Anti-tumor effects of a novel phenoxazinone, 2-amino-4,4-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx), which was synthesized by the reaction of 2-amino-5-methylphenol with bovine hemoglobin, were studied in terms of suppression of the proliferation of human lung carcinoma cells and apoptosis induction. When Phx was added to cultures of the human lung carcinoma cell lines A549 (adenocarcinoma) and H226 (squamous carcinoma), it caused the growth inhibition and the death of these cells. Phx also fragmented the DNA of these cells to oligonucleosomal-sized fragments, which is characteristic of the apoptosis, dependent on the dose and exposure time. The cellular death caused by the administration of Phx was partially reversed by the addition of Z-VAD-fmk, a caspase family inhibitor. Present results suggest that Phx demonstrates anti-cancer activity against human lung carcinoma cell lines A549 and H226, by inhibiting growth and inducing apoptosis.

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Ceramides (Cers) have recently been identified as key signaling molecules that mediate biological functions such as cell growth, differentiation, senescence, apoptosis, and autophagy. However, the functions of Cer accumulation in necrotic cell death remain unknown. The aim of this study was to clarify the relationship between Cer accumulation with inhibition of the conversion pathway of Cer and concomitant necrotic cell death. In order to minimize the effect of apoptosis against necrotic cell death, A549 cells having the inhibiting effect of caspase 9 by survivin were used in this study. Consequently, Cer accumulation in A549 cells would likely be associated with a pathway other than the mitochondrial caspase-dependent pathway of apoptosis. Here, we showed that the dual addition of a glucosyl-Cer synthase inhibitor and a ceramidase inhibitor to A549 cell culture induced palmitoyl-Cer accumulation with Cer synthase 5 expression and necrotic cell death with lysosomal rupture together with leakage of cathepsin B/alkalization after 2–3 h, although it is unknown in this study whether the necrotic cell death was caused by the lysosomal rupture. This Cer accumulation was followed by a steep increase in sphinganine base levels via the activation of serine palmitoyltransferase activity brought about by the increase in palmitoyl-coenzyme A concentration as a substrate after 5–6 h. The increase in palmitoyl-coenzyme A concentration was achieved by activation of the fatty acid synthetic pathway from acetyl coenzyme A.

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