Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis by targeting NFATc1, NF-κB, and AP-1 activation in RAW264.7 cells

MOLECULAR CARCINOGENESIS

Silibinin Inhibits Prostate Cancer Cells- andRANKL-Induced Osteoclastogenesis by TargetingNFATc1, NF-kB, and AP-1 Activation in RAW264.7 Cells

Chandagirikoppal V. Kavitha,1 Gagan Deep,1,2 Subhash C. Gangar,1 Anil K. Jain,1 Chapla Agarwal,1,2

and Rajesh Agarwal1,2*1Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,University of Colorado Denver, Aurora, Colorado2University of Colorado Cancer Center, Aurora, Colorado

Currently, there are limited therapeutic options against bone metastatic prostate cancer (PCA), which is primarily

responsible for high mortality and morbidity in PCA patients. Enhanced osteoclastogenesis is an essential featureassociated with metastatic PCA in the bone microenvironment. Silibinin, an effective chemopreventive agent, is inphase II clinical trials in PCA patients but its efficacy against PCA cells-induced osteoclastogenesis is largely unknown.

Accordingly, here we examined silibinin effect on PCA cells-induced osteoclastogenesis employing human PCA(PC3MM2, PC3, and C4-2B) and murine macrophage RAW264.7 cells. We also assessed silibinin effect on receptoractivator of nuclear factor kB ligand (RANKL)-induced signaling associated with osteoclast differentiation in RAW264.7cells. Further, we analyzed silibinin effect on osteomimicry biomarkers in PCA cells. Results revealed that silibinin (30–

90 mM) inhibits PCA cells-induced osteoclast activity and differentiation in RAW264.7 cells via modulating expressionof several cytokines (IGF-1, TGF-b, TNF-a, I-TAC, M-CSF, G-CSF, GM-CSF, etc.) that are important in osteoclastogene-sis. Additionally, in RAW264.7 cells, silibinin decreased the RANKL-induced expression and nuclear localization of

NFATc1, which is considered the master regulator of osteoclastogenesis. Furthermore, silibinin decreased the RANKL-induced DNA binding activity of NFATc1 and its regulators NF-kB and AP1, and the protein expression of osteoclastspecific markers (TRAP, OSCAR, and cathepsin K). Importantly, silibinin also decreased the expression of osteomimicry

biomarkers (RANKL, Runx2, osteocalcin, and PTHrP) in cell culture (PC3 and C4-2B cells) and/or in PC3 tumors.Together, our findings showing that silibinin inhibits PCA cells-induced osteoclastogenesis, suggest that silibinin couldbe useful clinically against bone metastatic PCA. � 2012 Wiley Periodicals, Inc.

Key words: prostate cancer; osteoclastogenesis; silibinin; RANK ligand; NFATc1

INTRODUCTION

Most patients with prostate cancer (PCA) die notbecause of the tumor growth at the primary site, butbecause it has spread to the other sites. Bone is themost preferred site for PCA metastasis and virtuallyall patients dying of PCA experience extensivebone metastasis [1]. Patients with bone metastasisroutinely suffer extreme bone pain, spinal-cordcompression, and fractures [2–4]. In addition,replacement of bone marrow by growing PCA cellsdisrupts normal hematopoiesis resulting in anemiaand increased susceptibility to infections in PCApatients [3]. Therefore, bone metastasis is associatedwith significant morbidity and mortality in ad-vanced PCA.Once settled in bone, PCA cells alter the delicate

balance of bone remodeling orchestrated by twotypes of bone cells namely osteoclast (involved inbone degradation) and osteoblast (involved in boneformation) [3–6]. PCA cells promote osteoclastmaturation and activation; thereby enhance boneresorption and liberation of several growth factorsincluding transforming growth factor beta (TGF-b),insulin-like growth factors (IGFs), Bone morphogenetic

proteins (BMPs), PDGF, etc. [2–5]. Bone degradationprovides PCA cells the initial space to expand,and the released growth factors promote PCA cellssurvival and proliferation [3,5]. The growth factors

Additional supporting information may be found in the online ver-sion of this article.

Abbreviations: PCA, prostate cancer; TGF-b, transforming growthfactor beta; IGF, insulin-like growth factor; RANKL, receptor activatorof nuclear factor kB ligand; NFATc1, nuclear factor of activated T-cells; NF-kB, nuclear factor-kappa B; Runx2, Runt-related transcrip-tion factors 2; OSCAR, osteoclast-associated receptor; TRAP, tartrateresistant acid phosphatase; CCM, control conditioned media; SBCM,silibinin-treated conditioned media; DMSO, dimethyl sulfoxide;EMSA, electrophoretic mobility shift assay; PTHrP, parathyroid hor-mone-related protein; TNFa, tumor necrosis factor alpha; M-CSF,macrophage-colony stimulating factor; G-CSF, granulocyte-colonystimulating factor; GM-CSF, granulocyte macrophage-colony stimu-lating factor; IGFBP-3, insulin-like growth factor binding protein 3;AP1, activator protein 1.

