Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner

Recenti, Raffaella and Leone, Giuseppe and Simi, Lisa and Orfei, Marco and Pinzani, Pamela and Pieraccini, Giuseppe and Moneti, Gloriano and Carossino, Anna Maria and Franchi, Alessandro and Bartolucci, Gianluca and Carbonell Sala, Silvia and Ginanneschi, Mauro and Tanini, Annalisa and Brandi, Maria Luisa (2007) Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner. Clinical cases in mineral and bone metabolism, 4 (2). pp. 146-155. ISSN 1971-3266

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Abstract

Background. Clodronate is used in high bone resorption diseases. Its action was defined as ?cytotoxic? based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As ?in vitro? bioeffects of clodronate are reversible, this compound should not be defined as ?cytotoxic?. Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R2 side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a ?-?-methylene analog of ATP that induces inhibition of the ADP/ATP translocase. Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiat ve modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex ?matrix metalloproteinase 9/tissue inhibitor of metalloproteinase? (MMP9/TIMP1) by RT-PCR analysis based on ?TaqMan? technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5?-[?-?-dichloromethylene] triphosphate (AppCCl2p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney ?U? test was conducted for statistical analysis. Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl2p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage. Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl2p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP

Item Type:	Article
Uncontrolled Keywords:	Bisphosphonates, clodronate, osteoclasts, ATP, bone resorption
Subjects:	600 Tecnologia - Scienze applicate > 610 Medicina e salute (Classificare qui la tecnologia dei servizi medici) > 612 Fisiologia umana > 612.7 Sistema muscoloscheletrico, tegumento
Depositing User:	Danilo Dezzi
Date Deposited:	20 Jan 2014 08:50
Last Modified:	20 Jan 2014 08:50
URI:	http://eprints.bice.rm.cnr.it/id/eprint/4948

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