Dasatinib as a Bone-Modifying Agent: Anabolic and Anti- Resorptive Effects

15 Pages·2012·1.17 MB·English

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-
Resorptive Effects
Antonio Garcia-Gomez1,2,3, Enrique M. Ocio1,3, Edvan Crusoe1,3, Carlos Santamaria2, Pilar Herna´ndez-
Campo2, Juan F. Blanco3, Fermin M. Sanchez-Guijo2,3, Teresa Herna´ndez-Iglesias1, Jesu´s G. Brin˜o´n4,
Rosa M. Fisac-Herrero5, Francis Y. Lee6, Atanasio Pandiella1,3, Jesu´s F. San Miguel1,2,3,
Mercedes Garayoa1,2,3*
1CentrodeInvestigacio´ndelCa´ncer,IBMCC,UniversidaddeSalamanca-CSIC,Salamanca,Spain,2CentroenReddeMedicinaRegenerativayTerapiaCelulardeCastillay
Leo´n, Salamanca, Spain, 3Hospital Universitario de Salamanca-IBSAL, Salamanca, Spain, 4Departamento de Biolog´ıa Celular y Patolog´ıa, Facultad de Medicina,
UniversidaddeSalamanca,Salamanca,Spain,5ServiciodeHematolog´ıa,HospitalGeneraldeSegovia,Segovia,Spain,6Bristol-MyersSquibbPharmaceuticalResearch
Institute,Princeton,NewJersey,UnitedStatesofAmerica
Abstract
Background:Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or
reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to
identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of
dasatinib(BMS-354825),amultitargetedtyrosinekinaseinhibitor,onosteoblastandosteoclastdifferentiationandfunction.
Methods:For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the
hMSC-TERTandtheMG-63celllineswereemployed.Osteoclastsweregeneratedfromperipheralbloodmononuclearcells
(PBMC)ofhealthy volunteers. Skeletally-immature CD1mice wereused inthe invivomodel.
Results:Dasatinib inhibited the platelet derived growth factor receptor-b (PDGFR-b), c-Src and c-Kit phosphorylation in
hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt
signaling.TreatmentofMSCsfromhealthydonors,butalsofrommultiplemyelomapatientswithlowdosesofdasatinib(2–
5nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also
observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation
markers,andincreasedtrabecularmicroarchitectureandnumberofosteoblast-likecells.Byinvitroexposureofhemopoietic
progenitors to a similar range of dasatinib concentrations (1–2 nM), novel biological sequelae relative to inhibition of
osteoclastformationandresorptivefunctionwereidentified,includingF-actinringdisruption,reducedlevelsofc-Fosand
ofnuclearfactorofactivatedTcells1(NFATc1)inthenucleus,togetherwithloweredcathepsinK,aVb3integrinandCCR1
expression.
Conclusions:Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which
suggestsitspotentialuseforthetreatmentofbonediseasessuchasosteoporosis,osteolyticbonemetastasisandmyeloma
bonedisease.
Citation: Garcia-Gomez A, Ocio EM, Crusoe E, Santamaria C, Herna´ndez-Campo P, et al. (2012) Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-
ResorptiveEffects.PLoSONE7(4):e34914.doi:10.1371/journal.pone.0034914
Editor:MicahLuftig,DukeUniversityMedicalCenter,UnitedStatesofAmerica
ReceivedJuly13,2011;AcceptedMarch8,2012;PublishedApril23,2012
Copyright:(cid:2)2012Garcia-Gomezetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbygrantsfromtheSpanishMinistryofScienceandInnovation –ISCIII(PI081825);MutuaMadrilen˜aMedicalResearch
Foundation(AP27262008);CentroenRedofRegenerativeMedicineandCellularTherapyfromCastillayLeo´n,Consejer´ıadeSanidadJCyL–ISCIII;theCooperative
ResearchThematicNetworkinCancer(RTICC;RD06/0020/0006andRD03/0020/0041);andSpanishFIS(PS09/01897).AG-GandCSaresupportedbytheCentroen
RedofRegenerativeMedicineandCellularTherapyfromCastillayLeo´nProject.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:FYLisanemployeeofBristol-MyersSquibb.Thisdoesnotaltertheauthors’adherencetoallthePLoSONEpoliciesonsharingdataand
materials.Therearenopatentsorproductsindevelopmenttodeclare.
*E-mail:[email protected]
Introduction metastasis andmultiple myelomabonedisease.In osteoporosis,a
prevalent disease of postmenopausal womenand elderly patients,
Bone mass is regulated by the balance of bone formation and
bone resorption exceeds that of bone formation resulting in a
bone resorptive rates. Alteration of this balance by increased
systemic impairment of bone mass, strength, and microarchitec-
number and activity of bone-resorbing osteoclasts (OCs) and/or
ture[1].Thishighlyincreasesthepropensityoffragilityfractures,
reduced differentiation and impaired activity of bone-forming
most commonly occurring in the spine, hip or wrist. Also, many
osteoblasts(OBs),leadstopathologicalstatesofboneloss.Thatis
solid tumors (prostate, breast, lung, colon, renal), commonly
the case of bone diseases such as osteoporosis, osteolytic bone
metastasize to bone. When this occurs, tumor cells mobilize
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BoneEffectsofDasatinib
cellular and extracellular matrix bone components to ultimately present study we provide in vitro evidences of the effect of low
promoteboneinvasionandenhancetumorgrowth,whichleadsto dasatinibconcentrationsinenhancingdifferentiationandfunction
deregulated bone remodeling and as a consequence, devastating of mesenchymal osteoprogenitors from both healthy donors, and
skeletal complications [2,3]. Furthermore, multiple myeloma interestingly, also from myeloma patients. This anabolic bone
(MM) is a hematological malignancy primarily developing within effect of dasatinib was also observed in the in vivo setting after
thebonemarrowasaconsequenceoftheabnormalexpansionof administration of relatively low dasatinib doses to skeletally-
clonalplasmacells.Interestingly,onemajor(inupto80%ofMM immaturemicetoavoidtheinhibitoryeffectsoftheagentonOCs
patients) clinical symptom associated with this disease is the and OC precursors and thus targeting endogenous osteoprogeni-
development of osteolytic lesions as a result of increased bone tor cells. Besides, within the same low nanomolar range of
resorption and marked impairment of bone formation. The dasatinib concentrations, we show in vitro data of additional
interactions of myelomatous cells with the bone marrow mechanisms of dasatinib inhibitory effect on OC differentiation
microenvironment are thought to be critical in the development [diminished expression of c-Fos and reduced levels of nuclear
of MM bone disease, and the diverse interplaying cellular and factor of activated T cells 1 (NFATc1) in the nuclear compart-
molecularcomponentshavebeenextensivelystudiedrecently[4– ment], and on OC function (F-actin ring disruption and lowered
6]. Of interest, isolated mesenchymal osteoprogenitor cells from aVb3 integrin, CCR1 and cathepsin K expression). Taken
thebonemarrowofmyelomapatientshavebeenreportedtoshow together, our data support the overall bone anabolic effects of
distinct gene expression profile and also reduced osteogenic dasatinib, with a double component of enhancement of OB
potentialascomparedtothosefromhealthydonors[7,8].Allthese differentiation and function together with inhibition of osteoclas-
lowbonemasspathologies(osteoporosis,osteolyticbonemetastasis togenesis and bone resorption, exerted within a similar concen-
and myeloma bone disease) cause skeletal fragility and are tration range. Potential therapeutic implications of dasatinib for
commonly associated to skeletal related events including patho- thetreatment of specific bonedisorders are alsodiscussed.
