Breast Cancer Res Treat (2015) 149:705–714 DOI 10.1007/s10549-015-3290-x
PRECLINICAL STUDY
Quetiapine inhibits osteoclastogenesis and prevents human breast cancer-induced bone loss through suppression of the RANKLmediated MAPK and NF-jB signaling pathways Hongkai Wang • Weiwei Shen • Xu Hu • Ying Zhang • Yunyun Zhuo • Tao Li • Feng Mei • Xinmin Li • Lan Xiao • Tongwei Chu
Received: 8 December 2014 / Accepted: 27 January 2015 / Published online: 10 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and lung cancer. Extensive research has revealed that the receptor activator of NF-jB ligand (RANKL), which is considered to be a key factor in osteoclast differentiation, plays an important role in cancer-associated bone resorption. Therefore, agents that can suppress this bone loss have therapeutic potential. In this study, we detected whether quetiapine (QUE), a commonly used atypical antipsychotic drug, can inhibit RANKL-induced osteoclast differentiation in vitro and prevent human breast cancerinduced bone loss in vivo. RAW 264.7 cells and bone marrow-derived macrophages (BMMs) were used to detect inhibitory effect of QUE on osteoclastogenesis in vitro. Mouse model of breast cancer metastasis to bone was used to test suppressive effect of QUE on breast cancer-induced bone loss in vivo. Our results show that QUE can inhibit RANKL-induced osteoclast differentiation from RAW 264.7 cells and BMMs without signs of cytotoxicity. Moreover, QUE reduced the occurrence of MDA-MB-231 Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3290-x) contains supplementary material, which is available to authorized users. H. Wang W. Shen X. Hu Y. Zhang Y. Zhuo T. Chu (&) Department of Orthopaedics, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China e-mail: [email protected] T. Li F. Mei L. Xiao (&) Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, China e-mail: [email protected] X. Li Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton T6G 2B7, Canada
cell-induced osteolytic bone loss by suppressing the differentiation of osteoclasts. Finally, molecular analysis revealed that it is by inhibiting RANKL-mediated MAPK and NF-jB signaling pathways that QUE suppressed the osteoclast differentiation. We demonstrate, for the first time, the novel suppressive effects of QUE on RANKLinduced osteoclast differentiation in vitro and human breast cancer-induced bone loss in vivo, suggesting that QUE may be a potential therapeutic drug for osteolysis treatment. Keywords Quetiapine Breast cancer Bone metastases Osteoclast Bone resorption
Introduction Breast cancer, along with lung and prostate cancers and multiple myeloma, is likely to metastasize to bone [1]. Once bone metastases have occured, the chance of survival is slim, and the quality of life of the patient dramatically drops, with a clinical outcome characterized by intractable pain, nerve compression syndromes, increased risk of fractures, and hypercalcemia [2]. Breast cancer bone metastases have a generally osteolytic nature and rely on the ability of tumor cells to activate osteoclast-induced bone resorption, which is a crucial event that contributes to the disruption of bone and the creation of physical space into which the tumor cells intrude [3, 4]. Breast cancer cells can produce factors that induce osteoclastogenesis, such as PTHrP and interleukins (IL)-1, IL-6, and IL-11. These factors act on osteoblasts to increase the production of RANKL [5, 6]. RANKL can bind to its receptor RANK which is located on the membrane of the osteoclast precursor cells and subsequently activate
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RANK [7]. The activation of RANK on osteoclast progenitor cells leads to the activation of TNF receptor-associated factors (TRAFs) and the subsequent stimulation of several downstream signaling molecules, including nuclear factor-kappa B (NF-jB), mitogen-activated protein kinases (MAPKs), activating protein 1 (AP-1), nuclear factor of activated T cells 1 (NFATc1), and phosphatidylinositol 3-kinase (PI3K)/Akt, resulting in the differentiation of osteoclast progenitor cells into multinucleated, bone-resorbing osteoclasts [8]. Osteolytic lesions, characterized by excessive bone resorption, are mainly induced by abnormal osteoclast differentiation. Therefore, suppressing the functional differentiation of osteoclasts is one of the main strategies for osteolysis treatment [9]. Some drugs, including bisphosphonates, estrogen, teriparatide, synthetic calcitonin, and denosumab, have been used for the treatment of osteolysis. But most of these drugs have many limitations or side effects, such as osteonecrosis, thromboembolism, osteosarcoma, and esophageal irritation [10–12]. Therefore, developing or discovering new drugs is required to improve bone therapy. Previously, we found that quetiapine (QUE), a commonly used atypical antipsychotic drug, can promote the differentiation of neural progenitors of the oligodendrocyte lineage through the MAPK signaling pathway [13]. Other studies have also demonstrated that QUE exerts anti-inflammatory [14] and antioxidant [15] effects, and modulates the NF-jB [16] and MAPK [17] pathways, both of which have been implicated in osteoclastogenesis [8, 18]. Thus, we asked whether QUE can inhibit osteoclastogenesis and potentially prevent cancer-induced bone loss. Our results demonstrate for the first time that QUE can inhibit osteoclastogenesis in vitro and prevent human breast cancer-induced bone loss in vivo through the suppression of the RANKL-mediated MAPK and NF-jB signaling pathways, suggesting that QUE is a potential lead compound for the development of novel drugs for osteolysis treatment.
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Precipitated cells were suspended in a-MEM supplemented with 10 % FBS and cultured for 24 h. Non-adherent cells were collected and cultured for 3 days in the presence of M-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ, USA). Floating cells were discarded, and adherent cells were used as BMMs. RAW 264.7 cells were seeded in 24-well plates at a density of 2 9 104 cells/well and placed in the CO2 incubator overnight. After 24 h, the culture medium was replaced with medium containing RANKL (50 ng/ml, PeproTech, Rocky Hill, NJ, USA) with various concentrations of QUE for 5 days. BMMs were plated at a density of 4 9 105 cells/well in a 24-well plate in the presence of RANKL (50 ng/ml) and M-CSF (20 ng/ml) with various concentrations of QUE for 7 days. Medium was changed every 2 or 3 days. Cytotoxicity assay Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cells (2 9 103 cells/well) were seeded on 96-well plates and cultured with or without QUE (AstraZeneca, Wilmington, DE) for expected time. After incubation, 10 ll CCK-8 solution was added. The plates were incubated for 4 h, after which, absorbance was measured at 450 nm using a microplate reader. TRAP staining of RAW 264.7 cells and BMMs To confirm the generation of multinucleated, osteoclastlike cells, the cultured cells were stained for tartrate-resistant acid phosphatase (TRAP) using the TRAP Staining Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. TRAP-positive multinucleated (3 or more nuclei) osteoclasts were visualized by light microscopy and photographed. Each osteoclast formation assay was performed at least 3 times. RNA isolation and RT-PCR analysis
Materials and methods Cell culture RAW 264.7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10 % FBS (Gibco, NY, USA) and antibiotics (100 IU penicillin/ ml and 100 mg streptomycin/ml). Bone marrow-derived macrophages (BMMs) were obtained from 4- to 6-week-old C57BL/6 mice. The tibias of mice were obtained and flushed with a-MEM. Cells were then centrifuged for 5 min at a speed of 1,000 rpm.
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Total RNA was isolated from cells with TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) following the instructions of the manufacturer. RNA was quantified using a spectrophotometer set (Nanodrop, Thermo Fisher Scientific Inc., USA). The same amount of total RNA from each sample was reverse transcribed to cDNA in a final volume of 20 ll using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time PCR was performed using FastStart Universal SYBR Green Master (Roche, shanghai, China). Quantification was normalized to the amount of glyceraldehyde-3phosphate dehydrogenase (Gapdh) cDNA. The specific primer sequences are listed in Table S1.
