Compared to normal cells, cancer cells tend to have higher concentrations of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) due to an accelerated cellular metabolism. The high ROS content leaves cancer cells increasingly susceptible to oxidative stress-induced cell death. This susceptibility can be manipulated in selective cancer therapy by further increasing production of ROS or inhibiting peroxide removal systems or a combination of the two. Pharmacological ascorbate (high-dose intravenous ascorbate) has been shown to sensitize pancreatic cancer to ionizing radiation (IR) by increasing production of ROS such as H2O2. Glutathione reductase (GR) and thioredoxin reductase (TrxR) are both important enzymes in peroxide removal systems. GR and TrxR function to recycle key electron donors in the cellular removal of H2O2. We hypothesized that inhibiting the peroxide removal systems via inhibition of GR and TrxR would enhance ascorbate-induced cytotoxicity in pancreatic cancer cells. Inhibition of TrxR activity enhanced ascorbate-induced cytotoxicity in MIA PaCa-2 pancreatic cancer cells. Additionally, knockdown of GR protein expression in combination with pharmacological ascorbate treatment increased MIA PaCa-2 pancreatic cancer cell sensitivity to IR. In MIA PaCa-2 and 403 F1 patient-derived pancreatic cancer cells, inhibition of both TrxR and GR activity combined with pharmacological ascorbate enhanced radiosensitivity. However, this effect was not seen in 339 patient-derived pancreatic cancer cells treated with the same dose of ascorbate. In conclusion, inhibition of TrxR activity, GR activity, or both enhances radiosensitivity and ascorbate-induced cytotoxicity in some, but not all, pancreatic cancer cell lines. Treatments combining ascorbate with inhibition of H2O2 removal may be an effective strategy for treatment of pancreatic adenocarcinoma.

With new insights on mechanism, there is renewed interest in the use of pharmacological ascorbate (AscH-) in cancer therapy. The generation of H2O2 with AscH- acting as an electron donor to O2 is central to AscH- -induced cytotoxicity. We hypothesized that catalytic manganoporphyrins (MnPs) would increase the rate of oxidation of AscH- thereby increasing the flux of H2O2, resulting in increased cytotoxicity. We tested three different MnPs: MnTBAP, MnT2EPyP, and MnT4MPyP, which represent a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP had the greatest effect on increasing the rate of oxidation of AscH-, as seen by the concentration of ascorbate radical [Asc*-] and rate of oxygen consumption. MnPs and AscH-, when combined at concentrations that had minimal effects alone, synergistically increased the cytotoxicity as seen by decreased clonogenic survival in human pancreatic cancer cell lines. Catalase, but not superoxide dismutase, reversed the cytotoxicity of AscH- and MnP, consistent with an H2O2-mediated mechanism. In addition, there was a marked increase in the steady-state concentration of ascorbate radical upon the addition of MnPs to whole blood ex vivo from mice infused with ascorbate as well as from patients treated with pharmacologic ascorbate. The combination of MnT4MPyP with ascorbate inhibited in vivo tumor growth. We conclude that MnPs can increase the rate of oxidation of AscH-, leading to an increased flux of H2O2 resulting in increased ascorbate-induced cytotoxicity