logicalfractures,severebonepain,hypercalcemiaandspinalcord
andnervecompression.Theseeventscanseverelycompromisethe
Methods
quality of life of patients and even result in significant mobidity
and increased risk of death. This emphasizes the need toidentify Participants
and develop new bone-targeted pharmacological agents which Samples from the bone marrow of 10 healthy donors and 10
mayprevent,reduceorevenreversethesepathologicalconditions newly diagnosed MM patients (stages I to III) were used in this
of bonelossin theabovementioned diseases. studyafterinformedandwrittenconsentofparticipants.Approval
Specifictyrosinekinaseshavebeenproposedaspotentialtargets ofthestudywasgrantedbytheInstitutionalReviewBoardofthe
for anti-tumor therapy. Imatinib mesylate (STI-571) is a tyrosine CIC, IBMCC (University of Salamanca-CSIC, Spain), and
kinase inhibitor which was originally approved as a first-line research was conducted following principles in the Declaration
treatmentforchronicmyeloidleukemiabecauseofitscapacityto of Helsinki.
inhibit the Bcr-Abl kinase activity of Philadelphia+ cells [9].
Additionaltyrosinekinaseswithoncogenicpotentialalsoinhibited
Reagents and immunochemicals
by imatinib include c-Kit, the platelet-derived growth factor
Dasatinib was provided by Bristol-Myers Squibb Company
receptors: PDGFR-a and PDGFR-b, and the c-Fms receptor
(Stamford, CT, USA). For in vitro assays, dasatinib was
[9,10], which account for the anti-tumor effect of imatinib in
reconstitutedindimethylsulfoxide(DMSO;Merck,Hohenbrunn,
several types of solid tumors. Interestingly, evidence has
Germany) at a stock concentration of 100mM and stored at
accumulated for a direct effect of imatinib in the skeleton with
220uC; further dilutions were made in tissue culture medium at
increased trabecular bone volume and bone mineral density in
the time of use. Recombinant human PDGF-BB, macrophage
imatinib-treated patients [11,12]. In vitro studies showed that
colony-stimulating factor (M-CSF) and receptor activator of NF-
imatinib suppressed OB proliferation and stimulated osteogenic
kB ligand (RANKL) were purchased from Peprotech (London,
geneexpressionandmineralizationmajorlybyinhibitingPDGFR
UK),whilestemcellfactor(SCF)wasobtainedfromStrathmann
function[11,13].Moreover,imatinibhasapotentinhibitoryeffect
(Hamburg, Germany). Primary antibodies for immunoblotting,
on OC bone resorption and stimulates apoptosis of mature OCs
immunohistochemical and flow cytometry analyses were directed
[14].
against: PDGFR-b, phospho-PDGFR-b (Tyr857), Erk1/2, phos-
Dasatinib(BMS-354825)isanoveloralbioactivemultitargeted
pho-Erk1/2(Thr202/Tyr204),NFATc1,histoneH1andcathep-
tyrosine kinase inhibitor which was developed as a second-
sin K, purchased from Santa Cruz Biotechnology (Santa Cruz,
generation drug rationally designed for the use against imatinib-
CA, USA); phospho-c-Fms (Tyr723), phospho-c-Kit (Tyr719), c-
resistant leukemias [15]. The target tyrosine kinase profile of
Src, phospho-Src (Tyr416), p38 MAPK, phospho-p38 MAPK
dasatinib partially overlaps that of imatinib but presenting much
(Thr180/Tyr182), Akt, phospho-Akt(Ser473), phospho-b-catenin
higher potency, and is also broader, including the Src family
(Thr41/Ser45), PU.1 and c-Fos, from Cell Signaling Technology
kinases[10].DasatinibisnowbeingevaluatedinPhaseIItrialsin
(Danvers,MA,USA);CD51/61andCD191,fromR&DSystems
avarietyoftumortypes,includingprostate,breast,colorectaland
(Minneapolis, MN, USA); c-Kit and nucleoporin p62, from BD
lung cancer [16]. However, taking into account the aforemen-
Biosciences (Bedford, MA, USA); a-tubulin, from Calbiochem
tioned skeletal effects of imatinib, it was expected that dasatinib
(Darmstadt, Germany); dephospho-b-catenin (aa 35–50), from
might be even more effective in inhibiting osteoclastogenesis and
Enzo Life Sciences (Plymouth Meeting, PA, USA), and T-cell
promoting bone formation. In fact, it has already been reported
factor4(Tcf4),fromUpstate(Millipore,Billerica,MA,USA).All
that dasatinib inhibits OC formation and resorption capacity,
cell culture media and reagents were purchased from Gibco
mainly by its potent inhibition of c-Fms on OC progenitors
[17,18]. Also, recent data of dasatinib effect enhancing osteo- (Paisley, UK). Trypan Blue Solution 0.4% was delivered by
blastogenesis from mesenchymal progenitors have been reported Sigma-Aldrich(St.Louis,MO,USA),andthealamarBluereagent
[19-21];otherauthors,however,haveclaimedaninhibitoryeffect by Invitrogen (Carlsbad, CA,USA).
onOBdifferentiationforthisagentinsimilarsettings[22].Inthe
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BoneEffectsofDasatinib
Cell lines period, cells were trypsinized and incubated with phycoerythrin
Thehumanmesenchymalstemcell(MSC)lineimmortalizedby (PE)conjugatedAnnexin-Vand7-amino-actinomycinD(7-AAD)
expression of the telomerase reverse transcriptase gene (hMSC- [Becton Dickinson (BD) Biosciences, Bedford, MA, USA] for
TERT)wasagenerousgiftfromDrDCampana(Departmentof complementary apoptosis/necrosis information. The cells were
Oncology and Pathology, St Jude Children’s Research Hospital, acquired using a FACSCalibur flow cytometer, and data were
Memphis, TN, USA) [23]. The human osteosarcoma cell line analyzed using the ModFit program to determine the number of
MG-63wasobtainedfromtheAmericanTypeCultureCollection cell divisions and the percentage of cells in each division
(CRL-1427; LGC Promochem, London, UK), and used as an (compared with undivided colcemid-treated cultures) or the
osteoblast-likecellline[24].CelllinesweregrowninRPMI1640 Paint-A-Gate program for percentages of apoptotic cells (BD
Biosciences).
