J Bone Miner Metab (2009) 27:24–35 DOI 10.1007/s00774-008-0013-y
ORIGINAL ARTICLE
Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners Masahiro Takahashi Æ Toshihide Mizoguchi Æ Shunsuke Uehara Æ Yuko Nakamichi Æ Shuhua Yang Æ Hiroko Naramoto Æ Teruhito Yamashita Æ Yasuhiro Kobayashi Æ Minoru Yamaoka Æ Kiyofumi Furusawa Æ Nobuyuki Udagawa Æ Takashi Uematsu Æ Naoyuki Takahashi
Received: 15 January 2008 / Accepted: 21 April 2008 / Published online: 13 December 2008 Ó The Japanese Society for Bone and Mineral Metabolism and Springer 2008
Abstract Osteoclasts are formed from the monocytemacrophage lineage in response to receptor activator of nuclear factor jB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D3 plus prostaglandin E2. Docetaxel at 10-8 M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10-6 M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited Electronic supplementary material The online version of this article (doi:10.1007/s00774-008-0013-y) contains supplementary material, which is available to authorized users. M. Takahashi S. Yang H. Naramoto M. Yamaoka K. Furusawa T. Uematsu Department of Oral Maxillofacial Surgery, Matsumoto Dental University, 1780 Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan T. Mizoguchi Y. Nakamichi T. Yamashita Y. Kobayashi N. Takahashi (&) Institute for Oral Science, Matsumoto Dental University, 1780 Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan e-mail:
[email protected] S. Uehara N. Udagawa Department of Biochemistry, Matsumoto Dental University, 1780 Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan
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osteoclast formation when added for the final 3 days. Docetaxel at 10-8 M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D3 plus prostaglandin E2 in osteoblasts was not affected by docetaxel even at 10-6 M. Docetaxel at 10-6 M, but not at 10-8 M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and L-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10-6 M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10-8 M and of osteoclast function at 10-6 M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Keywords Docetaxel Bone resorption Osteoclast Osteoblast Microtubule
Introduction Bone is a common metastatic site for breast carcinoma [1, 2]. Osteoclastic bone resorption plays important roles in bone invasion and metastasis of tumor cells [1–5]. The taxane-derived compounds paclitaxel (Taxol) and docetaxel (Taxotere) are chemotherapeutic agents used for the treatment of malignant diseases, including breast and oral cancers [6–8]. Combination treatment with trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor 2 (HER-2), and taxane-based chemotherapy has become the standard of care for women with HER2-positive metastatic breast cancer [9, 10]. Both paclitaxel and docetaxel bind to tubulin, the basic structural component of microtubules, and induce stabilization of microtubules [11, 12]. The antineoplastic effect
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of taxanes is believed to be derived from this mechanism. However, it is reported that taxanes have multiple cellular effects, such as stimulation of mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 mRNA expression, and apoptosis [13–16]. In addition, paclitaxel mimics the action of lipopolysaccharide (LPS), a bacterial component, in mice but not in humans [17, 18]. Toll-like receptor 4 (TLR4) is a signaling receptor for LPS [19]. Paclitaxel induces signals through mouse TLR4/myeloid differentiation protein-2 (MD-2) but not via human TLR4/MD-2 [20]. Thus, paclitaxel has two effects on murine cells, namely, inducing microtubule stabilization and LPS-like action. Osteoclasts, bone-resorbing multinucleated cells (MNCs), are differentiated from monocyte-macrophage lineage cells under the tight regulation of osteoblasts [21–23]. Osteoclasts are formed in cocultures of mouse osteoblasts and hematopoietic cells in the presence of osteotropic factors such as 1a,25-dihydroxyvitamin D3 [1,25(OH)2D3] and prostaglandin E2 (PGE2) [22]. Osteoblasts express two cytokines essential for osteoclast differentiation: receptor activator of NF-jB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) [22, 23]. M-CSF is constitutively expressed by osteoblasts, whereas the expression of RANKL is upregulated by the osteotropic factors [22, 23]. Osteoclast precursors express RANK and c-Fms, receptors of RANKL and M-CSF, respectively, and differentiate into osteoclasts in the presence of both cytokines. Two characteristic features of osteoclasts are the presence of ruffled borders and sealing zones [24, 25]. The resorbing area under the ruffled borders is acidic, which favors dissolution of bone mineral. The sealing zone, which serves for the attachment of osteoclasts to the bone surface, is observed as a ringed structure of F-actin dots (actin ring). Disruption of sealing zones results in the suppression of bone-resorbing activity of osteoclasts [24, 25]. Interaction between F-actin and the microtubule cytoskeleton has been shown in functioning osteoclasts [26]. Osteoclasts eliminate bone degradation products through transcytosis [24, 27, 28]. Transcytotic vesicles, which include the degradation products, move through the cell on the microtubule network from the ruffled border domain to the apical plasma membrane domain. Morimoto et al. [29] reported that osteoclasts expressed vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent secretion of L-glutamate in neurons [30]. VGLUT1 was localized in transcytotic vesicles in which L-glutamate was accumulated. Osteoclasts simultaneously secreted L-glutamate and the bone degradation products [29]. These results suggest that the secretion of L-glutamate reflects the function of osteoclasts. Paclitaxel has been shown to inhibit pit-forming activity of rat authentic osteoclasts [31]. Inoue et al. [32] reported
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that docetaxel at 10-7 M inhibited pit-forming activity of mouse osteoclasts, and the inhibitory effect was enhanced by simultaneously adding minodronate, a bisphosphonate. In the present study, we examined in detail the effects of docetaxel on osteoclast differentiation and function. Docetaxel inhibited bone resorption in two different manners: inhibition of osteoclast formation through inhibiting the proliferation of osteoclast precursors, and inhibition of osteoclast function through inhibiting actin ring formation and transcytosis.
