Disulfiram attenuates osteoclast differentiation in vitro: a potential antiresorptive agent.
Disulfiram (DSF), a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC) differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL)-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC-mediated disorders.
Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia.
Hua
H
Ying
Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia; Department of Orthopaedics, The Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
An
A
Qin
Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia.
Tak S
TS
Cheng
Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia.
Nathan J
NJ
Pavlos
Harry Perkins Institute of Medical Research, Perth, Australia; Department of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Perth, Australia.
Sarah
S
Rea
Department of Orthopaedics, The Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Kerong
K
Dai
Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia.
Ming H
MH
Zheng
D000818:Animals; D015153:Blotting, Western; D001862:Bone Resorption / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D004221:Disulfiram / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects*; D053245:RANK Ligand / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016328:NF-kappa B; D050778:NFATC Transcription Factors; D053245:RANK Ligand; D004221:Disulfiram