medium (hMSC-TERT) or DMEM medium (MG-63) supple-
mented with 10% heat-inactivated fetal bovine serum (FBS),
100U/mLpenicillinand100mg/mLstreptomycin.Allcelltypes Western blotting analyses
wereculturedat37uCinahumidifiedatmosphereinthepresence Proteinlysatesweregeneratedandwesternblottingprocedures
of 5%CO –95% air. were performed as previously described [27]. For subcellular
2
fractionation of proteomic samples, the Qproteome Cell Com-
Primary mesenchymal stem cells and osteoprogenitor partment kit was used(Qiagen GmbH,Hilden, Germany).
Detection of PDGFR-b, phospho-PDGFR-b (Tyr857), c-Kit,
cells
phospho-c-Kit (Tyr719), c-Src, phospho-Src (Tyr416), Erk1/2,
PrimaryMSCsfromBMsamplesofhealthydonors(n=10)and
phospho-Erk1/2 (Thr202/Tyr204), p38 MAPK, phospho-p38
MMpatients (n=10)were generatedasdescribedby Garayoa et
MAPK(Thr180/Tyr182),Akt,phospho-Akt(Ser473),phospho-c-
al. [25]. Briefly, mononuclear cells from bone marrow samples
Fms (Tyr723), PU.1, NFATc1, c-Fos, cathepsin K, phospho-b-
wereisolatedusingFicoll-Paquedensitygradientcentrifugation(r
catenin (Thr41/Ser45), dephospho-b-catenin (aa 35–50), histone
1.073; GE Healthcare, Uppsala, Sweden), cultured in DMEM
H1 and a-tubulin was performed by a standard procedure, using
with 10% FBS, 100U/mL penicillin, 100mg/mL streptomycin
primary and appropriate horseradish peroxidase-conjugated
and 2 mM L-glutamine for four days and selected by their
secondary antibodies and a luminol detection system with p-
adherencetoplasticware.Theculturemediumwasreplacedtwice
iodophenolenhancement for chemiluminescence.
weeklyuntilMSCcultureswereapproximately90%confluentor
ToanalyzetheeffectofdasatinibonPDGFR-b,c-Kitandc-Src
hadbeenincultureforamaximumof21days;atthatpoint,cells
tyrosinekinases,thehMSC-TERTandMG-63celllineswerefirst
were trypsinized (0.05% Trypsin-EDTA) and expanded in a 1:3
incubated with different concentrations of dasatinib for 6 hours
ratio.Atpassage3,selectedMSCsfrombothoriginsweretestedto
and then treated with 10ng/mL PDGF-BB or 50nM SCF for
meet definition criteria according to the recommendations of the
20minutespriortoproteinisolation.Totesttheeffectofdasatinib
International Society for Cellular Therapy [26] and experiments
on c-Fms, c-Kit and c-Src, OC progenitors were incubated with
were performed. dasatinibfor2 hoursandthentreatedwith50ng/mLM-CSFor
ToinduceexvivodifferentiationtoOBs,thegrowthmediumof 50nMSCF for20minutesprior toprotein isolation.
MSCs at 80–90% confluence was replaced by an osteogenic
differentiation medium consisting of a-MEM supplemented with Alkaline phosphatase and Runx2 activities and
10%FBS,10mMb-glycerolphosphate,50mg/mLascorbicacid
mineralization assay
and 10nM dexamethasone (all additives from Sigma-Aldrich).
PrimaryMSCswereculturedin12-wellplatesinMSCmedium
MSCsweregrownintheosteogenicmediumfor7(earlystageof
untilreaching,80%confluency.Cellswerethenchanged tothe
OB differentiation), 14 (pre-OB stage) or 21days (fully differen-
osteogenic differentiation medium in the presence or absence of
tiated OBs), replacing the medium every 3 or 4days, in the
dasatinib(2or5nM)for7or21days,atwhichtimesthealkaline
absence or presenceof specified concentrationsof dasatinib.
phosphatase (ALP) activity, or the Runx2 activity and minerali-
zationassays were performed.
Cell proliferation and viability assays
To measure ALP activity, cells were washed in phosphate-
To test whether dasatinib affected the growth capacity of the buffered saline (PBS), lysed in ice-cold lysis buffer and protein
MSC/OB lineage, the hMSC-TERT and MG-63 cell lines were content determined using the Micro BCA assay kit (Pierce,
seeded in 6-well plates at 104 cells/cm2 or 2.56103 cells/cm2, Rockford,IL).ALPactivitywasdeterminedbyspecifichydrolysis
respectively,andincubated for7 daysintheabsenceorpresence ofp-nitrophenylphosphateintop-nitrophenol(Sigma-Aldrich)and
of different dasatinib concentrations. Cells were then trypsinized quantified by OD reading at 405nm in triplicate using a
(0.05%Trypsin-EDTA)andcountedusingaTrypanBluesolution microplatereader(AsysUVM340,Biochrom,Eugendorf,Austria).
and a haemocytometer. The alamarBlue reagent was used to Valueswere referred tothetotal proteincontent ofthesample.
examine cell viability of the hMSC-TERT and primary MSCs When determining Runx2 activity, protein nuclear extracts
from myeloma patients at different time points and dasatinib were prepared using the Qproteome Cell Compartment kit.
concentrations along the osteogenic differentiation process, as by Quantification of Runx2 activation was performed with the
manufacturers instructions. ELISAbasedTrans-AM(AML-3/Runx2)kitaspermanufacturer
In addition,tocheckwhetherchangesinthenumberofviable instructions.
