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The aim of the present study was to determine whether phenoxazines such as 2-amino-4,4-alpha-dihydro-4alpha-phenoxazine-3-one (Phx-1) and 2-aminophenoxazine-3-one (Phx-3) may suppress the proliferation of human neuroblastoma cell line, NB-1 that is refractory to chemotherapeutic agents, inducing apoptosis through the activation of caspase pathway or not. Phx-1 and Phx-3 suppressed the proliferation of NB-1 cells extensively dependent on dose and time. The IC50 of Phx-1 and Phx-3 was about 20 microM and 0.5 microM, respectively, when the cells were treated with Phx-1 or Phx-3 for 72 h. Phx-1 and Phx-3 caused the mixed types of cell death-apoptosis and necrosis-in NB-1 cells, which was detected by flow cytometry. The induction of apoptosis/necrosis caused by these phenoxazines seemed to be correlated dominantly with the caspase independent pathway, because the increased activity of effector caspase 3/7 in NB-1 cells caused by 50 microM Phx-1 or 20 microM Phx-3 was completely cancelled by the addition of z-VAD-fmk, a pan-caspase inhibitor, but such phenoxazines-suppressed viability of NB-1 cells was not recovered to normal levels by this inhibitor. The results of this study demonstrate that Phx-1 and Phx-3 have antitumor activity against the neuroblastoma cell line, NB-1, though the IC50 was extremely low for Phx-3, inducing the mixed types of cell death, apoptosis and necrosis, caspase-independently.

biological and pharmaceutical bulletin

Phenoxazine Derivatives 2-Amino-4,4.ALPHA.-dihydro-4.ALPHA.-phenoxazine-3-one and 2-Aminophenoxazine-3-one-Induced Apoptosis through a Caspase-Independent Mechanism in Human Neuroblastoma Cell Line NB-1 Cells