title: Apoptosis induction preceded by mitochondrial depolarization in multiple myeloma cell line U266 by 2-aminophenoxazine-3-one.
abstract: 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.
authors.affiliation: Laboratory of Physiological Sciences, Faculty of Human Sciences, Waseda University, Tokorozawa, Saitama 359-1192, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Shirato
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Imaizumi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyazawa
authors.forename: Akira
authors.initials: A
authors.lastname: Takasaki
authors.forename: Junichiro
authors.initials: J
authors.lastname: Mizuguchi
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Che
authors.forename: Shinichi
authors.initials: S
authors.lastname: Akiyama
authors.forename: Akio
authors.initials: A
authors.lastname: Tomoda
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000473:pathology; D009336:Necrosis; D010078:Oxazines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D010078:Oxazines; C005512:3-aminophenoxazone; D053148:Caspase 3