Suppressive effect of genistein on rat bone osteoclasts: Apoptosis is induced through Ca2+ signaling

Abstract

The effect of genistein on osteoclast-like multinucleated cells (MNCs) from rat femoral tissues was investigated. When rat marrow cells were cultured for 7 d in a medium containing either vehicle, parathyroid hormone (10-8 M) or prostaglandine E2 (10-6 M), the formation of tartrate- resistant acid phosphatase (TRACP)-positive MNCs was significantly increased. This increase was markedly weakened by the presence of genistein (10-6 or 10-5 M). The bone cells isolated from rat femoral tissues were cultured for 48 h in an α-minimal essential medium (5% fetal bovine serum) containing either vehicle or genistein (10-8 to 10-5 M). Osteoclasts were estimated by staining for TRACP, a marker enzyme of osteoclasts. The presence of genistein caused a significant decrease in the number of osteoclasts. Such a decrease was also seen in the presence of calcitonin (10-10 to 10-8 M), dibutyryl cyclic adenosine 5'-monophosphate (DcAMP; 10-6 and 10-5 M), calcium chloride (10-4 and 10-3 M) or daidzein (10-7 to 10-5 M). The suppressive effect of genistein (10-5 M) was not further enhanced in the presence of calcitonin (10-8 M), DcAMP (10-5 M), or calcium chloride (10-3 M), and was completely abolished by the presence of dibucaine (10-6 M) or staurosporine (10-7 M), both of which are inhibitors of Ca2+- dependent protein kinases. Ca2+ ionophore (A23187; 10-6 M) induced a remarkable decrease in the number of osteoclasts in the absence or presence of genistein (10-5 M) or calcium chloride (10-3 M). The present study demonstrates that genistein has a direct suppressive effect on osteoclasts in vitro, suggesting that the isoflavone may induce apoptosis which is mediated through the pathway of intracellular Ca2+ signaling.