plumbagininhibitsosteoclastogenesisandreduceshuman breast cancer-induced osteolytic … · with...

Therapeutic Discovery

Plumbagin Inhibits Osteoclastogenesis and Reduces HumanBreast Cancer-Induced Osteolytic Bone Metastasis inMice through Suppression of RANKL Signaling

Bokyung Sung1, Babatunde Oyajobi2, and Bharat B. Aggarwal1

AbstractBone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and

multiple myeloma; agents that can suppress this bone loss have therapeutic potential. Extensive research within

the last decadehas revealed thatRANKL, amemberof the tumornecrosis factor superfamily, plays amajor role in

cancer-associated bone resorption and thus is a therapeutic target. We investigated the potential of vitamin K3

analogue plumbagin (derived from Chitrak, an Ayurvedic medicinal plant) to modulate RANKL signaling,

osteoclastogenesis, and breast cancer-induced osteolysis. Plumbagin suppressed RANKL-induced NF-kBactivation inmousemonocytes, an osteoclast precursor cell, through sequential inhibition of activation of IkBakinase, IkBa phosphorylation, and IkBa degradation. Plumbagin also suppressed differentiation of these cells

into osteoclasts induced either by RANKL or by human breast cancer or humanmultiplemyeloma cells.When

examined for its ability to prevent human breast cancer-induced bone loss in animals, plumbagin (2 mg/kg

body weight) administered via the intraperitoneal route significantly decreased osteolytic lesions, resulting in

preservation of bone volume in nude mice bearing human breast tumors. Overall, our results indicate that

plumbagin,avitaminKanalogue, is apotent inhibitorof osteoclastogenesis inducedby tumorcells andofbreast

cancer-induced osteolytic metastasis through suppression of RANKL signaling.Mol Cancer Ther; 11(2); 350–9.

�2011 AACR.

Introduction

Bonemetastasis is a common complication of advancedcancers such as breast cancer and prostate cancer.Depending on the site of primary tumor, metastasis tobone occurs in as many as 70% of patients with metastaticdisease and often results in skeletal morbidity (1, 2).Sequelae of bone metastasis include hypercalcemia ofmalignancy, severe bone pain, and debilitating skeletalmorbidity (1, 2). Bone metastases are commonly charac-terized as osteolytic, osteoblastic/osteosclerotic, ormixed. Although breast cancer is most often associatedwith osteolytic or mixed metastases, osteoblastic metas-tases are common inprostate cancer.Osteolytic lesions aredue to a marked increase in osteoclast number withreduced osteoblastic activity. Parathyroid hormone-relat-

ed protein (PTHrP) is known as a major player betweentumor and bone cells and induces the osteolytic process inpart through activation of the RANKL pathway (3). Incontrast to osteolytic lesions, osteosclerotic metastases aredefined by adramatic increase in newbone formation, butthey always possess a resorption component (4, 5).

Breast cancer cells produce factors that induce osteo-clastogenesis, PTHrP and interleukins (IL)-1, -6 and -11,which act on bone-forming osteoblasts to increaseproduc-tionof anessentialosteoclast stimulator, receptoractivatorof nuclear factor-kappa B (RANK) ligand (RANKL;refs. 4, 6, 7). RANKL-induced osteoclastogenesis is medi-ated through the cell surface receptor RANK. The inter-action of RANKLwith RANK leads to recruitment of TNFreceptor-associated factor to cell surface receptor RANKand then activate NF-kB signaling pathways (8). Indeed,secretion of RANKLby various tumor cells induces osteo-clastogenesis (9–12). Therefore, selective modulation ofRANKL signaling pathwaysmay have therapeutic poten-tial for cancer-inducedbone lossandbone-relateddiseasessuchasosteoporosis andosteoarthritis.A fullyhumanizedmonoclonalneutralizingantibody toRANKL,denosumab[also called Prolia (Amgen)], has been approved by the U.S. Food and Drug Administration for use in postmeno-pausal women with risk of osteoporosis.

Identification of novel targets for traditional medicineprovides a reverse pharmacology or bedside to benchapproach for novel drug discovery. We report here our

Authors' Affiliations: 1Cytokine Research Laboratory, Department ofExperimental Therapeutics, The University of Texas MD Anderson CancerCenter, Houston; 2Department of Cellular and Structural Biology, TheUniversity of Texas Health Science Center at San Antonio, San Antonio,Texas

Note: B.B. Aggarwal is the Ransom Horne, Jr., Professor of CancerResearch.

