title: Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.
abstract: Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.
authors.affiliation: Department of Oral Diagnosis and Oral Medicine, Hokkaido University                     Graduate School of Dental Medicine, Kita-ku, Sapporo 060-8586, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Abe
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Deyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikuiri
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Kanchu
authors.initials: K
authors.lastname: Tei
authors.forename: Hisashi
authors.initials: H
authors.lastname: Shinoda
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Suzuki
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Kitagawa
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001681:Biological Assay; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D053938:DNA Fragmentation / Q000187:drug effects; D004164:Diphosphonates / Q000737:chemistry / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D015726:Giant Cells / Q000166:cytology / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors
chemical_list: D015415:Biomarkers; D004164:Diphosphonates; D007527:Isoenzymes; D012333:RNA, Messenger; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D053148:Caspase 3; D053179:Caspase 7