Calebin A downregulates osteoclastogenesis through suppression of RANKL signalling

Archives of Biochemistry and Biophysics 593 (2016) 80e89

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Calebin A downregulates osteoclastogenesis through suppression of RANKL signalling Amit K. Tyagi a, Sahdeo Prasad a, *, Muhammed Majeed b, Bharat B. Aggarwal a, ** a b

Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Sabinsa Corporation, East Windsor, NJ, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 October 2015 Received in revised form 28 January 2016 Accepted 7 February 2016 Available online 11 February 2016

Osteoporosis is a bone disease that is exacerbated by aging and age-associated chronic diseases such as cancer. Cancer-induced bone loss is usually treated with bisphosphonates or denosumab, an antibody against receptor activator of nuclear factor (NF)-kB ligand (RANKL). Because these drugs are expensive and have numerous side effects and high rates of toxicity, safer, more effective, and more affordable therapies for osteoporosis are still needed. We identified a compound, calebin A (CA), derived from turmeric (Curcuma longa) that affects osteoclastogenesis through modulation of the RANKL signalling pathway. The CA's effect on NF-kB activation was examined by electrophoretic mobility shift assay. Using mouse macrophages in vitro model, we found that CA suppressed RANKL-induced osteoclast differentiation of macrophages into osteoclasts, and downregulate RANKL-induced osteoclastogenesis-related marker gene expression, including NFATc-1, TRAP, CTR, and cathepsin K. CA also suppressed the osteoclastogenesis induced by multiple myeloma and breast cancer cells. This effect of CA was correlated with suppression of the phosphorylation and degradation of inhibitor of kB and, thus, inhibition of NF-kB activation. Furthermore, we found that an NF-kB-specific inhibitory peptide blocked RANKL-induced osteoclastogenesis, demonstrating that the NF-kB signalling pathway is mandatory for RANKL-induced osteoclastogenesis. Our results conclusively indicate that CA downmodulates the osteoclastogenesis induced by RANKL and by tumour cells through suppression of NF-kB pathway. © 2016 Elsevier Inc. All rights reserved.

Keywords: Calebin A Osteoclastogenesis Cancer RANKL NF-kB

1. Introduction Osteoclast-related diseases are highly prevalent in old age and present huge economic costs. In 2002, an estimated 44 million people aged 50 years or older in the United States were at risk for fractures owing to osteoporosis or low bone mass. This number is projected to reach 61 million by 2020 because, as yet, there is no widely implemented and effective treatment. According to the American Academy of Orthopaedic Surgeons, the projected cost of osteoporosis care over the next 2 decades will be approximately $474 billion [1]. Bone remodelling is regulated by a highly dynamic process involving bone-forming osteoblasts, bone-degrading osteoclasts, and mechanical-sensing osteocytes. Coordination between

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], [email protected] (S. Prasad), [email protected] (B.B. Aggarwal). http://dx.doi.org/10.1016/j.abb.2016.02.013 0003-9861/© 2016 Elsevier Inc. All rights reserved.

osteoclasts and osteoblasts is critical for normal bone remodelling. Osteoclastogenesis is also known to important during skeletal development in childhood and skeletal maintenance in adulthood [1]. Osteoclastogenesis takes place in several stages, including commitment, differentiation, multinucleation, and activation of immature osteoclasts; the process is regulated by both systemic hormones and cytokines in the bone microenvironment, which contains stromal cells, osteoblasts, and other local factors [2]. One of the key factors for osteoclast differentiation is the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) [4], which is a major osteoclastogenic cytokine of the tumour necrosis factor (TNF) family. It is reported that osteoblastic cells express RANKL on their surface and promotes the differentiation of monocytes into osteoclasts [5,6]. Indeed, mice deficient in the rankl gene have been shown to display severe osteopetrosis, stunted growth, defective tooth eruption, and osteoblasts that fail to support osteoclastogenesis [7]. RANKL induced osteoclast formation is mediated through stimulation of RANK, which further interacts with the adaptor molecule TNF receptor-associated factor 6 (TRAF6) [2]. TRAF6 sequentially activates the IkB kinases (IKKs),

