphosphonates

canberescuedbyparalleltreatmentwiththesubstrateFPPforRAS

orGGPPfortheothermodifiedsmall-GTPases

AsalreadyshowninFig.1andSupplementalFig.1,treatmentof

MC3T3-E1cellswith50mMibandronatereducedthecellnumber

byabout90%.Afterthesameculturetime10mMGGPPbyitself

attenuated cell multiplication by about 50% and could only partiallyrescuetheeffectofibandronateonthesecells(Fig.7A).In U-2 OS cells 50mM ibandronate significantly reduced cell multiplication AlthoughGGPPitselfreduced cellmultiplication

by about25%,it couldrescue theeffectcaused byibandronate (Fig.7B).Similarly,inCCL-51cellsGGPPcouldrescueibandronate attenuatedcellmultiplicationbut,differentially,didonly margin-ally influence cell multiplication (Fig 7C) In MC3T3-E1 cells neitheribandronatenorGGPPinfluencedFasexpression(Fig.7D) Again, inboth otherstudiedcell linesibandronate inducedFAS expression.GGPPalonehadnosignificanteffectonFASexpression, butdownregulatedibandronateinducedFASexpressioninboth

U-2OS(Fig.7E)aswellasinCCL-51celllines(Fig.7F).Regarding DNMT1expression,againallthreetreatments(ibandronate,GGPP

or combination of both) showed no effect in MC3T3-E1 cells (Fig 7G) However in both tumor cell lines, ibandronate, as expected, down regulatedexpressionof DNMT1.GGPP counter-actedthiseffectandhadnoeffectwhenadministeredsingularly (Fig.7HandI).Thissupportsourhypothesisthatsmall-GTPases induceDNMT1expressionisinhibitedbybisphosphonatesleading

topromoterdemethylationandFASexpression

NextwetestedtheeffectsofFPP(40mM)oncellmultiplication (Fig 8).Inthe immortalizedMC3T3-E1 cellline (Fig.8A),FPP reducedcellnumberto63%,inU-2OScellsto71%(Fig.8B)whileit didnotaffectthisparameterinCCL-51cells(Fig.8C).However,in contrasttoGGPP,FPPcouldnotrescuethedown-regulationofcell multiplicationbyibandronate(Fig.8A–C).TheeffectsofFPPon FASexpressionwerecomparabletothatofGGPP:nosignificant effectsinMC3T3-E1cells(Fig.8D),whileinbothneoplasticcell lines(Fig.8 andF)40mMFPPrescuedtheibandronatemediated up-regulation of FAS Comparable effects were also found on DNMT1expressionwithnoeffects inMC3T3-E1cells(Fig.8G), while in U-2 OS FPP rescued the down-regulation of DNMT1 expressionbyibandronate(Fig.8H).Differentially,FPPdidnot change the attenuation of Dnmt1 expression in CCL-51 cells (Fig.8I).FPPalonedidnotaffectDNMT1expressioninanyoneof thesecelllines

4 Discussion

A growing body of evidence attributes an anti-tumorigenic capacitytobisphosphonates.Severalstudieshaveshownthatin patientsaffectedbydiverse malignances,bisphosphonate thera-piesshowapositiveoutcomewithregardtotumorgrowthand metastaticactivity[40–46].However,themechanismsunderlying thoseeffectsremainpoorlyunderstood

Byactivation,theRassuperfamilyofsmallGTPasesattachesto cellular membranes and, among many other cell regulatory functions,promotescellproliferation,differentiationandsurvival

activitybyinhibitingthemevalonatepathway.Bythisway,these drugs prevent prenylation of small-GTPases as well as their attachmenttothecellularmembranesandtherebytheirfunction

(clodronate and etidronate) can be considered as secured, the modeofactionofaminobisphosphonatesoncellsisnotasclear

[9,11,49,50].Initially,J774macrophageshavebeenconsideredasa model of aminobisphosphonates’ action because this cell line matchedtheorderofpotencyofthediversebisphosphonatesfor inhibiting bone resorption and inducing apoptosis [7,51–53] Howeverusingprimaryrabbitorhumanderivedosteoclasts,ithas beendemonstratedthatinhibitionofboneresorptionby bispho-sphonatesdoesnotrequireosteoclastapoptosis[54–56].Taking intoaccountthat J744arelymphoma-derivedtumorigeniccells

[57]onecanconcludethatbisphosphonatesactintumorcellsina differentwayascomparedtonormalosteoclasts