cells were due to diminished proliferative capacity or apoptotic Forquantitativeanalysisofalizarinredstaining(ARS),weused
effects of the drug, the hMSC-TERT cell line was stained with the method described by Gregory et al. [28]. Briefly, cells were
PKH67 (Sigma-Aldrich), a green fluorescent cell tracker that is fixed with 10% ice-cold phosphate-buffered formaldehyde for
retainedinthecellmembraneandthuscanbeusedformonitoring 10minutes, rinsed with distilled water and stained with 40mM
proliferation based on dye dilution with each cell division. After alizarin red (pH 4.2) for 20minutes at room temperature. After
PKH67labeling,cellswereseededin6-wellplatesat104cells/cm2 several washes to reduce non-specific ARS, stained cultures were
andincubatedfor7 daysintheosteogenicdifferentiationmedium photographedwithanOlympusDP70cameraonanOlympus31
inthepresenceorabsenceofdasatinib.Attheendoftheculture invertedmicroscope.Dyewasextractedbyaceticacidincubation
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BoneEffectsofDasatinib
and sample heating, and measured in triplicate at 405nm in 96- To assess bone morphology and microarchitecture, 10%
well plates. formalin-fixed femurs were analyzed by a micro-CT system
(MicroCATII; Siemens, Knoxville, TN, USA) at 75.0 kVp and
Real-time RT-PCR analysis 250.0uA.SevenhundredXRayprojectionswereacquiredduring
To evaluate the effect of dasatinib on the expression of bone a200urotationaroundthesample,with1250mscameraexposure
formation markers throughout their osteogenic differentiation, time per projection at full resolution (5126512). The reconstruc-
MSCsfromMMpatientsorhealthydonorswereculturedfor7or tion of the 3D image was done using COBRA V6.1.8 (Exxim
14daysintheosteogenicdifferentiationmediuminthepresenceor Computing Corporation, Pleasanton, CA, USA) with a final
absenceofthedrug.TotalRNAwasisolatedusingtheRneasyMini resolution of 10.4 mm/voxel. The post-processing, rendering and
kit (Qiagen GmbH, Hilden, Germany). Reverse transcription was generation of the cross sections of the samples was done using
performedwith 1.0mg RNA inthepresenceof randomhexamers Amira (Visage Imaging, San Diego, CA, USA). Analysis of
and 100U ofSuperScript RNaseH reverse transcriptase(Invitro- microarchitecturaltrabecularbonemorphologyinthedistalfemur
gen).ForPCRreactionsweusedtheStepOnePlusReal-TimePCR was performed using CT-Analyser software (SkyScan, Kontich,
SystemandTaqManGeneExpressionAssays(AppliedBiosystems, Belgium). Quantitative bone determined parameters were the
Foster City, CA, USA) according to manufacturer’s instructions. bone perimeter per area ratio (B Pm/B Ar; mm21), trabecular
AssayIDswere:ALP,Hs00758162_m1;COL1A1,Hs01076777_m1, number (Tb N;mm21) andtrabecular separation (TbSp;mm).
Osterix, Hs00541729_m1, and Runx2, Hs01047976_m1. Experi- Inparallel,otherfemurswerealsoprocessedforhistologicand/
ments were performed in duplicate for both the target and the or immunohistochemical studies following standard procedures.
endogenous gene (GAPDH) used for normalization. Relative Briefly,specimenswerefixedin10%formalinfor24h,decalcified
quantification of the target gene expression was calculated by the inOsteosoftbonedecalcifyingsolution(MerckKGaA,Darmstadt,
comparative threshold cycle (Ct) method: 2–DDCt where DCt= Germany)for5 daysandembeddedinparaffin.Sampleswerecut
Ct – Ct and DDCt=DCt – into3-mm-thicksectionsandstainedwithH&Eforbonehistologic
target gene GAPDH dasatinib-treated samples
DCt evaluationoreitherusedforimmunohistochemicalstudies.Inthe
samplesinabsenceofdasatinib.
latter case, antigen retrieval was carried out in a Pascal pressure
In vivo model chamber(Dako,Glostrup,Denmark)at90uCfor20minutesusing
a Tris-EDTA buffer pH 9.0, and then tissue endogenous
For in vivo studies, dasatinib powder was dissolved in sterile
peroxidase activity was quenched with a 3% H O solution for
80mMcitricacidpH 2.1tomakea10mg/mLstocksolutionand 2 2
10minutes. Sections were incubated overnight with an anti-Tcf4
then further dilutions were made in 80mM sodium citrate
pH 3.1.Thirty5-week-oldfemaleCD1healthymicewerehoused antibodyat4uCand1:20workingdilution,followedbyincubation
with EnVision anti-mouse complexes (Dako). The peroxidase
at our Animal Care Facility. At this age, healthy CD1 mice are
activity was shown using 3,39-diaminobenzidine+ (DAB+; Dako)
skeletally immature and show very active bone formation and
as a chromogen. Finally, sections were washed in water, lightly
minimal bone resorption, and this model was chosen so that the
counterstained with hematoxylin, dehydrated and mounted in
effectofdasatinibonbonecouldbemajorlyascribedtoitsaction
DPX.Histologicandimmunostainedsectionswereobservedwith
on OBs and not to inhibition of OC formation and function.
anOlympusBX51microscopeandphotographedwithaOlympus
Animals were divided into three groups (n=10 in each group)
DP70 camera. Tcf4 is an activating transcription factor which
receiving: a) 80mM sodium citrate pH 3.1 as vehicle; b) a lower
cooperativelyinteractswithRunx2/Cbfa1tostimulateosteoblast-
dasatinib dose of 2.5mg/kg, and c) a higher dasatinib dose of
specific osteocalcin expression [29], and thus can be used as a
10mg/kg. Treatment was administered by oral gavage in
bonafide marker forOBcells.
0.01 mL volume, in a BID regimen (twice a day), 5 days/week
in an attempt to maintain the dasatinib concentration range
throughout the day. Serum samples were collected at the Osteoclast differentiation and pit formation assays
beginning of the experiment, and also after 3 and 7 weeks of Peripheral blood monocuclear cells (PBMCs) from 6 healthy
dasatinib treatment (n=7 per group). Three to 5 animals per donors were obtained by density gradient centrifugation using
groupweresacrificedafter3 and7 weeksoftreatment,andboth Ficoll-Paque(r1.073;GEHealthcare),andculturedovernightat
femursweredissectedformicrotomographicimaging(micro-CT), 0.56106 cells/cm2 in a-MEM medium supplemented with 10%
histological and immunohistochemical analyses. All animal FBS and 100U/mL penicillin and 100mg/mL streptomycin.
experimentswereconductedaccordingtoInstitutionalGuidelines After removal of non-adherent cells, the remainder were
fortheUseofLaboratoryAnimalsoftheUniversityofSalamanca, maintained in the same medium but with additional 50ng/mL
after acquiring permission from the local Ethical Committee for RANKL and 25ng/mL M-CSF in the absence or presence of
AnimalExperimentation,andinaccordancewithcurrentSpanish dasatinibatindicatedtimesanddoses.Themediumwasreplaced
laws on animalexperimentation. twiceweeklyandculturesundereachconditionwereperformedin
Osteocalcin and ALP (as markers of new bone formation) as quadruplicate fromPBMCs ofat least three different donors.
