**2. Molecular Mechanisms Induced by Vitamin C**

In our previous study, vitamin C at concentrations of 0.25–2.0 mM significantly induced apoptosis in AML cell lines. Vitamin C induced oxidation of glutathione (GSH) to its oxidized form (GSSG). As a result, H2O2 accumulated in a concentration-dependent manner, in parallel with the induction of apoptosis. The direct role of H2O2 in the induction of apoptosis in AML cells was demonstrated by the finding that catalase could completely abrogate vitamin C-induced apoptosis [17].

A 30-min incubation of NB4 cells with 0–10 mM vitamin C resulted in its uptake in a concentration-dependent manner [17,19]. In accordance with its proposed pro-oxidant activity, vitamin C treatment reduced the GSH/GSSG ratio, which correlated with increased intracellular H2O2 in the NB4 cell line. Levine *et al.* suggested that the effect was due only to extracellular and not intracellular ascorbate, and that ascorbate-mediated cell death was probably due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as the electron donor [13].

Although H2O2 induced by ascorbate was first generated extracellularly, it is possible that it could diffuse across the plasma membrane into the intracellular space. Although studies in yeast and bacteria have shown that diffusion of H2O2 across membranes is limited, some reports have suggested that selected aquaporin homologues from plants and mammals can channel H2O2 across these membranes [20–25]. The susceptibility of cancer cells to ascorbate treatment might therefore be related to the permeability of hydrogen peroxide.

In our studies, vitamin C dramatically increased intracellular GSH oxidation and reactive oxygen species (ROS) levels within 3 h in a concentration-dependent manner. However, this GSH oxidation and the ROS accumulation did not last for a long period of time. After 3 h, the increase in GSH has been observed and hypothesized to be a defense mechanism [18]. No additional ROS accumulation resulted from the change in GSH. However, based upon our studies, the dramatic changes of intracellular oxidation state within 3 h seem to be enough to induce intracellular signaling. The studies showed that treatment with 1 mM vitamin C for only 30–60 min, followed by removal when replacing media, could induce apoptosis in both HL-60 and NB4 cells. This observation is consistent with initiation of apoptosis, resulting from generation of H2O2 after treatment with ascorbate. However, treatment with As2O3 resulted in less ROS accumulation than with vitamin C, and it was not in a concentration-dependent manner. However, ROS accumulation increased up to 24 h, with a long-lasting effect. Treatment with vitamin C combined with As2O3 increased ROS accumulation, as well as sustained the effect for up to 24 h. These results are consistent with cellular data showing that apoptosis induced by As2O3 is evident even at three days [26,27].
