We investigated whether the effect of β-cryptoxanthin (CRP) on osteoclastic cells formed in the mouse marrow culture system in vitro is enhanced by culture with zinc. Bone marrow cells were isolated from mice. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL; 50 ng/ml) for 96 h. The osteoclastic cells formed were further cultured for 24 or 72 h in a medium containing either vehicle, CRP, zinc sulfate (zinc), or CRP plus zinc with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml). The number of osteoclastic cells was significantly decreased after culture with the combination of CRP (10−7 M) and zinc (10−5 M) in the presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for CRP or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with CRP (10−7 M) plus zinc (10−5 M) for 24 or 72 h in the presence of M-CSF and RANKL, indicating that the combination of the two chemicals induces apoptotic cell death. CRP plus zinc-induced decrease in osteoclastic cells was significantly inhibited in the presence of caspase-3 inhibitor (10−8 or 10−7 M). Culture with CRP (10−7 M) plus zinc ((10−5 M) for 24 or 72 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL as compared with the value for each chemical alone. CRP plus zinc-induced increase in caspase-3 mRNA expression was completely inhibited in the presence of cycloheximide (10−7 M), an inhibitor of protein synthesis, or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DBR; 10−6 M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) and cathepsin K was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with CRP or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased after culture with CRP plus zinc in the presence or absence of M-CSF and RANKL for 72 h as compared with each chemical alone, while NF-κB mRNA expression was not significantly changed. This study demonstrated that the combination of CRP and zinc has potent suppressive effects on osteoclastic cells in vitro.

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