Corresponding Author: Bharat B. Aggarwal, The University of Texas MDAnderson Cancer Center, Unit 1950, Houston, TX 77030. Phone: 713-794-1817; Fax: 713-745-6339; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0731

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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Published OnlineFirst November 16, 2011; DOI: 10.1158/1535-7163.MCT-11-0731

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investigation of the potential of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; Fig. 1A), derived from theroot of an Ayurvedic plant, Plumbago zeylanica L. (alsoknown as Chitrak), to modulate RANKL signaling andbone loss. This compound has also been identified inJuglans regia (English walnut), Juglans cinerea (butternutand white walnut), and Juglans nigra (black walnut;ref. 13). Plumbagin has been shown to exert antibacterial(14), antifungal (15), antiatherosclerotic (16), and antican-cer effects (17–19) through a variety of mechanisms,including induction of ROS (20); suppression of NF-kB(13), AKT/mTOR (21), and STAT3 (22); induction of p53and JNK (23); activation of the NRF2-ARE pathway (24)and direct inhibition of histone acetyltransferase p300(25). In animal studies, it has been shown to be chemo-preventive for colon cancer (26) and to exhibit antitumoractivity in prostate cancer (27).We investigated whether plumbagin can suppress the

RANKL-induced NF-kB activation pathway and osteo-clastogenesis. Whether this vitamin K analogue can affectosteoclast differentiation induced by tumor cells was alsoexamined. Moreover, we investigated whether plumba-gin could prevent human breast cancer-induced bone lossin vivo. The results show that plumbagin inhibited theRANKL-inducedNF-kB activation pathway by inhibitingIkBa kinase (IKK); this effect correlated with suppressionof osteoclastogenesis induced by RANKL or by breastcancer or multiple myeloma cells. Finally, plumbagininhibited human breast cancer-induced osteolytic lesionsin nude mice.

Materials and Methods

ReagentsPlumbagin (purity >97%) was purchased from Sigma-

Aldrich. A 10 mmol/L solution of plumbagin was pre-pared in dimethyl sulfoxide, stored as small aliquots andthen diluted as needed in cell culture medium. DulbeccoModified Essential Medium (DMEM)/F12, RPMI 1640,DMEM, 0.4% trypan blue vital stain, and antibiotic-anti-mycotic mixture were obtained from Invitrogen. Fetalbovine serum (FBS) was purchased from Atlanta Biolo-gicals, Inc. Antibodies against IkBa, IKKa, and IKKbwereobtained from Imgenex. Antibody against phospho-IkBa(Ser32/36) was purchased from Cell Signaling Technolo-gy. Goat antirabbit and goat antimouse horseradish per-oxidase conjugates were purchased from Bio-Rad. Anti-body against b-actin and the leukocyte acid phosphatasekit (387-A) for tartrate-resistant acid phosphatase (TRAP)staining were purchased from Sigma-Aldrich. Protein A/G-agarose beads were obtained from Pierce. [g-32P]ATPwas purchased from MP Biomedicals.

Cell linesRAW 264.7, MDA-MB-231, MCF-7, MM.1S, and U266

cells were obtained from American Type Culture Collec-tion. RAW 264.7 cells were cultured in DMEM/F12 sup-plementedwith 10% FBS and antibiotics. This cell line is a

well-established osteoprogenitor cell system that has beenshown to express RANK and differentiate into functionalTRAP-positive osteoclasts when cultured with solubleRANKL (28). MDA-MB-231 and MCF-7 cells were cul-tured in DMEM, andMM.1S and U266 cells in RPMI 1640with 10% FBS. The above-mentioned cell lines were pro-cured more than 6 months ago and have not been testedrecently for authentication in our laboratory.

Electrophoretic mobility shift assays for NF-kBNuclear extractswerepreparedasdescribedpreviously

(29).

Western blot analysisTo determine the levels of protein expression in the

cytoplasm or nucleus, we prepared extracts (30) andfractionated them by 10% SDS-PAGE. After electropho-resis, theproteinswere electrotransferred tonitrocellulosemembranes, blotted with each antibody, and detected byenhanced chemiluminescence reagent (GE Healthcare).