Chandagirikoppal V. Kavitha and Gagan Deep contributed equallyto this work and share first authorship.

*Correspondence to: Skaggs School of Pharmacy and Pharma-ceutical Sciences, C-238, University of Colorado Denver, Aurora, CO.

Received 29 May 2012; Revised 20 August 2012; Accepted 23August 2012

DOI 10.1002/mc.21959

Published online in Wiley Online Library(wileyonlinelibrary.com).

� 2012 WILEY PERIODICALS, INC.

released by bone degradation as well as additionalfactors secreted by PCA cells such as endothelin-1,BMPs, Wnts promote osteoblast maturation, andformation of new bone [3–5,7]. Mature osteoblastsalso produce growth factors which further promotePCA cells growth in bone [3–5,7]. This vicious cycleinvolving PCA cells, osteoclasts, and osteoblasts pro-motes bone degradation as well as deposition ofnew ‘‘woven type bone’’ (uneven/immature/embry-onic), thus compromising the bone health and leadsto bone complications in PCA patients. WhereasPCA bone metastasis predominantly exhibits osteo-blastic features, histological findings, and analysesof bone turnover markers confirm that extensivebone degradation by activated osteoclasts is also anintegrated and essential phenomenon. Therefore,targeting osteoclast activity is considered one of thepreventive and therapeutic options against PCAbone metastasis.

Osteoclasts are highly specialized cells originatingfrom monocyte/macrophage lineage precursors. Inaddition to their critical role in skeletal develop-ment and maintenance, they are also implicated inthe pathogenesis of various bone diseases such as os-teoporosis, rheumatoid arthritis, and cancer metas-tasis as detailed above [8,9]. Hence, efforts to treatand/or prevent the skeletal-related events in PCAand other cancers have focused on the inhibition ofosteoclast activation [10,11]. Receptor activator ofnuclear factor kB ligand (RANKL) is considered as amain driver of osteoclast formation, survival, andfunction [9]. PCA cells also express RANKL and/orpromote RANKL expression in neighboring osteo-blast and stromal cells in the bone microenviron-ment, thereby enhance osteoclastogenesis [12].RANKL exerts its effect by binding to its receptorRANK present on osteoclast or its precursors, andactivates several major signaling pathways (nuclearfactor of activated T-cells (NFATc1), NF-kB, Akt, mi-togen activated protein kinase (MAPKs), etc.) re-quired for osteoclast differentiation and activity[13,14]. Considering the central role of RANKL inosteoclast differentiation, the identification of non-toxic agents that could target RANKL-induced sig-naling pathways and inhibit osteoclastogenesiswould have translational relevance against bonemetastatic PCA.

Silibinin, a flavonoid from milk thistle seed ex-tract, is one of the most-widely consumed dietarysupplements for its hepatoprotective efficacy[15,16]. Silibinin has shown remarkable efficacyboth in vitro and in vivo against PCA cells, and itsefficacy is currently being evaluated in PCA patients[17–22]. In the present study, we report silibinin ef-fect on PCA cells- and RANKL-induced osteoclasto-genesis in RAW264.7 cells, which is a well-established system to study osteoclastogenesis incell culture. We also demonstrate silibinin effect onimportant regulators of osteoclastogenesis and

osteomimicry biomarkers in PCA cells andxenografts.

MATERIALS AND METHODS

Cell Lines and Reagents

Human prostate carcinoma PC3 cells and murinemacrophage RAW264.7 cells were obtained fromthe ATCC (Manassas, VA). C4-2B cells were fromViroMed Laboratories. PC3MM2 cells were kind giftfrom Dr. Isaiah J. Fidler (University of Texas M.D.Anderson Cancer Center). Anti-rabbit peroxidaseconjugated secondary antibody was from Cell Sig-naling (Beverly, MA). a-Tubulin antibody was fromNeomarkers (Fremont, CA). Primary antibodies forRANKL, Runx2 (Runt-related transcription factors2), osteocalcin, TRAP, osteoclast-associated receptor(OSCAR), and biotinylated anti-rabbit secondary an-tibody were from Santa Cruz Biotechnology (SantaCruz, CA). NFATc1 antibody was from BD Bioscien-ces (New Bedford, MA). PTHrP, parathyroid hor-mone-related protein (PTHrP) and cathepsin Kantibodies were from Abcam (Cambridge, MA).Harris hematoxylin was from Sigma (St. Louis, MO)and 3,3-diaminobenzidine (DAB) was from Vectorlaboratories (Burlingame, CA). ECL detection kitand anti-mouse HRP conjugated secondary antibodywere from GE Healthcare (Buckinghamshire, UK).Bio-Rad detergent-compatible protein assay kit wasfrom Bio-Rad Laboratories (Hercules, CA). All otherreagents were obtained in their highest purity gradeavailable commercially.

