Zerumbone Abolishes RANKL-Induced NF-κB Activation, Inhibits Osteoclastogenesis, and Suppresses Human Breast Cancer–Induced Bone Loss in Athymic Nude Mice

Bone remodeling is controlled by the balance between bone formation and resorption (1). Many bone-related diseases, such as osteoporosis, rheumatoid arthritis, Paget's disease, and cancer-induced osteolysis, are characterized by excessive bone resorption by osteoclast, multinucleated cells derived from hematopoietic cells (1). The differentiation of osteoclast precursors to osteoclast is regulated by the bone microenvironment, which includes stromal cells, osteoblasts, and other local factors (2). Perhaps two most proteins crucial for osteoclast differentiation and activation are receptor activator of nuclear factor-κB (NF-κB; RANK) and its ligand, RANKL. The latter is a member of the tumor necrosis factor (TNF) superfamily of cytokines (3), and its expression is regulated by several factors that regulate bone resorption, including vitamin D3, glucocorticoids, interleukin (IL)-1, IL-6, and TNFα (2, 4, 5). The significance of RANK-RANKL pathway is supported by studies that show that disruption in either rank or rankl in mice produced severe osteopetrosis and defective tooth eruption resulting from a complete lack of osteoclasts (6). In contrast, genetic deletion of osteoprotegerin, a decoy receptor for RANKL and an inhibitor of RANK, produced osteoporosis resulting from an increased number of osteoclast (7).

Bone metastases represent a common cause of morbidity in patients with many types of cancer, occurring in up to 70% of patients with advanced breast or prostate cancer and in about 15% to 30% of patients with lung, colon, kidney, thyroid, and stomach carcinoma (8). Bone metastases can cause hypercalcemia, bone fragility, pathologic fractures, and spinal compression and are usually associated with severe bone pain due to osteolysis, which can be intractable (9). Besides solid tumors, osteolysis is present in other pathologies, such as psoriatic arthritis, periodontal disease, and multiple myeloma (10–12).

Above description thus indicates that agents that can suppress RANKL signaling have potential for treatment of osteoporosis. In the present report, we investigated whether zerumbone, a sesquiterpene derived from tropical ginger Zingiber zerumbet, can modulate RANKL-induced signaling and osteoclastogenesis. Zerumbone has been shown to suppress azoxymethane-induced rat aberrant crypt foci (13), inhibit dextran sodium sulfate–induced colitis in mice (14), suppress skin tumor initiation and promotion in mice (15), inhibit tumor growth in mice (16), and reduce cholecystokinin-induced pancreatitis in rats (17). Previously, we have shown that this agent can down-regulate NF-κB activation induced by various carcinogens and inflammatory agents and suppress NF-κB–regulated gene products (18). The current report is based on the hypothesis that zerumbone would also suppress RANKL signaling and RANKL-induced osteoclastogenesis. We found that zerumbone can suppress RANKL-induced NF-κB activation through inhibition of IκBα kinase (IKK) and inhibits osteoclastogenesis induced by RANKL and by breast cancer and multiple myeloma cells.

Reagents. Zerumbone (Fig. 1A), isolated as described (19), was prepared as a 50 mmol/L solution in DMSO and then further diluted in cell culture medium. DMEM/F12, RPMI 1640, DMEM, fetal bovine serum, 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Invitrogen. Rabbit polyclonal antibodies to IκBα were purchased from Santa Cruz Biotechnology. Antibody against phospho-IκBα (Ser^32/36) was purchased from Cell Signaling Technology. Anti-IKKα and anti-IKKβ antibodies were kindly provided by Imgenex. Goat anti-rabbit and goat anti-mouse horseradish peroxidase conjugates were purchased from Bio-Rad. Antibody against β-actin and leukocyte acid phosphatase kit (387-A) for tartrate-resistant acid phosphatase (TRAP) staining were purchased from Sigma-Aldrich. Protein A/G-agarose beads were obtained from Pierce. [γ-³²P]ATP was purchased from ICN Pharmaceuticals.