IKK assayTo determine the effect of plumbagin on RANKL-

induced IKK activation, IKK assay was done by amethoddescribed previously (30).

Osteoclast differentiation assayRAW 264.7 cells were cultured in 24-well dishes at a

density of 5 � 103 cells per well and allowed to adhereovernight. The medium was then replaced, and the cellswere treated with 5 nmol/L RANKL for 5 days. All celllines were subjected to TRAP staining using a leukocyteacid phosphatase kit. For coculture experiments withtumor cells, RAW 264.7 cells were seeded at 5 � 103 perwell and allowed to adhere overnight. The following day,U266 or MDA-MB-231 cells at 1 � 103 per well, wereadded to theRAW264.7 cells, treatedwith plumbagin andcocultured for 5 days before subjected to TRAP staining.

Bone resorption pit assayThe effect of plumbagin on bone resorption by pit for-

mation assay was carried out as described elsewhere (31).

In vivo osteolytic bone metastasis assayThe estrogen-independent human breast cancer cell

line MDA-MB-231 was cultured and resuspended in PBSsolution to give a final concentration of l � 105/100 mL.Five-week-old female BALB/c nu/nu mice (Harlan) wereinoculatedwithMDA-MB-231 cells (l� 105/100mL)directinto the left ventricle via a percutaneous approach. Themice were then randomly assigned to 1 of 2 groups,treated with vehicle (0.9% sodium chloride, n ¼ 5) orplumbagin (2 mg/kg body weight in vehicle, n ¼ 9) byintraperitoneal injection 5 times a week for 28 days andthen sacrificed. Radiographs (Faxitron Radiographicinspection unit; Kodak) were obtained at baseline andjust before sacrifice. Areas of osteolytic bone metastasesof tibiae and femorae, recognized as well-circumscribed

Plumbagin Abolished Osteoclastogenesis In Vitro and In Vivo

www.aacrjournals.org Mol Cancer Ther; 11(2) February 2012 351




Figure1. RANKL inducesNF-kBactivation andplumbagin abolishesRANKL-inducedNF-kBactivation. A, chemical structure of plumbagin.B, left, RAW264.7cells (1 � 106 cells) were incubated with or without the indicated concentrations of plumbagin for 4 hours, treated with RANKL (10 nmol/L) for a further 30minutes, and then tested for NF-kB activation by EMSA. Right, RAW 264.7 cells (1 � 106 cells) were incubated with plumbagin (5 mmol/L) for the indicatedtimes, treated with RANKL (10 nmol/L) for a further 30 minutes, and assayed for NF-kB activation by EMSA. Fold value is based on the value for medium(control), arbitrarily set at 1.C,RAW264.7 cells (1�106 cells)were incubatedwith plumbagin (5mmol/L) for 4 hours and then treatedwithRANKL (10nmol/L) forthe indicated times and assayed for NF-kBactivation by EMSA. Fold value is based on the value for medium (control), arbitrarily set at 1. D, cells (1� 106 cells)were incubated with plumbagin (5 mmol/L) for 4 hours and then treated with RANKL (10 nmol/L) for the indicated times. Cytoplasmic extracts wereprepared, fractionated by 10%SDS-PAGE, and electrotransferred to nitrocellulosemembranes.Western blot analysiswas carried outwith anti-IkBa. E, RAW264.7 cells (1� 106 cells) were pretreatedwith plumbagin (5 mmol/L) for 4 hours, incubatedwith ALLN (50 mg/mL for 30minutes), and then treatedwith RANKL(10 nmol/L) for 15 minutes. Cytoplasmic extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to nitrocellulose membranes.Western blot analysis was carried out using either antiphospho-IkBa (top) or anti-IkBa (bottom). F, RAW 264.7 cells (3 � 106 cells) were pretreatedwith plumbagin (5 mmol/L) for 4 hours and then incubated with RANKL (10 nmol/L) for the indicated times. Whole-cell extracts were immunoprecipitatedusing antibody against IKKa and analyzed by an immune complex kinase assay using recombinant GST-IkBa as described in Materials and Methods. Toexamine the effect of plumbagin on the level of IKKproteins, whole-cell extractswere fractionated by 10%SDS-PAGE and examined byWestern blot analysisusing anti-IKKa (middle) and anti-IKKb (bottom) antibodies.

