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Purpose: Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH-; intravenous administration achieving mM plasma concentrations) selectively enhances H2O2-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH- could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues.Methods and Materials: Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH-. Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy × 3) ± the ATM inhibitor KU60019 ± P-AscH-. Intestinal injury, oxidative damage, and transforming growth factor β immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH-. Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H2O2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene.Results: KU60019 with P-AscH- enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H2O2-dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH- markedly reduced intestinal toxicity and oxidative damage with KU60019.Conclusions: We provide evidence that redox modulating drugs, such as P-AscH-, may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues. [ABSTRACT FROM AUTHOR]

Determine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage. Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue. To assess free radical production as a result of traumatic injury, we measured biomolecular free radical generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2 J/cm2 impact to swine articular cartilage explants. This technique allows visualization of free radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant increases with impact injury, particularly at sites of cartilage cracking. Increases remain in the absence of live chondrocytes but are diminished; thus, they appear to be a cell-dependent and -independent feature of injury. We then modulated the extracellular environment with a pulse of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology. Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study directly confirms the production of biomolecular free radicals from articular trauma, providing a rigorous characterization of their formation by injury. [Display omitted] • Injury creates free radicals at sites of cartilage cracking. • These free radicals are cell dependent and independent. • Disrupting heparin binding concentrates this radical damage along the crack. [ABSTRACT FROM AUTHOR]

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50–150 nM) combined with physiological levels of Cu (15 μM CuSO 4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O 2 , relative to 4 or 21% O 2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O 2 vs. 21% O 2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism. Image 1 • Disulfiram causes selective toxicity in cancer cells vs. normal cells. • Disulfiram toxicity is enhanced at hypoxia. • Disulfiram causes selective radio-chemo-sensitization in cancer cells and hypoxia. • O 2 •-, H 2 O 2 , lipid oxidation, and Cu retention/efflux (via ATP7B) govern toxicity. [ABSTRACT FROM AUTHOR]