Interpreting Intracellular Hydrogen Peroxide in Cancer Cells to Understand Cancer Susceptibility to Pharmacological Ascorbate Therapy

The intravenous delivery of pharmacological ascorbate (P-AscH-) has recently been demonstrated to be a successful adjuvant in the treatment of some cancers. Administered as a series of infusions, P-AscH- generates high fluxes of extracellular hydrogen peroxide (H2O2), which is toxic to certain cancer cells while not affecting normal. In vitro studies indicate that cancer cells have a wide range in susceptibility to P-AscH- and subsequently to extracellular H2O2. The resulting intracellular H2O2 concentration is believed to accumulate differently in susceptible cancer cells as compared to non-susceptible cells. It is hypothesized that intracellular H2O2 concentration has a steady-state value that is significant for cell susceptibility and independent of cell type. Although this has been alluded to, this value has yet to be quantified. Further, the variations in cell parameters (i.e. membrane permeability via peroxiporins, catalase activity, etc.) for various cells are expected to be significant enough to alter intracellular H2O2 concentration, thereby impacting cell susceptibility. A steady-state model was developed which elucidates the parameter contribution to intracellular H2O2 accumulation. The intracellular H2O2 concentrations during P-AscH- therapy was quantified for pancreatic normal (H6c7; ascorbate non-responding), adenocarcinoma (MIA PaCa-2; ascorbate susceptible) and glioblastoma U-87 (non-responding), T98G (moderately susceptible) and LN-229 (highly susceptible) cell lines. Recognizing that MIA PaCa-2 has an enhanced expression of aquaporin-3 (AQP3) and the significance of AQP3 to plasma membrane permeability to H2O2, silenced AQP3 was also investigated. Interestingly, an increase in surviving fraction was observed for the silenced cells in clonogenic studies using therapeutic H2O2 concentrations. These results imply that cell-susceptibility to ascorbate therapy is significantly coupled to the plasma membrane permeability to H2O2, and in particular, elevated expressions of peroxiporins. Ultimately, this work provides insight to what targets are appropriate for improving P-AscH- therapy. Further, our mathematical results contradict the hypothesis that a unique intracellular H2O2 was sufficient for a specific clonogenic response. This aligns with recent work revealing that the combination of redox-active labile iron and high intracellular H2O2 concentration is the necessary and sufficient condition for cellular ascorbate-susceptibility. Quantifying the relationship of this combination to the clonogenic response is the subject of future research