Fig 3 Ibandronate down regulated cell viability/proliferation and up regulated FAS,

which could be rescued by transfection of FAS siRNA (A) After 3 days of treatment

with ibandronate (7.2mM, EC50) cell viability/proliferation was down regulated to

73.6% (control) After electroporation without siRNA ibandronate down regulated

cell viability/proliferation to 54.4% (Electrop.) a comparable value, which was found

after electroporation of a control siRNA (56.4%, Co siRNA) Electroporation of FAS

siRNA resulted in attenuation of down-regulation of cell viability/proliferation to

87.0% demonstrating the FAS regulation on this parameter The difference of 13.0%

suggests a minor second mechanism on regulation of cell viability/proliferation.

Bars represent mean  SD; **P  0.01; ***P  0.001; untreated vs Iban treated;

+++

P  0.001; Co siRNA vs FAS siRNA; n = 3 (B) The immune-blot with anti-FAS

antibody demonstrates that Ibandronate up regulated FAS protein expression as well.

Transfection with FAS siRNA down regulated ibandronate stimulated FAS protein

expression having no considerable effect on basal FAS expression.

Fig 4 Ibandronate down regulated DNMT1 expression in U-2 OS and CCL-51 but not in MC3T3-E1 cells (A and B) At 100mM ibandronate down regulated DNMT1 expression

in U-2 OS already after 24 h (C) while in CCL-51 attenuation did not reach significance until 72 h (E) In both cell lines the regulation was dose dependent, which reached significance already at 20mM in U-2 OS (D) and but not until 100mM in CCL-51 (F) DNMT1 was also down regulated at the protein level as demonstrated by immuno-blot Intensity measurements of 3 immune-blots revealed a significant down-regulation only for U-2 OS and CCL-51 cells but not for MC3T3-E1 cells (G) A representative blot is shown in (H) Bars represent mean  SD; *P  0.05; **P  0.01; ***P  0.001; n = 3.

non neoplastic immortalized mouse preosteoblastic MC3T3-E1

cells,ibandronateattenuatedcellmultiplicationby

down-regula-tion of the cell cycle as suggested by decreased expression of

cyclinsCcn2aandCcn2d(notshown).Moreover,down-regulation

of caspase 3/7 and caspase 8 in MC3T3-E1 cells assigned to

aminobisphosphonatesananti-apoptoticactivityasfoundin

MLO-Y4osteocytes[58]whileintumorigenicRAW264.7macrophages

mostbisphosphonatesinducedapoptosis[59].Inprimaryhuman

osteoblastsatconcentrationshigherthan107Mzoledronatedose

dependentlyreducedcell proliferation,although,at thehighest

concentrationapoptosiswasfoundaswell[37].Treatmentoffetal immortalized osteoblasts with pamidronate and zoledronate resultedinadosedependentdown-regulationofcellproliferation with increased differentiation [60] However, increased cell proliferationofhumanprimarytrabecularbonecells[61]andof humanbonemarrowstromalcellsbyalendronateandrisedronate hasbeenreported as well[62] Summarizingthesefindings,in normal mesenchymal bone cells, bisphosphonates decrease proliferationbutapoptosiswasfoundonlyatveryhigh concen-trations,whileintumorigeniccellsamino-bisphosphonatesinduce apoptosis

Here we show that treatment of tumor cell lines with ibandronatedownregulatedgenesoftheDNACpG-methylation apparatus (Dnmt1, Dnmt3b, Hells), which resulted in reduced bindingofDNMT1atthepromoterofFASinbothtumorcelllines AbsenceoftheDNA-methyltransferasesatthepromoterresultsin reduced CpG-methylation of the promoter during further cell divisions,whichisfollowedbyincreasedexpressionofthegene

activityofcaspase3/7andcaspase8,whichisaclassicalsignof

Table 2

Methylation levels (ratio methylated vs unmethylated) of the FAS promoters in the

studied cell lines.

