Phenoxazine derivatives 2-amino-4,4α-dihydro-4α-phenoxazine-3- one and 2-aminophenoxazine-3-one-induced apoptosis through a caspase-independent mechanism in human neuroblastoma cell line NB-1 cells

The aim of the present study was to determine whether phenoxazines such as 2-amino-4,4-α-dihydro-4α-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 μM and 0.5 μM, 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 μM Phx-1 or 20 μM 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.