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

The latency of peroxisomal catalase in terms of effectiveness factor for pancreatic and glioblastoma cancer cell lines in the presence of high concentrations of H2O2: Implications for the use of pharmacological ascorbate in cancer therapy.

Previous research has identified variation in cancer cell line response to high levels of extracellular H2O2 (eH2O2) exposure. This directly contributes to our understanding cellular efficacy of pharmacological ascorbate (P-AscH-) therapy. Here we investigate the factors contributing to latency of peroxisomal catalase of a cell and the importance of latency in evaluating cell exposure to eH2O2. First, we develop a mathematical framework for the latency of catalase in terms of an effectiveness factor, ηeff, to describe the catalase activity in the presence of high levels of eH2O2. A simplified relationship emerges, [Formula: see text] when mprp/Dij≪1, where mp,rp, and [Formula: see text] are the experimentally determined peroxisome permeability, average peroxisome radius, and the pseudo first-order reaction rate constant, respectively. [Formula: see text] is the catalase concentration in the peroxisome and k2=1.7x107M-1s-1. Next, previously published parameters are used to determine the latency effect of the cell lines: normal pancreatic cells (H6c7), pancreatic cancer cells (MIA PaCa-2), and glioblastoma cells (LN-229, T98G, and U-87), all which vary in their susceptibility to exposure to high eH2O2. The results show that effectiveness is not significantly different except for the most susceptible, MIA PaCa-2 cell line, which is higher when compared to all other cell lines. This result is counterintuitive and further implies that latency, as a single parameter, is ineffective in forecasting cell line susceptibility to P-AscH- therapy equivalent eH2O. Thus, further research remains necessary to identify why cancer cells vary in susceptibility to P-AscH- therapy

The latency of peroxisomal catalase in terms of effectiveness factor for pancreatic and glioblastoma cancer cell lines in the presence of high concentrations of H2O2: Implications for the use of pharmacological ascorbate in cancer therapy.

Previous research has identified variation in cancer cell line response to high levels of extracellular H2O2 (eH2O2) exposure. This directly contributes to our understanding cellular efficacy of pharmacological ascorbate (P-AscH-) therapy. Here we investigate the factors contributing to latency of peroxisomal catalase of a cell and the importance of latency in evaluating cell exposure to eH2O2. First, we develop a mathematical framework for the latency of catalase in terms of an effectiveness factor, ηeff, to describe the catalase activity in the presence of high levels of eH2O2. A simplified relationship emerges, [Formula: see text] when mprp/Dij≪1, where mp,rp, and [Formula: see text] are the experimentally determined peroxisome permeability, average peroxisome radius, and the pseudo first-order reaction rate constant, respectively. [Formula: see text] is the catalase concentration in the peroxisome and k2=1.7x107M-1s-1. Next, previously published parameters are used to determine the latency effect of the cell lines: normal pancreatic cells (H6c7), pancreatic cancer cells (MIA PaCa-2), and glioblastoma cells (LN-229, T98G, and U-87), all which vary in their susceptibility to exposure to high eH2O2. The results show that effectiveness is not significantly different except for the most susceptible, MIA PaCa-2 cell line, which is higher when compared to all other cell lines. This result is counterintuitive and further implies that latency, as a single parameter, is ineffective in forecasting cell line susceptibility to P-AscH- therapy equivalent eH2O. Thus, further research remains necessary to identify why cancer cells vary in susceptibility to P-AscH- therapy

Peroxiporin Expression Is an Important Factor for Cancer Cell Susceptibility to Therapeutic H2O2: Implications for Pharmacological Ascorbate Therapy.

Cancer cell toxicity to therapeutic H2O2 varies widely depending on cell type. Interestingly, it has been observed that different cancer cell types have varying peroxiporin expression. We hypothesize that variation in peroxiporin expression can alter cell susceptibility to therapeutic H2O2 concentrations. Here, we silence peroxiporin aquaporin-3 (AQP3) on the pancreatic cancer cell line MIA PaCa-2 and compare clonogenic survival response to the wild-type. The results showed a significantly higher surviving fraction in the clonogenic response for siAQP3 MIA PaCa-2 cells at therapeutic H2O2 doses (P < 0.05). These results suggest that peroxiporin expression is significant in modulating the susceptibility of cancer cells to ascorbate therapy

Calculated cell-specific intracellular hydrogen peroxide concentration: Relevance in cancer cell susceptibility during ascorbate therapy.

The high extracellular hydrogen peroxide (H2O2) concentrations generated during pharmacological ascorbate (P-AscH-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H2O2 concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H2O2 concentration values. The steady-state variation in intracellular H2O2 concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H2O2 concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H2O2 concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H2O2 concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H2O2 concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H2O2 concentration plays a key role in cellular susceptibility to P-AscH- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex

Silencing AQP3 with siRNA reduces AQP expression by a factor of 10 for MIA PaCa-2 cells.

<p>Verification for silencing of peroxiporin AQP3 on the plasma membrane of cancer cells is confirmed by the observed shift in the AQP3 apparent signal. An AQP3 specific signal is shown to exhibit a positive frequency of 91.7 (orange curve) for the unmodified MIA PaCa-2 cancer cells sampled (8,036 cells). After silencing AQP3 for MIA PaCa-2 cancer cells (siAQP3 MIA PaCa-2), the signal shifts to display a positive signal frequency of 59.8 (red curve) for sampled cells (8,067 cells). The peak of the unmodified MIA PaCa-2 cells (orange) displays an average positive signal around 200 whereas the siAQP3 MIA PaCa-2 cells (red) displays an average around 20. This shift in AQP3 signal demonstrates a decrease by a factor of 10 in AQP expression for the silenced MIA PaCa-2 cells when compared to unmodified MIA PaCa-2. The blue curve is the negative IgG control. Data were generated by immunofluorescence tagging, detection through flow cytometry, and analyzed through FlowJo (Treestar, Inc., Ashland, OR, USA).</p