Renewed interest in using pharmacological ascorbate (AscH−) to treat cancer has prompted interest in leveraging its cytotoxic mechanism of action. A central feature of AscH− action in cancer cells is its ability to act as an electron donor to O2 for generating H2O2. We hypothesized that catalytic manganoporphyrins (MnPs) would increase AscH− oxidation rates, thereby increasing H2O2 fluxes and cytotoxicity. Three different MnPs were tested (MnTBAP, MnT2EPyP, and MnT4MPyP) exhibiting a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP exerted the greatest effect on increasing the rate of AscH− oxidation as determined by the concentration of ascorbate radical [Asc•−] and the rate of oxygen consumption. At concentrations that had minimal effects alone, combining MnPs and AscH− synergized to decrease clonogenic survival in human pancreatic cancer cells. This cytotoxic effect was reversed by catalase, but not superoxide dismutase, consistent with a mechanism mediated by H2O2. MnPs increased steady-state concentrations of Asc•− upon ex vivo addition to whole blood obtained either from mice infused with AscH− or patients treated with pharmacologic AscH−. Lastly, tumor growth in vivo was inhibited more effectively by combining MnT4MPyP with AscH−. We concluded that MnPs increase the rate of oxidation of AscH− to leverage H2O2 flux and ascorbate-induced cytotoxicity.

Labile iron, i.e. iron that is weakly bound and is relatively unrestricted in its redox activity, has been implicated in both the pathogenesis as well as treatment of cancer. Two cancer treatments where labile iron may contribute to their mechanism of action are pharmacological ascorbate and ionizing radiation (IR). Pharmacological ascorbate has been shown to have tumor-specific toxic effects due to the formation of hydrogen peroxide. By catalyzing the oxidation of ascorbate, labile iron can enhance the rate of formation of hydrogen peroxide; labile iron can also react with hydrogen peroxide. Here we have investigated the magnitude of the labile iron pool in tumor and normal tissue. We also examined the ability of pharmacological ascorbate and IR to change the size of the labile iron pool. Although a significant amount of labile iron was seen in tumors (MIA PaCa-2 cells in athymic nude mice), higher levels were seen in murine tissues that were not susceptible to pharmacological ascorbate. Pharmacological ascorbate and irradiation were shown to increase the labile iron in tumor homogenates from this murine model of pancreatic cancer. As both IR and pharmacological ascorbate may rely on labile iron for their effects on tumor tissues, our data suggest that pharmacological ascorbate could be used as a radio-sensitizing agent for some radio-resistant tumors.

Pharmacological ascorbate (AscH−) selectively induces cytotoxicity in pancreatic cancer cells vs. normal cells via the generation of extracellular hydrogen peroxide (H2O2), producing double-stranded DNA breaks and ultimately cell death. Catalytic manganoporphyrins (MnPs) can enhance ascorbate-induced cytotoxicity by increasing the rate of AscH− oxidation and therefore the rate of generation of H2O2. We hypothesized that combining MnPs and AscH− with the chemotherapeutic agent gemcitabine would further enhance pancreatic cancer cell cytotoxicity without increasing toxicity in normal pancreatic cells or other organs. Redox active MnPs were combined with AscH− and administered with or without gemcitabine to human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin pentachloride) and MnT4MPyP (Mn(III)tetrakis(N-methylpyridinium-4-yl) porphyrin pentachloride) were investigated. Clonogenic survival was significantly decreased in all pancreatic cancer cell lines studied when treated with MnP + AscH− + gemcitabine, whereas non-tumorigenic cells were resistant. The concentration of ascorbate radical (Asc•−, an indicator of oxidative flux) was significantly increased in treatment groups containing MnP and AscH−. Furthermore, MnP + AscH− increased double stranded DNA breaks in gemcitabine treated cells. These results were abrogated by extracellular catalase, further supporting the role of the flux of H2O2. In vivo growth was inhibited and survival increased in mice treated with MnT2EPyP, AscH−, and gemcitabine without a concomitant increase in systemic oxidative stress. These data suggest a promising role for the use of MnPs in combination with pharmacologic AscH− and chemotherapeutics in pancreatic cancer.

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<p>PDF file - 102K, Table 1. AscH- and MnT4MPyP are synergistic as seen by Combination Index (CI) and Dose Reduction Index (DRI). Table 2. Summary statistics for tumor volume measurements in the generalized estimating equations analysis.</p>

<p>PDF file - 123K, Supplementary Figure 1. AsPC-1 and Panc-1 cells treated with MnT2EPyP or MnTBAP in combination with AscH-.</p>