Guggulsterone Inhibits Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor-KB Ligand and by Tumor Cells by Suppre

Cancer Prevention

Guggulsterone Inhibits Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor-KB Ligand and by Tumor Cells by Suppressing Nuclear Factor-KBActivation Haruyo Ichikawa and Bharat B. Aggarwal

Abstract Bone resorption is commonly associated with aging and with certain types of cancer, including multiple myeloma and breast cancer.What induces bone resorption is not fully understood, but the role of osteoclasts is well established. Recently, receptor activator of nuclear factor-nB (NF-nB) ligand (RANKL), a member of the tumor necrosis factor superfamily, was implicated as a major mediator of bone resorption, suggesting that agents that can suppress RANKL signaling have the potential to inhibit bone resorption or osteoclastogenesis. Guggulsterone [4,17(20)- pregnadiene-3,16-dione], isolated from the guggul tree Commiphora mukul and used to treat osteoarthritis and bone fractures, was recently shown to antagonize the farnesoid X receptor, decrease the expression of bile acid ^ activated genes, and suppress the NF-nB activation induced by various carcinogens.We investigated whether guggulsterone could modulate RANKL signaling and osteoclastogenesis induced by RANKL or tumor cells.We found that treatment of monocytes with guggulsterone suppressed RANKL-activated NF-nB activation (as indicated by gel-shift assay) and that this suppression correlated with inhibition of InBa kinase and phosphorylation and degradation of InBa, an inhibitor of NF-nB. Guggulsterone also suppressed the differentiation of monocytes to osteoclasts in a dose-dependent and time-dependent manner. Suppression of osteoclastogenesis by the NF-nB-specific inhibitory peptide implies a link between NF-nBand osteoclastogenesis. Finally, differentiation to osteoclasts induced by coincubating human breast tumor cells (MDA-MB-468) or human multiple myeloma (U266) cells with monocytes was also completely suppressed by guggulsterone. Collectively, our results indicate that guggulsterone suppresses RANKL and tumor cell ^ induced osteoclastogenesis by suppressing the activation of NF-nB.

Osteoclasts are multinucleated cells belonging to the mono- ligand (5). RANKL, which is expressed on the surface of cyte macrophage lineage that form through the fusion of their osteoblastic/stromal cells, is directly involved in the differen- mononuclear precursors. This multistep differentiation process tiation of monocyte macrophages into osteoclasts (3, 5, 6). is under the control of the bone microenvironment, which Mice with disruptions in the RANKL gene show a lack of includes stromal cells, osteoblasts, and local factors (1). One of osteoclasts, severe osteopetrosis, and defective tooth eruption, the key factors mediating osteoclastogenesis is receptor indicating that RANKL is essential for osteoclast differentiation activator of nuclear factor-nB (NF-nB) ligand (RANKL; ref. 2), (7). RANKL-induced osteoclastogenesis is mediated through a member of the tumor necrosis factor (TNF) family that has the cell surface receptor RANK. The interaction of RANKL with also been called osteoclast differentiation factor (3), TNF- RANK leads to recruitment of TNF receptor–associated factor related activation–induced cytokine (4), and osteoprotegerin adapter proteins and activation of NF-nB signaling pathways (8–11). Selective modulation of RANKL signaling pathways may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption, Authors’ Affiliation: Cytokine Research Laboratory, Department of Experimental such as osteoporosis, osteoarthritis, and cancer-induced bone Therapeutics,The University ofTexas M.D. Anderson Cancer Center, Houston,Texas Received 8/10/05; revised 11/1/05; accepted 11/11/05. loss. Thus, agents that can suppress RANKL signaling may be Grant support: Clayton Foundation for Research, Department of Defense U.S. able to suppress osteoclastogenesis-induced bone loss. Army Breast Cancer Research Program grant BC010610, NIH grant P01CA91844 A phytochemical that has aroused considerable interest is on lung chemoprevention, and NIH P50 Head and Neck Specialized Programs of guggulsterone [4,17 (20)-pregnadiene-3,16-dione], a plant Research Excellence (B.B. Aggarwal). sterol derived from the gum resin (guggulu) of the tree The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Commiphora mukul. The resin has been used in Ayurvedic with 18 U.S.C. Section 1734 solely to indicate this fact. medicine for centuries to treat a variety of ailments, including Requests for reprints: Bharat B. Aggarwal, Cytokine Research Laboratory, obesity, bone fractures, arthritis, inflammation, cardiovascular Department of Experimental Therapeutics, Box 143, The University of Texas M.D. disease, and lipid disorders (12, 13). The anti-arthritic and anti- Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-3503. ext. 6459; Fax: 713-794-1613; E-mail: [email protected]. inflammatory activity of gum guggul was shown as early as F 2006 American Association for Cancer Research. 1960 by Gujral et al. (14) followed by a report of activity in doi:10.1158/1078-0432.CCR-05-1749 experimental arthritis induced by mycobacterial adjuvant (15)