Materials and methods Animals and drugs Seven-week-old male and newborn ddY mice were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan). Recombinant human RANKL and M-CSF (Leukoprol) were purchased from PeproTech (London, UK) and Kyowa Hakko (Tokyo, Japan), respectively. 1,25(OH)2D3 and PGE2 were obtained from Wako Pure Chemical Industries (Osaka, Japan). LPS (Escherichia coli O26) was purchased from Sigma Chemical (St Louis, MO, USA). Rhodamine-conjugated phalloidin was from Molecular Probes (Eugene, OR, USA). Docetaxel (Taxotere) and paclitaxel (Taxol) were from Sanofi-Aventis (France) and Sigma Chemical (St. Louis, MO, USA), dissolved in dimethylsulfoxide and ethanol, respectively, and stored at 4°C. Rabbit antimouse phospho-extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204) antibody, rabbit antimouse ERK 1/2 antibody, rabbit antimouse inhibitor of nuclear factor-jB (NF-jB) (I-jB)a antibody, and rabbit anti-mouse b-actin antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Polymerase chain reaction (PCR) primers for RANKL, osteoprotegerin (OPG), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were synthesized by Invitrogen Life Technologies (Tokyo, Japan). All procedures for animal care were approved by the Animal Management Committee of Matsumoto Dental University. Responsiveness of mouse macrophages to docetaxel and paclitaxel Bone marrow cells obtained from tibiae of 7- to 9-week-old male mice (5 9 106 cells) were cultured in a-minimal essential medium (a-MEM) containing 10% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA) in the presence of M-CSF (100 ng/ml) on 60-mm-diameter dishes [33]. Nonadherent cells were harvested and further cultured for 2 days with M-CSF (50 ng/ml). The adherent cells were used as bone marrow macrophages. For western blot
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analysis, bone marrow macrophages were incubated with various concentrations of LPS, docetaxel, or paclitaxel for 30 min and then treated with the 0.1% NP-40 lysis buffer [33]. Whole cell extracts were electrophoresed and transferred onto polyuridine fluoride membranes. The membranes were then incubated with anti-phospho-ERK 1/2 antibody (1:1,000), anti-ERK antibody (1:1,000), antiI-jB antibody (1:700), or anti-b-actin antibody (1:2,000). The bound antibodies were visualized by enhanced chemiluminescence followed by exposure to X-ray film [33]. For the tumor necrosis factor-a (TNF-a) assay, bone marrow macrophages were incubated with various concentrations of LPS, docetaxel, or paclitaxel for 2 days. The amounts of TNF-a in the culture medium were measured using a TNF-a enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Osteoclast formation in mouse culture system Primary osteoblasts prepared from newborn mouse calvariae (1.0 9 104 cells/well) were cocultured for 6 days with bone marrow cells (1.0 9 105 cells/well) in the presence of 1,25(OH)2D3 (10-8 M) and PGE2 (10-6 M) in a-MEM containing 10% FBS in 96-well plates (0.3 ml/ well) [34]. Cocultures were treated with various concentrations of docetaxel for an entire culture period of 6 days, for the first 3 days, or for the final 3 days. After culturing for 6 days, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts [34]. TRAP-positive MNCs containing more than three nuclei were counted as osteoclasts. Mouse bone marrow macrophages (BMM/) were prepared as osteoclast precursors as described previously [33]. BMM/ (1.5 9 105 cells/well) were cultured in 48-well plates with RANKL (100 ng/ml) and M-CSF (50 ng/ml). BMM/ cultures were treated with various concentrations of docetaxel for an entire culture period of 6 days, for the first 3 days, or for the final 3 days. After culturing for 6 days, cells were fixed and stained for TRAP. The number of TRAP-positive osteoclasts was counted as already described.