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Intramedullary injection of tumor cells
4 °C. After washing with PBS, the secondary antibody, donkey anti-rabbit Alexa Fluor 568 (Invitrogen, Grand Island, NY, USA) was added and samples were incubated for 2 h in the dark at room temperature. The cells were observed under confocal microscopy (Olympus, Tokyo, Japan).
Six-week-old female immunodeficient BALB/c-nu/nu mice were obtained from the Animal Center of the Third Military Medical University and maintained under sterile conditions. All experiments were performed in accordance with the Health Guidelines for the Care and Use of Laboratory Animals with the approval of the Third Military Medical University Committee on Animal Care (permission NO: SCXKJUN-2007-015). The breast cancer cell line MDA-MB-231 was cultured and resuspended in PBS to a final concentration of 106 cells/ml. Mice were anesthetized with intraperitoneal injection of pentobarbital (60 mg/kg). A syringe with a 26 1/2 G needle was subsequently inserted in the proximal end of the tibia, and tumor cells (104 cells in 10 lL PBS) were injected into the intramedullary space. The mice were then randomly assigned to two groups and treated with vehicle (ddH2O, n = 8) or QUE (10 mg/kg, n = 8). Previous studies of our group demonstrated similar effects of oral administration and intraperitoneal injection about the method of QUE given [13, 19]. To reduce the chance of infection of mice which were used in our study, QUE was dissolved in ddH2O and was given through intraperitoneal injection once a day for 6 weeks until the end of the experiment, and the body weight of mice was recorded every 3 days. Assessment of osteolytic lesions in vivo To further characterize osteolytic bone lesions, mice were anesthetized and subjected to X-ray system (Kodak, In Vivo Imaging System FX Pro; 30 kVp for 1 min), scanned, and evaluated without knowledge of the experimental groups. For histological examination, tibias were dissected, cleaned of soft tissues, and fixed in 4 % paraformaldehyde in 0.1 M PBS for 48 h. The samples were then decalcified in EDTA and embedded in paraffin. Sections were cut and stained with H&E to evaluate the displacement of bone marrow by tumor cells. The sections were also stained for TRAP to observe the activation of osteoclasts and were then subjected to histomorphometric analysis. Immunofluorescent staining RAW 264.7 cells were stimulated with RANKL in the presence of QUE (50 lM) or the vehicle control for 0, 5, 15, 30, 60, and 120 min. The cells were fixed with 4 % paraformaldehyde for 10 min. This step was followed by permeabilization with 0.2 % Triton-X 100 for 5 min. After blocking with 1 % BSA in PBS for 20 min, the cells were stained with rabbit anti-p65 antibody (Cell Signaling Technology, USA) in 1 % BSA and incubated overnight at
Western blot Cells were cultured in the presence of RANKL with or without QUE (50 lM) for 0, 5, 15, 30, 60, and 120 min. Subsequently, cells were lysed with RIPA buffer plus PMSF (1 mM). Protein was separated on 8 % SDS-PAGE gels and transferred to PVDF membranes. The MAPK family antibody sampler kit, the phospho-MAPK family antibody sampler kit, the NF-jB pathway sampler kit, and the phospho-Akt antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The Akt antibody was purchased from Proteintech (Wuhan, Hubei, China), and the antibody against b-actin was purchased from Santa Cruz Biotechnology (CA, USA). Statistical analysis All experiments were repeated at least three times, each time in triplicate. Student’s t test was used for determining the significance of differences between two groups, whereas one-way analysis of variance (ANOVA) followed by a post hoc Tukey’s test was used for multiple comparisons using SPSS 17.0. All data are expressed as the mean ± SD. P values less than 0.05 (p \ 0.05) were accepted as statistically significant.
Results QUE suppresses osteoclastogenesis of RAW 264.7 cells and BMMs induced by RANKL without cytotoxicity Before examining the effect of QUE on osteoclast differentiation, we detected the cytotoxicity of QUE on RAW 264.7 cells and BMMs using a CCK-8 assay. As shown in Fig. 1, after treatment for 48 h, QUE showed no cytotoxicity on RAW 264.7 cells in the concentration range between 1 and 50 lM. Furthermore, there was no cytotoxicity on RAW 264.7 cells and BMMs after treatment by QUE (50 lM) for 5 or 7 days (Fig. S1). Therefore, QUE at the concentrations of 25 and 50 lM were used in subsequent in vitro experiments. To investigate the effect of QUE on osteoclastogenesis, RAW 264.7 cells were treated with different doses of QUE (1–50 lM) in the presence of RANKL. After 5 days of incubation, the cells were TRAP stained. As seen in Fig. 2,
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(50 lM) in the presence of RANKL and M-CSF. After 7 days of incubation, the cells were TRAP stained, and multinucleated cells were counted. The results show that QUE also suppresses the osteoclastogenesis of BMMs induced by RANKL and M-CSF (Fig. 2c, d). QUE acts at the early step of RANKL-induced osteoclastogenesis
QUE suppressed osteoclast formation in a dose-dependent manner. Treatment with 25 and 50 lM of QUE decreased the number of osteoclasts formed (Fig. 2a, b). These results indicate that QUE significantly suppresses the osteoclastogenesis of RAW 264.7 cells induced by RANKL. To further determine the suppressive effect of QUE on osteoclastogenesis, BMMs were also treated with QUE
Osteoclastogenesis is a very complicated process which involves cell proliferation, differentiation, activation, and survival [20–22]. To further determine at which stage QUE is most effective in inhibiting osteoclast differentiation of RAW 264.7 cells induced by RANKL, QUE (50 lM) was added to osteoclast differentiation cultures beginning at day 1, day 2, day 3, and day 4, respectively. The results showed that QUE inhibited osteoclastogenesis maximally when added from the initiation of RANKL treatment (Fig. 3a). The exposure of precursor cells to QUE at later stages (after day 3) was less effective in suppressing osteoclastogenesis (Fig. 3b). It is noted that, although QUE can suppress osteoclast differentiation at the very early differentiation stage, it has substantially no suppressive effect when osteoclast differentiation has come to the late stage.
Fig. 2 QUE inhibits osteoclast differentiation in RANKL-stimulated RAW 264.7 cells and BMMs. a RAW 264.7 cells dosed with 50 ng/ ml RANKL and treated with or without QUE (1, 10, 25, or 50 lM) for 5 days. c BMMs dosed with 20 ng/ml M-CSF and 50 ng/ml RANKL with or without QUE (50 lM) for 7 days. b, d TRAPpositive multinucleated cells with three or more nuclei were
considered as osteoclasts, and the number of osteoclasts was counted after taking images of random fields in different areas of each well. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Arrows osteoclasts; scale bar 100 lm; **p \ 0.01 compared with RANKL treatment
Fig. 1 Effect of QUE on cell viability. CCK-8 assay was performed after incubation of RAW 264.7 cells with QUE (0, 1, 10, 25, 50, and 100 lM) for 48 h. Values are expressed as the mean ± SD of triplicate experiments. *p \ 0.05
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Fig. 3 Inhibition of RANKL-induced osteoclastogenesis by QUE is an early event. a RAW 264.7 cells were incubated with RANKL (50 nmol/L), and then QUE (50 lM) was added on day 1, day 2, day 3, and day 4, respectively. At the end of 4 days, cells were stained for TRAP expression. b TRAP-positive multinucleated cells with three or more nuclei were considered as osteoclasts, and the number of
osteoclasts was counted after taking images of random fields in different areas of each well. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Arrows osteoclasts; scale bar 100 lm; *p \ 0.05, **p \ 0.01 compared with RANKL treatment
QUE inhibits the expression of osteoclastogenesisrelated genes in RAW 264.7 cells and BMMs induced by RANKL
QUE inhibits osteoclastogenesis induced by tumor cells in vivo
We also assessed the effect of QUE on osteoclastogenesis by evaluating the expression levels of osteoclastogenesisrelated genes. Throughout the differentiation process, osteoclasts express some markers, such as TRAP, calcitonin receptor (CalcR), cathepsin K (CtsK), and MMP-9 (gelatinase B) which, together with multinucleation and resorption, characterize the osteoclast phenotype [23]. Our results show that TRAP mRNA expression levels were significantly higher in the cells treated with RANKL. Moreover, QUE treatment inhibited the increased TRAP expression induced by RNAKL in a dose-dependent manner: 1 and 10 lM of QUE treatment had no effect while a decrease in TRAP expression was found in cells treated with 25 and 50 lM of QUE. Similar results were found for CalcR, CtsK, and MMP-9 mRNA expression levels (Fig. 4).