Collection of Conditioned Media

PCA cells (PC3, PC3MM2, and C4-2B) were treatedat 30–40% confluency with dimethyl sulfoxide(DMSO) or silibinin (30, 60, and 90 mM) for 72 h incomplete media (RPMI1640 with 10% fetal bovineserum and 1% penicillin–streptomycin). Thereafter,media was removed and cells were washed twicewith 0.5% serum media and incubated for another12 h with 0.5% serum media. Subsequently, theconditioned media was collected, centrifuged, andlabeled as CCM (control conditioned media), or30SBCM, 60SBCM, and 90SBCM for 30, 60, and90 mM silibinin-treated conditioned media, respec-tively, and stored at �808C until further use. Cellnumber in each group was counted using a hemocy-tometer. In the co-culture experiments, the volumeof the conditioned media was normalized with re-spective cell number, and a ratio of 50% condi-tioned media (CCM/SBCM) and 50% RAW264.7media with 0.5% FBS was used.

Osteoclastogenesis Assay

RAW264.7 cells were incubated with CCM orSBCM collected from PC3MM2, PC3, and C4-2Bcells in the presence of 5 ng/ml RANKL. Media wasreplaced every 48 h and after 5 d, cells were stained

2 KAVITHA ET AL.

Molecular Carcinogenesis

for tartrate resistant acid phosphatase (TRAP) usingTRAP staining kit (Takara, Shiga, Japan). The num-ber of TRAP positive cells (a measure of osteoclastactivity) was determined using a light microscope.Cells were processed further as per the protocol anddifferentiated osteoclasts (with four or more nuclei)were counted. RAW264.7 cells supplemented with5 ng/ml of RANKL only served as negative control.In another experiment, we supplemented silibinin(10, 20, and 30 mM) exogenously in the CCM andanalyzed its effect on osteoclast activity and differ-entiation. RAW264.7 cells were also treated withRANKL (100 ng/ml) together with DMSO or silibinin(10, 20, and 30 mM) and analyzed for osteoclast ac-tivity and differentiation.

Cytokine Array

Cytokine level in CCM and SBCM was comparedusing Human Cytokine Antibody Array C series1000from RayBio Technology (Norcross, GA) followingvendor’s protocol. Briefly, array membranes weresequentially blocked, incubated with CCM/90SBCM,biotin-conjugated antibodies and HRP-conjugatedstreptavidin. Cytokines expression on the arraymembranes was visualized by ECL detection systemfollowed by exposure to X-ray film. Expression ofeach cytokine (in duplicate) was quantifiedthrough scanning the X-ray film using Adobe Photo-shop 6.0 (Adobe Systems Inc., San Jose, CA) fol-lowed by densitometry analyses using Scion Imageprogram (National Institutes of Health, Bethesda,MD) and RayBio Analysis Tool. Cytokine locationon the array 6 and 7 is illustrated in SupplementaryFigure 1.

Direct Co-Culture Assay

In direct co-culture assay, PC3MM2 and RAW264.7cells were co-cultured (1:5 ratio) and treated withDMSO or silibinin (5, 10, 20, and 30 mM). The mediawas replaced every 48 h, and after 5 d, osteoclast ac-tivity and differentiation were determined as de-scribed above.

Western Blotting

At the end of each treatment, whole-cell lysates ornuclear and cytoplasmic extracts were prepared, andWestern blotting was performed as described earlier[23–26]. In each case, blots were subjected to multi-ple exposures on the film to ensure that the banddensity was in the linear range. Few blots were mul-tiplexed or stripped and re-probed with differentantibodies including a-tubulin antibody to confirmequal protein loading. Autoradiograms/bands werescanned using Adobe Photoshop 6.0.

Confocal Microscopy

RAW264.7 cells were treated with DMSO or silibi-nin (30 mM) in 0.5% serum media with or withoutRANKL (10 ng/ml). After 24 h, cells were processed

for confocal microscopy as described before [27] todetect NFATc1 level and localization.