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plates. After culturing for 6 h, osteoblasts were removed by the treatment of cells with phosphate-buffered saline (PBS) containing trypsin (0.05%) and ethylenediaminetetraacetic acid (EDTA) (0.53 mM) for 5 min [34]. The purity of osteoclasts in this preparation was about 95%. Purified osteoclasts were further cultured for 48 h in the presence or absence of RANKL (100 ng/ml), docetaxel (10-6 M), or both. Cells were then fixed and stained for TRAP [34]. TRAP-positive MNCs were counted as surviving osteoclasts. Pit formation and actin ring formation by osteoclasts For the pit formation assay, aliquots of the osteoclast preparation (0.1 ml) were cultured on dentine slices [34]. After culturing for 48 h, the cells were removed from the dentine slices, the slices were stained with Mayer’s hematoxylin (Sigma) to identify resorption pits, and the number of resorption pits was counted. For the actin ring formation assay, aliquots of the osteoclast preparation (0.1 ml) were cultured on dentine slices [34]. After culturing for 48 h, cells were fixed with 3.7% formaldehyde in PBS for 10 min. The cells were permeabilized with 0.1% Triton-X 100 in PBS for 5 min and incubated with rhodamine-conjugated phalloidin to visualize F-actin. The distribution of F-actin was detected using a fluorescence microscope. In some experiments, cells on dentine slices were fixed and stained for TRAP. TRAP-positive cells were counted as living TRAP-positive cells. Measurement of L-glutamate secretion from osteoclasts Purified osteoclasts prepared as described above (2 9 105 cells/dish) were washed three times with Ringer’s solution and then incubated for 30 min at 37°C [29]. The cells were incubated in Ringer’s solution and stimulated for 20 min by the addition of 50 mM KCl. The conditioned medium was collected, and the amount of L-glutamate was determined by high pressure liquid chromatography (HPLC) with a RESOLVE C18 column (4.6 mm 9 150 mm; Waters) as described previously [29].
Survival assay of mature osteoclasts
Cell proliferation assay
Coculturing was performed in the presence of 1,25(OH)2D3 (10-8 M) and PGE2 (10-6 M) in 10-cm-diameter dishes precoated with type I collagen gel (Nitta Gelatin, Osaka, Japan) [34]. After culturing for 6 days, all the cells were recovered as an osteoclast preparation from the dishes by treatment with a-MEM containing 0.2% collagenase (Wako) (10 ml/dish). The purity of osteoclasts in the osteoclast preparation was about 5%. Aliquots of the osteoclast preparation (0.1 ml) were cultured in 48-well
Cell viability was determined by the Alamar blue assay (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions. Mouse bone marrow cells (1.5 9 105 cells) or primary osteoblasts (1.5 9 104 cells) were cultured in 96-well plates with various concentrations of docetaxel for an entire 6 days, for the first 3 days, or for the final 3 days. After the indicated periods, the Alamar blue reagent was added to each well. The fluorescence was measured at wavelengths of 570 and 600 nm.
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PCR amplification of reverse-transcribed mRNA
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Mouse primary osteoblasts (1.0 9 10 cells) were cultured in 10-cm dishes for 3 days in a-MEM containing 10% FBS, and further cultured for 3 days with or without 1,25(OH)2D3 (10-8 M) plus PGE2 (10-6 M) in the presence or absence of docetaxel (10-6 M) [35]. Total RNA was extracted from osteoblasts using TRIzol solution (Invitrogen) for reverse transcription-polymerase chain reaction (RT-PCR) analysis of RANKL and OPG mRNAs. The first-strand cDNA was synthesized from total RNA with oligo-(dT)12–18 primers and was subjected to PCR amplification with EX Taq polymerase (Takara Biochemicals, Shiga, Japan) using the following PCR primers: mouse RANKL, 50 -CGCTCTGTTCCTGTACTTTCGAG CG-30 (forward, nucleotides 195–219) and 50 -TCGTGCT CCCTCCTTTCATCAGGTT-30 (reverse, nucleotides 757– 781); mouse OPG, 50 -TGGAGATCGAATTCTGCTTG-30 (forward, nucleotides 195–219) and 50 -TCAAGTGCTTGA GGGCATAC-30 (reverse, nucleotides 757–781); mouse GAPDH, 50 -ACCACAGTCCATGCCATCAC-30 (forward) and 50 -TCCACCACCCTGTTGCTGTA-30 (reverse). The PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining with UV light illumination. The sizes of the PCR products for mouse RANKL, OPG, and GAPDH are 587, 721, and 452 bp, respectively. Statistical analysis The results were expressed as the mean ± SD of three to five samples. Statistical analyses were performed using Student’s t test. Each experiment was repeated at least three times, and similar results were obtained.