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which phosphorylate and degrade IkBa, the inhibitor of NF-kB [2]. Upon IkBa degradation, NF-kB translocates to the nucleus, where it activates the transcription of several osteoclastogenesis-related genes [8]. Besides RANKL, M-CSF is also a key factor in the differentiation and activation of osteoclasts. M-CSF induces the proliferation, survival, and RANK- upregulation of osteoclast precursor cells [3]. Bone metastasis is a common cause of morbidity in patients with advanced multiple myeloma or advanced breast, prostate, lung, colon, kidney, thyroid, and stomach carcinomas [9,10]. Multiple myeloma and breast, prostate, and lung cancer, in particular, have a remarkable tendency to metastasize to the bones [11,12]. Because RANKL has previously been found to play a critical role in bone metastasis [11], agents that suppress RANKL signalling may have potential as inhibitors of osteoclastogenesis and, therefore, bone metastasis. Extensive evidences have shown that many natural compounds have chemopreventive and therapeutic efficacy against various chronic diseases. Turmeric, a rhizome of Curcuma longa, is one of the oldest traditionally used folk medicines in Asia. It has been shown to have numerous biological activities, including antioxidant, anti-inflammatory, anticancer, anti-arthritic, anti-atherosclerotic, antidepressant, anti-aging, antidiabetic, antimicrobial, wound healing, and memory-enhancing effects [13]. However, most research (over 7500 articles) on turmeric has focused on only one of its components, curcumin [14]. Calebin A (CA) (Fig. 1A), which is isolated from curcumin-free turmeric also has anticancer effects through the inhibition of several cell signalling pathways [15]. Whether this anticancer property of CA leads to the suppression of cancer associated bone loss, has not been investigated yet. In the present study, we hypothesized that CA can downmodulate RANKL-induced NF-kB activation and inhibit both RANKL- and cancer-induced osteoclastogenesis.

2. Materials and methods 2.1. Reagents CA was kindly provided by Sabinsa Corporation, East Windsor, NJ. A 50-mM solution of CA in dimethyl sulfoxide was prepared and stored in small aliquots at
20  C and appropriately diluted in the culture medium just before use. Dulbecco modified Eagle's medium (DMEM), DMEM/F-12, RPMI 1640, fetal bovine serum (FBS), an antibiotic-antimycotic mixture, and 0.4% trypan blue vital stain were obtained from Mediatech, Inc. (Manassas, VA). Recombinant RANKL protein was kindly provided by Dr. Bryant Darnay of The University of Texas MD Anderson Cancer Center (Houston, TX). Antibodies against IKKa, IKKb, and IkBa were purchased from Imgenex (San Diego, CA). Antibodies against phospho-IkBa (Ser32/ 36), cFms, TRAP, Cathepsin K and Calcitonin R were purchased from Cell Signalling Technology (Danvers, MA). Antibodies against RANK, extracellular signal-related kinase 2 (ERK2) and phospho-ERK1/2 (Thr202/Tyr204) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). NFATc1 antibody was obtained from Addgene (Cambridge, MA, USA), M-CSF was from R&D Systems (Minneapolis, MN, USA). Goat anti-rabbit and goat anti-mouse horseradish peroxidase conjugates were purchased from Bio-Rad (Hercules, CA). b-actin antibody and a leukocyte acid phosphatase kit (387-A) for tartrate-resistant acid phosphatase (TRAP) staining were purchased from Sigma-Aldrich (St. Louis, MO). The cell-permeable NFkB essential modulator (NEMO; also called IKKg)-binding domain peptide (NBP) was purchased from Imgenex. [g-32P]ATP was purchased from MP Biomedicals (Solon, OH).

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2.2. Cell lines RAW 264.7 mouse macrophage cells were kindly provided by Dr. Bryant Darnay and cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics. The RAW 264.7 cell line is a well-established osteoclastogenic cell system that has been shown to express RANK and to differentiate into functional TRAP-positive osteoclasts when cultured with soluble RANKL. In addition, RANKL has been shown to activate NF-kB in RAW 264.7 cells. MDA-MB-231 (human breast adenocarcinoma) and U266 (human multiple myeloma) cells were obtained from the American Type Culture Collection (Manassas, VA). The MDA-MB-231 cells were cultured in DMEM and the U266 cells in RPMI 1640 with 10% FBS. 2.3. Osteoclast differentiation assay To determine the effects of CA on RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured in 24-well plates at a density of 5  103 cells per well and allowed to adhere overnight. The medium was then replaced, and the cells were treated with 5 nM RANKL for 5 days. All cells were subjected to TRAP staining using the leukocyte acid phosphatase kit. For co-culture experiments with cancer cells, RAW 264.7 cells were seeded at a density of 5  103 cells per well and allowed to adhere overnight. The following day, U266 or MDA-MB-231 cells, at a density of 1  103 cells per well, were added to the RAW 264.7 cells, treated with CA, and co-cultured for 5 days before being subjected to TRAP staining. For conditioned medium experiments, RAW 264.7 cells were seeded at a density of 5  103 cells per well and allowed to adhere overnight. The following day, the medium was replaced with 4/5 of RAW 264.7 medium (DMEM/F12) plus 1/5 of conditioned medium from MDA-MB-231 or U266 cells. For that procedure, the supernatant of cultured cancer cells that had been centrifuged was used. Finally, the RAW 264.7 cells were cultured for 5 days and subjected to TRAP staining and an electrophoretic mobility shift assay (EMSA). 2.4. Cell proliferation assay The effects of CA on the proliferation of RAW 264.7 cells were determined by measuring mitochondrial dehydrogenase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) as the substrate [16]. In brief, 3000 RAW 264.7 cells (in 0.1 ml media) per well were incubated with various concentrations of CA, in triplicate, in 96-well plates at 37  C for 1, 3, or 5 days. At each interval, MTT solution was added to each well, and the plates were incubated for 2 h at 37  C. An extraction buffer (100 mM) comprised of 20% sodium dodecyl sulphate (SDS) and 50% dimethyl formamide was added, and the cells were incubated overnight at 37  C to dissolve the formazan formed during the reaction. The absorbance of the coloured product was then measured at 570 nm using a 96-well multiscanner (MRX Revelation, Dynex Technologies, Chantilly, VA). 2.5. Western blot analysis To determine protein expression levels, we prepared cytoplasmic and whole cell extracts and fractionated them using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE). After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with the relevant antibodies, and detected with enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ).

