title: Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner.
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 R(2)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 β-γ-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 differentiative 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'-[ β - γ -dichloromethylene] triphosphate (AppCCl(2)p) 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 AppCCl(2)p, 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 AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.
authors.affiliation: Internal Medicine.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Recenti
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Leone
authors.forename: Lisa
authors.initials: L
authors.lastname: Simi
authors.forename: Marco
authors.initials: M
authors.lastname: Orfei
authors.forename: Pamela
authors.initials: P
authors.lastname: Pinzani
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pieraccini
authors.forename: Gloriano
authors.initials: G
authors.lastname: Moneti
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Carossino
authors.forename: Alessandro
authors.initials: A
authors.lastname: Franchi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bartolucci
authors.forename: Silvia
authors.initials: S
authors.lastname: Carbonell Sala
authors.forename: Mauro
authors.initials: M
authors.lastname: Ginanneschi
authors.forename: Annalisa
authors.initials: A
authors.lastname: Tanini
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Brandi