Electrophoretic Mobility Shift Assay (EMSA)

EMSA was performed as described earlier [28].Briefly, NFATc1, nuclear factor-kappa B (NF-kB), andactivator protein 1 (AP1) specific oligonucleotideswere end-labeled with [g-32P]ATP using T4 polynu-cleotide kinase in 10� kinase buffer as per the man-ufacturer’s protocol (Promega, Madison, WI).Approximately 8 mg of nuclear extract was incubat-ed with 5� gel shift binding buffer and then withg32P end-labeled consensus oligonucleotides for20 min at 378C. DNA–protein complex was resolvedon 6% DNA retardation gel followed by gel dryingand autoradiography. Supershift assay was per-formed using specific antibodies (NFATc1; p50 andp65 for NF-kB; c-Jun and c-Fos for AP1) in nuclearextract from RANKL-treatment group and competi-tion assay was performed using excess unlabeledcold probe in the reaction mixture.

Immunohistochemistry (IHC)

We used archived paraffin-embedded PCA xeno-graft tissues from our previously completed PC3orthotopic xenograft study [18] to determine in vivoeffect of silibinin administration on the levels ofosteomimicry biomarkers following IHC proceduresdetailed earlier [29]. All the microscopic IHC analy-ses were performed using a Zeiss Axioscope 2 micro-scope (Carl Zeiss Microscopy, Thornwood, NY) andphotographs were captured (at 400�) with a CarlZeiss AxioCam MrC5 camera with Axiovision Rel4.5 software.

Statistical Analyses

Statistical analysis was performed using SigmaStat2.03 software (Jandel Scientific, San Rafael, CA).Data was analyzed using one-way ANOVA followedby Tukey or Bonferroni t-test and a statistically sig-nificant difference was considered at P � 0.05.

RESULTS

Silibinin Inhibits PCA Cells-Induced OsteoclastDifferentiation and Activity in RAW264.7 Cells

To examine the effect of silibinin on the PCAcells-induced osteoclastogenesis, we analyzed theeffect of CCM and SBCM on osteoclast differentia-tion and activity using RAW264.7 cells. We added5 ng/ml RANKL in CCM and SBCM to boost theosteoclastogenesis. After 5 d, we observed differenti-ated osteoclasts in CCM treated RAW264.7 cellswhile significantly lesser differentiated osteoclastswere formed in RAW264.7 cells treated with30SBCM, 60SBCM, and 90SBCM (Figure 1A). Negli-gible, if any, differentiated osteoclast was observedin RAW264.7 cells treated with 5 ng/ml RANKLalone. Osteoclast activity determined by TRAP

SILIBININ INHIBITS PCA- AND RANKL-INDUCED OSTEOCLASTOGENESIS 3


staining revealed that compared to CCM, SBCMtreated RAW264.7 cells have significantly lesserTRAP positive cells (Figure 1B). Silibinin effect wasdose-dependent and consistent in all the three PCAcell lines, but its effect was most prominent inPC3MM2 cells (Figure 1A and B). The representativeimages of TRAP positive RAW264.7 cells (purplishred cells marked by arrows) in different treatmentgroups (RANKL only or CCM and 90SBCM fromPC3MM2 cells) are shown in Figure 1C. Taken to-gether, these results showed that silibinin inhibitsPCA cells-induced osteoclastogenesis.

Silibinin Modulates the Level of Cytokines Secreted by

PCA Cells

Cytokines secreted by PCA cells in the bone mi-croenvironment play an important role in promot-ing osteoclastogenesis [3,4,6]. Since we observedsignificantly lesser osteoclastogenesis with SBCMcompared to CCM, next we compared the condi-tioned media (CCM and 90SBCM from PC3MM2

cells) for the expression of cytokines. Cytokinearrays analyses revealed the expression of 120 cyto-kines in the PCA conditioned media (CCM andSBCM) suggesting the complexity of interactioninvolved in the CCM-induced osteoclastogenesis.Array results showed that cytokines have differentialexpression in SBCM compared to CCM. The exactrole of individual changes in the cytokine expres-sion on osteoclastogenesis would require morefocused studies; however, we have listed few cyto-kines whose expression was increased (angiogenin,granulocyte macrophage-colony stimulating factor(GM-CSF), IL-6, insulin-like growth factor bindingprotein 3 (IGFBP-3), tissue inhibitor of metallopro-teinase (TIMPs), etc.) or decreased (IFN-g, IGF,TGF-b, tumor necrosis factor alpha (TNFa), macro-phage-colony stimulating factor (M-CSF), granulo-cyte-colony stimulating factor (G-CSF), etc.) bysilibinin treatment, and these cytokines directlyor indirectly affect osteoclastogenesis (Figure 2)[930–34]. We have circled and alphabetically labeled