Clin Cancer Res 2006;12(2) January 15, 2006 662 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Guggulsterone Suppresses Osteoclastogenesis and another on the effectiveness of guggul for treating oligonucleotide (15 Ag of protein with 16 fmol of DNA) from the HIV osteoarthritis of the knee (16). Recent studies have shown that long terminal repeat 5V-TTGTTACAAGGGACTTTCCGCTGGGGACTTTC- n guggulsterone is an antagonist for the bile acid receptor CAGGGAGGCGTGG-3V(boldface indicates NF- B binding sites) for 30 j farnesoid X receptor (17, 18). Other studies have shown that minutes at 37 C, and the DNA-protein complexes formed were separated from free oligonucleotide on 6.6 % native polyacrylamide guggulsterone enhances transcription of the bile salt export gels. A double-stranded mutated oligonucleotide, 5V-TTGTTACAACT- pump (19), thereby regulating cholesterol homeostasis. Our CACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3V,wasusedto laboratory reported that guggulsterone suppressed the DNA examine the specificity of binding of NF-nB to the DNA. The specificity binding of NF-nB induced by various carcinogens and of binding was also examined by competition with the unlabeled inflammatory agents (20). oligonucleotide. For supershift assays, nuclear extracts from TNF-treated Whether guggulsterone can suppress RANKL-induced NF-nB cells were incubated with antibodies against either p50 or p65 of NF-nB activation and osteoclastogenesis induced by RANKL and by for 15 minutes at 37jC before the complexes were analyzed by tumor cells was investigated. We show that guggulsterone electrophoretic mobility shift assays. The dried gels were visualized, suppressed the RANKL-induced NF-nB activation pathway by and radioactive bands were quantified by a Phosphorimager (Molecular inhibiting InBa kinase (IKK); moreover, this effect correlated Dynamics, Sunnyvale, CA) equipped with ImageQuant software. Western blotting analysis. To determine the levels of protein with the suppression of osteoclastogenesis induced by RANKL expression in the cytoplasm or nucleus, we prepared extracts (24) and or by breast cancer or multiple myeloma cells. fractionated them by SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each Materials and Methods antibody, and detected with enhanced chemiluminescence reagent (Amersham, Piscataway, NJ). The bands obtained were quantified by Materials. The rabbit polyclonal antibodies to InBa p50 and p65 using NIH Imaging software (Bethesda, MD). were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IKK assay. To determine the effect of guggulsterone on TNF-induced Antibody against phospho-InBa was purchased from Cell Signaling IKK activation, we used a method described elsewhere (25). Briefly, the Technology (Beverly, MA). Anti-IKK-a and anti-IKK-h antibodies were IKK complex from whole-cell extracts (400 Ag) was precipitated with kindly provided by Imgenex (San Diego, CA). Goat anti-rabbit antibody against IKK-a followed by treatment with protein A/G- horseradish peroxidase conjugate was purchased from Bio-Rad Sepharose beads (Pierce). After 2 hours of incubation, the beads were (Hercules, CA); goat anti-mouse horseradish peroxidase and BioCoat washed with lysis buffer and assayed in a kinase assay mixture Osteologic Bone Cell Culture System were from BD Biosciences (San containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L 32 Jose, CA); and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium DTT, 20 mCi [g- P]ATP, 10 mmol/L unlabeled ATP, and 2 Agof bromide was from Sigma-Aldrich (St. Louis, MO). Guggulsterone was substrate glutathione S-transferase (GST)/InBa (amino acids 1-54). After obtained from Steroid, Inc. (Newport, RI) and dissolved in DMSO as a incubation at 30jC for 30 minutes, the reaction was terminated by 100 mmol/L stock solution and stored at À20jC. DMEM/F12 medium, boiling with SDS sample buffer for 5 minutes. Finally, the protein was fetal bovine serum, 0.4% trypan blue vital stain, and an antibiotic- resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands antimycotic mixture were obtained from Invitrogen (Carlsbad, CA). were visualized with a PhosphorImager. To determine the total amounts Protein A/G-Sepharose beads were obtained from Pierce (Rockford, IL). of IKK-a and IKK-h in each sample, 50 Ag of the whole-cell protein was [g-32P]ATP was from ICN Pharmaceuticals (Costa Mesa, CA). Highly resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose purified recombinant murine TNF-a was provided by Genentech membrane, and blotted with anti-IKK-a or anti-IKK-h antibody. (South San Francisco, CA). The p65 peptide conjugated with the delivery peptide was kindly provided by Imgenex. Cell lines. The mouse macrophage cell line RAW 264.7 was Results obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/F12 medium supplemented with 10% fetal The aim of this study was to investigate the effect of bovine serum and antibiotics. This cell line has been shown to express guggulsterone on RANKL-induced activation of the NF-nB RANK and, when cultured with soluble RANKL, to differentiate into pathway and on osteoclastogenesis induced by the cytokine tartrate resistance acid phosphatase (TRAP)–positive, functional and by tumor cells. The murine monocytic cell line RAW 264.7 osteoclasts (9). RANKL has also been shown to activate NF-nBin was used because these cells have been shown to undergo these cells (21). TRAP staining was done by using a leukocyte acid differentiation to osteoclasts when exposed to RANKL. phosphatase kit (387-A) from Sigma-Aldrich. MDA-MB-468 (human Guggulsterone inhibits RANKL-induced NF-kBactivation. breast adenocarcinoma) cells were obtained from the American Previous studies from our laboratory have indicated that Type Culture Collection and cultured in MEM containing 10% fetal n a bovine serum, 100 Amol/L nonessential amino acids, 1 mmol/L stable transfection of RAW cells with a super-repressor I B n pyruvate, and 6 mmol/L L-glutamine. U266 (multiple myeloma) cells plasmid abolishes both NF- B activation and osteoclasto- were cultured in RPMI 1640 with 10% fetal bovine serum. Culture genesis induced by RANKL (26), thus implicating NF-nBin media were supplemented with 100 units/mL penicillin and 100 Ag/mL osteoclastogenesis. To investigate whether guggulsterone mod- streptomycin. ulates RANKL-induced NF-nB activation in RAW 264.7 cells, we Osteoclast differentiation assay. RAW 234.7 cells were cultured in incubated these cells with guggulsterone and RANKL, prepared 4 24-well dishes at a density of 1 Â 10 per well and allowed to adhere nuclear extracts, and assayed NF-nB activation by electropho- overnight. The medium was then replaced, and the cells were treated retic mobility shift assays in the nuclei. RANKL activated NF-nB with 5 nmol/L (100 ng/mL) RANKL. At day 5, cultures were stained for maximally within 30 minutes, and guggulsterone completely TRAP expression (22) by using an acid phosphatase kit, and the total n z abrogated the RANKL-induced NF- B activation (Fig. 1A). The number of TRAP-positive, multinucleated ( 3 nuclei) osteoclasts per n well were counted. extent of inhibition of NF- B by guggulsterone increased Electrophoretic mobility shift assays. We used electrophoretic with dose, with maximum inhibition observed at 50 Amol/L mobility shift assays to determine NF-nB activation as described guggulsterone (Fig. 1B). Treatment of cells with 50 Amol/L previously (23). Briefly, nuclear extracts from TNF-treated cells were guggulsterone for 4 hours had no effect on the cell viability as incubated with 32P-end–labeled, 45-mer double-stranded NF-nB determined by trypan blue exclusion method.

