The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat

Abstract

Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox's anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

Keywords: RANKL; ROS; doxorubicin; multiple myeloma; osteoclastogenesis; ovariectomy.

Figure 1

Febuxostat (Febu) inhibits receptor activator of NF-κB ligand (RANKL)-induced reactive oxygen species (ROS) production and osteoclast (OC) formation. (A) RAW264.7 cells were seeded onto a glass-bottom 96-well plate and cultured in quadruplicate with indicated concentrations of RANKL for 30 min followed by staining with the ROS detector CellRox green. Data are expressed as fold changes from controls (mean ± SD). (B) RAW264.7 cells were seeded onto a glass-bottom 96-well plate and cultured with RANKL (50 ng/mL) with or without Febu at 10 μM for 30 min. The number of RAW264.7 cells per field was counted under bright-field using a fluorescence microscope (BZ-X800) and ROS-expressing cells were defined as fluorescence DCF-positive cells (green). Data are expressed as % distribution of ROS-expressing cells (per field), mean ± SD. Representative photos are shown. Original magnification, ×200. Bar, 100 μm. (C) RAW264.7 cells were seeded onto a glass-bottom 96-well plate and cultured in triplicate with RANKL (50 ng/mL) with or without the indicated concentrations of Febu for 30 min, followed by CellRox green staining. Data are expressed as fold changes from controls (mean ± SD). (D) RAW264.7 cells and bone marrow monocyte–macrophage lineage cells (BMMs) were cultured in quadruplicate for 24 h and 10 days, respectively. Febu was added at the indicated concentrations. Cell viability was measured using the 2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-8) assay. Data are expressed as mean ± SD. (E) BMMs were cultured with macrophage colony-stimulating factor (M-CSF) (10 ng/mL) and RANKL (50 ng/mL) in the presence or absence of Febu (60 μM). Cell lysates were collected at 48 and 96 h. c-Fos, cathepsin K (CTSK), and NFATc1 protein levels were analyzed by Western blotting. β-actin served as a loading control. The band sizes of NFATc1 were densitometrically compared to those of a control after normalization to those of β-actin. (F) BMMs were cultured in quadruplicate with M-CSF (10 ng/mL) and RANKL (50 ng/mL) in the presence or absence of indicated concentrations of Febu for 10 days. TRAP-positive cells containing three or more nuclei per well were counted (left). Bone resorption activity was also analyzed and results are expressed as % resorbed area (right). Data are presented as mean ± SD. * p < 0.05. Representative photos are shown. Original magnification, ×100. Bar, 100 μm.

Figure 2

ROS production and osteoclastogenesis by Dox and RANKL in combination. (A) RAW264.7 cells were cultured in quadruplicate with indicated dose of doxorubicin (Dox) in the presence or absence of febuxostat (Febu) at 60 μM for 30 min. ROS expression was detected by CellRox green staining. Data are expressed as fold changes from controls (mean ± SD). (B) RAW264.7 cells were cultured in quadruplicate with Dox and/or RANKL as indicated for 30 min, and ROS expression was detected by CellRox green staining. Data are expressed as fold changes from controls (mean ± SD). (C) RAW264.7 cells were cultured with indicated reagents for 48 h. NFATc1 levels were analyzed by Western blotting. β-actin served as a loading control. The band sizes of NFATc1 were densitometrically compared to those of a control after normalization to those of β-actin. (D) RAW264.7 cells were cultured in quadruplicate with indicated reagents for 30 min and ROS expression was detected by CellRox green staining (left). Data are expressed as fold changes from controls (mean ± SD). * p <0.05. RAW264.7 cells were cultured with indicated reagents for 48 h. NFATc1 protein levels were analyzed by Western blotting (right). β-actin served as a loading control. (E) Bone marrow macrophages (BMMs) were cultured in quadruplicate with M-CSF (10 ng/mL) and indicated reagents for 14 days. The numbers of TRAP-positive multinucleated cells with 3-5 nuclei (white bars) or 6 or more nuclei (black bars) per well were counted. Bone resorption activity was analyzed and the results are presented as % resorbed area. Data are expressed as mean ± SD. * p < 0.05. Representative photos are shown. Original magnification, ×100. Bar, 100 μm.