Fig. 4 Effects of the RANKL and QUE treatments on mRNA expression levels of TRAP, CalcR, CtsK, and MMP-9. a Expression levels of TRAP, CalcR, CtsK and MMP-9 in RAW 264.7 cells treated with or without QUE for 5 days. b Expression levels of TRAP, CalcR, CtsK, and MMP-9 in BMMs treated with or without QUE for
Bone loss is one of the most common complications in patients with breast cancer [24]. There is evidence showing that bone destruction at sites of bone metastases is the result of abnormal osteoclast activity rather than of tumor cells per se [25, 26]. We investigated whether QUE could suppress osteolytic bone lesions induced by tumor cells in vivo. Firstly, MDA-MB-231 cells were treated with QUE (50 lM) for 1, 3, or 5 days respectively, and the results revealed that there was no cytotoxicity on MDAMB-231 cells after treatment by QUE for indicated time points (Fig. 5a), and then MDA-MB-231 cells (104 cells in 10 lL) were injected into the intramedullary space of mice, and the mice were treated with QUE (10 mg/kg) or vehicle control for 6 weeks. To identify osteolytic bone lesions, we analyzed bone tissue by X-ray analysis. As seen in Fig. 5b, osteolytic bone destruction of cortices was observed in the MDA-MB-231 tumor-bearing control mice. In contrast,
7 days. Data represent fold changes of target genes normalized to Gapdh mRNA and are expressed as a percentage of RANKL-dosed cells not treated with QUE, which were set to 100 %. Values represent the mean ± SD (n = 4). **p \ 0.01
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Fig. 5 QUE prevents human breast cancer-induced bone loss in vivo. a CCK-8 assay was performed to test effect of QUE (50 lM) on MDA-MB-231 cell viability. Cells were treated for 1, 3, or 5 days respectively. b X-ray images of the tibias from mice subjected to injection of MDA-MB-231 cells and treated with or without QUE for 6 weeks. c H&E and TRAP staining of one representative tibia from
each group. d Histomorphometric analysis of osteoclast numbers in the bone metastases of MDA-MB-231 cells. Data are expressed as number of osteoclasts/mm at the tumor-bone interface. The values are expressed as the mean ± SD. * tumor mass; arrows osteoclasts; # bone; scale bar 50 lm; **p \ 0.01 compared with the vehicle group
there was very little osteolysis in the QUE-treated mice. Interestingly, histomorphometric analysis performed on tibia sections stained with H&E or for the osteoclast marker TRAP revealed a significant reduction in the osteoclast number in mice treated with QUE versus vehicle (Fig. 5c, d). All mice survived until the end of the experiment and body weight of mice with or without QUE treatment showed no significant difference (Fig. S2). This shows that QUE can suppress osteolytic bone lesions induced by tumor cells in vivo by inhibiting osteoclast differentiation.
time points and QUE-treated cells were pretreated with QUE for 30 min. The phosphorylation states of MAPKs and Akt were detected by Western Blot analysis. Interestingly, QUE treatment significantly decreased the levels of phospho-p38 MAPK (Fig. 6b, f), phospho-ERK1/2 (Fig. 6c, g) and phospho-JNK (Fig. 6d, h), but had no effect on RANKL-induced phosphorylation states of Akt (Fig. 6e, i). Next, NF-jB pathway activation was examined. The activation of NF-jB pathway is accompanied by the degradation of IjBa and the nuclear translocation of NFjB subunit p65 [27]. RAW 264.7 cells were stimulated with RANKL and QUE as mentioned above. The level of IjBa decreased immediately between 5 and 60 min after RANKL treatment and recovered at 120 min. Upon QUE treatment, QUE was found to significantly suppress the RANKL-induced degradation of IjBa (Fig. 7a). Furthermore, when the level of phosphorylated p65 (phosphop65), one of the subunits of NF-jB, was examined, QUE treatment clearly suppressed the phospho-p65 level compared with cells treated with the vehicle control (Fig. 7a). This result was confirmed by a localization study using immunofluorescent staining of p65. In RANKL-stimulated control cells, p65 was found exclusively in the cytoplasm at the start of RANKL stimulation. After 15–30 min of RANKL stimulation, most cells exhibited nuclear localization of p65 (Fig. 7c). This trend was reversed 60 min
QUE suppresses RANKL-induced activation of the MAPK and NF-jB pathways during osteoclast differentiation The activation of the MAPK, PI3 K/Akt, and NF-jB pathways is critical for RANKL-induced osteoclastogenesis [8, 18]. In order to elucidate the molecular mechanisms by which QUE suppresses osteoclast differentiation, the activation of the MAPK, PI3 K/Akt, and NF-jB pathways was assessed. Firstly, we investigated the presence of phospho-p38 MAPK, phospho-ERK1/2 (p44/42), phosphoJNK, and phospho-Akt in QUE-treated or vehicle control cells upon RANKL stimulation. Accordingly, RAW 264.7 cells were stimulated with RANKL (50 ng/ml) in the presence of QUE or the vehicle control at the indicated
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Fig. 6 QUE suppresses the activation of MAPK signaling pathway induced by RANKL in RAW 264.7 cells. a Time course of the phosphorylations of MAPKs and Akt in RANKL-stimulated RAW 264.7 cells. Cells were stimulated with 50 ng/ml RANKL at the indicated time points. Phosphorylations of MAPKs and Akt were detected by western blot analysis, protein expression of p-p38, p-ERK, p-JNK and p-Akt was normalized against b-Actin. b, c, d, e, f, g, h, i RAW 264.7 cells were stimulated with RANKL (50 ng/ml)
in the presence of QUE (50 lM) or the vehicle control at the indicated time points, QUE-treated cells were pretreated with QUE for 30 min. p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Akt, and Akt were analyzed using western blot analysis (b, c, d, e). Protein expression of p-p38, p-ERK, p-JNK, and p-Akt was normalized against total p38, ERK, JNK and Akt (f, g, h, i). Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. *p \ 0.05, **p \ 0.01
Fig. 7 QUE suppresses the activation of NF-jB signaling pathway induced by RANKL in RAW 264.7 cells. RAW 264.7 cells were stimulated with RANKL (50 ng/ml) in the presence of QUE (50 lM) or the vehicle control at the indicated time points, QUE-treated cells were pretreated with QUE for 30 min. a, b Effects of QUE on IjBa, p-p65, and p65 protein levels in RANKL-stimulated RAW 264.7 cells. The protein levels were determined by western blot analysis (a),
and protein expression was normalized against b-Actin (b). c RAW 264.7 cells were treated as mentioned above and then stained for the NF-jB subunit p65. The cells were observed under a confocal microscope. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Scale bar 100 lm; **p \ 0.01
after stimulation. In contrast, QUE treatment completely abrogated the nuclear localization of NF-jB p65 at any time point (Fig. 7c). Therefore, QUE suppresses RANKLinduced NF-jB activation by inhibiting the degradation of
IjBa and abrogating the nuclear localization of NF-jB p65. Accordingly, QUE suppresses the activation of the MAPK and NF-jB pathways during osteoclast
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differentiation, cooperatively contributing to its anti-osteoclastogenic activity.