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Guggulsterone inhibits RANKL-induced IkBa phosphorylation and degradation through inhibition of IKK activity. Activation of NF-nB by most agents requires phosphorylation and degradation of its inhibitory subunit InBa. To investigate the mechanism involved in the inhibition of NF-nB activation by guggulsterone, we first checked the effects of guggulsterone treatment on the levels of InBa by using Western blot analysis. In cells treated with RANKL, InBa levels dropped within 10 minutes and returned to normal within 60 minutes (Fig. 2A, left). In contrast, cells pretreated with guggulsterone showed suppressed RANKL-induced degradation of InBa (Fig. 2A, right). Next, we investigated the effect of guggulsterone on the RANKL-induced phosphorylation of InBa, which occurs before its ubiquitination, and degradation of InBa (27). We used the proteasome inhibitor ALLN to prevent RANKL-induced degradation of InBa (28). Western blot analysis for phospho-InBa in Fig. 2B clearly indicates that RANKL induced InBa phosphorylation in RAW 264.7 cells, and that guggulsterone eliminated this effect. Treatment of cells with guggulsterone alone did not result in phosphorylation of InBa. Notably, the content of InBa in the guggulsterone-treated samples was less than that in the control; quantification of the InBa/h-actin ratio indicated that guggulsterone treatment down-regulated the expression of InBa and inhibited the RANKL-induced degradation of InBa. Because IKK phosphorylates InBa (29), we next checked whether guggulsterone alters the activity or the levels of IKK.