Figure 1. Effect of silibinin on PCA cells-induced osteoclast differ-entiation and activity in RAW264.7 cells. (A and B) PCA cells(PC3MM2, PC3, and C4-2B) were treated with silibinin (30–90 mM)and conditioned media was collected as detailed in Materials andMethods’ Section. RAW264.7 cells were treated with CCM or SBCMin presence of RANKL (5 ng/ml). In each case, media was replacedafter 48 h, and after 5 d, cells stained for TRAP. Differentiated

multinucleated osteoclasts (four or more nuclei) and TRAP positivecells were counted. (C) Representative images from osteoclastogene-sis assay with TRAP positive RAW264.7 cells (marked by arrows) areshown. CCM, control conditioned media; SBCM, silibinin-treatedconditioned media; �P � 0.001; #P � 0.01; $P � 0.05. [Color figurecan be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/mc]

4 KAVITHA ET AL.


Figure 2. Effect of silibinin on cytokines secretion in PC3MM2cells. Conditioned media was collected from DMSO or silibinintreated PC3MM2 cells, and processed for cytokine antibody array(volume normalized with respective cell number). (A) Expression ofcytokines in CCM and SBCM on Array-6, and (B) expression of cyto-kines in CCM and SBCM on Array-7. Osteoclastogenesis relevant

cytokines, whose expression was modulated with silibinin treatment,were labeled alphabetically on the array 6 and 7. (C and D) Thechange in the densitometry values of selected cytokines in 90SBCMversus CCM in Array-6 and Array-7 are presented. CCM, controlconditioned media; 90SBCM, 90 mM silibinin-treated conditionedmedia



these selected cytokines on the arrays (Figure 2Aand B) and presented name and changes in theirexpression (SBCM/CCM densitometric values) in thetables (Figure 2C and D).

Silibinin Directly Inhibits PCA Conditioned Media- andRANKL-Induced Osteoclast Differentiation and Activity in

RAW264.7 Cells

The above detailed experiment established an in-direct effect of silibinin on osteoclastogenesis, thatis, through targeting the secretion of various cyto-kines by PCA cells, which play a critical role in oste-oclast differentiation and activity. Based on theseresults, next, we assessed whether silibinin could di-rectly target the osteoclast differentiation and activi-ty. First, we incubated RAW264.7 cells with CCMfrom PC3MM2, PC3, and C4-2B cells together withsilibinin at 10, 20, and 30 mM for 5 d and analyzedthe effect on osteoclast differentiation and activity.Silibinin treatment inhibited the CCM-induced oste-oclast differentiation in a dose-dependent manner(Figure 3A). TRAP positive cells induced by CCMwere also significantly reduced upon silibinin addi-tion (Figure 3B). In another experiment, we stimu-lated RAW264.7 cells with RANKL (100 ng/ml) andtreated with DMSO or silibinin (10, 20, and 30 mM).As shown in Figure 3C, silibinin treatment stronglyinhibited RANKL-induced osteoclast differentiationand activity. Together, these results suggested thatsilibinin could also directly inhibit osteoclast differ-entiation and activity.

Silibinin Inhibits Osteoclast Differentiation and Activity in

RAW264.7 Cells Co-Cultured With PC3MM2 Cells

Next, to more closely simulate bone microenvi-ronment condition, we examined whether silibinincould inhibit osteoclast formation when PCA cellsare in direct contact with RAW264.7 cells. As shownin Figure 3D (bar diagram and photomicrographs),the presence of PC3MM2 cells induced the osteo-clast differentiation and activity in RAW264.7 cells,which was inhibited by silibinin treatment; whileTRAP positive cells were not observed in RAW264.7or PC3MM2 cells cultured alone.

Silibinin Suppresses RANKL-Induced NFATc1 Expression in

RAW264.7 Cells

NFATc1, a member of the NFAT family of tran-scription factors, is strongly up-regulated duringRANKL-induced osteoclastogenesis [35]. It has beenreported that NFATc1-deficient embryonic stemcells fail to differentiate into osteoclasts, and ectopicexpression of NFATc1 causes precursors to undergoefficient osteoclast differentiation even in the ab-sence of RANKL [35]. Hence, NFATc1 has beencoined as ‘‘master regulator of osteoclastogenesis.’’To understand the mechanism/s responsible for theinhibitory effect of silibinin on osteoclastogenesis,we next analyzed its effect on RANKL-induced

NFATc1 expression in RAW264.7 cells. As shown inFigure 4A, silibinin (30 mM) inhibited the RANKL-induced NFATc1 expression after 6, 12, and 24 h oftreatment. Furthermore, silibinin treatment stronglydecreased the RANKL-induced NFATc1 protein ex-pression in both nuclear and cytoplasmic fractions(Figure 4A). These results were further confirmed byimmunofluorescence, where RANKL stimulation(24 h) increased the NFATc1 expression (Alexa Fluor488-green staining) in both nucleus and cytoplasm,while silibinin treatment decreased the overallRANKL-induced NFATc1 expression (Figure 4B).NFATc1 binds to specific region of the DNA and