Discussion Almost 90 % of cancer-associated deaths result from tumor metastasis. Lung, liver, lymph node, and bone are some of the major organ sites where tumor metastasis is prone to occur. Bone metastasis is commonly associated with prostate cancer, lung cancer, and breast cancer. Breast cancer metastasis to bone leads to osteolytic lesions, and no currently available treatment is sufficient to treat bone metastasis induced by breast cancer. In the present study, we demonstrate for the first time that QUE, an FDA-approved chemical compound, can inhibit osteoclastogenesis and prevent bone loss induced by human breast cancer through suppression of the MAPK and NF-jB signaling pathways. Bisphosphonate drugs, denosumab, and teriparatide (synthetic parathyroid hormone) are the most widely used agents for preventing bone loss. Drugs of the bisphosphonate family have been used for many years as a standard of care by virtue of their ability to reduce the occurrences of skeletal-related events (SREs), relieve bone pain, and improve the quality of life of patients with cancer [28–30]. However, these drugs have many limitations in their use due to serious side effects including renal failure, gastrointestinal reactions, and osteonecrosis of the jaw [31]. Denosumab, a monoclonal antibody against RANKL, can inhibit osteoclast differentiation by effectively preventing the binding of RANKL with RANK. Denosumab has been approved by FDA for the prevention of SREs in patients with tumors, including breast cancer [31]. Compared with drugs of the bisphosphonate family, denosumab is more effective at reducing the risk of developing SREs as well as delaying the time to develop SREs in patients with breast cancer [29]. Unfortunately, as is the case with bisphosphonate drugs, denosumab is associated with a low incidence of osteonecrosis of the jaw and does not make any significant difference in overall patient survival [29]. Another drug, teriparatide, in contrast to bisphosphonate drugs and denosumab, acts on osteoblasts to stimulate bone formation. At first glance, it would seem ideal to use bisphosphonate drugs or denosumab in combination with teriparatide because bisphosphonate drugs and denosumab block bone resorption while teriparatide stimulates bone deposition. However, teriparatide is found to be associated with an increased risk of osteosarcoma and exacerbation of skeletal metastases because it is likely to lead to bone turnover [32]. Thus, an effective method or reagent for inhibiting osteoclastogenesis is in need for the treatment of bone metastasis. Our data indicated that QUE, a commonly
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used atypical antipsychotic drug (APD), has such a capacity. Osteoclasts are multinuclear giant cells derived from monocyte-macrophage lineage cells. Osteoclast differentiation from precursor cells can be induced by RANKL, which also controls the survival and function of mature osteoclasts. Therefore, RANKL, as a convincing stimulator, is used in studies on differentiation and function of osteoclasts. In this study, both RAW 264.7 cells and BMMs were used and dosed with RANKL to build models of osteoclast differentiation. In order to understand the effects of QUE on osteoclast differentiation, different doses of QUE were used in our experiment, and the results revealed that QUE treatments significantly reduced the number of TRAP-positive multinuclear osteoclasts without any signs of cytotoxicity, which mainly affected the early stage of osteoclast differentiation. We also assessed the effect of QUE on the expression of osteoclastogenesis-related genes. Throughout the differentiation process, osteoclasts express some markers, such as TRAP, CalcR, CtsK and MMP-9, which, together with multinucleation and resorption, characterize the osteoclast phenotype. TRAP is highly expressed in mature osteoclasts and is used as a marker of osteoclast function [33]. Mature osteoclasts also express the CalcR on their surface. In fact, CalcR has been considered as the best differentiation marker for osteoclasts [34]. CtsK and MMP-9 have been confirmed to play a key role in the resorption process [35]. Our results showed that QUE (25 and 50 lM) reduced the RANKL-induced expression of the osteoclast phenotypic markers TRAP, CalcR, CtsK, and MMP-9 in a dose-dependent manner. These results suggested that QUE suppressed osteoclast formation, at least partially, by inhibiting the expression of RANKL-induced osteoclastrelated genes. More importantly, our results show that QUE also suppresses osteolysis induced by breast cancer cells in vivo without affecting the body weight of mice, and histological TRAP staining of decalcified bone sections reveals a significant decrease in the number of osteoclasts relative to bone surface area in mice treated with QUE, suggesting the potential utility of QUE in the treatment of cancer-induced bone lesions. Previous studies have demonstrated that QUE exerts various effects such as anti-inflammatory [14], antioxidant [15] effects, and modulates the NF-jB [16] and MAPK pathways [17]. In the current study, we demonstrate that QUE can decrease the phosphorylation level of MAPKs including p38, ERK, and JNK, which were found to be activated by RANKL stimulation in RAW 264.7 cells. Among these MAPKs, p38 MAPK has an ability to regulate the microphthalmia-associated transcription factor and is thus a signaling molecule that plays an important
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role in the early stage of osteoclast differentiation [36], while dominant-negative JNK prevents RANKL-induced osteoclastogenesis [37]. In comparison, ERK has been confirmed to induce c-Fos expression [38], and inhibition of ERK protein kinase has been shown to suppress RANKL-induced osteoclast formation [39]. Thus, in combination with our experimental results, we can conclude that QUE has suppressive effect on osteoclastogenesis, at least partially, by inhibiting MAPK signaling. Apart from MAPK signaling, activation of the NF-jB pathway is also thought to be a crucial role in RANKL-induced osteoclast differentiation [40, 41]. The activation of NF-jB signaling pathway involves IjB phosphorylation, and the latter relies on the regulation of IjB kinase (IKK). RANKL can stimulate IKK activation and result in IjBa phosphorylation. Then, the polyubiquitinated IjBa is degraded by proteasomes, and the dissociative NF-jB subunit p65 enters into nucleus and binds to the targeted position of DNA, and starts the gene transcription [42]. Our results showed that QUE could inhibit nuclear localization of NF-jB p65 by suppressing RANKL-mediated IjBa degradation. These results indicate that inhibition of the NF-jB pathway is one of the mechanisms involved in the anti-osteoclastogenic effect of QUE. Moreover, we also excluded the possibility of the involvement of PI3 K/Akt pathway in the effect of QUE on RANKL-induced osteoclast differentiation. As an atypical antipsychotic drug, QUE is a multiple receptor antagonist with low affinity for D2 and high affinity for 5HT2A, 5HT1A, a-1, and a-2 adrenergic and histamine H1 receptors [43]. A recent study provides evidence that QUE may be linked to the epidermal growth factor receptor (EGFR) [44], which has been documented to play an important role in regulating osteoclast differentiation and survival through cross-talking with RANK signaling [45]. Our results suggest that suppressing the activation of the MAPK and NF-jB pathways during osteoclast differentiation may cooperatively contribute to the anti-osteoclastogenic activity of QUE. However, the molecular mechanism by which QUE inhibits RANKL-mediated MAPK and NF-jB pathway activation awaits further study. Although our data from in vitro and in vivo experiments illustrate QUE-mediated inhibition of osteoclast differentiation, it is possible that QUE might also target additional cells in vivo. Therefore, additional further analyses of other potential cellular and molecular targets of QUE during long-term treatment in vivo should be conducted when developing potential QUE-based therapeutic applications.
Conclusions Our findings that QUE inhibits osteoclastogenesis through the suppression of the MAPK and NF-jB pathways suggest
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that QUE may have great potential in the development of novel therapeutic drugs for disorders associated with bone loss. Acknowledgments This study was partly supported by grants from the National Natural Science Foundation of China (81271979), and National Basic Research Program of China (973 Program, No. 2010CB529400). The authors thank Yan Yin for carefully proofreading the manuscript and providing valuable comments. The authors also thank Dr. Yue Zhou and Dr. Jianqin Niu for their direction, advice, and teachings. Conflicts of interest of interest.