Fig. 1. RANKL induces NF-nB activation and guggulsterone inhibits it in a dose-dependent and time-dependent manner. A, RAW 264.7 cells (1 Â10 6) were incubated with guggulsterone (50 Amol/L) for 4 hours, treated with 10 nmol/L RANKL for the indicated times, and tested for nuclear NF-nB by electrophoretic mobility shift assay. B, RAW 264.7 cells (1 Â10 6) were incubated without or with the indicated concentrations of guggulsterone (GS) and RANKL (10 nmol/L) and tested for nuclear NF-nB by electrophoretic mobility shift assay.

n Supershift assay of NF- B-DNA probe binding showed that Fig. 2. Guggulsterone inhibits RANKL-induced InBa phosphorylation and RANKL-activated NF-nB consisted of p65 and p50 subunits degradation through inhibition of IKK activity. RAW 264.7 cells (1 Â10 6) were incubated with guggulsterone (GS ;50Amol/L) for 4 hours and treated with (Fig. 1C). The specificity of the RANKL-induced formation of 10 nmol/L RANKL for the indicated times. Cytoplasmic extracts were prepared to the NF-nB/DNA complexes was further confirmed by showing check the level of InBa (A); the level of phosphorylated InBa byWestern blot that binding was abolished by the presence of a 100-fold excess analysis (B); IKK activity (C, top); immunoprecipitated IKK and did the kinase n assay, and total IKK-a and IKK- h proteins byWestern blot analysis (middle and of unlabeled B-oligonucleotides but not by the mutated bottom) in cytoplasmic extracts. Quantification of InBa after normalization with oligonucleotide (Fig. 1C). h-actin (A).

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had no effect on the growth and survival of cells as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Guggulsterone acts early in the pathway leading to RANKL- induced osteoclastogenesis. It normally takes up to 5 days for RAW 264.7 cells to differentiate into osteoclasts in response to RANKL. To determine how early in this pathway guggulsterone acts, we treated the RAW 264.7 cells with RANKL, added guggulsterone on different days, and then checked its effect on osteoclast formation. Guggulsterone inhibited osteoclastogenesis even when the cells were exposed 24 hours after the RANKL treatment (Fig. 4A). However, the inhibitory effect decreased significantly when cells were treated with guggulsterone 4 days after RANKL treatment (Fig. 4B). Activation of NF-kB is critical for RANKL-induced osteoclastogenesis. Besides NF-nB activation, RANKL is known to activate several other signals in the cell. It is possible that guggulsterone inhibits RANKL-induced osteoclastogenesis by suppressing signals other than NF-nB. To establish that guggulsterone

Fig. 3. Guggulsterone inhibits RANKL-induced osteoclastogenesis. RAW 264.7 cells (1 Â 10 4) were incubated with or without RANKL (5 nmol/L) and with or without the indicated concentration of guggulsterone (GS) for 5 days and stained for TRAP expression. A, photograph of TRAP-positive cells. Original magnification, Â100. B, multinucleated (three nuclei) osteoclasts were counted.

With immunoprecipitation followed by in vitro IKK assay, cells treated with RANKL showed a sharp increase in IKK activity as indicated by the phosphorylation of InBa within 10 minutes. In contrast, cells pretreated with guggulsterone could not phosphorylate GST-InBa upon RANKL treatment (Fig. 2C, top). To check whether the apparent loss of IKK activity was due to the loss of IKK protein expression, the expression levels of the IKK subunits IKK-a and IKK-h were tested by Western blot analysis. Results in Fig. 2C clearly showed that guggulsterone treatment did not alter the expression of IKK-a and IKK-h. Guggulsterone inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells. Next, we investigated the effect of guggulsterone on osteoclastogenesis. RAW 264.7 cells were incubated with different concentrations of guggulsterone in the presence of RANKL and allowed to grow and differentiate into osteoclasts. Figure 3A illustrates that RANKL induced osteoclasts in the absence of guggulsterone, but its presence significantly decreased the differentiation. Quantitation revealed that the Fig. 4. Guggulsterone effectively inhibits RANKL-induced osteoclastogenesis number of osteoclasts decreased with increasing concentration 24 hours after stimulation. RAW 264.7 cells (1Â10 4) were incubated with or of guggulsterone (Fig. 3B). These results show that 5 Amol/L without RANKL (5 nmol/L), and guggulsterone (GS ;5Amol/L) was added either at the same time or after the indicated intervals. Cells were cultured for 5 days after guggulsterone was sufficient to suppress osteoclastogenesis by RANKL treatment and stained forTRAP expression. A, photographs of cells. Original >90%. Treatment of cells with 5 Amol/L guggulsterone for 5 days magnification, Â100. B, multinucleated (three nuclei) osteoclasts were counted.

