Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular Ca2+ increase and generation of
reactive
oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular Ca2+ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a Ca2+
activated
protein, and Ca2+/ calmodulin-dependent protein kinase Ⅱ in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of Ca2+/calmodulin-dependent
protein kinase Ⅱ (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed
by
intracellular Ca2+ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin
and
Ca2+/calmodulin-dependent protein kinase II in HepG2 cells.
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