Fig 5 DNMT1 bound to the FAS promoter, which resulted in change of the DNA methylation status in U-2 OS and CCL-51 but not in MC3T3-E1 cells (A and B) 50mM ibandronate significantly reduced binding of DNMT1 to the promoter of FAS in U-2 OS cells (C) which resulted in a decreased ratio of methylated by unmethylated cytosines already after 24 h (D) In CCL-51 cells ibandronate attenuated binding of DNMT1 to the Fas promoter as well (E) but it decreased methylation not until 48 h treatment (F) Bars

ofFasexpressionwasfoundinnon-neoplasticMC3T3-E1cells.The verylowbasalmethylationlevelofFaspromoterinthesecellscan explainthis fact.Wehaverecentlydemonstratedthatculturing these cells on culture dishes that are not covered by collagen results in down-regulation of DNMT1 and HELLS followed by demethylationoftheFaspromoterandincreasedexpressionofthe gene[19]

Small-GTPasesplayacentralroleinbisphosphonates’activity These drugs inhibit the activity of the enzymes, which are responsibleforsynthesisofFPPandGGPP,whicharetransferred

totheaminoacidsmotifCAAXanchoringofthesmall-GTPasesto thecellmembrane,whichisaprerequisitefortheiraction.Effects

ofbisphosphonateson small-GTPasesmediatedsignal transduc-tioncanberescuedbyco-treatmentwithGGPPorFPP,wherein generalFPPrescuesRAS-activityandGGPPtheactivityofthe RHO-and RAB-family [66,67] Again, transformed cell lines behaved differentially;inCCL-51andU-2OS,GGPPrescuedibandronate attenuatedcellmultiplication, upregulatedDNMT1and, conse-quently,downregulatedFASexpression.GGPP,however,couldnot rescuetheimmortalizednon-neoplasticcelllineMC3T3-E1from theeffectsofibandronate.Wefoundnodown-regulationofDnmt1 andno up-regulationofFas.Moreover,GGPPalonesignificantly decreasedcellmultiplicationaswell.GGPPistheposttranslational modifieroftheRHO-andRAB-small-GTPases.Recently,ithasbeen demonstratedthatinhibitionofprenylationbybisphosphonates ratheractivatesthaninactivatestheRHOfamilyofsmall-GTPases including RAC, CDC42 and RHO [9] Interestingly, osteoblast-restrictedRac1deletionleadstodefectiveboneacquisitioninvivo and knockdown impairs growth and induces apoptosis in the osteoblast cell line OP9 [68] These data suggest that GGPP increases interaction of RAC1 with cellular membranes, which resultsinreducedactivatedRAC1leadingtodecreased prolifera-tionandincreasedapoptosis

A down-regulation of cell multiplication was also found in mesenchymalMC3T3-E1andU-2OScellsbyFPPbutnotinthe neoplastic cell line resulting from an epithelial tumor Down-regulationofcellmultiplicationbyFPPsuggeststheinvolvement

ofRAS.Aftertranslation,FASisfarnesylatedandtransportedtothe endoplasmaticreticulumwhereitispreparedforpalmitoylation andforwardedtotheGolgi-membranes.Thisinitiatesacycleof traffictoandfromtheplasmamembranes,whereitisactivated

[13,16].HighercellularconcentrationsofFPPcouldinfluencethe equilibrium of this cycle and therefore alter differentiation, proliferationandapoptosisrate

Unlike GGPP, FPP couldnot rescue down-regulation of cell multiplicationbyibandronateinU-2OSandCCL-51.Thissuggests that RHO- and RAB-small-GTPases are responsiblefor ibandro-nate’seffectonthisparameter.ThisissurprisingbecauseinU-2OS, FPP canceledtheeffectonDNMT1expressionandinhibited up-regulationofFAS.Anexplanationis down-regulationofthecell cyclebyibandronateviadown-regulationofcyclinsandapossible up-regulationof cellcycle inhibitors[68–71].From the rescue-experiments one can conclude that ibandronate inhibits cell multiplication by decreasing cell cycling and by increasing apoptosis Apart from apoptosis a second process was also suggestedbytransfectionofFASsiRNA,whichcouldnotcompletely recoveribandronate’sdown-regulationofcell viability/prolifera-tion.However, thisexperiment strengthensourhypothesis that up-regulationofFASisresponsibleforinductionofapoptosisvia caspasesandconfirmsrecentdataobtainedfromHMC1mastcell leukemiacellsthatweretreatedwithdemethylatingdrugs[34] AlthoughFPPandGGPPdifferentiallyinfluencedcell multipli-cation,unexpectedly,exceptforDnmt1expressioninCCL-51,no differenceswere foundin rescuingFASand DNMT1expression; bothisoprenoidscouldrecovertheeffectsofibandronate,whichis

Fig 6 Ibandronate translocated RAS from the membranes to the cytoplasm in U-2

OS (B) and CCL-51 (C) but not in MC3T3-E1 cells (A) Intensity measurements of 3

immune-blots revealed a significant change in the localization of RAS in U-2 OS and