Figure 3

Febuxostat does not compromise the cytotoxic effects of Dox on multiple myeloma (MM) cells. (A) The MM cell lines RPMI82266, U266, OPM-2, and MM.1S were cultured in triplicate with the indicated concentrations of Dox and febuxostat (Febu) for 48 h. The cell viability was measured by the WST-8 cell proliferation assay. Results are expressed as mean ± SD. (B) RPMI82266 and MM.1S MM cells were cultured with the indicated concentrations of Dox and Febu for 24 h. Caspase 3 and cleaved caspase 3 protein levels were analyzed by Western blotting. β-actin served as a loading control. (C) RPMI8226 and U266 MM cells were cultured in triplicate with the indicated concentrations of Dox and/or NAC for 48 h. The cell viability was analyzed by the WST-8 cell proliferation assay. Results are expressed as mean ± SD.

Figure 3

Febuxostat does not compromise the cytotoxic effects of Dox on multiple myeloma (MM) cells. (A) The MM cell lines RPMI82266, U266, OPM-2, and MM.1S were cultured in triplicate with the indicated concentrations of Dox and febuxostat (Febu) for 48 h. The cell viability was measured by the WST-8 cell proliferation assay. Results are expressed as mean ± SD. (B) RPMI82266 and MM.1S MM cells were cultured with the indicated concentrations of Dox and Febu for 24 h. Caspase 3 and cleaved caspase 3 protein levels were analyzed by Western blotting. β-actin served as a loading control. (C) RPMI8226 and U266 MM cells were cultured in triplicate with the indicated concentrations of Dox and/or NAC for 48 h. The cell viability was analyzed by the WST-8 cell proliferation assay. Results are expressed as mean ± SD.

Figure 4

Febuxostat suppresses osteoclastogenesis by MM cells. (A) Mouse whole bone marrow cells were seeded onto 24-well plates and cultured with M-CSF (10 ng/mL) for 3 days. After that, human MM cell lines (RPMI8226 and KMS-11) and the murine MM cell line 5TGM1 were added onto the cells. The cells were cultured in quadruplicate with or without febuxostat (Febu) (60 μM) for 14 days in the presence of M-CSF (10 ng/mL). The numbers of TRAP-positive multinucleated cells per well were counted. Data are expressed as mean ± SD. * p < 0.05. Representative photos are shown. Original magnification, ×200. Bar, 100 μm. (B) Human bone marrow stromal cells were cultured with RPMI 8226 and KMS-11 MM cell lines in the presence or absence of Febu at 60 μM for 24 h. After removing the MM cells, the expression of RANKL in the bone marrow stromal cells was detected by RT-PCR. GAPDH served as an internal control. (C) Human bone marrow stromal cells were cultured with Dox at 0.5 μM for 24 h. The expression of RANKL mRNA was detected by RT-PCR. GAPDH served as an internal control.

Figure 5

Febuxostat ameliorates bone loss in ovariectomized (OVX) mice. Sham-operated or OVX mice were treated for 3 weeks with or without febuxostat (Febu). μCT images were then taken. (A) Representative μCT images of sham and OVX mice with or without febuxostat (Febu) treatment are shown. (B) Bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb. Sp) were calculated based on μCT images of the tibiae (n = 4 for each group). Results are expressed as mean ± SD. * p < 0.05. (C) The sera of sham and OVX mice were collected and serum levels of TRACP-5b were measured. Data are expressed as mean ± SD (n = 4 for each group). * p < 0.05. (D) The tissue sections of tibiae (n = 4 for each group) were stained with anti-cathepsin-K antibody to identify OCs, and OC numbers per field were counted. Data are expressed as mean ± SD. * p < 0.05. Representative photos are shown. Original magnification, ×100.