Cell lines. RAW 264.7 (mouse macrophage), MDA-MB-231 (human breast adenocarcinoma), and U266 cells (human multiple myeloma) were obtained from the American Type Culture Collection. RAW 264.7 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and antibiotics. This cell line is a well-established osteoclastogenic cell system that has been shown to express RANK and differentiate into functional TRAP-positive osteoclasts when cultured with soluble RANKL (20). Moreover, RANKL has been shown to activate NF-κB in RAW 264.7 cells (21). MDA-MB-231 cells in DMEM and U266 cells were cultured in RPMI 1640 with 10% fetal bovine serum.

Electrophoretic mobility shift assays for NF-κB. Nuclear extracts were prepared as described previously (22). Briefly, nuclear extracts from RANKL-treated cells were incubated with ³²P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg protein with 16 fmol DNA) from the HIV long terminal repeat, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (boldface indicates NF-κB–binding sites), for 30 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. The dried gels were visualized with a Storm820 and radioactive bands were quantified using ImageQuant software (Amersham).

Western blot analysis. To determine the levels of protein expression in the cytoplasm or nucleus, we prepared extracts (22) and fractionated them by 10% SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each antibody, and detected by enhanced chemiluminescence reagent (GE Healthcare).

Reverse transcription-PCR analysis. RANKL mRNA expression was determined by reverse transcription-PCR (RT-PCR). MDA-MB-231 cells were treated with zerumbone for 24 h. Total RNA was extracted from cells using Trizol (Invitrogen). One microgram of total RNA was converted to cDNA by SuperScript reverse transcriptase and then amplified by Platinum Taq polymerase using SuperScript One-Step RT-PCR kit (Invitrogen). The primers include those for RANKL (forward primer, 5′-CGTTGGATCACAGCACATCAG-3′; reverse primer, 5′-AGTATGTTGCATCCTGATCCG-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; forward primer, 5′-GTCTTCACCACCATGGAG-3′; reverse primer, 5′-CCACCCTGTTGCTGTAGC-3′). Cycling conditions were 30-s denaturation at 94°C, 30-s annealing at 56°C, and 30-s elongation at 72°C for 35 cycles. PCR products were electrophoresed on 2% agarose gels and gel images were visualized under UV light and photographed.

Trypan blue exclusion assay. Cells were harvested from the 24-well plates by treatment with 0.2% trypsin-EDTA, centrifuged, and suspended with 1 mL culture medium. Cell suspension was mixed with equal volume of 0.4% isotonic trypan blue solution. Total cell number and fraction of nonviable, dye-accumulating cells were counted after 2 min in Fuchs-Rosenthal hemocytometer under light microscope.

IKK assay. To determine the effect of zerumbone on RANKL-induced IKK activation, IKK assay was done by a method described previously (22). Briefly, the IKK complex from whole-cell extracts (800 μg protein) was precipitated with antibody against IKKα followed by treatment with protein A/G-agarose beads. After 2 h of incubation, the beads were washed with lysis buffer and assayed in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl₂, 2 mmol/L DTT, 20 mCi [γ-³²P]ATP, 10 mmol/L unlabeled ATP, and 2 μg substrate glutathione S-transferase (GST)-IκBα (amino acids 1–54). After incubation at 30°C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with a PhosphorImager. To determine the total amounts of IKKα and IKKβ in each sample, the whole-cell protein was resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and blotted with anti-IKKα or anti-IKKβ antibody.

Osteoclast differentiation assay. RAW 264.7 cells were cultured in 24-well dishes at a density of 5 × 10³ per well and allowed to adhere overnight. The medium was then replaced, and the cells were treated with 5 nmol/L RANKL for 5 d. All cell lines were subjected to TRAP staining using leukocyte acid phosphatase kit (387-A).

