In this way, the effect of secreted proteins from PCa cells on bone cells or the effects of secreted proteins from bone cells on PCa cells could be investigated. Using co-culture, we tested the molecular alterations in osteoclasts or osteoblasts stimulated by PCa cells. Recent studies by other investigators showed that coculture of osteoclast and osteoblast could be useful for examining bone metabolism and osteoclastogenesis. These findings suggest that the co-culture system is very useful for investigating the molecular alterations when PCa cells are homing to the bone, which will allow for assessing alterations in the signal transductions between PCa cells and the local bone cells during PCa bone metastasis and bone remodeling as documented by our results. The differentiation of osteoclasts or osteoblasts is an important step during cancer metastasis to the bone and bone remodeling. By using our co-culture system, we found that pre-osteoclasts could differentiate in to mature osteoclast when co-cultured together with PCa cells. In addition, pre-osteoblasts could also differentiate in to mature osteoblast when co-cultured with PCa cells. Furthermore, we found that co-culture of osteoclasts and osteoblasts with PCa cells could increase 4-(Benzyloxy)phenol the differentiation of osteoclasts and osteoblasts. These findings suggest that the molecules produced by PCa cells could induce the differentiation of osteoclasts and osteoblasts. Importantly, we found that isoflavone and BR-DIM could inhibit the differentiation of osteoclasts and osteoblasts when co-cultured with PCa cells, suggesting that isoflavone and BR-DIM would be useful for the inhibition of PCa bone metastasis and bone remodeling. It has been known that PCa cells in bone metastasis stimulate bone remodeling while bone remodeling facilitates PCa bone metastasis and invasion in the bone, which is known as a vicious cycle of bone remodeling and bone metastasis. Our results showed that isoflavone and BR-DIM inhibited bone cell differentiation, suggesting that isoflavone and BR-DIM could inhibit PCa bone metastasis through disrupting bone remodeling. It has been reported that differentiation of osteoclasts occurs first during bone remodeling when PCa cells metastasize to the bone. Therefore,4-(Aminomethyl)benzoic acid inhibition of osteoclast differentiation is important for interrupting PCa bone metastasis and homing. It is well known that RANK/RANKL/OPG signaling is key regulator for osteoclast differentiation. Our results showed that RANKL treatment could significantly induce differentiation of osteoclast, which is consistent with published report. More importantly, we found that TGF-b could up-regulate the expression of RANKL, leading to the differentiation of osteoclasts. These results are also consistent with report from other investigator, suggesting that the activation of TGF-b, which is commonly seen in advanced PCa, could stimulate the differentiation of osteoclasts, leading to bone resorption and bone remodeling. Moreover, it is well known that TGF-b could also induce EMT, which facilitates PCa progression including invasion and bone metastasis. Importantly, our data showed that isoflavone could attenuate TGF-b induced osteoclast differentiation, and that isoflavone and BR-DIM could regulate the expression of EMT markers, suggesting that these natural agents could be useful for the prevention of PCa progression and bone metastasis. Emerging evidence suggest that microRNAs regulate many physiological and pathological processes. Our results showed that in addition to regulating osteoclasts, RANKL could also up-regulate the expression of miR-92a. The miR-92a has been found to down-regulate the expression of anti-tumor genes, resulting in cancer cell proliferation.