Genistein and zinc synergistically stimulate apoptotic cell death and suppress RANKL signaling-related gene expression in osteoclastic cells

'The effects of the combination of genistein and zinc, which have an anabolic effect on bone metabolism, on osteoclastic cells in mouse marrow culture system in vitro was investigated. 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-kB) ligand (RANKL; 50 ng/ml)for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle, genistein, zinc sulfate (zinc), or genistein plus zinc with or without M-CSF (10 ng/ml) and RANKL (50ng/ml) for 24 or 72 h. The number of osteoclastic cells was significantly decreased with culture of genistein (10(-6) M) plus zinc (10(-5) M) in presence or absence of M-CSF and RANKL for 24 or 72 h as compared with the value for genistein or zinc alone. Agarose gel electrophoresis showed the presence of low-molecular weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with genistein (10(-6) M) plus zinc (10(-5) M) for 24 or 72 h, indicating that the combination of two chemicals induces apoptotic cell death. Such an effect was not seen in the case of each chemical. Genistein plus zinc-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Culture with genistein (10(-6) M) plus zinc (10(-5) M) for 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. Genistein 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 (DRB; 10(-6) M), an inhibitor of transcription activity. The mRNA expression of tartrate-resistant acid phosphatase (TRACP) or cathepsin K was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 72 h as compared with genistein or zinc alone. Nuclear factor of activated T cells c1 (NFATc1) mRNA expression was significantly decreased with culture of genistein plus zinc in the presence of M-CSF and RANKL for 24 or 72 h as compared with each chemical alone, while NF-kB mRNA expression was not significantly changed. This study demonstrates that the combination of genistein and zinc has potent stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function.