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Mol Cancer Ther; 11(2) February 2012 Molecular Cancer Therapeutics352




radiolucent lesions on radiographs, were quantitatedusingMetaMorph imaging software (Molecular Devices).Radiographs were analyzed by investigators blinded tothe composition of the groups in the experiment or theexperimental protocol. Areal data for each group arepresented as mm2/lesion (mean � SEM).

Quantitative micro-computed tomography analysisA mCT40 scanner (SCANCO Medical AG) was used to

acquire tomographic images (100 slices) of formalin-fixedtibiae using energy settings of 55 kV and 145 mA, inte-gration timeof 250mswith 125projections per 180�, at a 12mm voxel size (isotropic). Images were reconstructedusing proprietary Scanco evaluation software. To obtainbone volume/total volume (BV/TV), trabecular number,trabecular thickness, and trabecular connectivity density,contours were drawn within the cortices around themetaphyseal regions, such that the total volume includedonly trabecular bone at 0.2 mm below the growth plateand extending 0.12 mm. Bone volume included all bonetissue that had a material density greater than 438.7mgHA/cm3, thereby giving a measure of bone vol-ume/total volume. The same threshold setting for theauto-contouring feature in the Scanco software was usedfor all bone samples. Cross-sectional images wereexported in tiff format and imported into AMIRA 3-Dgraphics software (Mercury Computer Systems). Using aconsistent threshold, AMIRA was used to generate 3-Drenderings of the metaphyses. All micro-computedtomography (micro-CT) image acquisitions and analyseswere carried out by an individual blinded to the compo-sition of the experimental groups.

Statistical analysisData are presented as mean� SEM and analyzed using

StatView software (version 5.0; SAS Institute, Inc.). Sta-tistical significance of differences was assessed using thenonparametric Mann–Whitney U test. Values of P � 0.05were considered statistically significant.

Results

The aim of this study was to investigate the effect ofvitamin K3 analogue plumbagin on osteoclastogenesisinduced by RANKL or by tumor cells and on cancer-induced bone loss in animals. We used the RAW 264.7cell (murinemacrophage) system, as it is awell-establishedmodel for osteoclastogenesis (32). Whether plumbagincouldmodulate osteoclastogenesis induced by tumor cellssuch as breast cancer and multiple myeloma was anotherfocus of these studies. We also investigated the effects ofplumbagin on cancer cell-mediated bone loss in athymicnude mice bearing human breast cancer xenograft.

Plumbagin represses RANKL-induced NF-kBactivation in RAW 264.7 cellsBinding of RANKL to its receptor RANK activates

TNF receptor-associated factor 6, which is linked to

activation of NF-kB. We investigated whether plumba-gin modulates RANKL-induced NF-kB activation inmonocytic RAW 264.7 cells. Cells were either pre-treated with plumbagin for 4 hours or left untreated,then exposed to RANKL at indicated concentrations;nuclear extracts were prepared and NF-kB activationdetermined by electrophoretic mobility shift assay(EMSA). As shown in Fig. 1B (left panel), RANKLactivated NF-kB; however, plumbagin suppressedRANKL-induced NF-kB activation in a dose-dependentmanner. We also investigated the duration of incuba-tion required for plumbagin to suppress RANKL-induced NF-kB activation. Cells were preincubatedwith 5 mmol/L plumbagin for the indicated timeperiod up to 4 hours and then exposed to RANKL.As shown by EMSA result in Fig. 1B (right panel),RANKL-induced NF-kB was inhibited by plumbaginwithin 4 hours. Plumbagin by itself did not activateNF-kB.

Plumbagin abolished RANKL-inducedphosphorylation and degradation of IkBa

Once cells are triggeredwith stimuli such as proinflam-matory cytokines (i.e., TNF and IL-1), these stimuli acti-vate the IKK complex, which phosphorylates IkB andthereby tags it for ubiquitination and degradation by theproteasome. The degradation of IkB thus allowsNF-kB totranslocate into the nucleus where it can act as a tran-scription factor.