All the authors state that they have no conflicts
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PRECLINICAL STUDY
Quetiapine inhibits osteoclastogenesis and prevents human breast cancer-induced bone loss through suppression of the RANKLmediated MAPK and NF-jB signaling pathways Hongkai Wang • Weiwei Shen • Xu Hu • Ying Zhang • Yunyun Zhuo • Tao Li • Feng Mei • Xinmin Li • Lan Xiao • Tongwei Chu
Received: 8 December 2014 / Accepted: 27 January 2015 / Published online: 10 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and lung cancer. Extensive research has revealed that the receptor activator of NF-jB ligand (RANKL), which is considered to be a key factor in osteoclast differentiation, plays an important role in cancer-associated bone resorption. Therefore, agents that can suppress this bone loss have therapeutic potential. In this study, we detected whether quetiapine (QUE), a commonly used atypical antipsychotic drug, can inhibit RANKL-induced osteoclast differentiation in vitro and prevent human breast cancerinduced bone loss in vivo. RAW 264.7 cells and bone marrow-derived macrophages (BMMs) were used to detect inhibitory effect of QUE on osteoclastogenesis in vitro. Mouse model of breast cancer metastasis to bone was used to test suppressive effect of QUE on breast cancer-induced bone loss in vivo. Our results show that QUE can inhibit RANKL-induced osteoclast differentiation from RAW 264.7 cells and BMMs without signs of cytotoxicity. Moreover, QUE reduced the occurrence of MDA-MB-231 Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3290-x) contains supplementary material, which is available to authorized users. H. Wang W. Shen X. Hu Y. Zhang Y. Zhuo T. Chu (&) Department of Orthopaedics, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China e-mail: [email protected] T. Li F. Mei L. Xiao (&) Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, China e-mail: [email protected] X. Li Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton T6G 2B7, Canada
cell-induced osteolytic bone loss by suppressing the differentiation of osteoclasts. Finally, molecular analysis revealed that it is by inhibiting RANKL-mediated MAPK and NF-jB signaling pathways that QUE suppressed the osteoclast differentiation. We demonstrate, for the first time, the novel suppressive effects of QUE on RANKLinduced osteoclast differentiation in vitro and human breast cancer-induced bone loss in vivo, suggesting that QUE may be a potential therapeutic drug for osteolysis treatment. Keywords Quetiapine Breast cancer Bone metastases Osteoclast Bone resorption
Introduction Breast cancer, along with lung and prostate cancers and multiple myeloma, is likely to metastasize to bone [1]. Once bone metastases have occured, the chance of survival is slim, and the quality of life of the patient dramatically drops, with a clinical outcome characterized by intractable pain, nerve compression syndromes, increased risk of fractures, and hypercalcemia [2]. Breast cancer bone metastases have a generally osteolytic nature and rely on the ability of tumor cells to activate osteoclast-induced bone resorption, which is a crucial event that contributes to the disruption of bone and the creation of physical space into which the tumor cells intrude [3, 4]. Breast cancer cells can produce factors that induce osteoclastogenesis, such as PTHrP and interleukins (IL)-1, IL-6, and IL-11. These factors act on osteoblasts to increase the production of RANKL [5, 6]. RANKL can bind to its receptor RANK which is located on the membrane of the osteoclast precursor cells and subsequently activate
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RANK [7]. The activation of RANK on osteoclast progenitor cells leads to the activation of TNF receptor-associated factors (TRAFs) and the subsequent stimulation of several downstream signaling molecules, including nuclear factor-kappa B (NF-jB), mitogen-activated protein kinases (MAPKs), activating protein 1 (AP-1), nuclear factor of activated T cells 1 (NFATc1), and phosphatidylinositol 3-kinase (PI3K)/Akt, resulting in the differentiation of osteoclast progenitor cells into multinucleated, bone-resorbing osteoclasts [8]. Osteolytic lesions, characterized by excessive bone resorption, are mainly induced by abnormal osteoclast differentiation. Therefore, suppressing the functional differentiation of osteoclasts is one of the main strategies for osteolysis treatment [9]. Some drugs, including bisphosphonates, estrogen, teriparatide, synthetic calcitonin, and denosumab, have been used for the treatment of osteolysis. But most of these drugs have many limitations or side effects, such as osteonecrosis, thromboembolism, osteosarcoma, and esophageal irritation [10–12]. Therefore, developing or discovering new drugs is required to improve bone therapy. Previously, we found that quetiapine (QUE), a commonly used atypical antipsychotic drug, can promote the differentiation of neural progenitors of the oligodendrocyte lineage through the MAPK signaling pathway [13]. Other studies have also demonstrated that QUE exerts anti-inflammatory [14] and antioxidant [15] effects, and modulates the NF-jB [16] and MAPK [17] pathways, both of which have been implicated in osteoclastogenesis [8, 18]. Thus, we asked whether QUE can inhibit osteoclastogenesis and potentially prevent cancer-induced bone loss. Our results demonstrate for the first time that QUE can inhibit osteoclastogenesis in vitro and prevent human breast cancer-induced bone loss in vivo through the suppression of the RANKL-mediated MAPK and NF-jB signaling pathways, suggesting that QUE is a potential lead compound for the development of novel drugs for osteolysis treatment.
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Precipitated cells were suspended in a-MEM supplemented with 10 % FBS and cultured for 24 h. Non-adherent cells were collected and cultured for 3 days in the presence of M-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ, USA). Floating cells were discarded, and adherent cells were used as BMMs. RAW 264.7 cells were seeded in 24-well plates at a density of 2 9 104 cells/well and placed in the CO2 incubator overnight. After 24 h, the culture medium was replaced with medium containing RANKL (50 ng/ml, PeproTech, Rocky Hill, NJ, USA) with various concentrations of QUE for 5 days. BMMs were plated at a density of 4 9 105 cells/well in a 24-well plate in the presence of RANKL (50 ng/ml) and M-CSF (20 ng/ml) with various concentrations of QUE for 7 days. Medium was changed every 2 or 3 days. Cytotoxicity assay Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cells (2 9 103 cells/well) were seeded on 96-well plates and cultured with or without QUE (AstraZeneca, Wilmington, DE) for expected time. After incubation, 10 ll CCK-8 solution was added. The plates were incubated for 4 h, after which, absorbance was measured at 450 nm using a microplate reader. TRAP staining of RAW 264.7 cells and BMMs To confirm the generation of multinucleated, osteoclastlike cells, the cultured cells were stained for tartrate-resistant acid phosphatase (TRAP) using the TRAP Staining Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. TRAP-positive multinucleated (3 or more nuclei) osteoclasts were visualized by light microscopy and photographed. Each osteoclast formation assay was performed at least 3 times. RNA isolation and RT-PCR analysis
Materials and methods Cell culture RAW 264.7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10 % FBS (Gibco, NY, USA) and antibiotics (100 IU penicillin/ ml and 100 mg streptomycin/ml). Bone marrow-derived macrophages (BMMs) were obtained from 4- to 6-week-old C57BL/6 mice. The tibias of mice were obtained and flushed with a-MEM. Cells were then centrifuged for 5 min at a speed of 1,000 rpm.
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Total RNA was isolated from cells with TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) following the instructions of the manufacturer. RNA was quantified using a spectrophotometer set (Nanodrop, Thermo Fisher Scientific Inc., USA). The same amount of total RNA from each sample was reverse transcribed to cDNA in a final volume of 20 ll using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time PCR was performed using FastStart Universal SYBR Green Master (Roche, shanghai, China). Quantification was normalized to the amount of glyceraldehyde-3phosphate dehydrogenase (Gapdh) cDNA. The specific primer sequences are listed in Table S1.
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Intramedullary injection of tumor cells
4 °C. After washing with PBS, the secondary antibody, donkey anti-rabbit Alexa Fluor 568 (Invitrogen, Grand Island, NY, USA) was added and samples were incubated for 2 h in the dark at room temperature. The cells were observed under confocal microscopy (Olympus, Tokyo, Japan).