In vivo osteolytic bone metastasis assay. The estrogen-independent human breast cancer cell line MDA-MB-231 was cultured and resuspended in PBS to give a final concentration of l × 10⁵/100 μL, which was injected directly into the left ventricle of 4- to 5-wk-old female BALB/c nu/nu mice (Harlan) via a percutaneous approach. The mice were then randomly assigned to one of three groups (n ≥ 9/group), treated with vehicle (0.9% sodium chloride) or zerumbone (20 or 100 mg/kg body weight in vehicle) by i.p. injection three times a week for 28 d, and then sacrificed. Radiographs (Faxitron radiographic inspection unit, Kodak) were obtained at baseline and just before sacrifice. Area of osteolytic bone metastases on X-rays of tibiae and femorae, recognized as well-circumscribed radiolucent lesions on radiographs, was quantitated using MetaMorph imaging software (Molecular Devices). Radiographs were analyzed by investigators blinded to the composition of the groups in the experiment or the experimental protocol and areal data for each group are presented as mm²/lesion (mean ± SE).

Immunohistochemistry. Bone histology and histomorphometric analysis of bone tissue sections were performed as previously described with minor modifications (23). Briefly, on day 28 after tumor cell inoculation, animals treated with vehicle or zerumbone were sacrificed; both hind limbs from each animal were harvested, fixed in 10% formalin, decalcified in EDTA, and embedded in paraffin; and 4- to 5-μm-thick sections of undecalcified long bones were cut with a Leica microtome. Sections were stained with H&E, orange G, and phloxine. Histomorphometric analysis for tumor burden was performed on H&E-stained sections of tibiae using the MetaMorph computerized image analysis system. Tumor burden is expressed as a percentage of the total area of medullary cavity (between the cortices and a fixed distance from the growth plate) that is occupied by tumor. Osteoclasts were detected in situ in serial sections as previously described by cytochemical staining for TRAP activity, a marker of osteoclasts (23).

Statistical analysis. Data are presented as mean ± SE and analyzed using StatView software (version 5.0; SAS Institute, Inc.). Statistical significance of differences was assessed using the nonparametric Mann-Whitney U test. Values of P ≤ 0.05 were considered statistically significant.

The aim of the present study was to investigate the effect of zerumbone on RANKL signaling that leads to osteoclastogenesis. Whether zerumbone could modulate osteoclastogenesis induced by tumor cells was another focus of these studies. We used the RAW 264.7 cell (murine macrophage) system, as it is a well-established model for osteoclastogenesis (24).

Zerumbone abrogates RANKL-induced NF-κB activation. To investigate whether zerumbone modulates RANKL-induced NF-κB activation in RAW 264.7 cells, cells were either pretreated with zerumbone for 12 hours or left untreated and then exposed to RANKL for indicated times, nuclear extracts were prepared, and NF-κB activation was assayed by electrophoretic mobility shift assay. As shown in Fig. 1B, RANKL activated NF-κB in a time-dependent manner; however, zerumbone completely abrogated RANKL-induced NF-κB activation (Fig. 1B).

To determine the minimum concentration of zerumbone required to suppress RANKL-induced NF-κB activation, cells were pretreated with various concentrations of zerumbone and then exposed to RANKL. Zerumbone almost completely suppressed NF-κB activation at 20 μmol/L (Fig. 1C). Treatment of cells with 20 μmol/L zerumbone for 12 hours had no effect on cell viability as determined by the trypan blue exclusion method.

Zerumbone inhibits RANKL-induced IκBα phosphorylation and degradation. Most cytokines activate NF-κB through phosphorylation and degradation of its inhibitory subunit IκBα (25). To identify the mechanism involved in the inhibition of NF-κB activation by zerumbone, we first checked the effect on zerumbone on level of IκBα by using Western blot analysis. In cells treated with RANKL, the IκBα level dropped within 5 minutes and returned to normal level within 60 minutes (Fig. 2A,, left). In contrast, cells pretreated with zerumbone showed suppressed RANKL-induced degradation of IκBα (Fig. 2A , right).