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Fig. 1. Calebin A (CA) inhibits receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. (A) The structure of CA. (B) RAW 264.7 cells (5  103 cells/mL) were incubated with RANKL (5 nM), or RANKL plus CA (0, 1, 2, 5 mM) for 5 days and stained to show osteoclastogenesis via tartrate-resistant acid phosphatase (TRAP) expression. TRAPpositive cells were photographed. Original magnification, 100. (C) Quantification of multinucleated osteoclasts (those containing 3 or more nuclei) after treatment with RANKL (5 nM) alone or RANKL plus CA (0, 1, 2, 5 mM) for 5 days. Mean of 3 measurements ± standard deviation (SD). (D) RAW 264.7 cells (3  103 cells per 100 ml) were incubated with medium only (Ctrl) or with 1, 2, or 5 mM CA for 1, 3, or 5 days. Cell proliferation was assessed with the MTT method.

2.6. EMSA for NF-kB

2.7. Transfection of p65 plasmid

To assess NF-kB activation, nuclear extracts were prepared and EMSA was carried out as described previously [15]. In brief, nuclear extracts from untreated and RANKL-treated RAW 264.7 cells were incubated with the 32P-end-labeled 45-mer double-stranded NF-kB oligonucleotide (15 mg protein with 16 fM DNA) from the HIV long terminal repeat, 50 -TTGTTACAAGGGACTTTCCGCTGGGGACTTTC CAGGGGGAGGCGTGG-30 (boldface indicates NF-kBebinding sites), for 30 min at 37  C. The DNA-protein complex formed was separated from free oligonucleotides on 6.6% native polyacrylamide gels. The dried gels were visualized with a Storm 820 molecular imager (Amersham, Piscataway, NJ, USA), and radioactive bands were quantified using a densitometer and Image Quant software (ImageJ software, NIH, USA).

To determine the role of NF-kB/p65 expression on CA-induced inhibition osteoclastogenesis, RAW 264.7 cells (5  105 cells/well) were plated in 6-well plates and transiently transfected by the calcium phosphate method with pNF-kB (0.5 mg) and control plasmid pCMVFLAG1 DNA for 24 h. The medium was then replaced, and the cells were treated with RANKL (5 nM) and or CA (5 mM) for 5 days. All cells were subjected to TRAP staining using the leukocyte acid phosphatase kit. 2.8. Statistical analysis Data were analysed using ImageJ software (NIH, USA) and are presented as means ± standard deviation (SD). The statistical

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Fig. 2. CA inhibits RANKL-induced osteoclastogenesis. (A) Schematic representation of treatment schedule. (B) RAW 264.7 cells (5  103 cells/mL) were incubated with 5 nM RANKL, 5 mM CA, or both for 3, 4, or 5 days and stained to show TRAP expression. Original magnification, 100. (C) RAW 264.7 cells (5  103 cells/mL) were incubated with either medium (indicated by the letter C) or RANKL (5 nM) along with the indicated concentrations of CA for 3, 4, or 5 days and then stained to show TRAP expression. Multinucleated osteoclasts (3 nuclei) were counted. Mean of 3 measurements ± SD.

significance of differences was assessed by student t-test. P values  0.05 were considered statistically significant.

3. Results Our results demonstrated that CA indeed suppressed the RANKL-induced NF-kB activation pathway by inhibiting the phosphorylation of IkBa, which is correlated with the suppression of osteoclastogenesis.

