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Background: Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have poor 5-year survival. Pharmacological ascorbate (P-AscH - , high dose, intravenous, vitamin C) has shown promise as an adjunct to chemotherapy. We hypothesized adding P-AscH - to gemcitabine and nab-paclitaxel would increase survival in patients with metastatic PDAC., Methods: Patients diagnosed with stage IV pancreatic cancer randomized 1:1 to gemcitabine and nab-paclitaxel only (SOC, control) or to SOC with concomitant P-AscH - , 75 g three times weekly (ASC, investigational). The primary outcome was overall survival with secondary objectives of determining progression-free survival and adverse event incidence. Quality of life and patient reported outcomes for common oncologic symptoms were captured as an exploratory objective. Thirty-six participants were randomized; of this 34 received their assigned study treatment. All analyses were based on data frozen on December 11, 2023., Results: Intravenous P-AscH - increased serum ascorbate levels from micromolar to millimolar levels. P-AscH - added to the gemcitabine + nab-paclitaxel (ASC) increased overall survival to 16 months compared to 8.3 months with gemcitabine + nab-paclitaxel (SOC) (HR = 0.46; 90 % CI 0.23, 0.92; p = 0.030). Median progression free survival was 6.2 (ASC) vs. 3.9 months (SOC) (HR = 0.43; 90 % CI 0.20, 0.92; p = 0.029). Adding P-AscH - did not negatively impact quality of life or increase the frequency or severity of adverse events., Conclusions: P-AscH - infusions of 75 g three times weekly in patients with metastatic pancreatic cancer prolongs overall and progression free survival without detriment to quality of life or added toxicity (ClinicalTrials.gov number NCT02905578)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ferroptosis is a form of cell death characterized by a pro-oxidative cellular milieu and iron-dependent lipid peroxidation. Ferroptosis has been implicated in various forms of liver injury, in keeping with the major role of the liver in iron metabolism. Limited research has addressed potential differences in ferroptosis mediators with age and sex, especially in an in vivo model. The goal of this investigation was to evaluate hepatic labile iron and mediators of ferroptosis with ageing in both sexes. Because female animals generally display greater antioxidant defences than males, we hypothesized that females would display a phenotype resistant to ferroptosis. Here, we determined iron contents, protein expression of ferroptosis mediators and measures of oxidative injury in liver samples from 12- and 24-month-old male and female Fischer 344 rats. In comparison to males, the livers of female rats at both ages contained more non-haem iron, which was associated with greater ferritin heavy chain expression and attenuated expression of transferrin receptor-1. In female rats, the 24-month-old group had higher contents of thiobarbituric acid reactive substances compared with their 12-month-old counterparts, yet similar contents of labile iron. These results suggest a disconnect between labile iron contents and oxidative injury with age. Female animals also displayed greater expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), a modulator of ferroptosis, and greater abundance of high molecular weight 4-hydroxnonenal-modified proteins. These results demonstrate clear differences in iron and ferroptosis mediators between sexes and suggest that female rats of this strain might be more susceptible to ferroptosis., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The toxicity of ionizing radiation limits its effectiveness in the treatment of pancreatic ductal adenocarcinoma. Pharmacologic ascorbate (P-AscH - ) has been shown to radiosensitize pancreatic cancer cells while simultaneously radioprotecting normal cells. We hypothesize that P-AscH - protects the small intestine while radiosensitizing pancreatic cancer cells partially through an oxidative stress mechanism. Duodenal samples from pancreaticoduodenectomy specimens of patients who underwent radio-chemotherapy ± P-AscH - and mouse tumor and jejunal samples treated with radiation ± P-AscH - were evaluated. Pancreatic cancer and non-tumorigenic cells were treated with radiation ± P-AscH - to assess lipid peroxidation. To determine the mechanism, pancreatic cancer cells were treated with selenomethionine or RSL3, an inhibitor of glutathione peroxidase 4 (GPx4). Radiation-induced decreases in villi length and increases in 4-HNE immunofluorescence were reversed with P-AscH - in human duodenum. In vivo, radiation-induced decreases in villi length and increased collagen deposition were reversed in P-AscH - -treated jejunal samples. P-AscH - and radiation increased BODIPY oxidation in pancreatic cancer cells but not in non-tumorigenic cells. Selenomethionine increased GPx4 protein and activity in pancreatic cancer and reversed P-AscH - -induced toxicity and lipid peroxidation. RSL3 treatment inhibited GPx4 activity and increased lipid peroxidation. Differences in oxidative stress may play a role in radioprotecting normal cells while radiosensitizing pancreatic cancer cells when treated with P-AscH - .