Results Responsiveness of mouse macrophages to docetaxel and paclitaxel Paclitaxel has been shown to activate MAPKs and NF-jB in mouse macrophages through TLR4/MD-2 [17, 20]. We first examined the dose–response effect of docetaxel on TNF-a production in bone marrow macrophages in comparison with those of LPS and paclitaxel (Fig. 1). Paclitaxel as well as LPS dose-dependently stimulated TNF-a production in bone marrow macrophages whereas docetaxel did not (Fig. 1a). Phosphorylation of ERK1/2 was also induced within 30 min in bone marrow macrophages treated with LPS (1 lg/ml) or paclitaxel (10-4 M) but not with docetaxel (10-4 M) (Fig. 1b). Degradation of I-jBa in bone marrow macrophages was induced within
TNF production (pg/ml)
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Fig. 1 Effects of docetaxel and paclitaxel on the function of mouse bone marrow macrophages. Bone marrow macrophages were prepared from bone marrow cells as described in ‘‘Materials and methods’’. a Bone marrow macrophages were treated for 12 h with increasing concentrations of lipopolysaccharide (LPS), docetaxel, or paclitaxel. The conditioned medium was then collected, and the tumor necrosis factor-alpha (TNF-a) concentration was determined. The results were expressed as the means ± SD of three cultures. *P \ 0.01 was significantly different from the control culture. b Bone marrow macrophages were treated for 0, 15, or 30 min with LPS (1 lg/ml), docetaxel (10-4 M), or paclitaxel (10-4 M). Cells were then lysed and subjected to Western blot analysis using antiphospho-ERK1/2 (p-ERK1/2) antibody or anti-ERK antibody. c Bone marrow macrophages were treated for 0, 15, or 30 min with LPS (1 lg/ml), docetaxel (10-4 M), or paclitaxel (10-4 M). The amounts of inhibitor of nuclear factor-jB (NF-jB) (I-jBa) in the cell lysates were determined by Western blot analysis
30 min in response to LPS and paclitaxel. Docetaxel failed to induce I-jBa degradation in bone marrow macrophages, suggesting that paclitaxel but not docetaxel stimulated NF-jB transcriptional activity in mouse macrophages (Fig. 1c). These results indicate that paclitaxel has two different effects on murine cells: an LPS-like effect and a microtubule-stabilizing effect. LPS has been shown to induce osteoclastic bone resorption [35]. Therefore, we
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used docetaxel, not paclitaxel, to evaluate the effect of taxane-derived compounds on osteoclastic bone resorption. Effects of docetaxel on osteoclast formation in the mouse coculture system Mouse osteoblasts and bone marrow cells were cocultured in the presence of 1,25(OH)2D3 (10-8 M) plus PGE2 (10-6 M) together with or without an increasing concentration of docetaxel (Fig. 2). Many TRAP-positive osteoclasts (MNCs) were formed in the culture within 6 days in response to 1,25(OH)2D3 plus PGE2 in the coculture (Fig. 2). Docetaxel added to the culture for the entire culture period (6 days) dose-dependently inhibited osteoclast formation induced by 1,25(OH)2D3 plus PGE2 (Fig. 2a). Complete inhibition of osteoclast formation was observed in the cocultures treated with docetaxel at 10-7 M. Docetaxel added to the culture for the first 3 days similarly inhibited osteoclast formation, with complete inhibition at 10-7 M (Fig. 2b). When docetaxel was added
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Fig. 2 Effects of docetaxel on osteoclast formation in mouse cocultures. Primary osteoblasts and bone marrow cells were cocultured in 96-well plates for 6 days in the presence of 1,25(OH)2D3 (10-8 M) plus prostaglandin E2 (PGE2) (10-6 M). Increasing concentrations of docetaxel or osteoprotegerin (OPG) (100 ng/ ml) were added to the coculture for an entire 6 days (a), for the first 3 days (b), or for the final 3 days (c). After culturing for 6 days, cells were fixed and stained for tartrate acid-resistant phosphatase (TRAP). TRAPpositive cells appeared as dark red cells. TRAP-positive multinucleated cells (MNCs) were counted as osteoclasts (left panels). The results were expressed as the means ± SD of three cultures. Three panels on the right for each period show TRAP staining of the cocultures. *P \ 0.01 was statistically significant. Bars 50 lm