To determine the effect of plumbagin on RANKL-induced IkBa degradation, we treated the cells withRANKL for different times in absence and presence ofpumbagin; and then examined for NF-kB and IkBa. Wefound that plumbagin inhibited RANKL-induced activa-tion of NF-kB (Fig. 1C) and RANKL-induced IkBa deg-radation (Fig. 1D). RANKL induced IkBa degradation incontrol cells as early as 5 minutes after treatment, but inplumbagin-pretreated cells RANKLhad no effect on IkBadegradation (Fig. 1D).

Next, we investigated the effect of plumbagin on thephosphorylation of IkBa. To determine this, we usedthe proteasome inhibitor N-acetyl-leu-leu-norleucinal(ALLN) to block RANKL-induced IkBa degradation.Cells were either treated with plumbagin, ALLN orRANKL and then examined for phosphorylation of IkBa.Because of rapid phosphorylation and degradation ofIkBa, no band was detected in cells treated with RANKLalone (Fig. 1E). Thus ALLN was required to see thephosphorylated band. Results clearly indicate thatRANKL induces the phosphorylation of IkBa (6th lane)and that plumbagin pretreatment inhibits the phosphory-lation (8th lane; Fig. 1E).

Plumbagin suppresses IKK activation induced byRANKL

Degradation of IkB is a tightly regulated event that isinitiated upon specific phosphorylation by activated IKK.Weexamined the effect of plumbaginonRANKL-induced






IKK activation using immune complex assays. The resultsindicated that RANKL activated IKK in a time-dependentmanner and thatplumbagin suppressedRANKL-inducedactivation of IKK (Fig. 1F, top panel). To check whetherthe apparent loss of IKK activity was due to downregula-tion of IKK protein expression, the levels of the IKKsubunits IKKa and IKKb were tested by Western blotanalysis. Neither RANKL nor plumbagin affected theexpression of IKKa or IKKb protein (Fig. 1E, middle andbottom panel).

Plumbagin inhibits RANKL-inducedosteoclastogenesis

Next, we examined the effect of plumbagin on osteo-clast differentiation induced by RANKL from osteoclastprecursor murine monocyte RAW 246.7 cells. Cells weretreated with 0.2, 0.5, or 1 mmol/L of plumbagin in thepresence of RANKL and allowed to differentiate intoosteoclasts. As indicated in Fig. 2A, RANKL-inducedformation of osteoclasts in the control cells at day 3. Bycontrast, the differentiation into osteoclasts induced byRANKL was significantly decreased in the presence ofplumbagin. The reduction of osteoclast formation corre-lated with increasing concentration of plumbagin(Fig. 2B). As little as 0.2 mmol/L plumbagin significantly

Figure 2. Plumbagin suppresses RANKL-induced osteoclastogenesis.A, RAW 264.7 cells (5 � 103 cells) were incubated with either mediumor RANKL (5 nmol/L) or RANKL and plumbagin (1 mmol/L) for 3, 4, or 5days and then stained for TRAP expression to examine osteoclastformation. TRAP-positive cells were photographed (originalmagnification,�100). B, cells (5� 103 cells) were incubated with eithermedium or RANKL (5 nmol/L) along with the indicated concentration ofplumbagin for 3, 4, or 5 days and then stained for TRAP expression.Multinucleated osteoclasts were counted. Cells exposed tomedium alone were used as a control (C). C, RAW264.7 cells(1 � 104 cells) were seeded into calcium phosphate apatite-coatedplates, treated with plumbagin (1 mmol/L) for 4 hours, and then treatedwith RANKL (5 nmol/L). After 5 days of incubation, cells weresubjected to lysis and images were obtained under light microscopy.Arrows, pit formation.

Figure 3. Inhibition of RANKL-induced osteoclastogenesis by plumbaginis an early event. A, RAW 264.7 cells (5 � 103 cells) were incubated withRANKL (5nmol/L) and thenplumbagin (1mmol/L)wasaddedeither onday0, after 1 day, after 2 days, after 3 days, or after 4 days. At the end of5 days, cells were stained for TRAP expression. B, multinucleatedosteoclasts (i.e., those containing 3 nuclei) were counted. Cells wereuntreated with plumbagin but exposed to RANKL, indicated as acontrol (C).

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suppressed RANKL-induced differentiation into osteo-clasts.Under these conditions, theviability of cellswasnotsignificantly affected (data not shown).