Purpose: Pharmacologic ascorbate (P-AscH-) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH- with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH- in humans., Patients and Methods: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker., Results: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7-26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems., Conclusions: P-AscH- combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255., (©2023 American Association for Cancer Research.)

Ascorbate (vitamin C) can rapidly oxidize in many near-neutral pH, aqueous solutions. We report on the stability of ascorbate solutions prepared for infusion into patients using standard pharmacy protocols, for example, 75 g of ascorbate/L in water for infusion. The concentration of ascorbate was monitored for changes over time using direct UV-Vis spectroscopy. The pH of the solution was about 5.7 with no significant change over 24 h. There was only an approximate loss of 1% per day over the first 3 days of storage. This information allows decisions on how far ahead of need such preparations can be made. We also provide laboratory approaches to minimize or control the rate of oxidation of ascorbate solutions for use in chemical and biochemical studies as well as preclinical animal studies. The goal is to have the amount of ascorbate intended to be used in experiments be the actual amount available., Competing Interests: Declaration of Competing Interest The authors declare no competing interests.

Nitric oxide (NO • ) generated by nitric oxide synthases is involved in many physiological and pathophysiological processes. However, non-enzymatic formation of NO • also occurs in vivo . Here we investigated the production of NO • from nitrite, as facilitated by ascorbate, over the pH range of 2.4-7.4. Using a nitric oxide electrode, we observed at low pH a rapid generation of NO • from nitrite and ascorbate that slows with increasing pH. The formation of NO • was confirmed by its reaction with oxyhemoglobin. In the ascorbate/nitrite system a steady-state level of NO • was achieved, suggesting that a futile redox cycle of nitrite-reduction by ascorbate and NO • -oxidation by dioxygen was established. However, at pH-values of around 7 and greater, the direct reduction of nitrite by ascorbate is very slow; thus, this route to the non-enzymatic production of NO • is not likely to be significant process in vivo in environments having a pH around 7.4. The production of nitric oxide by nitrite and ascorbate would be important only in areas of lower pH, e.g . stomach/digestive system, sites of inflammation, and areas of hypoxia such as tumor tissue. In patients receiving very large doses of ascorbate delivered by intravenous infusion, plasma levels of ascorbate on the order of 20 - 30 mM can be achieved. After infusion, levels of nitrate and nitrite in plasma were unchanged. Thus, in blood and tissue that maintain a pH of about 7.4, the reduction of nitrite to nitric oxide by ascorbate appears to be insignificant, even at very large, pharmacological levels of ascorbate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Recent studies have demonstrated an important role for vitamin C in the epigenetic regulation of cancer-related genes via DNA demethylation by the ten-eleven translocation (TET) methylcytosine dioxygenase enzymes. DNA methyltransferase (DNMT) reverses this, increasing DNA methylation and decreasing gene expression. Dual oxidase (DUOX) enzymes produce hydrogen peroxide (H 2 O 2 ) in normal pancreatic tissue but are silenced in pancreatic cancer (PDAC). Treatment of PDAC with pharmacologic ascorbate (P-AscH - , intravenous, high dose vitamin C) increases DUOX expression. We hypothesized that inhibiting DNMT may act synergistically with P-AscH - to further increase DUOX expression and cytotoxicity of PDAC. PDAC cells demonstrated dose-dependent increases in DUOX mRNA and protein expression when treated with DNMT inhibitors. PDAC cells treated with P-AscH - + DNMT inhibitors demonstrated increased DUOX expression, increased intracellular oxidation, and increased cytotoxicity in vitro and in vivo compared to either treatment alone. These findings suggest a potential therapeutic, epigenetic mechanism to treat PDAC.