controls the transcription of genes that are criticallyimportant in the osteoclastogenesis [9,36]. Accord-ingly, next, we examined silibinin effect on theDNA binding of NFATc1 by EMSA. Our results clear-ly showed that silibinin treatment strongly inhibitsthe NFATc1 DNA binding (Figure 4C, left panel).EMSA results were confirmed using the NFATc1antibody and competition with excess of coldprobe (Figure 4C, right panel). Furthermore, silibi-nin treatment strongly inhibited RANKL-causedincrease in the expression of osteoclast specificmarkers TRAP, cathepsin K, and OSCAR (Figure 4D),which are controlled by NFATc1 [35,37]. Together,these data suggest that silibinin effectively inhibitsRANKL-induced NFATc1 activation and its transcrip-tional targets that result in the inhibition ofosteoclastogenesis.

Silibinin Inhibits RANKL-Induced NF-kB and AP1 Activationin RAW264.7 Cells

Upon RANKL binding, RANK activates severalsignaling pathways including NF-kB and AP1,which directly or indirectly play a role inthe NFATc1 activation and osteoclastogenesis[9,13,14,38]. Therefore, next we examined silibinineffect on RANKL-induced activity of NF-kB and AP1.EMSA results clearly showed that RANKL increasedthe NF-kB DNA binding after 3, 6, and 12 h; andRANKL-induced NF-kB DNA binding was inhibitedby silibinin (30 mM) treatment (Figure 5A). RANKLalso increased the AP1 DNA binding at 3 and 6 htime-points, even though increase was not asstrong as NF-kB; and even a decrease in AP1 DNAbinding was observed after 12 h of RANKL exposure(Figure 5B, left panel). However, an increase inAP1 DNA binding with RANKL was evident again at24 h time-point (Figure 5B, right panel). Silibinin(30 mM) treatment strongly reduced the RANKL-induced DNA binding of AP1 at all time-points stud-ied except 12 h (Figure 5B). These results clearlyshowed the inhibitory effect of silibinin on RANKL-induced NF-kB and AP1 activation in RAW264.7cells. These results also suggested a time-dependentRANKL effect on AP1 activation and demands in-depth time-course studies to clearly establish RANKLeffect on AP1 DNA binding.

6 KAVITHA ET AL.


Figure 3. (A and B) Direct effect of silibinin on osteoclast differen-tiation and activity induced by PCA cells conditioned media andRANKL. RAW264.7 cells were treated with CCM from PC3MM2,PC3, and C4-2B cells in presence of DMSO or silibinin (10, 20, and30 mM) together with RANKL (5 ng/ml). After 5 d of treatment,osteoclast differentiation and activity was measured. (C) Effect ofsilibinin on RANKL-induced osteoclast differentiation and activity.RAW264.7 cells were treated with DMSO or silibinin (10, 20, and30 mM) in the presence of RANKL (100 ng/ml), and after 5 d oftreatment, osteoclast differentiation, and activity was measured.(D) Effect of silibinin on osteoclast differentiation and activity in

RAW264.7 cells co-cultured with PC3MM2 cells. PC3MM2 andRAW264.7 cells were co-cultured (1:5 ratio) and treated with DMSOor silibinin (5–30 mM). Media was replaced after 48 h, and after 5 dof treatment, osteoclast differentiation, and activity was measured.Representative microscopic images from direct co-culture assay withTRAP positive cells (marked by arrows) are shown. SB, silibinin;CCM, control conditioned media; SBCM, silibinin-treated condi-tioned media; �P � 0.001; #P � 0.01; $P � 0.05. [Color figure canbe seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/mc]



Subsequently, EMSA results were confirmed usingspecific antibodies (p50, p65, c-Jun, and c-Fos) insupershift assay and competition with excess of coldprobe (Figure 5C and D). A clear supershift in NF-kBwas observed with p50 antibody (Figure 5C).Though, we did not observe a supershift in AP1 inthe presence of c-Jun or c-Fos antibody but only adecrease in the gel shift (Figure 5D), and theseresults indirectly implied a partial presence of c-Junand c-Fos in the AP1–DNA complex. More studiesare required to identify the other possible constitu-ents of AP1 (JunB, JunD, Fra1-2, or ATF1-7) in AP1–DNA complex. Together, these results suggest thatsilibinin inhibits RANKL-induced signaling path-ways that have an important role in regulatingNFATc1 expression as well as osteoclastogenesis.