3.1. CA inhibits RANKL-induced osteoclastogenesis To determine whether CA inhibits RANKL-induced osteoclastogenesis, we treated RAW 264.7 cells with different concentrations of CA in the presence of RANKL and allowed the cells to differentiate into osteoclasts. Morphological observations clearly revealed that RANKL induced the cells differentiation into osteoclasts (Fig. 1B). However, RANKL-induced osteoclast formation decreased with exposure to increasing concentrations of CA, as measured by counting the number of TRAP-positive osteoclasts per well (Fig. 1C).

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osteoclastogenesis was time-dependent, we treated RAW 264.7 cells with RANKL and CA and incubated them for 3, 4, or 5 days to allow them to differentiate into osteoclasts (Fig. 2A). Morphological observation clearly showed that RAW 264.7 cells treated with RANKL differentiated into osteoclasts and that the addition of CA inhibited osteoclast differentiation (Fig. 2B). Moreover, RANKLinduced osteoclast differentiation was found to be timedependent, with the maximum number of TRAP-positive osteoclasts per well reached at day 5 (Fig. 2B). CA, however, decreased the number of TRAP-positive osteoclasts in a dose-dependent manner, with the strongest inhibition at a dose of 5 mM at all time points (Fig. 2C). 3.2. CA acts at an early step in the RANKL-induced osteoclastogenesis pathway Normally, RANKL-induced complete osteoclast differentiation in RAW 264.7 cells takes about 5 days. To determine the point at which CA acts in the differentiation pathway, we treated RAW 264.7 cells with RANKL and added CA 0, 1, 2, 3, and 4 days later. On day 5, the effects on osteoclast formation were measured (Fig. 3A). Microscopic observation (Fig. 3B) and counting of the number of TRAP-positive osteoclasts per well (Fig. 3C) showed that exposure to CA substantially inhibited osteoclast formation at 1e2 days after RANKL stimulation. However, at days 3 and 4 after RANKL exposure, CA no longer prevented osteoclast formation (Fig. 3B and C), indicating that it probably acts at an early step in the osteoclast differentiation pathway. 3.3. CA inhibits osteoclastogenesis induced by tumour cells Because increased osteoclastogenesis is commonly linked with breast cancer and multiple myeloma, we next investigated whether CA modulates osteoclastogenesis induced by these cancer cells in RAW 264.7 cells. When we co-incubated RAW 264.7 cells with MDAMB-231 breast cancer or U266 multiple myeloma cells and allowed them to differentiate for 5 days, we found that MDA-MB-231 cells induced differentiation of RAW 264.7 cells into osteoclasts and that CA inhibited this differentiation (Fig. 4A). A similar pattern was observed for U266 cells (Fig. 4B). These results show that CA suppresses the process of osteoclastogenesis induced by cancer cells. One major mechanism that has been associated with osteoclastogenesis is the NF-kB activation pathway. Therefore, we investigated whether conditioned medium from MDA-MB-231 and U266 cells induced NF-kB activation in RAW 264.7 cells. We found that conditioned medium indeed effectively activated NF-kB (Fig. 4C) in RAW 264.7 cells. These findings indicate that the NF-kB transcription factor is involved in osteoclast differentiation induced by cancer cells. 3.4. CA abrogates RANKL-induced NF-kB activation Fig. 3. CA inhibits RANKL-induced osteoclastogenesis 24 h after stimulation. (A) Schematic representation of treatment schedule. (B) RAW 264.7 cells (5  103 cells/mL) were incubated with medium, RANKL (5 nM), CA (5 mM), or RANKL and CA for the indicated times and stained for TRAP expression on day 5. Original magnification, 100. (C) Multinucleated osteoclasts (3 nuclei) were counted. C stands for cells incubated in medium alone. Mean of 3 measurements ± SD.

To exclude the possibility that this observation was attributable to a reduction in cell proliferation by CA, proliferation was assessed at the same concentrations of CA at days 1, 3, and 5. We found that CA did not significantly affect the proliferation of RAW 264.7 cells (Fig. 1D). To determine whether CA's inhibitory effect on

To further examine whether CA downmodulates RANKLinduced NF-kB activation, RAW 264.7 cells were either pretreated with various concentration of CA for 8 h (Fig. 5A, left panel) or CA (50 mM) for various time periods (Fig. 5A, right panel) and then exposed to RANKL for 30 min Fig. 5A. NF-kB activation was assayed using EMSA. As shown in Fig. 5A, RANKL activated NF-kB; however, CA completely abrogated RANKL-induced NF-kB activation. To test the effect of CA dose on NF-kB activation in RANKL-exposed cells, we pretreated RAW 264.7 cells with various concentrations of CA for 8 h and then exposed them to RANKL for 30 min. We found that CA completely suppressed NF-kB activation at a dose of 50 mM (Fig. 5B). Treatment of cells with 50 mM CA but no RANKL for 8 h had no effect on the activation of NF-kB.