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to the culture for the final 3 days, docetaxel even at 10-5 M only slightly inhibited osteoclast formation induced by 1,25(OH)2D3 plus PGE2 (Fig. 2c). OPG (100 ng/ml), a decoy receptor of RANKL, added to the coculture for either the entire culture period, the first 3 days, or the final 3 days strongly inhibited osteoclast formation induced by 1,25(OH)2D3 plus PGE2 (Fig. 2). We previously showed that osteoclast progenitors proliferated in the first half of the culture period and differentiated into osteoclasts during the latter half period of the cocultures [34, 36]. Therefore, these results suggest that the inhibitory effect of docetaxel on osteoclast formation is the result of inhibition of the proliferation of osteoclast progenitors or osteoblasts or both cell types. Effects of docetaxel on proliferation of osteoclast progenitors and osteoblasts The effects of docetaxel on the growth of primary osteoblasts and bone marrow cells in culture for 6 days were
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Fig. 3 Effects of docetaxel on cell viability of bone marrow cells and osteoblasts, and on receptor activator of nuclear factor jB ligand (RANKL) mRNA expression, in osteoblasts. Bone marrow cells or primary osteoblasts were cultured with increasing concentrations of docetaxel for an entire 6 days (a), for the first 3 days (b), or for the final 3 days (c) (left panels). After the indicated periods, Alamar blue reagent was added to each well. The fluorescence was measured at wavelengths of ex 570 nm (ex) and em 600 nm (em), and cell viability was determined. Osteoblasts were cultured for 3 days, and then treated for 3 days with or 1,25(OH)2D3 plus PGE2 in the presence or absence of docetaxel (10-6 M) (d). Total RNA was extracted from osteoblasts and subjected to polymerase chain reaction (PCR) analysis of RANKL, OPG, and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA expression. *P \ 0.01 was statistically significant
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examined using the Alamar blue assay (Fig. 3). When osteoblasts and bone marrow cells were independently treated with docetaxel for the entire culture period, the viability of both types of cells was decreased in response to docetaxel at 10-8–10-6 M (Fig. 3a). Docetaxel added for the first 3 days similarly decreased the viability of osteoblasts and bone marrow cells (Fig. 3b). In contrast, docetaxel added at 10-6 M for the final 3 days to osteoblast cultures showed no inhibitory effect on osteoblast viability (Fig. 3c). This finding suggests that the proliferation of osteoblasts was suppressed during the final 3 days of culture as a result of cell confluency. The viability of osteoblasts was not affected by treatment with
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1,25(OH)2D3 plus PGE2 in the presence or absence of docetaxel added for the final 3 days (data not shown). Treatment of osteoblasts with 1,25(OH)2D3 plus PGE2 upregulated RANKL mRNA expression in osteoblasts (Fig. 3d). Docetaxel at 10-6 M failed to affect the upregulation of RANKL mRNA expression in osteoblasts by 1,25(OH)2D3 plus PGE2. Marked changes in OPG mRNA expression were not observed among osteoblast cultures in the presence or absence of 1,25(OH)2D3 plus PGE2 together, with or without docetaxel. These results suggest that docetaxel suppressed osteoclast formation through inhibition of the proliferation of osteoclast progenitors but not their differentiation into osteoclasts.
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Fig. 4 Effects of docetaxel on osteoclast formation in mouse BMM/ cultures. Mouse BMM/ were cultured in 48-well plates with RANKL (100 ng/ml) and macrophage colony-stimulating factor (M-CSF) (50 ng/ml). Increasing concentrations of docetaxel were added to BMM/ cultures for an entire 6 days (a), for the first 3 days (b), or for the final 3 days (c). After culturing for 6 days, cells were fixed and stained for TRAP. TRAPpositive cells appeared as dark red cells. TRAP-positive MNCs were counted as osteoclasts (left panels). The results were expressed as the means ± SD of three cultures. Right panels show TRAP staining of the cocultures. *P \ 0.01 was statistically significant. Bar 50 lm
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osteoclast formation is caused by inhibition of the proliferation of osteoclast progenitors.