well as TRAP5b levels (the active isoform of TRAP, specifically ToevaluatetheeffectofdasatinibonOCformation,cellswere
expressed by OCs, and thus reflecting OC number and activity), stainedfortartrate-resistantacidphosphate(TRAP;leukocyteacid
were quantified in collected sera. Markers of bone metabolism phosphatase kit, Sigma), according to the manufacturer’s instruc-
were measured bydedicated ELISAsaccording tomanufacturers tionsafter21daysofculture.UsingaLeicaDMI6000Binverted
guidelines [Alkaline phosphatase (Sigma-Aldrich), Mouse osteo- microscope, TRAP+ cells containing three or more nuclei were
calcin (Biomedical Technologies, Stoughton, MA, USA) and enumerated with the aid of a 161 mm2 grid (five randomly
Mouse TRAP Assay (Immunodiagnostic Systems, Boldon, UK)]. selected fields/well) and photographed with a Leica DFC350FX
Forstatisticalanalysis,valuesforadeterminedserummarker(e.g. camera.
osteocalcin) at each point (control, 3 and 7 weeks of treatment) Totesttheeffectofdasatinibontheboneresorptioncapacityof
werenormalizedforeveryindividualanimaltoitsownosteocalcin OCs, PBMCs at 0.66106 cells/well were seeded on calcium-
level at the beginning of the experiment, and plotted as fold coated slides (Becton Dickinson) in 200ml of OC medium
change. supplemented with 5ng/mL TGF-b1 (Peprotech) and 1 mM
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BoneEffectsofDasatinib
dexamethasonefor17days[30].Themediumwaschangedtwice the absence or presence of escalating doses of dasatinib to check
weekly by semi-depletion in the absence or presence of indicated for potential differences in sensitivity due to their differentiation
concentrations of dasatinib. At the end of the assay, cells were status. As illustrated in Figure 1A, PDGF-BB exposure markedly
removed with a 0.1% Triton X-100 solution and resorption pits increased PDGFR-b tyrosine phosphorylation in both cell lines,
werephotographedusingaLeicaDFC350FXcameramountedon and dasatinib strongly diminished PDGF-stimulated PDGFR-b
aLeicaDMI6000Bmicroscope.Thetotalresorbedareaperwell activation.PDGF-BBtreatmentalsoincreasedbasalp-Y416ofc-
was calculated using the Adobe Photoshop histogram function Src,ascribabletoPDGFR-bactivationanditsinteractionwiththe
(AdobePhotoshopCS2,9.0.2).Assayswereperformedintriplicate Src family kinases [35]. Dasatinib treatment only partially
for eachcondition usingPBMCs fromfive healthy volunteers. abrogated both basal and PDGF-induced c-Src activation in the
hMSC-TERT, while inhibition of c-Src phosphorylation was
Flow cytometry on osteoclast cells more pronounced in the MG-63 cell line (Figure 1A). We also
show that both cell lines express c-Kit, the stem cell factor (SCF)
After 2 weeks of osteoclastogenic differentiation in the absence
receptor for which dasatinib also presents inhibitory activity
or presence of dasatinib, pre-OBs were trypsinized (0.25%
[10,36]. SCF-induced c-Kit phosphorylation is efficiently dimin-
Trypsin-EDTA) and resuspended in Dulbecco’s PBS. Cells were
ished bydasatinib inbothcelllines(Figure 1A).
stained with anti-CD51/61-PE (aVb3 integrin-PE conjugated
Togainsomeinsightintothemechanismsunderlyingtheeffect
Mouse IgG ) or anti-CD191-APC (CCR1-allophycocyanin con-
1
ofdasatinibonthesecells,weexploredtheactivationstatusofkey
jugated Mouse IgG ) for 15minutes and subsequently with 7-
2B
moleculesinsignalingpathwaysdownstreamtheaffectedtyrosine
AAD for 5 minutes. After washes, cells were acquired on a
kinases, such as:Erk1/2, Akt, and p38mitogen activated protein
FACSCalibur flow cytometer using the CellQuest program and
kinase (MAPK). As observed in Figure 1B, dasatinib inhibits
analyzed with the Infinicyt software 1.3 (Cytognos, Salamanca,
PDGF-induced Akt activation in the hMSC-TERT and even
Spain).SpecificstainingforCD51/61orCD191wasevaluatedon
7-AAD- viablecells. more effectively in the MG-63 cell line. Inhibition of PDGF-
induced PI3-kinase/Akt activation has been shown to promote
osteogenic differentiation and mineralized-matrix production in
Actin ring formation assay
osteogeniccultures[11,37],andasimilareffectcouldbeexpected
PBMCs were seeded at 0.56106 cells/cm2 on cover slips and
from dasatinib in our experimental settings. On the other hand,
cultured inthespecified osteoclastogenic medium for12–14days
dasatinib induced a remarkable inhibition of Erk 1/2 phosphor-
inthepresenceorabsenceofthedrug.Atthispoint,pre-OCswere
ylation only in the MG-63 cell line and failed to modulate p38
fixedin2%paraformaldehydefor20minutes,permeabilizedwith
activationinneithercellline.AlthoughactivationofErk1/2and
0.1%TritonX-100andblockedin5%bovineserumalbuminin
p38havebeenreportedtopromoteOBdifferentiationandmatrix
PBS.Coverslipswerethenincubatedwithrhodamine-conjugated
mineralization through Osterix [38,39], at least in our experi-
phalloidin (1:200; Invitrogen) for 20minutes to visualize F-actin,
mental settings, dasatinib does not seem to stimulate the
andstainedwithDAPItomakenucleievident.Afterafinalrinse,
osteogenic differentiation and function by similar molecular
cover slips were mounted with an aqueous mounting medium
mechanisms.
(VectorLaboratories,Burlingame,CA,USA),andthedistribution
of the F-actin ring was observed with a Leica DMI6000 B
Dasatinib inhibits osteoprogenitor cell and osteoblast
microscopeandphotographedwiththeLeicaDFC350FXcamera.
proliferation
Assays were performed in triplicate for each condition using
OBsarederivedfromprecursorMSCs,aprocesswhichimplies
PBMCs fromat least three different healthy volunteers.
reductionoftheirproliferativeandstemcellself-renewalcapacities
whileacquisitionofOB-specificfeaturesandlineagecommitment
Statistical analysis
[31].Wethereforefirsttestedtheeffectofdasatinibonthegrowth
Each assay was performed at least three times. Quantitative
rateandviabilityofOBcellsandtheirmesenchymalprogenitors.
data were expressed as mean 6 SD or SEM, as specified.