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Fig. 4. CA suppresses osteoclastogenesis induced by tumour cells. (A) RAW 264.7 cells (5  103 cells/mL) were incubated in the presence of MDA-MB-231 cells (1  103 cells/mL), exposed to CA (5 mM) for 5 days, and stained to show TRAP expression (left panel). Original magnification, 100. Multinucleated osteoclasts were counted (right panel). Mean of 3 measurements ± SD. (B) RAW 264.7 cells (5  103 cells/mL) were incubated in the presence of U266 cells (1  103 cells/mL, exposed to CA (5 mM) for 5 days, and stained to show TRAP expression (left panel). Original magnification, 100. Multinucleated osteoclasts (3 nuclei) were counted (right panel). Mean of 3 measurements ± SD. (C) RAW 264.7 cells (1.5  106 cells per well) were incubated in the presence of conditioned medium from MDA-MB-231 or U266 cells for 24 h and then assessed for nuclear factor-kB (NF-kB) activity using an electrophoretic mobility shift assay (EMSA).

3.5. CA inhibits RANKL-induced IkBa phosphorylation and degradation Because nuclear translocation of NF-kB requires the proteolytic degradation of IkBa, we next examined whether CA's suppression of NF-kB was caused by inhibition of IkBa degradation. We used Western blotting to assay cytoplasmic IkBa degradation after RANKL stimulation at a range of time intervals (Fig. 5C). As shown in Fig. 5C, RANKL induced IkBa degradation in untreated control cells within 10 min, but IkBa levels returned to normal within 60 min. In contrast, cells pretreated with CA exhibited complete inhibition of IkBa degradation. IkBa phosphorylation is necessary for IkBa degradation, so we next

examined whether CA affects IkBa phosphorylation. We found that RANKL induced IkBa phosphorylation within 5 min. However, phosphorylation was completely inhibited by CA pretreatment. We also investigated the effect of CA on IkBa phosphorylation by using the proteasome inhibitor N-acetylleucyl-leucyl-norleucinal (ALLN), which prevents RANKL-induced IkBa degradation (Fig. 5D). Western blot analysis showed that cotreatment with RANKL and ALLN induced phosphorylation of IkBa at serines 32 and 36 in RAW 264.7 cells and that CA pretreatment inhibited the induction of phosphorylation. CA alone did not induce phosphorylation of IkBa (Fig. 5D). The phosphorylation, ubiquitination, and proteasome-mediated degradation of IkBa in response to RANKL is a rapid process. These results indicate that CA inhibits RANKL-induced NF-kB activation through

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Fig. 5. CA inhibits RANKL-induced NF-kB activation and IkBa degradation and phosphorylation. (A) RAW 264.7 cells (1.5  106 cells per well) were incubated with 50 mM of CA for 8 h and treated with 10 nM RANKL for 30 min. Nuclear extracts were examined for NF-kB activation using EMSA. (B) RAW 264.7 cells (1.5  106 cells per well) were treated with the indicated concentrations of CA for 8 h, treated with 10 nM RANKL for 30 min, and examined for NF-kB activation using EMSA. (C) RAW 264.7 cells (1.5  106 cells per well) were incubated with 50 mM of CA for 8 h and then treated with 10 nM RANKL for the indicated times (min). Cytoplasmic extracts were examined for IkBa degradation and phosphorylation by Western blotting with anti-IkBa and anti-phospho-IkBa antibodies. b-actin was used as a loading control. (D) RAW 264.7 cells (1.5  106 cells per well) were incubated with 50 mM of CA for 8 h, then incubated with ALLN (50 mg/mL) for 30 min, and finally treated with RANKL (10 nM) for 15 min. Cytoplasmic extracts were prepared and analysed by Western blotting using phospho-IkkBa antibody. The same membrane was reprobed with IkBa and b-actin antibodies. (E) RAW 264.7 cells (1.5  106 cells per well) were pre-incubated with CA for 8 h and then exposed to RANKL (10 nM) for the indicated times. Whole-cell extracts were analysed by Western blotting using phospho-extracellular signal-related kinase (p-ERK) 1/2 antibody. ERK 1/2 was used as a loading control.

suppression of IkBa degradation and phosphorylation. 3.6. Inhibition of osteoclastogenesis by CA is NF-kB-specific Because RANKL-induced osteoclastogenesis is triggered by 2 main signalling pathways, namely, the NF-kB and mitogenactivated protein kinase (MAPK) pathways, we investigated

whether CA pretreatment also affects the MAPK pathway. We examined the effect of CA on ERK1/2 activation. As shown in Fig. 5E, RANKL induced ERK1/2 phosphorylation in osteoclast precursor RAW 264.7 cells. However, pretreatment of cells with CA did not inhibit the RANKL-induced activation of ERK phosphorylation, indicating CA-induced inhibition of osteoclastogenesis is not mediated through MAPK pathway.