Pharmacological ascorbate (P-AscH - ; high dose given intravenously) generates H 2 O 2 that is selectively cytotoxic to cancer compared to normal cells. The RAS-RAF-ERK1/2 is a major signaling pathway in cancers carrying RAS mutations and is known to be activated by H 2 O 2 . Activated ERK1/2 also phosphorylates the GTPase dynamin-related protein (Drp1), which then stimulates mitochondrial fission. Although early generation of H 2 O 2 leads to cytotoxicity of cancer cells, we hypothesized that sustained increases in H 2 O 2 activate ERK-Drp1 signaling, leading to an adaptive response; inhibition of this pathway would enhance the toxicity of P-AscH - . Increases in phosphorylated ERK and Drp1 induced by P-AscH - were reversed with genetic and pharmacological inhibitors of ERK and Drp1, as well as in cells lacking functional mitochondria. P-AscH - increased Drp1 colocalization to mitochondria, decreased mitochondrial volume, increased disconnected components, and decreased mitochondrial length, suggesting an increase in mitochondrial fission 48 h after treatment with P-AscH - . P-AscH - decreased clonogenic survival; this was enhanced by genetic and pharmacological inhibition of both ERK and Drp1. In murine tumor xenografts, the combination of P-AscH - and pharmacological inhibition of Drp1 increased overall survival. These results suggest that P-AscH - induces sustained changes in mitochondria, through activation of the ERK/Drp1 signaling pathway, an adaptive response. Inhibition of this pathway enhanced the toxicity P-AscH - to cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress., (Published by Oxford University Press on behalf of Nucleic Acids Research 2023.)

Ferumoxytol (FMX) is an FDA-approved magnetite (Fe 3 O 4 ) nanoparticle used to treat iron deficiency anemia that can also be used as an MR imaging agent in patients that can't receive gadolinium. Pharmacological ascorbate (P-AscH-; IV delivery; plasma levels ≈ 20 mM) has shown promise as an adjuvant to standard of care chemo-radiotherapy in glioblastoma (GBM). Since ascorbate toxicity mediated by H 2 O 2 is enhanced by Fe redox cycling, the current study determined if ascorbate catalyzed the release of ferrous iron (Fe 2+ ) from FMX for enhancing GBM responses to chemo-radiotherapy. Ascorbate interacted with Fe 3 O 4 in FMX to produce redox-active Fe 2+ while simultaneously generating increased H 2 O 2 fluxes, that selectively enhanced GBM cell killing (relative to normal human astrocytes) as opposed to a more catalytically active Fe complex (EDTA-Fe 3+ ) in an H 2 O 2 - dependent manner. In vivo, FMX was able to improve GBM xenograft tumor control when combined with pharmacological ascorbate and chemoradiation in U251 tumors that were unresponsive to pharmacological ascorbate therapy. These data support the hypothesis that FMX combined with P-AscH- represents a novel combined modality therapeutic approach to enhance cancer cell selective chemoradiosentization in the management of glioblastoma., Competing Interests: Declaration of competing interest This manuscript has not been published and is not under consideration for publication in other journals. We have no conflicts of interest to disclose., (Published by Elsevier B.V.)

At pharmacological levels, ascorbate (P-AscH - ) acts as a pro-oxidant by generating H 2 O 2 , depleting ATP in sensitive cells leading to cell death. The aim of this study was to determine the role of ATP production by oxidative phosphorylation or glycolysis in mechanisms of resistance to P-AscH - induced cell death. Pancreatic cancer cells were used to generate ρ 0 cells by mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase or Herpes Simplex Virus protein. The ρ 0 phenotype was confirmed by probing for mitochondrial DNA, mitochondrial DNA-encoded cytochrome c oxidase subunit 2, and monitoring the rate of oxygen consumption. In ρ 0 cells, glycolysis accounted for 100% of ATP production as there was no mitochondrial oxygen consumption. Even though the activities of H 2 O 2 -removing antioxidant enzymes were similar in both the parental and ρ 0 clones, P-AscH - -induced clonogenic cell death in ρ 0 cells showed more resistance than the parental cell line. In addition, P-AscH - induced more DNA damage and more consumption of NAD + and greater decreases in the production of ATP in the parental cell line compared to the ρ 0 cells. Thus, cancer cells that largely use oxidative phosphorylation to generate ATP may be more sensitive to P-AscH - compared with cells that are glycolysis-dependent., (© 2022. The Author(s).)

Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1 -/- mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1 -/- neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1 -/- neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming., (©2022 Society for Leukocyte Biology.)

Objective: 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/cm 2 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., (Published by Elsevier Inc.)

Objectives: Pharmacological ascorbate (P-AscH - , high-dose, intravenous vitamin C) has shown promise as an adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC) treatment. The objective of this study was to determine the effects of P-AscH - when combined with PDAC chemotherapies., Methods: Clonogenic survival, combination indices, and DNA damage were determined in human PDAC cell lines treated with P-AscH - in combination with 5-fluorouracil, paclitaxel, or FOLFIRINOX (combination of leucovorin, 5-fluorouracil, irinotecan, oxaliplatin). Tumor volume changes, overall survival, blood analysis, and plasma ascorbate concentration were determined in vivo in mice treated with P-AscH - with or without FOLFIRINOX., Results: P-AscH - combined with 5-fluorouracil, paclitaxel, or FOLFIRINOX significantly reduced clonogenic survival in vitro. The DNA damage, measured by γH2AX protein expression, was increased after treatment with P-AscH - , FOLFIRINOX, and their combination. In vivo, tumor growth rate was significantly reduced by P-AscH - , FOLFIRINOX, and their combination. Overall survival was significantly increased by the combination of P-AscH - and FOLFIRINOX. Treatment with P-AscH - increased red blood cell and hemoglobin values but had no effect on white blood cell counts. Plasma ascorbate concentrations were significantly elevated in mice treated with P-AscH - with or without FOLFIRINOX., Conclusions: The addition of P-AscH - to standard of care chemotherapy has the potential to be an effective adjuvant for PDAC treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Platinum-based chemotherapy with or without immunotherapy is the mainstay of treatment for advanced stage non-small cell lung cancer (NSCLC) lacking a molecular driver alteration. Pre-clinical studies have reported that pharmacological ascorbate (P-AscH-) enhances NSCLC response to platinum-based therapy. We conducted a phase II clinical trial combining P-AscH- with carboplatin-paclitaxel chemotherapy., Experimental Design: Chemotherapy naïve advanced stage NSCLC patients received 75 g ascorbate twice per week intravenously with carboplatin and paclitaxel every three weeks for four cycles. The primary endpoint was to improve tumor response per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 compared to the historical control of 20%. The trial was conducted as an optimal Simon's two-stage design. Blood samples were collected for exploratory analyses., Results: The study enrolled 38 patients and met its primary endpoint with an objective response rate of 34.2% (p = 0.03). All were confirmed partial responses (cPR). The disease control rate was 84.2% (stable disease + cPR). Median progression-free and overall survival were 5.7 months and 12.8 months, respectively. Treatment-related adverse events (TRAE) included one grade 5 (neutropenic fever) and five grade 4 events (cytopenias). Cytokine and chemokine data suggest that the combination elicits an immune response. Immunophenotyping of peripheral blood mononuclear cells demonstrated an increase in effector CD8 T-cells in patients with a progression-free survival (PFS) ≥ 6 months., Conclusions: The addition of P-AscH- to platinum-based chemotherapy improved tumor response in advanced stage NSCLC. P-AscH- appears to alter the host immune response and needs further investigation as a potential adjuvant to immunotherapy., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ferumoxytol (FMX) is an iron oxide nanoparticle that is FDA approved for the treatment of iron deficiency anemia. FMX contains an Fe3O4 core. Currently, the redox chemistry of Fe3O4 nanoparticles remains relatively unexplored. FMX has recently gained interest as an anti-cancer agent. Ionizing radiation (IR) is a treatment modality employed to treat several types of cancer. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the products produced from the radiolysis of water can oxidize the Fe3O4 core of FMX. Because of the limited diffusion of the HO2• and HO• produced, these highly oxidizing species have little direct effect on FMX oxidation. We have determined that H2O2 is the primary oxidant of FMX. In the presence of labile Fe2+, we found that reducing species generated from the radiolysis of H2O are able to reduce the Fe3+ sites of the Fe3O4 core. Importantly, we also have shown that IR stimulates the release of ferric iron from FMX. Because of its release of iron, FMX may serve as an adjuvant to enhance radiotherapy., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Interest in the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s. Recently, pharmacological ascorbate has been used in preclinical and early-phase clinical trials as a selective radiation sensitizer in cancer. The results of these studies are promising. This review summarizes data on pharmacological ascorbate (1) as a safe and efficacious adjuvant to cancer therapy; (2) as a selective radiosensitizer of cancer via a mechanism involving hydrogen peroxide; and (3) as a radioprotector in normal tissues. Additionally, we present new data demonstrating the ability of pharmacological ascorbate to enhance radiation-induced DNA damage in glioblastoma cells, facilitating cancer cell death. We propose that pharmacological ascorbate may be a general radiosensitizer in cancer therapy and simultaneously a radioprotector of normal tissue.