Mouse BMM/ were cultured in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) together with or without increasing an concentration of docetaxel (Fig. 4). TRAP-positive osteoclasts (MNCs) were formed in the culture within 6 days in response to RANKL plus M-CSF (Fig. 4). Docetaxel added to the culture for the entire culture period (6 days) dose-dependently inhibited osteoclast formation induced by RANKL plus M-CSF with complete inhibition at 10-7 M (Fig. 4a). Docetaxel added to the culture for the first 3 days similarly inhibited osteoclast formation (Fig. 4b). When docetaxel was added to the culture for the final 3 days, docetaxel even at 10-6 M slightly inhibited osteoclast formation induced by RANKL plus M-CSF (Fig. 4c). These results confirm the previous finding that the inhibitory effect of docetaxel on
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We next examined effects of docetaxel on osteoclast function (Fig. 5). We reported previously that purified osteoclasts spontaneously died by apoptosis within 36 h and that RANKL promoted the survival of osteoclasts [37]. We therefore examined the effect of docetaxel on the survival of purified osteoclasts. When about 1,000 TRAPpositive osteoclasts (MNCs) were cultured for 36 h in the absence of osteoblasts, most of the osteoclasts died spontaneously (Fig. 5a). RANKL (100 ng/ml) added to the culture supported the survival of osteoclasts. Docetaxel (10-6 M) showed no effect on the survival of osteoclasts in the presence or absence of RANKL (Fig. 5a). We next examined the effects of docetaxel on the pit-forming
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10-6
10-5
Docetaxel (M)
Docetaxel
(c)
Number of osteoclasts/slice
(10-5 M)
800 *
600 400
TRAP
Pit
Control
200 0
Control
2x10-5
2x10-7
Docetaxel (M) Number of pits/slice
Fig. 5 Effects of docetaxel on survival and pit-forming activity of osteoclasts. a Effects of docetaxel on the survival of osteoclasts in plastic dishes. Purified osteoclasts were incubated for 48 h in the presence or absence of RANKL (100 ng/ml), docetaxel (10-6 M), or both. Cells were then fixed and stained for TRAP (right panels). TRAP-positive MNCs were counted as surviving osteoclasts (left panel). Bar 50 lm. b Effect of docetaxel on pit-forming activity of osteoclasts. The osteoclast preparation was cultured on dentine slices with increasing concentrations of docetaxel. After culturing for 48 h, cells were removed from dentine slices, and the slices were stained with Mayer’s hematoxylin to identify resorption pits (right panels). The number of resorption pits was counted (left panel). The results were expressed as the means ± SD of three cultures. *P \ 0.01 was statistically significant. Bar 50 lm. c Effect of docetaxel on the survival of osteoclasts on dentine slices. The osteoclast preparation was cultured on dentine slices with docetaxel at 2 9 10-7 M or 2 9 10-5 M. After culturing for 48 h, some dentine slices were stained for TRAP, and TRAPpositive cells were counted (left upper panel). Cells were also removed from dentine slices to identify resorption pits, and the number of resorption pits was counted (left lower panel). Right panels show TRAP staining and resorption pit staining of the slices
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Docetaxel (2x10-7 M)
1600 1200
Docetaxel
800
(2x10-5 M)
400 0
*
Control
2x10-5
2x10-7
Docetaxel (M)
activity of osteoclasts placed on dentine slices. When the osteoclast preparation was cultured on dentine slices for 48 h, many resorption pits were formed on the dentine slices (Fig. 5b). Docetaxel at 10-6–10-5 M significantly inhibited the pit-forming activity of osteoclasts (Fig. 5b). The inhibitory effects of docetaxel on the pit-forming
activity may be caused by the toxic effects on osteoclasts. Then we examined effects of docetaxel on the survival of osteoclasts on dentine slices. A similar number of TRAPpositive cells was observed on dentine slices in cultures with and without treatment with docetaxel even at 2 9 10-5 M for the entire culture period of 48 h, although
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Number of osteoclasts having actin rings
(a)
Control
600 *
400
200
*
Docetaxel (10-7 M)
0 Control
10-8
10-7
10-6
10-5
Docetaxel (M) Docetaxel (10-5 M)
(b) L-Glutamate release (pmol / 104cells)
Fig. 6 Effects of docetaxel on actin ring formation and Lglutamate secretion in osteoclasts. a Effects of docetaxel on actin ring formation in osteoclasts cultured on dentine slices in the presence of increasing concentrations of docetaxel. After culturing for 48 h, cells were fixed with formaldehyde, permeabilized with Triton-X 100, and incubated with rhodamine-conjugated phalloidin. F-actin was detected using a fluorescence microscope (right panels). Osteoclasts having actin rings were counted on the whole surface of a dentine slice. b Effects of docetaxel on L-glutamate secretion in osteoclasts plated on 35-mm dishes. Purified osteoclasts were prepared by the removal of osteoblasts. Osteoclasts were treated for 20 min with or without 50 mM KCl in the presence of increasing concentrations of docetaxel. The amount of Lglutamate in the culture medium was determined. Results were expressed as the means ± SD of three cultures. *P \ 0.01 was statistically significant