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Fig. 6. A peptide that targets the NF-kB essential modulator (NEMO)-binding domain inhibits RANKL-induced osteoclastogenesis. (A) RAW 264.7 cells (5  103 cells/mL) were pretreated with 100 mM of NEMO-binding domain peptide (NBP) for 2 h. The medium was then changed and RANKL (5 nM) was added for 5 days. The cells were then stained to show TRAP expression. Original magnification, 100. (B) Multinucleated osteoclasts (3 nuclei) were counted. Mean of 3 measurements ± SD. (C) RAW 264.7 cells (1.5  106 cells per well) were incubated with 100 mM of NBP for 2 h and then incubated with 10 nM RANKL for 30 min. The cells were examined for NF-kB activation using EMSA. (D) Overexpression of NF-kB/p65 reduces inhibitory effects of CA on RANKL-induced osteoclastogenesis. RAW 264.7 cells (5  103 cells/well) were first transfected with control and p65 plasmids. After 24 h cells were treated with 5 mM of CA and 5 nM of RANKL for 5 days and then stained to show TRAP expression. Original magnification, 100.

3.7. Inhibition of NF-kB abrogates osteoclastogenesis We further examined whether RANKL-induced osteoclastogenesis is caused by the activation of NF-kB using a specific inhibitor of the regulatory subunit of the IKK complex, which is also known as NF-kB essential modulator (NEMO). NEMO binding peptide (NBP blocks the activation of NF-kB). To determine the effect of NBP on RANKL-induced osteoclastogenesis, we pretreated RAW 264.7 cells with 100 mM NBP for 2 h and then with RANKL for 5 days. Our results showed that RANKL induced osteoclastogenesis and that NBP inhibited it (Fig. 6A and B). Furthermore, we found that nuclear extracts from RAW 264.7 cells treated with 100 mM NBP for 2 h and then with RANKL for 30 min completely inhibited NF-kB activation (Fig. 6C). These results confirm that NF-kB activation was responsible for osteoclast differentiation in RAW 264.7 cells and that inhibition of NF-kB with either CA or NBP prevented osteoclastogenesis. 3.8. Overexpression of NF-kB/p65 abolishes osteoclastogenesis inhibitory effects of CA To determine whether CA inhibits RANKL-induced osteoclatogenesis mediated through NF-kB activation, p65 was overexpressed in cells and then effect of CA on RANKL-induced osteoclatogenesis was studied. As shown in Fig. 6D, CA minimally inhibited RANKLinduced osteoclastogenesis in p65 transfected RAW 264.7 cells, however it almost completely inhibited in cells transfected with

control plasmid. This result indicates that p65 plays a role in RANKL induced osteoclastogenesis and thus inhibition of NF-kB/p65 by CA inhibits osteoclastogenesis. 3.9. CA abrogates M-CSF-induced osteoclast differentiation markers Further to examine the possible role of CA in the regulation of osteoclast differentiation in early stage, the RAW 264.7 cells were pre-treated with CA and induced by M-CSF for 24 h. As shown in Fig. 7A, Expression of RANK was upregulated by M-CSF. However, CA downmodulates M-CSF-induced expression of RANK. There was no significant variation in c-Fms-expression treated by M-CSF as well as CA. On the basis of these results, it can be concluded that CA inhibits M-CSF-induced RANK expression. 3.10. CA inhibits RANKL-induced NFATc1 and other osteoclastogenesis-related marker gene expression Because NFATc1 is also play a role in the regulation of osteoclastogenesis, the effect of CA on RANKL-induced NFATc1 expression were determined. As indicated in Fig. 7B, NFATc1 expression was upregulated when exposure to RANKL, and CA pretreatment can abolish RANKL-induced NFATc1 expression. Basically, activation and nuclear translocation of NFATc1 and osteoclast differentiation are mediated by the expression of large number of marker genes, including TRAP, cathepsin K and calcitonin R, regulated by NFATc1. These genes are activated by the

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Fig. 7. CA inhibits M-CSF-induced RANK expression and osteoclastogenesis-related gene expression. (A) RAW 264.7 cells (1.5  106 cells per well) were incubated with 50 mM of CA for 8 h and treated with 20 ng/mL M-CSF for 24 h. Cell lysates were examined for immunoblotting with RANK and c-Fms antibodies. b-actin was used as a loading control. (B) RAW 264.7 cells (1.5  106 cells per well) were incubated with 50 mM of CA for 8 h and treated with 10 nM RANKL for 24 h. Cell lysate were examined for different osteoclastogenesis-related genes as indicated by Western blotting. b-actin was used as a loading control.

