β‐cryptoxanthin stimulates apoptotic cell death and suppresses cell function in osteoclastic cells: Change in their related gene expression

The effect of beta-cryptoxanthin, a kind of carotenoid, 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 NF-kappaB ligand (RANKL; 25 ng/ml) for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle or beta-cryptoxanthin (10(-8)-10(-6) M) with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 24-72 h. Osteoclastic cells were significantly decreased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) with or without M-CSF and RANKL for 24, 48, or 72 h. beta-Cryptoxanthin (10(-8) M)-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h, indicating that the carotenoid induces apoptotic cell death. Apoptosis-related gene expression was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL, while Bcl-2 and Apaf-2 mRNA expressions were significantly increased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) without M-CSF and RANKL for 24 or 48 h. Akt-1 mRNA expression was not significantly changed with culture of the carotenoid (10(-7) or 10(-6) M) for 24 or 48 h. Moreover, tartrate-resistant acid phosphatase (TRACP) activity, or TRACP and cathepsin K mRNA expressions were significantly decreased with culture of beta-cryptoxanthin (10(-6) M) in the presence or absence of M-CSF and RANKL for 48 h. This study demonstrates that beta-cryptoxanthin has stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function.