Plumbagin reducesRANKL-inducedbone resorptionWealso investigatedwhether the inhibition of RANKL-

induced osteoclastogenesis by plumbagin leads to inhi-bition of bone resorption. RAW264.7 cells were seededinto calciumphosphate apatite-coated plates, treatedwithplumbagin along with RANKL and incubated for 5 days.After incubation, resorption pit formation was analyzed.RANKL induced pit formation (Fig. 2C, arrows), whereasplumbagin significantly inhibited RANKL-induced pitformation (Fig. 2C). These results suggest that plumbaginsuppressed not only osteoclastogenesis but also boneresorption.

Plumbagin acts at an early step of osteoclastogenesisinduced by RANKLTo determine at which stage plumbagin inhibits

osteoclastogenesis, the vitamin K analogue was added

to osteoclast differentiation cultures beginning at day 0,1 day later, 2 days later, 3 days later, or 4 days later.Plumbagin inhibited osteoclastogenesis maximallywhen added from the beginning with RANKL treat-ment (Fig. 3A and B). The exposure of precursor cells toplumbagin at later stages (after 3 days) was less effec-tive in preventing osteoclastogenesis (Fig. 3B, fifth col-umn). Because the RAW 264.7 cells exposed to RANKLfor 3 days have already committed to osteoclast differ-entiation, it is reasonable to suggest that plumbagin canblock osteoclast differentiation but cannot reverse thedifferentiation process once cells have committed to theosteoclast.

Plumbagin inhibits osteoclastogenesis induced bytumor cells

Bone loss is one of the most common complications inpatients with breast cancer (33) or multiple myeloma (34).Whether this vitamin K3 analogue, plumbagin, alsoblocks tumor cell-induced osteoclastogenesis of RAW264.7 cells was investigated. As shown in Fig. 4, coculture

Figure 4. Plumbagin blocksosteoclastogenesis induced bytumor cells. RAW 264.7 cells wereincubated in the presence of U266(A), MM.1S (B), MCF-7 (C), or MDA-MB-231 cells (D) for 24 hours,exposed to plumbagin (1 mmol/L) for5 days, and finally stained for TRAPexpression. Multinucleatedosteoclasts (i.e., those containing 3nuclei) in cocultures were counted.






of RAW 264.7 cells with human multiple myeloma U266(Fig. 4A) or MM.1S cells (Fig. 4B) induced osteoclastdifferentiation, and plumbagin suppressed this differen-tiation in adose-dependentmanner.We found,moreover,that human breast cancer cells, such as MDA-MB-231(Fig. 4C) and MCF-7 (Fig. 4D), also induced formationof osteoclasts, and plumbagin suppressed this differenti-ation (Fig. 4C and D). These results indicate that osteo-clastogenesis induced by tumor cells, is significantlysuppressed by plumbagin.

Plumbagin suppresses osteolysis in MDA-MB-231breast cancer tumor-bearing mice

Breast cancer is one of the most common cancers affect-ing women in western countries, including the UnitedStates. In breast cancer patients, the frequency of bonemetastasis is much higher than that of lung and livermetastases (1, 4). To determine whether plumbagin couldsuppress osteolytic bone metastasis, we injected nudemicewith humanbreast cancerMDA-MB-231 cells,whichare triple negative (negative for estrogen receptor, pro-gesterone receptor, and human epidermal growth factorreceptor 2), and treated them with plumbagin (2 mg/kg)or vehicle control for 4 weeks. To identify osteolytic bonemetastasis, we analyzed osteolytic lesions by microradi-ography andmicro-CT analysis. As seen in representativeradiographs in Fig. 5A, osteolytic bone metastasis anddestruction of cortices (arrows) were observed in theMDA-MB-231 tumor-bearing control mice (left panel). Incontrast, there was very little osteolysis in the plumbagin(2 mg/kg)-treated mice, and the cortices remained intact(left panel). Quantitative analysis of the radiographsmea-suring osteolytic lesion area and number confirmed thisobservation (Fig. 5B and C).

Due to extensive bone destruction, a decrease in resid-ual trabecular/cancellous bone volume is consideredcharacteristic of tumor-induced osteolytic bone disease.Micro-CT analyses of proximal tibiae (Fig. 6A) showedthat treatment with plumbagin resulted in a significantpreservation of cancellous/trabecular bone volume (Fig.6B), which was accompanied by a significant increase intrabecular connectivity density in plumbagin-treatedtumor-bearing mice compared with the vehicle-treatedtumor-bearing mice (Fig. 6C). Although there was a smallincrease in trabecular thickness in plumbagin-treatedmice, this was not statistically significant (data notshown).