Next, we investigated the effect of zerumbone on the phosphorylation of IκBα, which occurs before its ubiquitination and degradation (25). We used the proteasome inhibitor N-acetyl-leu-leu-norleucinal (ALLN) to prevent RANKL-induced IκBα degradation. Western blot analysis for phospho-IκBα in Fig. 2B clearly indicates that RANKL induced IκBα phosphorylation in RAW 264.7 cells and that zerumbone abrogated this effect. Zerumbone alone did not result in phosphorylation of IκBα.

Zerumbone inhibits RANKL-induced IKK activation. Because zerumbone inhibits the phosphorylation and degradation of IκBα, we next checked whether zerumbone alters the activity or the levels of IKK, which leads to IκBα phosphorylation. Immunocomplex kinase assay on cells treated with RANKL showed a sharp increase in IKK activity as indicated by the phosphorylation of GST-IκBα within 3 minutes. In contrast, cells pretreated with zerumbone could not phosphorylate GST-IκBα on RANKL treatment (Fig. 2C,, top). To check whether the apparent loss of IKK activity was due to the loss of IKK protein expression, the levels of the IKK subunits IKKα and IKKβ were tested by Western blot analysis. Results in Fig. 2C clearly show that zerumbone treatment did not alter the expression of IKKα and IKKβ.

Zerumbone inhibits RANKL-induced osteoclastogenesis. Next, the effect of zerumbone on RANKL-induced osteoclastogenesis was examined. RAW 264.7 cells were incubated with different concentrations of zerumbone in the presence of RANKL and allowed to differentiate into osteoclasts. As shown in Fig. 3A, RANKL induced osteoclasts in the absence of zerumbone at day 4. By contrast, the differentiation into osteoclast was significantly decreased in the presence of zerumbone. In addition, the formation of osteoclast decreased with increasing concentration of zerumbone (Fig. 3B). As little as 0.5 μmol/L zerumbone had a significant effect on RANKL-induced osteoclast formation. Under these conditions, the viability of cells was not significantly affected (Supplementary Fig. S1).

Zerumbone acts at an early step 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 elucidate how early zerumbone acts in this pathway, RAW 264.7 cells were treated initially with RANKL, zerumbone was added 1, 2, 3, and 4 days after RANKL addition, and then its effect on osteoclast formation was determined. Zerumbone inhibited osteoclastogenesis even when the cells were exposed 24 hours after the RANKL treatment. However, the inhibitory effect decreased significantly in a time-dependent manner when cells were treated with zerumbone after RANKL treatment (Fig. 4).

Zerumbone inhibits osteoclastogenesis induced by tumor cells. Osteoclastogenesis is commonly linked with breast cancer (26) and multiple myeloma (27) through the activation of NF-κB (28). Whether zerumbone also inhibits tumor cell–induced osteoclastogenesis of RAW 264.7 cells was investigated. As shown in Fig. 5A and B, incubating RAW 264.7 cells with breast cancer MDA-MB-231 cells or multiple myeloma U266 induced osteoclast differentiation, and zerumbone suppressed this differentiation. These results indicate that osteoclastogenesis induced by tumor cells is significantly suppressed by the presence of zerumbone.

Whether human breast cancer or multiple myeloma cells express RANKL was examined by RT-PCR. We found that human multiple myeloma U266 cells do express RANKL but human breast cancer cells (MDA-MB-231) do not (Fig. 5C).

Zerumbone decreases osteolysis in MDA-MB-231 breast cancer tumor-bearing mice. To determine the effect of zerumbone on bone loss, we used a xenotransplant mouse model. Human breast cancer cells (MDA-MB-231) were injected directly into the left ventricle. At day 28, osteolytic bone metastases were detectable by radiography in the majority of the animals in different groups. As shown in Fig. 6A, there was no significant difference in body weight between control tumor-bearing mice and zerumbone-administrated groups. This result indicates that zerumbone is nontoxic even at the higher dose (100 mg/kg). The data presented in Fig. 6B showed that zerumbone (20 and 100 mg/kg) suppressed osteolytic bone metastases compared with vehicle-treated human breast tumor-bearing mice. The effect was more pronounced at 100 mg/kg than at 20 mg/kg dose of zerumbone. Therefore, as indicated in Fig. 6C, human breast cancer induced osteolysis (represented by radiolucency) and the destruction of cortices (arrows) in bone of tumor-bearing mice (left). In contrast, there was very little osteolysis in bone from the higher dose zerumbone (100 mg/kg)–treated groups and the cortices remained intact (right).

