Shirato Ken, Imaizumi Kazuhiko, Miyazawa Keisuke, Takasaki Akira, Mizuguchi Junichiro, Che Xiao-Fang, Akiyama Shinichi, Tomoda Akio
Laboratory of Physiological Sciences, Faculty of Human Sciences, Waseda University, Tokorozawa, Saitama 359-1192, Japan.
Biol Pharm Bull. 2008 Jan;31(1):62-7. doi: 10.1248/bpb.31.62.
The aim of the present study was to investigate the mechanism of apoptosis in human multiple myeloma cell line, U266, caused by 2-aminophenoxazine-3-one (Phx-3). Flow-cytometrical and morphological analyses showed that Phx-3 increased the population of annexin V-positive cells including early stage apoptotic cells and late stage apoptotic cells and induced DNA fragmentation or apoptotic body formation in U266 cells, indicating that Phx-3 induced the apoptosis of U266 cells. Activity of caspase-3 was extensively increased in U266 cells treated with Phx-3 time-dependently within 24 h, but this Phx-3-stimulated activity of the enzyme in the cells was completely cancelled by the addition of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. The addition of z-VAD-fmk almost blocked the apoptotic effect of Phx-3 against U266 cells, indicating that Phx-3-induced apoptosis of U266 cells was dependent on a caspase signaling pathway. Moreover, the apoptosis of U266 cells occurred after the induction of cell cycle arrest of the cells in the S and G(2)/M phase, the loss of mitochondrial membrane potential, and activation of caspase-3 reached maximum, which were caused by Phx-3 within 24 h. These results support the views that the apoptosis of U266 cells caused by Phx-3 may be preceded by the cell cycle arrest, depolarization of mitochondria and activation of caspase-3. These results support the view that Phx-3 may be utilized in future as chemotherapeutic agent against multiple myeloma which is extremely refractory to chemotherapy.
本研究的目的是探究2-氨基吩恶嗪-3-酮（Phx-3）诱导人多发性骨髓瘤细胞系U266凋亡的机制。流式细胞术和形态学分析表明，Phx-3增加了膜联蛋白V阳性细胞的比例，包括早期凋亡细胞和晚期凋亡细胞，并诱导U266细胞出现DNA片段化或凋亡小体形成，这表明Phx-3诱导了U266细胞凋亡。在用Phx-3处理的U266细胞中，半胱天冬酶-3的活性在24小时内随时间依赖性显著增加，但通过添加泛半胱天冬酶抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（z-VAD-fmk），可完全消除Phx-3刺激的该酶在细胞中的活性。添加z-VAD-fmk几乎阻断了Phx-3对U266细胞的凋亡作用，这表明Phx-3诱导的U266细胞凋亡依赖于半胱天冬酶信号通路。此外，U266细胞的凋亡发生在细胞周期停滞于S期和G(2)/M期、线粒体膜电位丧失以及半胱天冬酶-3的激活达到最大值之后，这些都是由Phx-3在24小时内引起的。这些结果支持以下观点：Phx-3诱导的U266细胞凋亡可能先于细胞周期停滞、线粒体去极化和半胱天冬酶-3的激活。这些结果支持这样一种观点，即Phx-3未来可能被用作针对对化疗极度难治的多发性骨髓瘤的化疗药物。
