Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate.

AIMS: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. RESULTS: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3- and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. INNOVATION: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. CONCLUSIONS: The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects.

Anticancer therapeutic potential of Mn porphyrin/ascorbate system.

Ascorbate (Asc) as a single agent suppressed growth of several tumor cell lines in a mouse model. It has been tested in a Phase I Clinical Trial on pancreatic cancer patients where it exhibited no toxicity to normal tissue yet was of only marginal efficacy. The mechanism of its anticancer effect was attributed to the production of tumoricidal hydrogen peroxide (H2O2) during ascorbate oxidation catalyzed by endogenous metalloproteins. The amount of H2O2 could be maximized with exogenous catalyst that has optimized properties for such function and is localized within tumor. Herein we studied 14 Mn porphyrins (MnPs) which differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity. Such properties affect the in vitro and in vivo ability of MnPs (i) to catalyze ascorbate oxidation resulting in the production of H2O2; (ii) to subsequently employ H2O2 in the catalysis of signaling proteins oxidations affecting cellular survival pathways; and (iii) to accumulate at site(s) of interest. The metal-centered reduction potential of MnPs studied, E1/2 of Mn(III)P/Mn(II)P redox couple, ranged from -200 to +350 mV vs NHE. Anionic and cationic, hydrophilic and lipophilic as well as short- and long-chained and bulky compounds were explored. Their ability to catalyze ascorbate oxidation, and in turn cytotoxic H2O2 production, was explored via spectrophotometric and electrochemical means. Bell-shape structure-activity relationship (SAR) was found between the initial rate for the catalysis of ascorbate oxidation, vo(Asc)ox and E1/2, identifying cationic Mn(III) N-substituted pyridylporphyrins with E1/2>0 mV vs NHE as efficient catalysts for ascorbate oxidation. The anticancer potential of MnPs/Asc system was subsequently tested in cellular (human MCF-7, MDA-MB-231 and mouse 4T1) and animal models of breast cancer. At the concentrations where ascorbate (1mM) and MnPs (1 or 5 µM) alone did not trigger any alteration in cell viability, combined treatment suppressed cell viability up to 95%. No toxicity was observed with normal human breast epithelial HBL-100 cells. Bell-shape relationship, essentially identical to vo(Asc)oxvs E1/2, was also demonstrated between MnP/Asc-controlled cytotoxicity and E1/2-controlled vo(Asc)ox. Magnetic resonance imaging studies were conducted to explore the impact of ascorbate on T1-relaxivity. The impact of MnP/Asc on intracellular thiols and on GSH/GSSG and Cys/CySS ratios in 4T1 cells was assessed and cellular reduction potentials were calculated. The data indicate a significant increase in cellular oxidative stress induced by MnP/Asc. Based on vo(Asc)oxvs E1/2 relationships and cellular toxicity, MnTE-2-PyP(5+) was identified as the best catalyst among MnPs studied. Asc and MnTE-2-PyP(5+) were thus tested in a 4T1 mammary mouse flank tumor model. The combination of ascorbate (4 g/kg) and MnTE-2-PyP(5+) (0.2mg/kg) showed significant suppression of tumor growth relative to either MnTE-2-PyP(5+) or ascorbate alone. About 7-fold higher accumulation of MnTE-2-PyP(5+) in tumor vs normal tissue was found to contribute largely to the anticancer effect.