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osteoclastogenesis induced by tumor cells is significantly suppressed by the presence of guggulsterone. Treatment of cells with 5 Amol/L guggulsterone for 5 days had no effect on the growth and survival of cells as determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.

Discussion

Guggulsterone, commonly used to treat osteoarthritis, bone fractures, and arthritis, acts through a mechanism that is not understood. The present study was designed to investigate the role of guggulsterone on bone resorption. We showed that guggulsterone suppressed NF-nB activation, IKK, and phosphorylation and degradation of InBa induced by RANKL, a bone-resorbing cytokine. Guggulsterone also suppressed the RANKL-induced monocyte differentiation to osteoclast. Osteo- clastogenesis induced by human breast tumor cells or human multiple myeloma was also abrogated by guggulsterone. In the present study, we used a homogeneous, clonal population of murine monocytic cells RAW 264.7 to define the direct effect of guggulsterone on osteoclast development induced by RANKL. The advantage of this system is that it does not contain any osteoblast/bone marrow stromal cells or cytokine like macrophage-colony-stimulating factor and allows us to focus on RANKL signaling in preosteoclast cells. Our results indicate that RANKL activates NF-nB in osteoclastic precursor cells through the activation of IKK and subsequent

Fig. 5. The p65 inhibitory peptide suppresses RANKL-induced osteoclastogenesis. RAW 264.7 cells (1Â10 4) were exposed to RANKL (5 nmol/L), PTD, p65, and PTD-p65 (0.3 Amol/L) in various combinations. After 5 days, cells were stained for TRAP expression. A, photographs of cells. Original magnification, Â100. B, multinucleated (three nuclei) osteoclasts were counted. suppressed osteoclastogenesis by inhibiting NF-nB activation, we treated RAW 264.7 cells with the p65 inhibitory peptide recently described from our laboratory (30). Neither the delivery peptide (PTD) nor the p65 inhibitory peptide alone significantly inhibited osteoclastogenesis in RAW cells (Fig. 5A and B). However, the conjugate of the delivery peptide with the p65 inhibitory peptide (PTD-p65) significantly suppressed osteoclastogenesis. These results suggest that NF-nB activation has critical role in RANKL-induced osteoclastogenesis. Coincubation of monocytes with tumor cells leads to osteoclastogenesis. Osteoclastogenesis is commonly associated with breast cancer (31, 32) and with multiple myeloma (33). Both multiple myeloma U266 and breast cancer MDA-MB-468 cells are known to express constitutive NF-nB (34, 35) and express RANKL (36, 37). Whether these tumor cells can Fig. 6. Guggulsterone inhibits MDA-MB-468- and U266-induced induce osteoclastogenesis is not known. We found that osteoclastogenesis. RAW 264.7 cells (1 Â 10 4) were incubated with or without incubating monocytes with breast cancer MDA-MB-468 cells MDA-MB-468 cells (1 Â10 3; A) or U266 cells (1 Â 10 3; B), and guggulsterone (Fig. 6A) or with multiple myeloma U266 cells (Fig. 6B) (GS ;5Amol/L) was added either at the same time or after the indicated intervals. Cells were cultured for 5 days after coincubation and stained forTRAP expression. induced osteoclast differentiation, and that guggulsterone Right, photographs of cells. Original magnification, Â100. Left, multinucleated suppressed this differentiation. These results indicate that (three nuclei) osteoclasts were counted.

