The cytotoxic and pro-apoptotic effects of a single dose of 2-aminophenoxazine-3-one (Phx-3) or 2-deoxyglucose (2-DG) or of a combined dose of Phx-3 and 2-DG were studied in the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2 and the rat normal hepatocellular cell line RLN-10. The number of viable cells decreased in a dose-dependent manner, when dRLh-84, HepG2 or RLN-10 cells were treated with 2-DG (0.5-20 mM) or Phx-3 (1-50 µM) alone at 37˚C for 48 h. When these cells were treated with 10 mM 2-DG and different concentrations of Phx-3, the number of viable cells decreased dose-dependently and in an additive manner for these agents. A single dose of 2 or 10 µM Phx-3 induced apoptotic morphology characterized by nuclear condensation and cell shrinkage in dRLh-84, HepG2 and RLN-10 cells, while a single dose of 10 mM 2-DG did not. When Phx-3 (2 or 10 µM) treatment was combined with 2-DG (10 mM) treatment in these three cell lines, the cells with apoptotic morphology increased extensively, which was confirmed by flow cytometric analysis. In addition, autophagic morphology characterized by cytosolic vacuole formation was significantly increased in the hepatocellular carcinoma cell lines dRLh-84 and HepG2 but not in the normal hepatocellular cell line RLN-10 after a single dose of Phx-3 or 2-DG or a combined dose of Phx-3 and 2-DG. Furthermore, when dRLh-84 and HepG2 cells were treated with Phx-3 alone or a combined dose of Phx-3 and 2-DG, depolarization of the mitochondria was extensive, but that of the normal cell line RLN-10 was not. These results may imply that the mechanism for the apoptosis of hepatocellular carcinoma cells caused by Phx-3 alone or a combined dose of Phx-3 and 2-DG differs from that of the normal cell line RLN-10. The present results demonstrate that Phx-3 alone may be beneficial for targeting liver cancer and that its anticancer activity may be enhanced by 2-DG. However, a combined dose of Phx-3 and 2-DG may exert adverse effects on normal liver cells, as evidenced by the cytotoxic and pro-apoptotic effects of the combined treatment in the rat normal hepatocellular cell line RLN-10.

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