Mitochondrial depolarization and apoptosis associated with sustained activation of c-jun-N-terminal kinasein the human multiple myeloma cell line U266 induced by 2-aminophenoxazine-3-one

Abstract

Abstract.We investigated the involvement of c-jun-N-terminal kinase (JNK) in mitochondrial depolarization and apoptosis in a human multiple myeloma cell line, U266, treated with 2-aminophenoxazine (Phx-3). It was found that, with Phx-3 administration to U266 cells, JNK was phosphorylated 2 and 7.5-fold at 6 and 24 h, respectively, compared to the Phx-3-free control. This increasing activation of JNK in U266 cells with Phx-3 correlated with cellular disorders, such as mitochondrial depolarization and cellular apoptosis. When the JNKspecific inhibitor SP6000125 was administered to the U266 cells together with Phx-3, the number of cells exhibiting mitochondrial depolarization and cellular apoptosis was significantly reduced. These results suggest that JNK activation in human multiple myeloma U266 cells may be closely associated with mitochondrial depolarization and apoptosis. IntroductionIt is generally accepted that the loss of mitochondrial membrane integrity causes the release of cytochrome c, caspase-3 activation and cellular apoptosis (1-3). The agents employed to induce these cellular events in cancer cells may be favorable candidates for the treatment of cancer (4,5). We previously found that 2-aminophenoxazine-3-one (Phx-3), produced by the reactions of o-aminophenol and bovine hemoglobin solutions (6), induced apoptosis in various cancer cells, including the gastric cancer cell lines MKN45 and KATO III (7), the human glioblastoma cell lines A-172 and U-251 (8), human neuroblastoma NB-1 cells (9), and human multiple myeloma U266 cells (10), regardless of caspase-3-dependent or -independent pathways. We previously reported that Phx-3 induced mitochondrial depolarization and apoptosis in human myeloma U266 cells (10); the caspase-3-dependent apoptosis of the cells was preceded by mitochondrial depolarization, induced by Phx-3. However, the mechanism by which Phx-3 caused mitochondrial depolarization in the cells was not clarified.It has been demonstrated that c-jun-N-terminal kinase (JNK) plays a crucial role in the activation of the intrinsic apoptotic pathway mediated by mitochondria (11). Tsuruta et al (12) demonstrated that JNK promotes Bax translocation to the mitochondria, resulting in cytochrome c release and cellular apoptosis. JNK was activated in multiple myeloma cell lines, including U266 cells, when treated with arsenic trioxide (ATO). Its activation was sustained for a long time and was associated with the ATO-induced apoptosis of U266 cells (13). Therefore, it is conceivable that the Phx-3-induced mitochondrial depolarization and apoptosis in U266 cells that we observed previously (10) is associated with JNK activation. In the present study, we investigated the role of JNK in Phx-3-induced mitochondrial depolarization and cellular apoptosis in human multiple myeloma U266 cells. Materials and methodsPreparation of 2-aminophenoxazine-3-one. 2-Aminophenoxazine-3-one (Phx-3) was prepared according to the method described by Shimizu et al (6). The chemical structure of Phx-3 is shown in Fig. 1. Phx-3...

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