Six-week-old female immunodeficient BALB/c-nu/nu mice were obtained from the Animal Center of the Third Military Medical University and maintained under sterile conditions. All experiments were performed in accordance with the Health Guidelines for the Care and Use of Laboratory Animals with the approval of the Third Military Medical University Committee on Animal Care (permission NO: SCXKJUN-2007-015). The breast cancer cell line MDA-MB-231 was cultured and resuspended in PBS to a final concentration of 106 cells/ml. Mice were anesthetized with intraperitoneal injection of pentobarbital (60 mg/kg). A syringe with a 26 1/2 G needle was subsequently inserted in the proximal end of the tibia, and tumor cells (104 cells in 10 lL PBS) were injected into the intramedullary space. The mice were then randomly assigned to two groups and treated with vehicle (ddH2O, n = 8) or QUE (10 mg/kg, n = 8). Previous studies of our group demonstrated similar effects of oral administration and intraperitoneal injection about the method of QUE given [13, 19]. To reduce the chance of infection of mice which were used in our study, QUE was dissolved in ddH2O and was given through intraperitoneal injection once a day for 6 weeks until the end of the experiment, and the body weight of mice was recorded every 3 days. Assessment of osteolytic lesions in vivo To further characterize osteolytic bone lesions, mice were anesthetized and subjected to X-ray system (Kodak, In Vivo Imaging System FX Pro; 30 kVp for 1 min), scanned, and evaluated without knowledge of the experimental groups. For histological examination, tibias were dissected, cleaned of soft tissues, and fixed in 4 % paraformaldehyde in 0.1 M PBS for 48 h. The samples were then decalcified in EDTA and embedded in paraffin. Sections were cut and stained with H&E to evaluate the displacement of bone marrow by tumor cells. The sections were also stained for TRAP to observe the activation of osteoclasts and were then subjected to histomorphometric analysis. Immunofluorescent staining RAW 264.7 cells were stimulated with RANKL in the presence of QUE (50 lM) or the vehicle control for 0, 5, 15, 30, 60, and 120 min. The cells were fixed with 4 % paraformaldehyde for 10 min. This step was followed by permeabilization with 0.2 % Triton-X 100 for 5 min. After blocking with 1 % BSA in PBS for 20 min, the cells were stained with rabbit anti-p65 antibody (Cell Signaling Technology, USA) in 1 % BSA and incubated overnight at
Western blot Cells were cultured in the presence of RANKL with or without QUE (50 lM) for 0, 5, 15, 30, 60, and 120 min. Subsequently, cells were lysed with RIPA buffer plus PMSF (1 mM). Protein was separated on 8 % SDS-PAGE gels and transferred to PVDF membranes. The MAPK family antibody sampler kit, the phospho-MAPK family antibody sampler kit, the NF-jB pathway sampler kit, and the phospho-Akt antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The Akt antibody was purchased from Proteintech (Wuhan, Hubei, China), and the antibody against b-actin was purchased from Santa Cruz Biotechnology (CA, USA). Statistical analysis All experiments were repeated at least three times, each time in triplicate. Student’s t test was used for determining the significance of differences between two groups, whereas one-way analysis of variance (ANOVA) followed by a post hoc Tukey’s test was used for multiple comparisons using SPSS 17.0. All data are expressed as the mean ± SD. P values less than 0.05 (p \ 0.05) were accepted as statistically significant.
Results QUE suppresses osteoclastogenesis of RAW 264.7 cells and BMMs induced by RANKL without cytotoxicity Before examining the effect of QUE on osteoclast differentiation, we detected the cytotoxicity of QUE on RAW 264.7 cells and BMMs using a CCK-8 assay. As shown in Fig. 1, after treatment for 48 h, QUE showed no cytotoxicity on RAW 264.7 cells in the concentration range between 1 and 50 lM. Furthermore, there was no cytotoxicity on RAW 264.7 cells and BMMs after treatment by QUE (50 lM) for 5 or 7 days (Fig. S1). Therefore, QUE at the concentrations of 25 and 50 lM were used in subsequent in vitro experiments. To investigate the effect of QUE on osteoclastogenesis, RAW 264.7 cells were treated with different doses of QUE (1–50 lM) in the presence of RANKL. After 5 days of incubation, the cells were TRAP stained. As seen in Fig. 2,
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(50 lM) in the presence of RANKL and M-CSF. After 7 days of incubation, the cells were TRAP stained, and multinucleated cells were counted. The results show that QUE also suppresses the osteoclastogenesis of BMMs induced by RANKL and M-CSF (Fig. 2c, d). QUE acts at the early step of RANKL-induced osteoclastogenesis
QUE suppressed osteoclast formation in a dose-dependent manner. Treatment with 25 and 50 lM of QUE decreased the number of osteoclasts formed (Fig. 2a, b). These results indicate that QUE significantly suppresses the osteoclastogenesis of RAW 264.7 cells induced by RANKL. To further determine the suppressive effect of QUE on osteoclastogenesis, BMMs were also treated with QUE
Osteoclastogenesis is a very complicated process which involves cell proliferation, differentiation, activation, and survival [20–22]. To further determine at which stage QUE is most effective in inhibiting osteoclast differentiation of RAW 264.7 cells induced by RANKL, QUE (50 lM) was added to osteoclast differentiation cultures beginning at day 1, day 2, day 3, and day 4, respectively. The results showed that QUE inhibited osteoclastogenesis maximally when added from the initiation of RANKL treatment (Fig. 3a). The exposure of precursor cells to QUE at later stages (after day 3) was less effective in suppressing osteoclastogenesis (Fig. 3b). It is noted that, although QUE can suppress osteoclast differentiation at the very early differentiation stage, it has substantially no suppressive effect when osteoclast differentiation has come to the late stage.
Fig. 2 QUE inhibits osteoclast differentiation in RANKL-stimulated RAW 264.7 cells and BMMs. a RAW 264.7 cells dosed with 50 ng/ ml RANKL and treated with or without QUE (1, 10, 25, or 50 lM) for 5 days. c BMMs dosed with 20 ng/ml M-CSF and 50 ng/ml RANKL with or without QUE (50 lM) for 7 days. b, d TRAPpositive multinucleated cells with three or more nuclei were
considered as osteoclasts, and the number of osteoclasts was counted after taking images of random fields in different areas of each well. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Arrows osteoclasts; scale bar 100 lm; **p \ 0.01 compared with RANKL treatment
Fig. 1 Effect of QUE on cell viability. CCK-8 assay was performed after incubation of RAW 264.7 cells with QUE (0, 1, 10, 25, 50, and 100 lM) for 48 h. Values are expressed as the mean ± SD of triplicate experiments. *p \ 0.05
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Fig. 3 Inhibition of RANKL-induced osteoclastogenesis by QUE is an early event. a RAW 264.7 cells were incubated with RANKL (50 nmol/L), and then QUE (50 lM) was added on day 1, day 2, day 3, and day 4, respectively. At the end of 4 days, cells were stained for TRAP expression. b TRAP-positive multinucleated cells with three or more nuclei were considered as osteoclasts, and the number of
osteoclasts was counted after taking images of random fields in different areas of each well. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Arrows osteoclasts; scale bar 100 lm; *p \ 0.05, **p \ 0.01 compared with RANKL treatment
QUE inhibits the expression of osteoclastogenesisrelated genes in RAW 264.7 cells and BMMs induced by RANKL
QUE inhibits osteoclastogenesis induced by tumor cells in vivo
We also assessed the effect of QUE on osteoclastogenesis by evaluating the expression levels of osteoclastogenesisrelated genes. Throughout the differentiation process, osteoclasts express some markers, such as TRAP, calcitonin receptor (CalcR), cathepsin K (CtsK), and MMP-9 (gelatinase B) which, together with multinucleation and resorption, characterize the osteoclast phenotype [23]. Our results show that TRAP mRNA expression levels were significantly higher in the cells treated with RANKL. Moreover, QUE treatment inhibited the increased TRAP expression induced by RNAKL in a dose-dependent manner: 1 and 10 lM of QUE treatment had no effect while a decrease in TRAP expression was found in cells treated with 25 and 50 lM of QUE. Similar results were found for CalcR, CtsK, and MMP-9 mRNA expression levels (Fig. 4).