Overall tumor burden in tibiae assessed histomorphometrically was statistically not significantly different between the tumor-bearing (Fig. 6D,, a, 54 ± 9%, n = 15) and zerumbone-treated groups (Fig. 6D,, b, 49 ± 9%, n = 17). However, TRAP activity shows intense osteoclast activity with tumor-bearing mice at tumor-bone interface (Fig. 6D,, c, pink/red staining) and also at some distance away from the tumor around the growth plate. In contrast, osteoclast activity is markedly reduced in zerumbone-treated groups (Fig. 6D,, d). In vehicle-treated tumor-bearing animals, the osteoclasts presented at the tumor-bone interface along the cortico-endosteal surface where they are rarely seen (Fig. 6D,, e). In contrast, there are no osteoclasts present at the cortico-endosteal surface despite the tumor cells being in contact with bone and the comparable amounts of tumor in the two groups of tumor-bearing animals (Fig. 6D , f). These results indicate that zerumbone significantly suppressed the osteolysis in tumor-bearing mice.

Bone loss is one of the major problems associated with aging, arthritis, cancer, and other chronic inflammatory illnesses. Identification of inhibitors of bone loss that are safe, efficacious, and affordable is needed. Zerumbone is one such compound that is derived from shampoo ginger and has been shown to suppress inflammatory pathways. The goal of this study was to investigate the effect of zerumbone, a bioactive sesquiterpene, on RANKL-induced NF-κB activation and osteoclastogenesis induced by both RANKL and tumor cells.

Our results indicated that RANKL activated NF-κB in osteoclast precursor cells through the activation of IKK and subsequent IκBα phosphorylation and degradation. The RANKL-induced NF-κB activation signaling pathway differs from that of the TNF-induced pathway. For instance, NIK, which may function as an activator of IKKα, is necessary in RANKL-induced NF-κB activation (21); however, it is dispensable for TNF-induced NF-κB activation (29). Therefore, Novack and colleagues (30) reported that NIK-deficient osteoclast precursors did not respond to RANKL in an in vitro differentiation system devoid of osteoblasts. We also found that zerumbone inhibited RANKL-induced IKK activation, leading to the suppression of NF-κB activation.

A recent study established that IKKβ, but not IKKα, is essential for inflammation-induced bone loss and is required for osteoclastogenesis in vivo (31). Although the ability of zerumbone to suppress TNF-induced IKK activation has been reported (18), the current study is the first to report that zerumbone can suppress RANKL-induced IKK activation and consequently NF-κB activation. How zerumbone inhibits RANKL-induced IKK activation is not clear. Numerous kinases have been implicated in the activation of IKK, including AKT, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 1, 2, and 3, glycogen synthase kinase-3β, and NIK (32). Previously, our group has shown that NIK-induced NF-κB activation is blocked by zerumbone (18). Thus, it is possible that zerumbone inhibits RANKL-induced IKK activation through inhibition of NIK. However, other potential mechanisms cannot be ruled out based on the data presented here.