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The dysregulated host response and organ damage following systemic infection that characterizes a septic event predisposes individuals to a chronic immunoparalysis state associated with severe transient lymphopenia and diminished lymphocyte function, thereby reducing long-term patient survival and quality of life. Recently, we observed lasting production of reactive oxygen species (ROS) in mice that survive sepsis. ROS production is a potent mechanism for targeting infection, but excessive ROS production can prove maladaptive by causing organ damage, impairing lymphocyte function, and promoting inflammaging, concepts paralleling sepsis-induced immunoparalysis. Notably, we observed an increased frequency of ROS-producing immature monocytes in septic hosts that was sustained for greater than 100 days postsurgery. Recent clinical trials have explored the use of vitamin C, a potent antioxidant, for treating septic patients. We observed that therapeutic vitamin C administration for sepsis limited ROS production by monocytes and reduced disease severity. Importantly, we also observed increased ROS production by immature monocytes in septic patients both at admission and ∼28 days later, suggesting a durable and conserved feature that may influence the host immune response. Thus, lasting ROS production by immature monocytes is present in septic patients, and early intervention strategies to reduce it may improve host outcomes, potentially reducing sepsis-induced immunoparalysis., (Copyright © 2021 The Authors.)

There is a rapidly growing body of literature supporting the notion that differential oxidative metabolism in cancer versus normal cells represents a metabolic frailty that can be exploited to open a therapeutic window into cancer therapy. These cancer cell-specific metabolic frailties may be amenable to manipulation with non-toxic small molecule redox active compounds traditionally thought to be antioxidants. In this review we describe the potential mechanisms and clinical applicability in cancer therapy of four small molecule redox active agents: melatonin, vitamin E, selenium, and vitamin C. Each has shown the potential to have pro-oxidant effects in cancer cells while retaining antioxidant activity in normal cells. This dichotomy can be exploited to improve responses to radiation and chemotherapy by opening a therapeutic window based on a testable biochemical rationale amenable to confirmation with biomarker studies during clinical trials. Thus, the unique pro-oxidant/antioxidant properties of melatonin, vitamin E, selenium, and vitamin C have the potential to act as effective adjuvants to traditional cancer therapies, thereby improving cancer patient outcomes., (Copyright © 2021. Published by Elsevier B.V.)

Pancreatic cancer cells (PDACs) are more susceptible to an oxidative insult than normal cells, resulting in greater cytotoxicity upon exposure to agents that increase pro-oxidant levels. Pharmacological ascorbate (P-AscH - ), i.e., large amounts given intravenously (IV), generates significant fluxes of hydrogen peroxide (H 2 O 2 ), resulting in the killing of PDACs but not normal cells. Recent studies have demonstrated that P-AscH - radio-sensitizes PDAC but is a radioprotector to normal cells and tissues. Several mechanisms have been hypothesized to explain the dual roles of P-AscH - in radiation-induced toxicity including the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), RelB, as well as changes in bioenergetic profiles. We have found that P-AscH - in conjunction with radiation increases Nrf2 in both cancer cells and normal cells. Although P-AscH - with radiation decreases RelB in cancer cells vs. normal cells, the knockout of RelB does not radio-sensitize PDACs. Cellular bioenergetic profiles demonstrate that P-AscH - with radiation increases the ATP demand/production rate (glycolytic and oxidative phosphorylation) in both PDACs and normal cells. Knocking out catalase results in P-AscH - radio-sensitization in PDACs. In a phase I trial where P-AscH - was included as an adjuvant to the standard of care, short-term survivors had higher catalase levels in tumor tissue, compared to long-term survivors. These data suggest that P-AscH - radio-sensitizes PDACs through increased peroxide flux. Catalase levels could be a possible indicator for how tumors will respond to P-AscH - .