Fig. 4 Effects of the RANKL and QUE treatments on mRNA expression levels of TRAP, CalcR, CtsK, and MMP-9. a Expression levels of TRAP, CalcR, CtsK and MMP-9 in RAW 264.7 cells treated with or without QUE for 5 days. b Expression levels of TRAP, CalcR, CtsK, and MMP-9 in BMMs treated with or without QUE for
Bone loss is one of the most common complications in patients with breast cancer [24]. There is evidence showing that bone destruction at sites of bone metastases is the result of abnormal osteoclast activity rather than of tumor cells per se [25, 26]. We investigated whether QUE could suppress osteolytic bone lesions induced by tumor cells in vivo. Firstly, MDA-MB-231 cells were treated with QUE (50 lM) for 1, 3, or 5 days respectively, and the results revealed that there was no cytotoxicity on MDAMB-231 cells after treatment by QUE for indicated time points (Fig. 5a), and then MDA-MB-231 cells (104 cells in 10 lL) were injected into the intramedullary space of mice, and the mice were treated with QUE (10 mg/kg) or vehicle control for 6 weeks. To identify osteolytic bone lesions, we analyzed bone tissue by X-ray analysis. As seen in Fig. 5b, osteolytic bone destruction of cortices was observed in the MDA-MB-231 tumor-bearing control mice. In contrast,
7 days. Data represent fold changes of target genes normalized to Gapdh mRNA and are expressed as a percentage of RANKL-dosed cells not treated with QUE, which were set to 100 %. Values represent the mean ± SD (n = 4). **p \ 0.01
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Fig. 5 QUE prevents human breast cancer-induced bone loss in vivo. a CCK-8 assay was performed to test effect of QUE (50 lM) on MDA-MB-231 cell viability. Cells were treated for 1, 3, or 5 days respectively. b X-ray images of the tibias from mice subjected to injection of MDA-MB-231 cells and treated with or without QUE for 6 weeks. c H&E and TRAP staining of one representative tibia from
each group. d Histomorphometric analysis of osteoclast numbers in the bone metastases of MDA-MB-231 cells. Data are expressed as number of osteoclasts/mm at the tumor-bone interface. The values are expressed as the mean ± SD. * tumor mass; arrows osteoclasts; # bone; scale bar 50 lm; **p \ 0.01 compared with the vehicle group
there was very little osteolysis in the QUE-treated mice. Interestingly, histomorphometric analysis performed on tibia sections stained with H&E or for the osteoclast marker TRAP revealed a significant reduction in the osteoclast number in mice treated with QUE versus vehicle (Fig. 5c, d). All mice survived until the end of the experiment and body weight of mice with or without QUE treatment showed no significant difference (Fig. S2). This shows that QUE can suppress osteolytic bone lesions induced by tumor cells in vivo by inhibiting osteoclast differentiation.
time points and QUE-treated cells were pretreated with QUE for 30 min. The phosphorylation states of MAPKs and Akt were detected by Western Blot analysis. Interestingly, QUE treatment significantly decreased the levels of phospho-p38 MAPK (Fig. 6b, f), phospho-ERK1/2 (Fig. 6c, g) and phospho-JNK (Fig. 6d, h), but had no effect on RANKL-induced phosphorylation states of Akt (Fig. 6e, i). Next, NF-jB pathway activation was examined. The activation of NF-jB pathway is accompanied by the degradation of IjBa and the nuclear translocation of NFjB subunit p65 [27]. RAW 264.7 cells were stimulated with RANKL and QUE as mentioned above. The level of IjBa decreased immediately between 5 and 60 min after RANKL treatment and recovered at 120 min. Upon QUE treatment, QUE was found to significantly suppress the RANKL-induced degradation of IjBa (Fig. 7a). Furthermore, when the level of phosphorylated p65 (phosphop65), one of the subunits of NF-jB, was examined, QUE treatment clearly suppressed the phospho-p65 level compared with cells treated with the vehicle control (Fig. 7a). This result was confirmed by a localization study using immunofluorescent staining of p65. In RANKL-stimulated control cells, p65 was found exclusively in the cytoplasm at the start of RANKL stimulation. After 15–30 min of RANKL stimulation, most cells exhibited nuclear localization of p65 (Fig. 7c). This trend was reversed 60 min
QUE suppresses RANKL-induced activation of the MAPK and NF-jB pathways during osteoclast differentiation The activation of the MAPK, PI3 K/Akt, and NF-jB pathways is critical for RANKL-induced osteoclastogenesis [8, 18]. In order to elucidate the molecular mechanisms by which QUE suppresses osteoclast differentiation, the activation of the MAPK, PI3 K/Akt, and NF-jB pathways was assessed. Firstly, we investigated the presence of phospho-p38 MAPK, phospho-ERK1/2 (p44/42), phosphoJNK, and phospho-Akt in QUE-treated or vehicle control cells upon RANKL stimulation. Accordingly, RAW 264.7 cells were stimulated with RANKL (50 ng/ml) in the presence of QUE or the vehicle control at the indicated
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Fig. 6 QUE suppresses the activation of MAPK signaling pathway induced by RANKL in RAW 264.7 cells. a Time course of the phosphorylations of MAPKs and Akt in RANKL-stimulated RAW 264.7 cells. Cells were stimulated with 50 ng/ml RANKL at the indicated time points. Phosphorylations of MAPKs and Akt were detected by western blot analysis, protein expression of p-p38, p-ERK, p-JNK and p-Akt was normalized against b-Actin. b, c, d, e, f, g, h, i RAW 264.7 cells were stimulated with RANKL (50 ng/ml)
in the presence of QUE (50 lM) or the vehicle control at the indicated time points, QUE-treated cells were pretreated with QUE for 30 min. p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Akt, and Akt were analyzed using western blot analysis (b, c, d, e). Protein expression of p-p38, p-ERK, p-JNK, and p-Akt was normalized against total p38, ERK, JNK and Akt (f, g, h, i). Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. *p \ 0.05, **p \ 0.01
Fig. 7 QUE suppresses the activation of NF-jB signaling pathway induced by RANKL in RAW 264.7 cells. RAW 264.7 cells were stimulated with RANKL (50 ng/ml) in the presence of QUE (50 lM) or the vehicle control at the indicated time points, QUE-treated cells were pretreated with QUE for 30 min. a, b Effects of QUE on IjBa, p-p65, and p65 protein levels in RANKL-stimulated RAW 264.7 cells. The protein levels were determined by western blot analysis (a),
and protein expression was normalized against b-Actin (b). c RAW 264.7 cells were treated as mentioned above and then stained for the NF-jB subunit p65. The cells were observed under a confocal microscope. At least 4 wells were used for each tested reagent. Cells not treated with QUE but exposed to RANKL were used as a positive control, and the values are expressed as the mean ± SD. Scale bar 100 lm; **p \ 0.01
after stimulation. In contrast, QUE treatment completely abrogated the nuclear localization of NF-jB p65 at any time point (Fig. 7c). Therefore, QUE suppresses RANKLinduced NF-jB activation by inhibiting the degradation of
IjBa and abrogating the nuclear localization of NF-jB p65. Accordingly, QUE suppresses the activation of the MAPK and NF-jB pathways during osteoclast
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differentiation, cooperatively contributing to its anti-osteoclastogenic activity.
