Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro

Bone. 2001 May;28(5):465-73. doi: 10.1016/s8756-3282(01)00412-4.


Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Bone Diseases, Metabolic / drug therapy*
  • Bone Diseases, Metabolic / enzymology
  • Bone Diseases, Metabolic / physiopathology
  • Bone and Bones / drug effects*
  • Bone and Bones / enzymology
  • Bone and Bones / physiopathology
  • Caspase 3
  • Caspase 6
  • Caspase 7
  • Caspases / drug effects*
  • Caspases / metabolism
  • Diphosphonates / pharmacology*
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Dyes / pharmacokinetics
  • Humans
  • Nitrogen / metabolism
  • Osteoclasts / cytology
  • Osteoclasts / drug effects*
  • Osteoclasts / enzymology
  • Protein Prenylation / drug effects
  • Protein Prenylation / physiology
  • Rabbits
  • Tumor Cells, Cultured / cytology
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / enzymology


  • Diphosphonates
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • CASP3 protein, human
  • CASP6 protein, human
  • CASP7 protein, human
  • Caspase 3
  • Caspase 6
  • Caspase 7
  • Caspases
  • Nitrogen