RANKL-mediated signalling pathway. Therefore, whether CA modulates the expression of RANKL-induced osteoclastogenesisrelated marker genes, were also examined. As shown in Fig. 7B, RANKL induced the expression of TRAP, cathepsin K and calcitonin R. However, no effect of CA alone has been noticed in the expression of these marker proteins. These results indicate that CA can inhibit the RANKL-induced NFATc1, TRAP, cathepsin K and calcitonin R expression. 4. Discussion The aim of the present study was to investigate the effect of a novel bioactive compound, CA, which has been isolated from curcumin-free turmeric, on bone loss. Our results suggest that CA suppresses RANKL- and cancer cell-induced differentiation of mouse macrophages into osteoclasts. We also found that CA acts at an early point in the osteoclastogenic pathway. Finally, we found that the inhibition of osteoclastogenesis by CA was mediated through suppression of the RANKL signalling pathway. Osteoclastsdspecialized cells derived from the monocyte/ macrophage hematopoietic lineageddevelop and adhere to bone matrix. They play a crucial role in bone disease: a decrease or increase in the number of osteoclasts leads to osteosclerosis or osteoporosis, respectively [17,18]. Where CA, which is derived from turmeric, a non-toxic, affordable, safe, and traditionally used medicinal and culinary spice, has been shown to suppress inflammatory pathways and inhibit osteoclastogenesis. Systemic hormones and cytokines provide the molecular signals for the control of osteoclastogenesis and the maintenance of homeostasis [2]. The discovery of the RANK signalling pathway in osteoclasts has provided insight into the mechanisms of osteoclastogenesis: RANKL and NF-kB have been shown to be major mediators of osteoclastogenesis [8]. Specifically, it has been reported that an IkB super-suppressor inhibited osteoclast

differentiation and activation [19] and that a dominant-negative IkB protein lacking the NH2-terminal phosphorylation site lowered NF-kB activation and suppressed recruitment of osteoclasts [20]. Therefore, a compound that inhibits activation of NF-kB is very likely to be able to inhibit osteoclastogenesis. In this study, we determined that CA suppresses RANKL-induced osteoclastogenesis through inhibiting RANKL-induced NF-kB activation and that it does so at an early step in the osteoclast differentiation process. Our research group has previously found similar results for peripheral blood mononuclear cells stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL [21,22]. We found further confirmation that inhibition of the NF-kB signalling pathway functions to arrest the osteoclast differentiation process by examining NBP, which targets the IKKa and IKKb kinases and inhibits NF-kB activation. NBP has been shown to inhibit osteoclastogenesis in vivo and to delay the onset, lower the incidence, and decrease the severity of rheumatoid arthritis [23]. Moreover, it has also been reported that pharmacological or genetic inactivation of IKKa, IKKb, or both is sufficient for the inhibition of osteoclastogenesis, inflammation, and osteolytic-induced bone loss [24,25]. Our findings showed that CA completely inhibited RANKLinduced osteoclastogenesis in the same manner as NBP. The inhibitory effect of CA on NF-kB and osteoclastogenesis as well was as potent as that of NBP, at least in vitro. RANKL activates two major signalling pathways leading to osteoclastogenesis: (1) the NF-kB pathway, by recruiting the adapter proteins TRAF2, TRAF3, TRAF5, TRAF6, and NF-kB-inducing kinase via TRAF6; and (2) the MAPK pathway via activation of ERK and phosphorylation of c-Fos and c-Jun [26]. While ERK is responsible for osteoclast survival, NF-kB regulates osteoclast activation for bone resorption [27]. Our study has shown that CA does not affect the MAPK pathway, suggesting that the inhibitory effect CA exerts on osteoclastogenesis is specific to the NF-kB pathway. However previously reported that Calebin-A modulates the activities of MAPK family members, which includes decreased JNK, ERK and increased p38 activity [15]. These results suggest that Calebin-A might be an effective compound for the treatment of human gastric and other MDR cancers. As NFATc1 is also known as master transcription factor of osteoclastogenesis and regulated by the NF-kB [43], CA also have the potential to inhibit the RANKL-induced NFATc1 expression. This evaluation is consistent with previous observations where downregulation of NF-kB downmodulate the NFATc1 expression [44,45]. Besides NFATc1, CA Also down modulated the NFATc1-regulated gene expression including TRAP, calcitonin R, and cathepsin K. A major problem in metastatic breast cancer and multiple myeloma is osteoclast-mediated bone destruction [9,28]. Several studies have demonstrated that myeloma cells enhance osteoclast formation and activity through upregulation of RANKL [29,30] or by expressing RANKL themselves [31,32]. U266 multiple myeloma cells are known to express RANKL [33,34] and to exhibit NF-kB activation [21,35] but MDAMB231 breast cancer cells do not express RANKL [36]. Then how MDAMB231 cells induce osteoclastogenesis is not very much clear. However, cytokines such as macrophage colony-stimulating factor, IL-7 and TNF-a have been reported to induce osteoclastogenesis [37]. In the present study CA inhibited MDAMB-231 cells-induced osteoclastogenesis probably through suppression of these cytokines. Consistent with that finding, the present study has shown that CA inhibits osteoclastogenesis induced by MDA-MB-231 and U266 cells, suggesting that CA is an attractive potential agent for the treatment of patients with bone metastasis. Moreover, our findings indicated that the tumour cells secreted certain factors that stimulate osteoclast differentiation. Bisphosphonates are the main currently available treatment for patients with bone metastasis or cancer-related bone disease