As seen in Figure 2A, the number of cells in both the hMSC-
Statistical comparisons using the non-parametric Mann-Whitney
TERT and MG-63 cultures was progressively reduced with
U-testwereconsideredstatisticallysignificantforvaluesofp,0.05
increasing doses of dasatinib. We further studied the effect of
(SPSS Statistics 15.0,Chicago,IL, USA).
increasing dasatinib concentrations on the number of viable cells
at different times along the osteogenic differentiation process. A
Results doseandtime-dependentreductiononthenumberofviablecells
was observed as referred to the absence of the drug (control) at
Dasatinib inhibits PDGFR-b, c-Kit and c-Src
each time point, which was especially evident at the end of the
phosphorylation and modulates downstream signaling
differentiation period (21 days) and with dasatinib concentrations
on osteoprogenitor cells $10nM (Figure 2B). Also, OBs derived from primary MSCs
We first examined the effect of dasatinib on two tyrosine resultedmoresensitivetohigherdasatinibdosesthanOBsderived
kinases, PDGFR-b and c-Src, which are described targets for from the hMSC-TERT cell line (Figure 2B, left graph vs right
dasatinibandwithknowninvolvementinmesenchymalstemcell/ graph),withnosignificantdifferencesinthenumberofviableOBs
osteoblast (MSC/OB) proliferation and function. PDGF is derived fromMMpatientsand healthydonors (datanot shown).
mitogenictomesenchymalcellswhileinhibitingOBdifferentiation Itislikelythatthereducednumberofviablecellsinosteogenic
and bone matrix formation [31,32], and targeted deletion of cultures after dasatinib treatment could be majorly ascribed to
PDGFR-b in murine mesenchymal cells also stimulates OB reduced cell proliferation and/or increased apoptosis. Using the
differentiationandfunction[33].Ontheotherhand,knockdown hMSC-TERT cell line and a 7-day culture period, we show that
of Src expression has also been shown to enhance OB dasatinib effect until a 50nM dose was mainly due to a reduced
differentiationandfunction[19,34].Serum-starvedhMSC-TERT proliferative capacity (Figure 2C, left) as assessed by reduced
and MG-63 cell lines (respectively representing the multipotent numberofcelldivisionswhenincreasingdasatinibconcentration.
MSCsandthedifferentiatedOBs),wereexposedtoPDGF-BBin Thepercentageofapoptoticand/ornecrotichMSC-TERTcells,
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BoneEffectsofDasatinib
Figure1.DasatinibinhibitsPDGFR-b,c-Kitandc-Srcphosphorylationinmesenchymalandosteoblast-likecelllines.(A)Mesenchymal
(hMSC-TERT)andosteoblast-like(MG-63)celllineswerepretreatedwithdifferentconcentrationsofdasatinibfor6hoursandthenexposedtoPDGF-
BBorSCFfor20minutesbeforeproteinlysatesweregenerated.ImmunoblottingwithspecificantibodiesagainsttotalandphosphorylatedPDGFR-b,
c-Kitandc-Srcwereperformed.(B)Modulationofdownstreamsignalingafterdasatinibtreatment.Similarlytoexperimentalconditionsin(A),the
hMSC-TERT and the MG-63 cell lines were pretreated with 50nM dasatinib for 6hours, stimulated with PDGF-BB for 20minutes and then cell
harvested for protein isolation. Immunoblotting is shown for total and phosphorylated forms of PDGFR-b, c-Src, Erk 1/2, Akt and p38 mitogen
activatedproteinkinase(MAPK).
doi:10.1371/journal.pone.0034914.g001
however, was only slightly increased within the mentioned (Figure 3A), thereby supporting the osteoblastogenic therapeutic
dasatinib doses (Figure 2C, right). Our results are therefore potential of dasatinibin thisdisease.
consistent with those of other authors which have found that Wealsoexaminedthebiologicaleffectofdasatinibinpromoting
dasatinib inhibits osteoprogenitor cell proliferation [19] and osteogenicdifferentiationbymeasuringALPandthetranscription
induces apoptosiswith higherdoses ofthedrug ($100nM)[20]. factor Runx2/Cbfa1 activities as well as mineralized-matrix
Importantly, the number of viable cells in OBs derived from formation, in OBs differentiated from primary MSCs and from
primaryMSCs(eitherfromMMpatientsorhealthydonors)after the hMSC-TERT cell line. Dasatinib (at 2 nM or 5 nM) was
the 21-day differentiation period was more pronouncedly added to the osteogenic medium at the initiation of OB
diminished as compared to OBs derived from the hMSC-TERT differentiation, and ALP and Runx2/Cbfa1 activities were
cellline(Figure2B,leftgraphvsrightgraph).Thisissueshouldbe measured as surrogates of OB activity at day 7 and 14,
takenintoaccountifdasatinibisusedforthetreatmentofhuman respectively. Dasatinib significantly and dose-dependently in-
primary osteoprogenitor cells, in order to achieve a compromise creased ALP activity even in OBs derived from primary MSCs
betweendasatinibinhibitionofcellproliferationanditsosteogenic (Figure 3B, upper). Also Runx2/Cbfa1 activity measured in
potential. In fact, the use of high dasatinib concentrations might nuclear lysates of pre-OBs was augmented in the presence of
have been the reason why some authors failed to observe an dasatinib, although the increase was not statistically significant in
osteogeniceffectonhumanMSCswiththisdrug[22].Inthelight OBsderivedfromhealthydonors(Figure3B,lower).Toestimate
of these observations and for next experiments, we decided to matrixmineralization,alizarinredstaininganddyequantification
restrict the use of dasatinib concentrations to the low nanomolar wereperformedaftera21-dayosteogenicdifferentiationofMSCs.
range (#5 nM). Under the aforementioned conditions, OBs derived from the
hMSC-TERTcelllineunderwentacleardose-dependentincrease
inmineralization(Figure3C,left);aslightbutreproducibletrend
Low dasatinib concentrations promote osteogenic
towardsincreasedmatrixmineralizationwasalsoobservedinOBs
differentiation, alkaline phosphatase and Runx2/Cbfa1
derived from primary MSCs from healthy volunteers and
activities and matrix mineralization
myeloma patients, although it did not reach significance in the
Since OB maturation implies a balance between proliferation latter (Figure 3C,right).