J Bone Miner Metab (2009) 27:24–35
400 300 200
* *
*
*
*
100 0
KCl (50 mM) Docetaxel (M)
10-8
10-7
10-6
10-5
Nocodazole (5 M)
the number of pits on dentine slices was markedly suppressed in the presence of docetaxel (Fig. 5c). These results suggest that inhibition of the pit-forming activity of osteoclasts by docetaxel was not caused by the toxic effects on the cells (Fig. 5c). Effects of docetaxel on actin ring formation and transcytosis in osteoclasts It was reported that nocodazole, a microtubule depolymerizing reagent, disrupted actin rings and inhibited the pit-forming activity of osteoclasts cultured on dentine slices in a reversible manner [26, 38]. We then examined the dose–response effect of docetaxel on actin ring formation in osteoclasts cultured on dentine slices. Consistent with the effect on pit formation, docetaxel at 10-6–10-5 M disrupted actin rings in osteoclasts (Fig. 6a). Docetaxel at 10-8–10-7 M showed no effect on the actin rings. These results suggest that docetaxel inhibits osteoclast function through disruption of actin rings. Osteoclasts eliminate the degradation products through transcytosis, in which the microtubule network plays critical roles [24, 27, 28]. It was also reported that osteoclasts secrete L-glutamate through
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transcytosis [29]. We therefore assessed L-glutamate secretion from osteoclasts in the presence and absence of docetaxel (Fig. 6b). KCl depolarizes osteoclasts through voltage-gated K? channels [30]. The secretion of L-glutamate was induced in osteoclasts in response to 50 mM KCl (Fig. 6b). Docetaxel at 10-7–10-5 M inhibited the secretion of L-glutamate from osteoclasts treated with KCl, but did not inhibit it at 10-8 M. These results suggest that the organization of microtubules plays important roles in the function of osteoclasts.
Discussion Taxane-based compounds are widely used for the treatment of cancer [6–9]. Although the anticancer properties of taxanes are believed to result from interference with microtubule assembly, it has been reported that taxanes have other cellular effects, including stimulation of MAPK and apoptosis [12–15]. It was also shown that paclitaxel mimics the action of LPS in mice [16, 17, 20]. In our experiments, paclitaxel as well as LPS induced phosphorylation of ERK, degradation of I-jBa, and TNF-a
J Bone Miner Metab (2009) 27:24–35
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1,25(OH)2D3
a Osteoblasts
RANKL
Proliferation Effective concentrations of docetaxel
b
Bone marrow macrophages
Proliferation
Effective concentrations of docetaxel
>10-8 M
No effect (10-6 M)
>10-8 M
Osteoclast precursor cells
RANKL Expression
TRAP-positive mononuclear cells
Differentiation
No effect (10-7 M)
Multinucleated osteoclasts
Fusion & Survival No effect (10-6 M)
Active Osteoclasts
Function
>10-6 M - 10-5 M
Fig. 7 Schematic representation of the action of docetaxel on osteoblasts, osteoclast progenitors, and osteoclasts. a Effects of docetaxel on proliferation and function of osteoblasts. Docetaxel at 10-8 M inhibits the proliferation of osteoblasts. RANKL expression induced by 1,25(OH)2D3 plus PGE2 in osteoblasts is not affected by docetaxel even at 10-6 M. b Effects of docetaxel on the proliferation and differentiation of osteoclast progenitors and on the function of osteoclasts. Docetaxel at 10-8 M inhibits the proliferation of
osteoclast progenitors. In contrast, docetaxel even at 10-7 M failed to inhibit the differentiation of osteoclast progenitors into TRAPpositive mononuclear cells and their fusion to form MNCs. Docetaxel at 10-6 M inhibits the pit-forming activity of osteoclasts cultured on dentine slices. Docetaxel at 10-6 M inhibits actin ring formation and L-glutamate secretion in osteoclasts. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10-8 M, and inhibition of osteoclast function at 10-6 M