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InBa phosphorylation and degradation. These results are in suppression of NF-nB activation would play an important role agreement with those of Wei et al. (21). We also showed that in osteoclast formation. guggulsterone inhibits RANKL-induced IKK activation, leading It is possible that the inhibitory effect of guggulsterone on to the suppression of NF-nB activation. The mechanism of osteoclastogenesis is not mediated through suppression of NF-nB activation induced by RANKL differs from that of TNF. NF-nB. This is unlikely, however, as we found that p65 For instance NF-nB-inducing kinase (also called NIK), although inhibitory peptide, which inhibits activate NF-nB induced by required for RANKL-induced NF-nB activation (38), is dispens- various activators (48), inhibited osteoclastogenesis induced by able for TNF-induced NF-nB activation (39). Osteoclastogenesis RANKL in RAW 264.7 cells. Additionally, we have previously is regulated by primarily by canonical pathway. Although NIK shown that suppression of NF-nB by DN-InBa abrogates is needed for RANKL signaling, genetic deletion of NIK, p100, osteoclastogenesis (26). Thus, it is very likely that NF-nB or p52 has no effect on osteoclastogenesis, suggesting non- suppression shown in this study is involved in the inhibition of canonical pathway is less important. NF-nB p50À/À and p52À/À osteoclastogenesis by guggulsterone. double knockout mice, however, exhibit severe osteopetrosis Breast cancers commonly cause osteolytic metastases in bone, caused by failure of osteoclast formation (40, 41). Similar to a process that depends on osteoclast-mediated bone resorption our results, however, RANKL-induced osteoclastogenesis is (49), but the mechanism responsible for tumor-mediated impaired in NIK-deleted cells (42). osteoclast activation has not yet been clarified. We showed in More recent study showed that IKKs are potent regulators this study that breast cancer cell induced osteoclastogenesis, and of cytokine-induced osteoclatogenesis and inflammatory guggulsterone inhibited it. Bone resorption has also been arthritis (43). That guggulsterone can suppress RANKL- associated with multiple myeloma (49). We found that multiple induced IKK activation leading to suppression of NF-nB myeloma cell–induced osteoclastogenesis was also suppressed activation is in agreement with our previous report in which by the guggulsterone. Both of these tumors have been shown to we showed that this steroid can suppress TNF-induced IKK express RANKL (37, 50) and exhibit constitutive NF-nB activation (20). activation (51, 52). Thus, it is very likely that these tumors We found that suppression of RANKL-induced NF-nB activate osteoclastogenesis through RANKL expression. This activation by guggulsterone correlated with inhibition of implies that guggulsterone could be used in the treatment of osteoclastogenesis. The interaction of RANKL with RANK results secondary bone lesions associated with cancer and those in a cascade of intracellular events, including the activation of associated with nonmalignant diseases like postmenopausal NF-nB (8, 9, 43). NF-nB signaling has been shown to play an osteoporosis, Paget’s disease, and rheumatoid arthritis. important role in osteoclastogenesis (44). NF-nB is activated by RANKL both in RAW 264.7 cells and in monocytes (5, 9, 45, 46) and is required in vivo for osteoclast formation (41). p50 and Acknowledgments p52 expression are essential for RANK-expressing-osteoclast We thank Christine Wogan for her critical review of this article and Dr. Bryant + precursors to differentiate into TRAP osteoclasts in response to Darnay (Department of Experimental Therapeutics, The University of Texas M.D. RANKL and other osteoclastogenic cytokines (47). Therefore, Anderson Cancer Center, Houston,TX) for supplying the RANKL protein.

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Clin Cancer Res 2006;12(2) January 15, 2006 668 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Editor's Note Clinical Cancer Editor's Note: Guggulsterone Inhibits Research Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor-kB Ligand and by Tumor Cells by Suppressing Nuclear Factor-kB Activation

The Clinical Cancer Research editors are publishing this note to alert readers to a concern about this article (1). Institutional review determined that the same image was used to represent inhibition of RANKL-induced osteoclastogenesis by p65 and PTD in Fig. 5A. The authors were unable to provide original data for these images at the time of institutional review.

Reference 1. Ichikawa H, Aggarwal BB. Guggulsterone inhibits osteoclastogenesis induced by receptor activator of nuclear factor-kB ligand and by tumor cells by suppressing nuclear factor-kB activation. Clin Cancer Res 2006;12:662–8.

Published ﬁrst September 4, 2018. doi: 10.1158/1078-0432.CCR-18-2525 2018 American Association for Cancer Research.

www.aacrjournals.org 4347 Guggulsterone Inhibits Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor- κB Ligand and by Tumor Cells by Suppressing Nuclear Factor- κB Activation

Haruyo Ichikawa and Bharat B. Aggarwal

Clin Cancer Res 2006;12:662-668.

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