and differentiation, it could be hypothesized that inhibition of The Wnt/b-catenin signaling pathway is known to play a key
osteoprogenitorproliferationbydasatinibwouldcorrelatewithan role in the osteogenic differentiation of mesenchymal progenitors
enhanced osteogenic differentiation. We evaluated whether andinnormalskeletaldevelopment[41].Therefore,weexamined
dasatinib was capable of modulating osteogenic gene expression whether the activity of dasatinib in our experimental setting was
in OBs derived from primary MSCs at the selected low accompanied by downstream activation of the canonical Wnt
concentrations (2–5nM). Expression of bone-formation markers signal transduction. As observed in Figure 3D by immunoblot
suchasalkalinephosphatase(ALP),collagenItypeA1(COLIA1) analysis, dasatinib clearly induced accumulation of the active
and the transcription factors Runx2/Cbfa1 and Osterix was dephosporylated form of b-catenin in the nuclear compartment,
evaluated by real time RT-PCR and analyzed at either day7 or whereas levels of the inactive phosphorylated form in the
14 of the differentiation process [40]. Figure 3A shows that cytoplasm were reduced. Taken together, our data show that the
dasatinib clearly increased the expression of the osteogenic genes MSC/OBlineageexpressestyrosinekinasessuchasPDGFR-b,c-
tolevelshigherthanthoseobservedinthesameconditionsbutin Src,andc-Kit,whoseactivationcanbepartiallyinhibitedbylow
absenceofthedrug(control).Interestinglyenough,thiseffectwas doses of dasatinib (#5 nM). Within the same range of dasatinib
not restrained toMSCsfromhealthy volunteers,butMSCsfrom concentrations, these effects are associated with activation of
myeloma patients also responded to dasatinib in a similar way canonical Wntsignaling.
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BoneEffectsofDasatinib
Figure2.DasatinibreducesthenumberofviablecellsbyinhibitionofmesenchymalandOBcellproliferation.(A)Dasatinibdecreases
thenumberofMSCandOBcellsinculture.ThehMSC-TERTandtheMG-63celllineswereculturedfor7daysinmaintenancemediumintheabsence
or presenceof increasing dasatinibconcentrations, andthenthenumberofcells ateachconditionwascountedwithahaemocytometeranda
TrypanBluesolution.Representativemicrographsareshown.(B)Dasatinibreducedthenumberofviablecellsinosteogenicculturesinatimeand
concentration-dependent manner. MSCs were maintained in osteogenic medium for 7, 14 or 21days in the presence of different dasatinib
concentrations,andpercentageofviablecellswasevaluatedwiththealamarBlueassayonOBsderivedfromthehMSC-TERT(left)andfromprimary
MSCsfromMMpatients(right).Dataareexpressedasthemean6SDfromthreeexperiments.Statisticallysignificantdifferencesfromcontrolare
indicatedas*P,0.05.(C)Dasatinib(5–50nM)reducesthenumberofcelldivisionsinthehMSC-TERTcellline(left)butdoesnotinduceapoptosis
(right). MSCs were stained with PKH67 and cultured in osteogenic medium for 7days in the absence or presence of dasatinib; at the time of
collection,cellswerealsostainedwithAnnexin-V-PEand7-AADandanalyzedbyflowcytometry.
doi:10.1371/journal.pone.0034914.g002
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BoneEffectsofDasatinib
Figure3.DasatinibpromotesosteogenicdifferentiationofMSCsfromMMpatientsandhealthydonorsandofthehMSC-TERTcell
line.(A)Dasatinibupregulatestheexpressionofbone-formationmarkersintheosteogenicdifferentiationprocess.PrimaryMSCsfromMMpatients
andhealthydonorswereculturedinosteogenicmediuminthepresence(2–5nM)orabsenceofdasatinib,andtotalRNAwasisolatedondays7and
14.Real-timeqRT-PCRwasusedtodeterminetheexpressionofseveralOBrelatedmarkers:ALPwasdeterminedatday7,whereasthetranscription
factorsRunx2/Cbfa1andOsterix(Osx),andcollagenItypeA1(COLIA1)weremeasuredatday14.Expressionlevelsforeachgenewerenormalizedto
GAPDHexpressionandreferredtothoseintheabsenceofdasatinib.Graphsillustratemeanvaluesfromsamplesfrom5healthydonorsand5MM
patients6SEM(bars)*P,0.05.(B)DasatinibincreasesALPandRunx2/Cbfa1activitiesinosteoprogenitorcells.InthehMSC-TERTcelllineandin
primaryhMSCsderivedfromthreemyelomapatientsandthreehealthydonors,ALPactivitywasmeasuredatday7(uppergraph)andRunx2/Cbfa1
activitywasmeasuredatday14(lowergraph)aftertheadditionofdasatinibtotheosteogenicdifferentiationmedium.Dataarerepresentedasthe
mean6SDfromthreeexperiments.(C)Dasatinib(2–5nM)augmentsbonematrixmineralizationinOBsderivedfromthehTERT-MSCcellline(left)or
MSCsfromhealthydonorsandmyelomapatients(right),asassessedbyalizarinredstainingquantification.Dataarerepresentedasthemean6SD
fromthreeexperimentswiththehMSC-TERTcellline,andasthemean(5MMpatientsand5healthydonors)6SEMinexperimentswithprimary
MSCs.Statisticallysignificantdifferencesfromcontrolsareindicatedas*,whereP,0.05.Micrographsshowmatrixmineralizationafteralizarinred
stainingofcorrespondentdifferentiatedOBs.(D)Bothdephospho-andphospho-b-cateninlevelsweredeterminedbyimmunoblottingincytosolicor
nuclearlysatesofpre-OBsdifferentiatedfromthehMSC-TERTcelllineintheabsenceorpresenceofdasatinib.HistoneH1anda-tubulinwereusedas
loadingcontrolsfornuclearorcytosolicproteinfractions.
doi:10.1371/journal.pone.0034914.g003
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BoneEffectsofDasatinib
Dasatinib promotes osteoblast differentiation in vivo 14–21. As seen in Figure 5A, when dasatinib was present for
Consistent with our in vitro findings, we corroborated the bone 21days, it markedly reduced OC numbers in a dose-dependent
anabolic properties of dasatinib in the in vivo setting. To better fashion (IC50=2.16nM; P,0.05 at $1 nM vs. control). When
discern the putative bone formation effect of dasatinib from its dasatinib was added to early OC progenitors (day 7) or to
knowninhibitoryactivityonOCformationandfunction,weused committedOCprecursors(day 14)itwasalsoeffectiveinreducing
skeletally-immature 5 week-old healthy mice which present very osteoclastogenesis, although higher doses were required: IC50
activeboneformationandminimalboneresorption.Twodifferent =3.14nM; P,0.05 at $2.5nM vs control (7–21days); IC50
dosesofdasatinib(2.5mg/kgvs10mg/kg)administeredtwice-a- =5.62nM;P,0.05at$2.5 nMvscontrol(14–21days).Notably,
day (BID) and two periods of dasatinib treatment (3 vs 7weeks) the number of OCs was markedly reduced at higher doses of
were compared in order to evaluate potential osteogenic/anti- dasatinib (Figure 5A, e.g., $5 nM dasatinib, 1–21days). This
proliferative activities of thedrug on endogenous osteoprogenitor could be explained by a toxic effect of dasatinib on OC
cells,aswehadobservedinvitroforprimaryMSCs.BeingALPan progenitors at those doses, but it may well also reflect that
earlymarkerofboneformation,Figure4AshowsthatALPlevels dasatinib istargetingessential pathways forOC viability.