Silibinin Down-Regulates Expression of OsteomimicryMarkers in PCA Cells Both In Vitro and In Vivo

PCA cells have a unique characteristic of adaptingto the bone microenvironment through expressingseveral proteins whose expression is usually limitedto bone cells, a phenomenon termed as ‘‘osteomimi-cry’’ [39,40]. Accordingly, we also examined silibi-nin effect on osteomimicry biomarkers in PCA cellsincluding RANKL, Runx2, and PTHrP. As shown inFigure 6A, silibinin treatment down-regulated theexpression of these molecules in PC3 and C4-2Bcells with a strong effect observed only at 90 mMdose. Next, we confirmed these cell culture resultsin vivo by analyzing PC3 orthotopic xenograft tis-sues. Earlier, we have reported that silibinin feeding

Figure 4. Effect of silibinin on RANKL-induced NFATc1 activationand osteoclast specific markers in RAW264.7 cells. (A) RAW264.7cells were treated with or without RANKL (10 ng/ml) and silibinin(30 mM) for the indicated time in 0.5% serum media. Whole-celllysates or nuclear and cytoplasmic extracts were prepared and ana-lyzed for NFATc1 expression by Western blotting. (B) Effect of silibi-nin on the RANKL-induced expression and cellular localization ofNFATc1 was analyzed by immunofluorescence. Images were cap-tured at 1,000� magnification on a Nikon inverted confocal micro-scope using 488/405 nm laser wavelengths to detect Alexa Fluor

488 (green) and 40,6-diamidino-2-phenylindole (DAPI; blue) emis-sions, respectively. (C) Nuclear extracts were prepared after 24 h ofRANKL and silibinin treatment in RAW264.7 cells and analyzed forNFATc1 DNA binding activity by EMSA (left panel). Super shift andcompetition assays were performed using nuclear extract fromRANKL-treatment group to confirm the specificity of NFATc1 binding(right panel). (D) Effect of silibinin on osteoclast specific markers(TRAP, cathepsin K and OSCAR) was analyzed by Western blottingin whole-cell lysates. SB, silibinin.

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(100 mg/kg body weight for 7 wk) inhibits thegrowth of PC3 tumors in the prostate microenviron-ment [18]. IHC analyses of these PC3 tumor tissuesrevealed a significant reduction in the expression ofRANKL, Runx2, osteocalcin, and PTHrP in silibinin-treated group in comparison to control (Figure 6B).Taken together, these results clearly suggest that sili-binin inhibits osteomimicry markers in PCA cellsboth in vitro and in vivo.

DISCUSSION

PCA continues to be the most common non-cuta-neous cancer and the second leading cause of can-cer-related deaths among American men [41].According to American Cancer Society report, about241,740 incidences (29% of total estimated newcases) and 28,170 deaths (9% of total estimateddeaths) due to PCA are estimated in American menin 2012 [41]. Bone metastasis is considered primarilyresponsible for high morbidity and mortality in PCApatients. To alleviate pain, PCA patients with bonemetastasis are generally treated with bisphospho-nates which are osteoclast inhibitors [42]. Denosu-mab (trade name ‘‘Xgeva’’), a human monoclonalantibody against RANKL, has been reported to delaybone metastasis in men with PCA [11,43]. Recently,denosumab was approved for the prevention of skel-etal-related events in patients with breast or prostatecancer. But both bisphosphonates and denosumabhave considerable adverse effects such as osteonec-rosis and hypocalcaemia, and there are still doubtsabout their beneficial effect on the overall survivalof the patients [11]. Therefore, targeting PCA cells aswell as osteoclastogenesis through non-toxic naturalagents is relevant, and in the present study, we dem-onstrate the effect of one such natural agent, silibi-nin, on the PCA cells-induced osteoclastogenesis.In recent years, silibinin has been extensively

studied in pre-clinical models as well as clinically

for its efficacy against variety of cancers includingPCA [44]. Anti-cancer effects of silibinin havebeen shown by targeting proliferation, apoptosis,angiogenesis, epithelial–mesenchymal transition(EMT), and metastasis [44,45]. Silibinin also targetstumor microenvironment components such as en-dothelial and macrophage cells towards inhibitingangiogenesis in lung tumors [46]. There have beenearlier reports that silibinin inhibits osteoclastogen-esis while enhances osteoblastogenesis [30,34].Kim et al. [34] have shown that silibinin treatmentdoes not affect mature osteoclast function butinhibits the resorption pits formation via targetingosteoclastogenesis. Despite these earlier studies,the effect of silibinin on the PCA cells interactionwith bone microenvironment component (suchas osteoclast) remained unknown. Results frompresent study for the first time revealed thatsilibinin targets PCA cells-induced osteoclast differ-entiation and activity in RAW264.7 cells. Anotherhighlight of the present study is that we report in-hibitory effect of silibinin on several osteomimicrybiomarkers (RANKL, Runx2, osteocalcin, and PTHrP)in PCA cells. The inhibition of osteomimicry bio-markers’ expression might compromise PCA cells’ability to adapt to the bone microenvironmentand result in the inhibition of bone metastaticgrowth.