Osteoclastogenesis is dependent on RANKL under physiologic and pathologic conditions (1, 2). Although the mechanism by which RANKL induces osteoclastogenesis is not completely understood, it is known that RANKL-induced activation of NF-κB and MAPK pathways as well as up-regulation of NFAT2 expression are required for osteoclastogenesis (33). The importance of NF-κB in osteoclastogenesis is suggested by using gene deletion models. The mice lacking both NF-κB p50 and p52 or mice deficient in IKKβ exhibit severe osteopetrosis caused by failure of osteoclast formation (33, 34). Abu-Amer and colleagues (35) reported that IκB supersuppressor blocked osteoclast differentiation and activation. Moreover, the same group reported that the dominant-negative IκB protein, which lacks the NH₂-terminal phosphorylation site, lowered NF-κB activation and reduced recruitment of osteoclasts to bone erosion (36). Furthermore, IKK inhibitory peptide can suppress osteoclastogenesis and bone loss in a mouse model of arthritis (31). Thus, it is reasonable to suggest that zerumbone suppresses osteoclastogenesis through inhibition of NF-κB activation.

Malignant tumors with the skeleton as the primary site as well as metastatic bone lesions are considered a major health problem, affecting over 350,000 patients in the United States annually. Among them, 70% to 95% of patients with multiple myeloma and up to 75% of patients with advanced breast cancer or prostate cancer develop bone metastasis (37, 38). Osteoclast-mediated bone destruction is a major complication in metastatic breast cancer and multiple myeloma. In the present study, we showed that zerumbone inhibited the osteoclastogenesis induced by breast cancer and by multiple myeloma cells. Both breast cancer and multiple myeloma cells are known to express RANKL (27, 39) and to exhibit constitutive NF-κB activation (40, 41), thus implicating them in the induction of osteoclastogenesis via the expression of RANKL. We also found that breast cancer–induced bone loss in mice could be inhibited by zerumbone. Consequently, our results suggest that zerumbone could be used in the treatment of cancer-induced bone lesions. Bisphosphonate drugs (BP), potent inhibitors of osteoclast formation and activity, are the current standard of care and are most widely used drugs for treatment of cancer-induced osteolytic diseases. Their efficacy in abating pain and prolonging time to significant skeletal complications has been shown in the treatment of breast cancer–induced bone diseases (42). However, there is increasing recognition of the increased risk of severe osteonecrosis of the jaw in cancer patients receiving BPs (43–45) with devastating consequences for affected patients (46). Thus, it is therefore prudent to develop other antiresorptive strategies to combat lytic bone lesions in patients with metastatic cancer, including breast cancer metastases. At present, a fully humanized monoclonal neutralizing antibody to RANKL, denosumab (Amgen), is ongoing in clinical trials with multiple myeloma and breast cancer and recent data suggest that it is efficacious (47).

Zerumbone is derived from a tropical ginger and thus should have minimum toxicity, as it is used routinely for traditional medicine. It could be safely used in treatment of secondary bone lesions associated with various cancers (31, 48, 49), including breast cancer, and those associated with other diseases, such as osteoporosis, Paget's disease, and rheumatoid arthritis. Zerumbone suppresses osteoclast formation in vitro in a RANKL-dependent manner as shown in this article. We also found that this sesquiterpene is remarkably well tolerated with no toxicity effects on systemic administration to mice at 100 mg/kg daily for 4 weeks. In comparison with BPs or denosumab, ginger is very inexpensive and thus likely to have a superior cost-benefit ratio. Because humans consume large amounts of ginger regularly in certain parts of the world with no untoward effects, these properties of zerumbone suggest potential utility of the compound, at least as adjunct therapy, in breast cancer patients. However, there are currently no preclinical data to inform well-designed clinical trials and observational studies that will be needed to determine short- and long-term effects of zerumbone and to guide clinicians and patient on its use during breast cancer treatment. The data reported in this article thus provide rationale for extensive preclinical studies on zerumbone in breast cancer.

No potential conflicts of interest were disclosed.

Grant support: Clayton Foundation for Research (B.B. Aggarwal) and NIH/National Cancer Institute grants 1PO1-CA1248701-A2 (B.B. Aggarwal) and KO1CA104180 (B.O. Oyajobi). B.B. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Walter Pagel for editorial review of this manuscript and Dr. Bryant Darnay for providing the RANKL protein.