Discussion

Almost 90% of cancer-associated deaths are due totumor metastasis. Some of the major organ sites oftumor metastasis include lung, liver, lymph node, brain,and bone. Bone metastasis is commonly associated withprostate cancer, multiple myeloma, and breast cancer.Breast cancer metastasis to bone leads to osteolyticlesions, and no treatment is available. Because RANKL,a member of the TNF superfamily, has been closelylinked with bone loss, it is a novel therapeutic target.In the study presented here, we investigated the effect ofplumbagin, an analogue of vitamin K derived from anAyurvedic plant, for its potential to affect RANKLsignaling, osteoclastogenesis and breast cancer-inducedosteolytic lesions in mice.

We found that plumbagin exhibits numerous effects:first, it inhibited RANKL-mediated NF-kB activation;second, it inhibited RANKL-induced IkBa phosphoryla-tion and degradation; third, it blocked activation of IKK;

Figure 5. Plumbagin decreases breast cancer (MDA-MB-231)-induced bone loss in mice. Nude mice were injected with human breast cancer MDA-MB-231cells and then treated with 2 mg/kg of plumbagin (n ¼ 9; 5 times a week) or vehicle control (n ¼ 5); they underwent weekly radiographic imaging. A,representative radiographs of mice treated with vehicle (left) or plumbagin (right). Arrows, osteolytic bone lesions caused by injection of MDA-MB-231 cells;treatment with plumbagin reduced these osteolytic bone lesions. B and C, the effect of plumbagin on the number and area of osteolytic lesions inhumanMDA-MB-231 breast cancer-bearingmice. Quantitation of discrete lytic lesions usingMetaMorph, a computerized image acquisition and quantitationsoftware, indicates that treatment of tumor-bearing mice with plumbagin reduced the number (B) and average area (C) of osteolytic lesions in micebearing MDA-MB-231 tumors compared with vehicle-treated tumor-bearing mice.

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fourth, it suppressed RANKL-induced osteclastogenesis;fifth, it abrogated osteoclastogenesis induced by breastcancer and multiple myeloma; and sixth, when given toanimals, it suppressed human breast cancer-inducedosteolytic lesions in animals. All these observationstogether suggest the potential of this compound in sup-pression of cancer-associated bone loss.This is the first report to suggest that plumbagin can

abrogate RANKL signaling. The RANKL/RANK inter-action results in a cascade of intracellular events,including activation of NF-kB (28, 35). NF-kB signalinghas been shown to play an important role in osteoclas-togenesis (36). NF-kB p50�/� and p52�/� doubleknockout mice exhibit severe osteopetrosis caused byfailure of osteoclast formation (37, 38). Both p50 and p52expression are essential for RANK-expressing osteo-clast precursors to differentiate into TRAP-positiveosteoclasts in response to RANKL and other osteoclas-togenic cytokines (39). IKKb, a component of the NF-kBsignaling pathway, plays an essential role in inflamma-tion-induced bone loss and is required for osteoclasto-genesis (40). IKKb-deficient bone marrow cells do notdifferentiate to osteoclasts in vitro when stimulated byRANKL. These reports suggest that any drug that canblock RANKL signaling would play an important role insuppression of osteoclast formation. In this study, we

found that plumbagin indeed suppressed NF-kB acti-vation by RANKL and this suppression correlated withinhibition of osteoclastogenesis.

Breast cancer, prostate cancer, and multiple myeloma,all of which metastasize to bone frequently, express pro-teins that can migrate and anchor in bone, includingchemokines (e.g., CXCR4; ref. 41) and proteins that pro-mote pericellular proteolysis and invasion (e.g., matrixmetalloproteinases, MMP), angiogenesis (42) and osteo-clastogenesis (1). Previous reports from our laboratoryand others showed that plumbagin could suppress theexpression of MMPs (for invasion) and VEGF (for angio-genesis) in various types of cancer cell lines, includinghumanmultiplemyeloma (13, 22, 43).Aziz and colleaguesreported that plumbaginwas apotent inhibitor of prostatecancer cell invasion through suppression of cell invasionand metastasis marker protein MMP-9 and VEGF inprostate cancer cell lines and in athymicmicewith ectopicimplantation of hormone-refractory DU145 cells (27).These data are in agreement with our report that plum-bagin can inhibit metastasis of breast cancer MDA-MB-231 cells to bone, thus leading to decrease of osteolysis inmice.