Osteoclastogenesis is a complex phenomenon andin addition to RANKL, it is affected by severalgrowth factors and cytokines [31,32,34,47–57]. Cy-tokine array results showed that silibinin treatmentaltered the expression of several cytokines secretedby PCA cells (Figure 2); and these changes in cyto-kines level could be responsible for the significantinhibition of osteoclastogenesis observed withSBCM compared to CCM (Figure 1). For example,silibinin decreased the level of several cytokines(including IGF-1, TGF-b, TNF-a, HGF, thymus

Figure 5. Effect of silibinin on RANKL-induced NF-kB and AP1 activation in RAW264.7 cells. (A–D) RAW264.7cells were treated with silibinin (30 mM) for the indicated time in the presence of RANKL (10 ng/ml), and nucle-ar extracts were prepared and analyzed for (A) NF-kB and (B) AP1 DNA binding activity by EMSA. Super shiftand competition assays were performed using nuclear extract from RANKL-treatment group to confirm thespecificity of (C) NF-kB and (D) AP1 binding. SB, silibinin.



activation regulated cytokine (TARC), and IL-17)that are known to promote osteoclastogenesis[32,34,51,54–56]. Furthermore, silibinin treatmentenhanced the level of several cytokines (such asangiogenin, Acrp30, IGFBP-3, I-TAC, and TIMPs)that could directly or indirectly inhibit osteoclasto-genesis [48–50,52,53]. Similarly, silibinin treatmentdecreased the level of M-CSF and G-CSF, but

enhanced the level of GM-CSF. Importantly, bothM-CSF and G-CSF promote osteoclastogenesis, whileGM-CSF inhibits osteoclastogenesis [31,47,54,57].Together, cytokine array results suggested that silibi-nin could target multiple circuitries towards inhibit-ing osteoclastogenesis; however extensive studiesare required to validate the role of individual cyto-kine targeted by silibinin.

Figure 6. Effect of silibinin on osteomimicry biomarkers in PCAcells. (A) PC3 and C4-2B cells were treated with silibinin at indicateddoses for 72 h. At the end of the treatment, whole-cell lysates wereprepared and analyzed for the expression of RANKL, Runx2, andPTHrP by Western blotting. (B) Paraffin-embedded PCA xenografttissues from previously completed PC3 orthotopic xenograft studywere analyzed to determine effect of silibinin administration on the

expression of RANKL, Runx2, osteocalcin (OC), and PTHrP by IHC.Immunoreactivity (represented by brown staining) of these biomark-ers was scored as 0þ (no staining), 1þ (weak staining), 2þ (moder-ate staining), 3þ (strong staining), 4þ (very strong staining). Thedata shown in the bar diagram represents mean � SEM of four tofive samples for each group. SB, silibinin; �P � 0.001.

10 KAVITHA ET AL.


As mentioned above, RANKL-RANK signalingis the main regulator of osteoclast differentiationprocess [9,14]. RANKL binding to its receptorRANK results in the recruitment of adaptormolecule TRAF6, activation of NF-kB, and subse-quent initial induction of NFATc1 [9,13]. Thereafter,AP1 complex containing c-Fos plays a role in theauto-amplification of NFATc1, and enables a robustinduction of NFATc1 [9]. RANKL also activatesMAPKs which could affect NFATc1 expression viamodulating AP-1 activation [9,13,58]. NFATc1 coop-erates with other transcriptional factors (AP1, PU.1,MITF, etc.) to activate osteoclast-specific genes suchas TRAP, cathepsin K, OSCAR [9,36,59]. Results frompresent study showed that silibinin targeted multi-ple signaling molecules (NF-kB, AP-1, and NFATc1)towards inhibiting osteoclastogenesis. For example,silibinin strongly decreased the RANKL-inducedNFATc1 expression as well as its DNA binding activi-ty resulting in a decreased expression of NFATc1 reg-ulated genes (TRAP, cathepsin K, and OSCAR).Similarly, silibinin treatment inhibited the RANKL-induced DNA binding of NF-kB and AP1. However,more studies are needed in future to establish therelative importance of these signaling molecules insilibinin-caused inhibition of osteoclastogenesis.Overall, results from the present study clearly

established that silibinin inhibits PCA cells-inducedosteoclastogenesis. Molecular studies showed thatsilibinin inhibited multiple signaling molecules butNFATc1 seems to be the central target in osteoclas-togenesis inhibition by silibinin. Considering thefact that silibinin consumption is extremely safeand that it has already been tested in PCA patients,our results suggest translational utility of silibinin inPCA patients with advanced metastatic disease.

ACKNOWLEDGMENTS

This work was supported by NCI RO1 grantCA102514.

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Documents

Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis by targeting NFATc1, NF-κB, and AP-1 activation in RAW264.7 cells