Discussion Almost 90 % of cancer-associated deaths result from tumor metastasis. Lung, liver, lymph node, and bone are some of the major organ sites where tumor metastasis is prone to occur. Bone metastasis is commonly associated with prostate cancer, lung cancer, and breast cancer. Breast cancer metastasis to bone leads to osteolytic lesions, and no currently available treatment is sufficient to treat bone metastasis induced by breast cancer. In the present study, we demonstrate for the first time that QUE, an FDA-approved chemical compound, can inhibit osteoclastogenesis and prevent bone loss induced by human breast cancer through suppression of the MAPK and NF-jB signaling pathways. Bisphosphonate drugs, denosumab, and teriparatide (synthetic parathyroid hormone) are the most widely used agents for preventing bone loss. Drugs of the bisphosphonate family have been used for many years as a standard of care by virtue of their ability to reduce the occurrences of skeletal-related events (SREs), relieve bone pain, and improve the quality of life of patients with cancer [28–30]. However, these drugs have many limitations in their use due to serious side effects including renal failure, gastrointestinal reactions, and osteonecrosis of the jaw [31]. Denosumab, a monoclonal antibody against RANKL, can inhibit osteoclast differentiation by effectively preventing the binding of RANKL with RANK. Denosumab has been approved by FDA for the prevention of SREs in patients with tumors, including breast cancer [31]. Compared with drugs of the bisphosphonate family, denosumab is more effective at reducing the risk of developing SREs as well as delaying the time to develop SREs in patients with breast cancer [29]. Unfortunately, as is the case with bisphosphonate drugs, denosumab is associated with a low incidence of osteonecrosis of the jaw and does not make any significant difference in overall patient survival [29]. Another drug, teriparatide, in contrast to bisphosphonate drugs and denosumab, acts on osteoblasts to stimulate bone formation. At first glance, it would seem ideal to use bisphosphonate drugs or denosumab in combination with teriparatide because bisphosphonate drugs and denosumab block bone resorption while teriparatide stimulates bone deposition. However, teriparatide is found to be associated with an increased risk of osteosarcoma and exacerbation of skeletal metastases because it is likely to lead to bone turnover [32]. Thus, an effective method or reagent for inhibiting osteoclastogenesis is in need for the treatment of bone metastasis. Our data indicated that QUE, a commonly
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used atypical antipsychotic drug (APD), has such a capacity. Osteoclasts are multinuclear giant cells derived from monocyte-macrophage lineage cells. Osteoclast differentiation from precursor cells can be induced by RANKL, which also controls the survival and function of mature osteoclasts. Therefore, RANKL, as a convincing stimulator, is used in studies on differentiation and function of osteoclasts. In this study, both RAW 264.7 cells and BMMs were used and dosed with RANKL to build models of osteoclast differentiation. In order to understand the effects of QUE on osteoclast differentiation, different doses of QUE were used in our experiment, and the results revealed that QUE treatments significantly reduced the number of TRAP-positive multinuclear osteoclasts without any signs of cytotoxicity, which mainly affected the early stage of osteoclast differentiation. We also assessed the effect of QUE on the expression of osteoclastogenesis-related genes. Throughout the differentiation process, osteoclasts express some markers, such as TRAP, CalcR, CtsK and MMP-9, which, together with multinucleation and resorption, characterize the osteoclast phenotype. TRAP is highly expressed in mature osteoclasts and is used as a marker of osteoclast function [33]. Mature osteoclasts also express the CalcR on their surface. In fact, CalcR has been considered as the best differentiation marker for osteoclasts [34]. CtsK and MMP-9 have been confirmed to play a key role in the resorption process [35]. Our results showed that QUE (25 and 50 lM) reduced the RANKL-induced expression of the osteoclast phenotypic markers TRAP, CalcR, CtsK, and MMP-9 in a dose-dependent manner. These results suggested that QUE suppressed osteoclast formation, at least partially, by inhibiting the expression of RANKL-induced osteoclastrelated genes. More importantly, our results show that QUE also suppresses osteolysis induced by breast cancer cells in vivo without affecting the body weight of mice, and histological TRAP staining of decalcified bone sections reveals a significant decrease in the number of osteoclasts relative to bone surface area in mice treated with QUE, suggesting the potential utility of QUE in the treatment of cancer-induced bone lesions. Previous studies have demonstrated that QUE exerts various effects such as anti-inflammatory [14], antioxidant [15] effects, and modulates the NF-jB [16] and MAPK pathways [17]. In the current study, we demonstrate that QUE can decrease the phosphorylation level of MAPKs including p38, ERK, and JNK, which were found to be activated by RANKL stimulation in RAW 264.7 cells. Among these MAPKs, p38 MAPK has an ability to regulate the microphthalmia-associated transcription factor and is thus a signaling molecule that plays an important
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role in the early stage of osteoclast differentiation [36], while dominant-negative JNK prevents RANKL-induced osteoclastogenesis [37]. In comparison, ERK has been confirmed to induce c-Fos expression [38], and inhibition of ERK protein kinase has been shown to suppress RANKL-induced osteoclast formation [39]. Thus, in combination with our experimental results, we can conclude that QUE has suppressive effect on osteoclastogenesis, at least partially, by inhibiting MAPK signaling. Apart from MAPK signaling, activation of the NF-jB pathway is also thought to be a crucial role in RANKL-induced osteoclast differentiation [40, 41]. The activation of NF-jB signaling pathway involves IjB phosphorylation, and the latter relies on the regulation of IjB kinase (IKK). RANKL can stimulate IKK activation and result in IjBa phosphorylation. Then, the polyubiquitinated IjBa is degraded by proteasomes, and the dissociative NF-jB subunit p65 enters into nucleus and binds to the targeted position of DNA, and starts the gene transcription [42]. Our results showed that QUE could inhibit nuclear localization of NF-jB p65 by suppressing RANKL-mediated IjBa degradation. These results indicate that inhibition of the NF-jB pathway is one of the mechanisms involved in the anti-osteoclastogenic effect of QUE. Moreover, we also excluded the possibility of the involvement of PI3 K/Akt pathway in the effect of QUE on RANKL-induced osteoclast differentiation. As an atypical antipsychotic drug, QUE is a multiple receptor antagonist with low affinity for D2 and high affinity for 5HT2A, 5HT1A, a-1, and a-2 adrenergic and histamine H1 receptors [43]. A recent study provides evidence that QUE may be linked to the epidermal growth factor receptor (EGFR) [44], which has been documented to play an important role in regulating osteoclast differentiation and survival through cross-talking with RANK signaling [45]. Our results suggest that suppressing the activation of the MAPK and NF-jB pathways during osteoclast differentiation may cooperatively contribute to the anti-osteoclastogenic activity of QUE. However, the molecular mechanism by which QUE inhibits RANKL-mediated MAPK and NF-jB pathway activation awaits further study. Although our data from in vitro and in vivo experiments illustrate QUE-mediated inhibition of osteoclast differentiation, it is possible that QUE might also target additional cells in vivo. Therefore, additional further analyses of other potential cellular and molecular targets of QUE during long-term treatment in vivo should be conducted when developing potential QUE-based therapeutic applications.
Conclusions Our findings that QUE inhibits osteoclastogenesis through the suppression of the MAPK and NF-jB pathways suggest
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that QUE may have great potential in the development of novel therapeutic drugs for disorders associated with bone loss. Acknowledgments This study was partly supported by grants from the National Natural Science Foundation of China (81271979), and National Basic Research Program of China (973 Program, No. 2010CB529400). The authors thank Yan Yin for carefully proofreading the manuscript and providing valuable comments. The authors also thank Dr. Yue Zhou and Dr. Jianqin Niu for their direction, advice, and teachings. Conflicts of interest of interest.
All the authors state that they have no conflicts
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