Importantly, the expression of RANKL has also been found in some cancer cells as well as in activated Tcells. Therefore, RANKL signaling has been believed to be a therapeutic target for the inhibition of bone remodeling and bone metastasis. Moreover, several molecules including endothelin-1, BMP, and Wnt have been believed as the important regulators for osteoblast differentiation and bone formation. The histological studies have shown that osteoblastic lesions of PCa bone metastasis are characterized by deposition of new bones, which are produced by osteoblasts, unorganized and interlaced between foci of cancer cells. In osteoblasts, endothelin-1, BMP, and the molecules in Wnt signaling are highly expressed. In addition, elevated serum levels of bone-specific alkaline phosphatase, a marker of osteoblast differentiation and proliferation, are often observed in cancer patient with bone metastasis, suggesting the importance of these molecules in PCa bone metastasis. Therefore, targeting these osteoblastic molecules could inhibit bone remodeling and PCa bone metastasis. Recently, natural agents have received much attention in the area of cancer research. Isoflavone genistein mainly found in soybean has shown its ability to inhibit cancer cell growth in vitro and in vivo without toxicity. We have previously found that isoflavone genistein could inhibit NF-kB and Akt activation in cancer cells. Moreover, we have reported that dietary genistein could inhibit PCa in experimental bone metastasis in a SCID-human model and that genistein could potentiate apoptosis inducing effects of chemotherapeutic agents through down-regulation of NF-kB. 3,39-diindolylmethane is another natural agent and mainly found in the members of the family Cruciferae such as broccoli. We and others have found that DIM and its formulated product could downregulate the expression of AR, Akt and NF-kB,MI-538 leading to the inhibition of PCa growth and the induction of apoptosis in vitro and in vivo. DIM was also found to potentiate the therapeutic efficacy of chemotherapeutics. In this study, we investigated whether isoflavone mixture G2535 containing 70.5% genistein, and BR-DIM could inhibit the differentiation of osteoclasts and osteoblasts mediated through regulation of cellular signaling pathways that are involved in bone remodeling and PCa bone metastasis. Since we found that isoflavone and BR-DIM inhibited the differentiation of osteoclast, we further investigated the molecular mechanism of isoflavone and BR-DIM action on osteoclastgenesis. It is well known that PCa cells frequently metastasize to the bone. In the bone, metastasized PCa cells could utilize the nutrients from blood in the bone marrow,AZ191 interact with preosteoclasts and pre-osteoblasts, and stimulate bone remodeling. The interaction between PCa cells and pre-osteoclasts/pre-osteoblasts is a critical step for bone remodeling during PCa bone metastasis. Therefore, development of a co-culture system with PCa cells and pre-osteoclasts/pre-osteoblasts is important for investigating molecular interactions of signaling pathways during PCa bone metastasis and bone remodeling. Our data showed that androgen-insensitive PCa cells including PC-3 and C4-2B cells could grow nicely with pre-osteoclasts or preosteoblasts in the same culture dish with defined medium, which mimics in vivo environment with direct interaction of PCa cells and local bone cells. In our co-culture system, PCa cells, preosteoclasts, and pre-osteoblasts could also grow in individual chambers which were separated by a membrane allowing proteins distribution between the two chambers while separating different types of cells in individual chambers.