CCL-51 cells but not in MC3T3-E1 cells (A) For each cell line a representative blot is

shown Bars represent means normalized to the signal of untreated cells  SD;

*P  0.05; **P  0.01; n = 3.

in line with recent findings that both isoprenoids are able to

recover bisphosphonates-induced apoptosis There are several

possibleexplanationsforthis finding[11].Bisphosphonatesare,

withsomeexceptions[72,73],inhibitorsofFPP-synthasebutnotof

theGGPP-synthase.Therefore,whencellsweretreatedwithFPP

someof thecompoundswillbeconverted toGGPP, whichwill

modifyand rescue RHO-family small-GTPases as well, at least

partially[11].Recently,ithasbeendemonstratedthatinpresence

of inhibition of farnesyltransferase, geranylgeranylation occurs

insteadoffarnesylationofKRAS4A,BandNRASbutnotforHRAS

Moreover,geranylgeranyltransferasecanuseFPPassubstrateas

welltomodifyproteins of theRHO-family [74–80].As

bispho-sphonatesdepletebothFPPandGGPP,raisingtheconcentrationof

oneoftheisoprenoidsbyaddition,modificationwiththe‘‘wrong’’

modifiermayoccur.Thesefactsmaysupportacriticalviewonthe

implicationoftherescueexperiments,sothatno unambiguous

assignment of a small-GTPase can be made Even though the

presentedresultsclearlydemonstratetheinvolvementof

small-GTPases in the epigenetic DNA methylation process and FAS

mediatedapoptosis,adetailedanalysisofthelargefamilyof

small-GTPaseswillbenecessarytounravelthemechanism

Takentogether,ourexperimentssupportthecommon

knowl-edge that more members of the group of small-GTPases are

involvedintheregulationofcellmultiplicationandapoptosisand

suggest a different mode of action of bisphosphonatesin non-transformed vs neoplastic cell lines, as supported by previous findings[10,81]

As already mentioned above, most tumors or transformed neoplastic cells are characterized by an activated RAS/MAPK pathway[13–15],whichresultsinmethylationofcytosinesinthe promoteroftumorsuppressorgenesandofthepro-apoptoticFAS gene[12,24,30,32,33,82,83].Bothtumorcelllines,however,havea highlymethylatedFASpromoter,andtherefore,aninhibitedFAS expression.ThissuggeststhatRASmustbeactivatedandattached

tothecellmembranetoenablebisphosphonatestodisplaytheir epigenetic activity Ibandronate, as demonstrated by immune-blotting, prevented RAS from attachment to membranes and translocatedtheproteinintothecytoplasm;nochangewasfound

in MC3T3-E1 cells indicating that RAS is not attached to membranes, and therefore not or only marginally active, as generally found in normal, growth factor-unstimulated cells

differentially in response to bisphosphonates when compared withnormalcells.Itshouldbeemphasizedagainthatactivation RAS/MAPK pathway seems to be a prerequisite for bispho-sphonates’actionintumorcells.FeaturingtheRAS/MAPKpathway

is wellfounded bythe factthat epigenetic silencingof theFas promoter is a consequence from the signaling cascade of this

Fig 7 Effects of GGPP on ibandronate regulated cell multiplication and expression of FAS and DNMT1 Ibandronate down regulated cell multiplication in all three cell lines tested In MC3T3-E1 cells, GGPP itself attenuated cell multiplication but could not prevent ibandronate’s down-regulation (A) In U-2 OS (B) and CCL-51 (C) cells, GGPP rescued down-regulation of cell multiplication Moreover, the combination of both drugs increased cell multiplication in CCL-51 cells significantly (C) Neither ibandronate nor GGPP had an effect of Fas (D) and Dnmt1 (G) expression in MC3T3-E1 cells In U-2 OS cells, GGPP rescued ibandronate’s up-regulation of FAS (E) or down-regulation of DNMT1 (H), respectively Equally, in CCL-51 cells, GGPP rescued ibandronate’s up-regulation of Fas (F) or down-regulation of Dnmt1 (I), respectively Bars represent mean  SD; *P  0.05; **P  0.01; ***P  0.001 treatment vs Co; +++

P  0.001 GGPP vs Ibn; 

P  0.05; 

P  0.001 GGPP vs Ibn + GGPP; ###

P  0.001 Ibn vs Ibn + GGPP; n = 3.