in serum were significantly increased in mice treated with both Figure 5B shows the area of resorptive pits. Progressive
dosesofdasatinibafter3weeksoftreatment,whereasALPlevels substantial reductions of resorbed lacunae were observed with
remained unaffected with respect to vehicle-treated animals at increasing dasatinib concentrations, resorption being almost
longer treatment periods. Relative to osteocalcin (also a bone completelyabrogatedataconcentrationof2.5nM.Ofnote,this
formation marker, but expressed at later stages of OB differen- effectofdasatinibonOCsisachievedwithinsimilarlowdosesof
tiation),significantincreasesinserumwereobservedforbothdoses dasatinib(lownanomolarrange)asforitsactivityinpromotingin
of dasatinib after 3weeks and even further increments were vitro osteogenic differentiation from mesenchymal precursors.
attained in a 7-week period. Minimal differences were found in Thus, in vitro doses of 2–2.5 nM dasatinib on OCs are sufficient
osteocalcinserumlevelsbetweenthe2.5mg/kgBIDand10mg/ for inhibition of OC formation to a 20% of the control and to
kgBIDdoses,neitherafter3weeksnorafter7 weeksoftreatment, reduce the resorptive activity further to a 5% of the control, and
which probably reflects a near to plateau-effect on osteocalcin wouldnotinterferewiththeosteogenicactivityofthiscompound.
induction with the doses in our study. Due to the use of young
healthy mice with limited OC function, no changes on levels of Mechanism of action of dasatinib on osteoclasts
TRAP5b (a surrogate marker for OC number) were measured We first assessed that the low concentrations of dasatinib
betweenbaselineandafter3or7weeksoftreatment(Figure4C). capableofreducingOCformationandresorptioninourprevious
ThisisinaccordancewithaveryscarcepresenceofOCsobserved experiment (1–2nM) were also effective in inhibiting the
in the histological sections from femurs of control animals along activation of the M-CSF receptor, c-Fms, in OCs (as is shown in
theexperiment (datanot shown). Figure 6A). Since M-CSF and RANKL are the two main
The effects of both doses of dasatinib were also evaluated by proliferation and survival factors involved in osteoclastogenesis
quantitative micro-CT scanning of distal femurs of treated mice. from monocyte/macrophage precursors, the c-Fms kinase has
As observed in Figure 4D, dasatinib treatment led to a marked been consideredasa major target of dasatinibon OCs[17,18].
increase in trabecular microarchitecture of cancellous bone in a However,sincedasatinibiscapableofinhibitingOCformation
dose- and time-dependent manner. This effect was associated to whennotpresentthroughoutthedifferentiationprocessbutalsoat
significant increases of trabecular number (Tb N; mm21) and of later stages of OC differentiation (days7–21 and 14–21;
the ratio of bone perimeter per bone area (B Pm/B Ar; mm21), Figure 5A), together with the fact that OC resorption is more
together with decreased trabecular separation (Tb Sp; mm) effectively reduced than OC formation (Figure 5A vs 5B),
comparedwithvehicle-treatedanimals(Figures4E-G).Theeffects indirectly suggests that inhibition of additional tyrosine kinases
of dasatinib on increased trabecular structures were more otherthanc-Fms,arealsocontributingtodasatinibeffectsonthis
pronounced for the 10mg/kg BID and the 7-week period celltype.Figure6AshowsthatinOCprecursors,evenatdosesas
treatmentascomparedtotherestoftheexperimentalconditions. lowas1or2 nM,dasatinibiscapableofinhibitingtheactivation
The increased trabecular number was equally apparent by of two other tyrosine kinases, such as c-Src and c-Kit. The c-Src
histologic observation of newly formed trabeculae at the kinase is an essential molecule for OC resorption, intervening in
epiphyseal plate (Figure 4H), and also correlated with increased theaVb3integrinoutside-insignalinginthesealingzonebetween
number and intensity of staining of Tcf4 positive OB-like cells the OC and the bone matrix [43]; in accordance with these
lining the trabecular borders (Figure 4I). The transcriptional observations,c-Src-/-miceshowanosteopetroticphenotypewith
activationoftargetgenesbyTcftranscriptionfactorsmediatesthe OCsunabletoformruffledbordersdespiteanormalmorpholog-
activation of the canonical Wnt/b-catenin signalling pathway, ical appearance [44]. Thus, inhibition of this tyrosine kinase by
which is essential in OB differentiation [41]. Specifically, within dasatinib would greatly compromise OC functionality. On the
the Tcf family members, Tcf4 is the one most abundantly other hand, the ligand for c-Kit, the SCF, has been shown to be
expressed in OB cell lines and primary human MSCs [42]; mitogenicforOCprecursorsandtopromotematureOCactivity
therefore,theincreasednumberandintensityofTcf4positivecells [45]. Inhibition of signaling through c-Kit by dasatinib may
maywellreflectanincreasednumberofactiveOBsafterdasatinib therefore also play a role in inhibition of osteoclastogenesis and
treatment. diminished OCresorption.
Besides,whenanalyzingtheexpressionofseveralkeymolecules
Dasatinib inhibits osteoclast formation and activity implicated in OC commitment/differentiation/function, we were
We first confirmed the inhibitory effect of dasatinib in able to identify further and novel consequences of dasatinib
osteoclastogenesis (Figure 5A) and OC function (Figure 5B), as treatment on this cell type. As shown in Figure 6B, in early OC
hasalreadybeenreportedforthisdrug[17,18].Forthispurpose, progenitors (day 4 since initiation of in vitro differentiation)
PBMCs from healthy volunteers were incubated in an M-CSF/ dasatinib does not affect levels of PU.1, which is a transcription
RANKL-containing medium for 21days, and dasatinib was factorthatregulatesthecommitmentofmyeloidcellstocommon
added throughout the differentiation process or on days 7–21 or progenitorsformacrophagesandOCs[45].AtalaterstageofOC
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BoneEffectsofDasatinib
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