Combination of beta-cryptoxanthin and zinc has potent effects on apoptotic cell death and suppression of bone resorption-related gene expression in osteoclastic cells.
We investigated whether the effect of beta-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-kappaB (NF-kappaB) 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-beta-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-kappaB 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.
Yamaguchi
Masayoshi
M
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. yamamasa1155@yahoo.co.jp
Uchiyama
Satoshi
S
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001862:Bone Resorption; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D065366:Cryptoxanthins; D003513:Cycloheximide / Q000378:metabolism; D053938:DNA Fragmentation; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D053245:RANK Ligand / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D024341:Xanthophylls / Q000494:pharmacology*; D015032:Zinc / Q000494:pharmacology*
D016588:Anticarcinogenic Agents; D065366:Cryptoxanthins; D007527:Isoenzymes; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D053245:RANK Ligand; D024341:Xanthophylls; D016173:Macrophage Colony-Stimulating Factor; D003513:Cycloheximide; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002403:Cathepsins; D053148:Caspase 3; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D015032:Zinc