production in mouse macrophages, but docetaxel did not. We reported that LPS stimulated RANKL expression in osteoblasts [35]. However, docetaxel failed to affect RANKL expression in osteoblasts. These results suggest that docetaxel does not possess as strong an action on MAPK in murine cells as does LPS. Our results also suggest that if mouse assay systems are employed for the evaluation of taxanes, docetaxel should be used rather than paclitaxel. Docetaxel at 10-8 M inhibited osteoclast formation in the mouse cocultures. RANKL-induced osteoclast formation in mouse BMM/ cultures was also inhibited by 10-8 M docetaxel. The number of TRAP-positive mononuclear cells was parallel with the number of TRAPpositive multinucleated osteoclasts (data not shown). These results suggest that the inhibitory effect of docetaxel on osteoclast formation was caused by inhibiting the proliferation of osteoclast precursors and supporting osteoblasts (Fig. 7). Docetaxel at 10-6–10-5 M also inhibited the pitforming activity of osteoclasts. Thus, docetaxel showed inhibitory effects on bone resorption with different points of actions. These inhibitory effects did not seem to be due
to a cytotoxic effect, because the number of osteoclasts on dentine slices was not significantly reduced even after treatment with 2 9 10-5 M docetaxel for 48 h. Actin ring formation and L-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10-6 M. In contrast, RANKL expression in osteoblasts was not affected by docetaxel even at 10-6 M, suggesting that docetaxel does not harmfully influence the function of osteoblasts. These results suggest that docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10-8 M, and inhibition of osteoclast function at 10-6 M (Fig. 7). Hall et al. [31] reported that paclitaxel inhibited the pitforming activity of rat osteoclasts at 10-6–10-5 M. Consistent with their finding, docetaxel at similar concentrations inhibited the function of mouse osteoclasts. Docetaxel at 10-6–10-5 M disrupted actin rings in osteoclasts. Using confocal microscopy, we showed that microtubules overlapped the top of the F-actin dots in osteoclasts cultured on dentine slices [26]. Treatment of osteoclasts with cytochalasin D, an F-actin-depolymerizing reagent, induced perturbation of the microtubules in
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34
osteoclasts. Conversely, nocodazole disrupted actin rings and inhibited the pit-forming activity of osteoclasts [26]. Thus, not only depolymerization but also stabilization of microtubules affected the actin cytoskeleton in functioning osteoclasts. These findings suggest that the coordination of microtubules and the actin cytoskeleton is important for the function of osteoclasts. Osteoclasts eliminate bone degradation products through transcytosis via the microtubule network [24, 27, 28]. VGLUT1, a glutamate transporter essential for vesicle storage, has been shown to be localized in the transcytotic vesicles in osteoclasts [29]. Osteoclasts secrete L-glutamate as well as bone degradation products through transcytosis in response to 50 mM KCl. In the present study, the secretion of L-glutamate from osteoclasts was strongly inhibited by docetaxel at 10-7–10-5 M. These results suggest that microtubules also play important roles in the transcytosis of osteoclasts, and that docetaxel inhibits the process of transcytosis. It should be noted that osteoclast formation was inhibited by low concentrations of docetaxel (10-8 M). We have shown that CD14-positive (CD14?) cells prepared from human peripheral blood mononuclear cells differentiate into osteoclasts in the presence of RANKL and M-CSF [39]. Docetaxel inhibited human osteoclast formation in CD14? cells in culture in a dose-dependent manner, with a minimal effective dose of 10-8 M (data not shown). Docetaxel at 10-8–10-7 M has been reported to induce cell death of human breast cancer cells (MCF-7 and MDA-MB231) [8, 40]. The growth of PC-3 prostate cancer cells was inhibited by 10-9–10-8 M docetaxel [41]. Thus, osteoclast formation and the growth of cancer cells were markedly suppressed by the similar concentrations of docetaxel (10-8–10-7 M). These results suggest that taxanes are strong inhibitors not only of the growth of cancer cells but also of osteoclast formation. Patients with cancer metastatic to the skeleton often develop osteolytic bone lesions [1–5]. Many cancers secrete bone resorption-stimulating cytokines. In turn, factors released from the bone matrix during bone resorption can stimulate tumor growth [2, 3]. This is a so-called ‘‘vicious cycle,’’ in which osteoclasts play central roles. In addition, tumor-induced hypercalcemia results from the increase in osteoclastic bone resorption [42]. Multiple sites are targets for bone-directed therapies. It is well known that high doses of taxane compounds induce serious side effects on various organs and tissues by affecting tubulin structures [43, 44]. We have shown here that docetaxel at relatively low concentrations (10-8–10-7 M) was also effective in inhibiting osteoclast differentiation. These results suggest that taxane-based therapy may have beneficial side effects, such as inhibition of tumor-induced hypercalcemia and osteolytic bone metastasis.
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