In the study reported here, we show that plumbaginsuppressed the osteoclastogenesis induced by breastcancer and by multiple myeloma cells. Both breast

Figure 6. Plumbagin reducesosteolysis and preserves trabecular/cancellous bone in humanMDA-MB-231 breast cancer-bearing mice. A,three-dimensional computerreconstructions of residual bone bymicro-CT showing significant boneloss in proximal tibia of a vehicle-treated tumor-bearing mouse andinhibition of bone loss in a tumor-bearing mouse treated withplumbagin. Each computerrendering is from the mouse with themedian BV/TV in that group. B andC,graphical representation ofmicro-CTdata. Tumor-bearing mice treatedwith plumbagin exhibit greatertrabecular/cancellous bone volume(B) and significantly greater relativetrabecular connectivity density (C)than tumor-bearingmice treatedwithvehicle.






cancer and multiple myeloma are known to produceRANKL (12, 34) and exhibit constitutive NF-kB activa-tion (44, 45). This NF-kB activation can cause inductionof osteoclastogenesis via expression of RANKL. Inbreast cancer, osteolytic lesions occur in 80% of patientswith stage IV metastatic disease (46). These lesions haveincreased osteoclast activity and net bone destruction(47). Our results suggest that plumbagin may havepotential in the treatment of cancer-induced bonelesions.

The most widely used agents for prevention of boneloss include synthetic calcitonin, bisphosphonate drugs,raloxifene, and teriparatide (synthetic parathyroid hor-mone). The bisphosphonates, potent inhibitors of osteo-clast formation and activity, are the current standard ofcare and are the most used drugs for treatment of cancer-induced osteolytic diseases. Several reports have indicat-ed, however, that bisphosphonates increase the risk ofsevere osteonecrosis of the jaw in cancer patients (48),withdevastating consequences for affected patients (49). It isprudent, therefore, to develop safer antiresorptive strat-egies to combat lytic and mixed bone lesions in patientswith metastatic cancer in the skeleton.

Plumbagin, a naphthoquinone derived fromAyurvedicmedicine, has been used routinely in traditional medicineand is quite safe. Numerous reports indicate that it exhi-bits anticancer activities against a variety of tumorsthrough activation of multiple cell signaling pathways(13, 21, 22, 27, 43, 50). Comparedwith bisphosphonates ordenosumab, plumbagin is very inexpensive and has min-imal side effects.Ourdataprovide the rationale for further

animal and human studies with plumbagin for breastcancer-associated bone loss.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

B. Sung, B.O. Oyajobi, and B.B. Aggarwal took part in the studydesign. B. Sung and B.O. Oyajobi conducted the study. Data analysiswas done by B. Sung and B.O. Oyajobi. Data interpretation was done byB. Sung and B.O. Oyajobi. Drafting of manuscript was done by B. Sungand B.O. Oyajobi. Approving of final version of manuscript was doneby B.B. Aggarwal.

Acknowledgments

The authors thank Kathryn Hale for carefully proofreading themanuscript and providing valuable comments. The authors also thankSteve Munoz of the Bone Center at Vanderbilt University MedicalCenter for assistance with the micro-CT analyses.

Grant Support

This work was supported by a core grant from the NIH (CA-16 672), aprogram project grant fromNIH (NIH CA-124787-01A2), a grant from theCenter for Targeted Therapy of the University of Texas MD AndersonCancer Center and institutional funds from theUniversity of TexasHealthScience Center at San Antonio.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 14, 2011; revised November 2, 2011; acceptedNovember 7, 2011; published OnlineFirst November 16, 2011.

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2012;11:350-359. Published OnlineFirst November 16, 2011.Mol Cancer Ther Bokyung Sung, Babatunde Oyajobi and Bharat B. Aggarwal Suppression of RANKL SignalingCancer-Induced Osteolytic Bone Metastasis in Mice through Plumbagin Inhibits Osteoclastogenesis and Reduces Human Breast

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