Estrogen analogues are promising drugs for postmenopausal osteoporosis, but because of their possible side effects such as increased risk of cancer, estrogens which exert their estrogenic effects selectively on bone are desired. It has been shown that rhein inhibits osteoclast formation and bone resorption activity and has an antitumor role in several types of cancers. Having found that rhein had high affinity for the bone mineral, we synthesized estrone-rhein hybrid compounds and confirmed that one of these hybrid compounds, LC, exhibited a selective profile in the bone and prevented bone loss but had no effect on endometrium growth in ovariectomized rats. However, the mechanisms underlying its actions on human bone cells have not been well defined. Here we show that LC concurrently stimulates osteoprotegerin (OPG) and inhibits receptor activator of nuclear factor-κB ligand (RANKL) and Interleukin-6 (IL-6) production by human osteoblastic MG-63 cells containing two estrogen receptor (ER) isotypes. Treatment with the ER antagonist ICI 182,780 abrogates the above actions of LC on osteoblast-derived cells. Using small interfering double-stranded RNAs (siRNA) technology, we further demonstrate that the effects of LC on IL-6 production are mediated by both ERα and ERβ but those on OPG and RANKL expression primarily by ERα. Furthermore, we also demonstrate that LC functions at least partially through activation of the classic estrogen response element (ERE) pathway as well as Ras/MEK/ERK and PI3K/Akt signaling. The effect of LC on bone is due to not only its estrogenic activity but also action of its rhein moiety. Also, this compound shows much weaker effect on breast epithelial cell growth than that of estrone. Therefore, using rhein for conjugating compounds is a promising method of effectively targeting estrogens to the bone.
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