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[38,39]. However, bisphosphonates have adverse effects and are not effective for all patients [40]. Recently, the US Food and Drug Administration approved the RANKL antibody denosumab (Prolia) for the treatment of post-menopausal osteoporosis and bone loss in patients with hormone-treated prostate or breast cancer [41]. However, denosumab carries black box warnings due to severe side effects and is very expensive. Celecoxib, a nonsteroidal antiinflammatory drug and inhibitor of COX-2, has also been used for osteoclastogenesis inhibition [42,46] but also has some undesirable side effects. Previous studies have shown that CA, which is derived from turmeric, is non-toxic [13] and therefore has potential for the treatment of secondary bone lesions not only associated with cancer, but also with nonmalignant diseases such as postmenopausal osteoporosis, Paget disease, and rheumatoid arthritis. Further studies are needed, however, to confirm whether CA can suppress osteoclastogenesis using clinically relevant animal models before proceeding to in vivo trials in cancer patients. Conflict of interest The authors have no conflicts of interest related to this manuscript. Acknowledgments We thank Amy Ninetto for editorial review of this manuscript and Prof V. Craig Jordan (Father of Tamoxifen) for providing the facility and required reagents to revise the manuscript. This work was supported by a grant from Jarrow Formulas, Inc., and Center for Targeted Therapy of MD Anderson Cancer Center. References [1] Z. Jin, X. Li, Y. Wan, Mol. Endocrinol. 29 (2015) 172e186. [2] M.P. Yavropoulou, J.G. Yovos, J. Musculoskelet. Neuronal Interact. 8 (2008) 204e216. [3] F. Arai, T. Miyamoto, O. Ohneda, T. Inada, T. Sudo, K. Brasel, T. Miyata, D.M. Anderson, T. Suda, J Exp Med 8 (1999) 1741e1754. [4] D.M. Anderson, E. Maraskovsky, W.L. Billingsley, W.C. Dougall, M.E. Tometsko, E.R. Roux, M.C. Teepe, R.F. DuBose, D. Cosman, L. Galibert, Nature 390 (1997) 175e179. [5] D.L. Lacey, E. Timms, H.L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sullivan, N. Hawkins, E. Davy, C. Capparelli, A. Eli, Y.X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G. Senaldi, J. Guo, J. Delaney, W.J. Boyle, Cell 93 (1998) 165e176. [6] S.L. Teitelbaum, Science 289 (2000) 1504e1508. [7] Y.Y. Kong, H. Yoshida, I. Sarosi, H.L. Tan, E. Timms, C. Capparelli, S. Morony, A.J. Oliveira-dos-Santos, G. Van, A. Itie, W. Khoo, A. Wakeham, C.R. Dunstan, D.L. Lacey, T.W. Mak, W.J. Boyle, J.M. Penninger, Nature 397 (1999) 315e323. [8] J. Xu, H.F. Wu, E.S. Ang, K. Yip, M. Woloszyn, M.H. Zheng, R.X. Tan, Cytokine Growth Factor Rev. 20 (2009) 7e17. [9] R.E. Coleman, Cancer Treat. Rev. 27 (2001) 165e176. [10] R.E. Coleman, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 12 (2006) 6243se6249s. [11] T. Onishi, N. Hayashi, R.L. Theriault, G.N. Hortobagyi, N.T. Ueno, Nat. Rev. Clin. Oncol. 7 (2010) 641e651. [12] J. Sturge, M.P. Caley, J. Waxman, Nat. Rev. Clin. Oncol. 8 (2011) 357e368.

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