Dihydroartemisinin (DHA) is an FDA-approved antimalarial drug that has been repurposed for cancer therapy because of its preferential antiproliferative effects on cancer versus normal cells. Mitochondria represent an attractive target for cancer therapy based on their regulatory role in proliferation and cell death. This study investigates whether DHA conjugated to innately fluorescent N-alkyl triphenylvinylpyridinium (TPVP) perturbs mitochondrial functions resulting in a differential toxicity of cancer versus normal cells. TPVP-DHA treatments resulted in a dose-dependent toxicity of human melanoma and pancreatic cancer cells, whereas normal human fibroblasts were resistant to this treatment. TPVP-DHA treatments resulted in a G 1 -delay of the cancer cell cycle, which was also associated with a significant inhibition of the mTOR-metabolic and ERK1/2-proliferative signaling pathways. TPVP-DHA treatments perturbed mitochondrial functions, which correlated with increases in mitochondrial fission. In summary, TPVP mediated mitochondrial targeting of DHA enhanced cancer cell toxicity by perturbing mitochondrial functions and morphology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pharmacological ascorbate (P-AscH - ) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe 3+ ) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe 2+ ). Because paramagnetic Fe 3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH - -induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe 3+ or Fe 2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH - plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH - infusion, post-P-AscH - infusion, and post-radiation (3-4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe 3+ and Fe 2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe 3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe 2+ phantoms. Compared to pre-P-AscH - , GBM T2* and QSM imaging were significantly changed post-P-AscH - infusion consistent with conversion of Fe 3+ to Fe 2+ . No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH - inducing greater changes in tumor labile iron pools compared to the normal tissue. CONCLUSIONS: T2* and QSM MR-imaging responses are consistent with P-AscH - reducing Fe 3+ to Fe 2+ , selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH - -induced changes in redox-active iron., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5-16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma., Competing Interests: The authors have declared that no competing interests exist.

Previous research has identified variation in cancer cell line response to high levels of extracellular H 2 O 2 (eH 2 O 2 ) 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 eH 2 O 2 . First, we develop a mathematical framework for the latency of catalase in terms of an effectiveness factor, η eff , to describe the catalase activity in the presence of high levels of eH 2 O 2 . A simplified relationship emerges, [Formula: see text] when m p r p /D ij ≪1, where m p ,r p , 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 k 2 =1.7x10 7 M -1 s -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 eH 2 O 2 . 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 eH 2 O. Thus, further research remains necessary to identify why cancer cells vary in susceptibility to P-AscH - therapy., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Selenium (Se) is an essential trace nutrient required for optimal human health. It has long been suggested that selenium has anti-cancer properties. However, clinical trials have shown inconclusive results on the potential of Se to prevent cancer. The suggested role of Se in the prevention of cancer is centered around its role as an antioxidant. Recently, the potential of selenium as a drug rather than a supplement has been uncovered. Selenium compounds can generate reactive oxygen species that could enhance the treatment of cancer. Transformed cells have high oxidative distress. As normal cells have a greater capacity to meet oxidative challenges than tumor cells, increasing the flux of oxidants with high dose selenium treatment could result in cancer-specific cell killing. If the availability of Se is limited, supplementation of Se can increase the expression and activities of Se-dependent proteins and enzymes. In cell culture, selenium deficiency is often overlooked. We review the importance of achieving normal selenium biology and how Se deficiency can lead to adverse effects. We examine the vital role of selenium in the prevention and treatment of cancer. Finally, we examine the properties of Se-compounds to better understand how each can be used to address different research questions., Competing Interests: Authors disclosed the following: J.M.S., R.G., J.C.S, and G.R.B.: These authors declare no conflict of interest. Y.Z.: Advisory Board: Amgen, Roche Diagnostics, Novartis, Jansen, Eisai, Exelixis, Castle Bioscience, Array, Bayer, Pfizer, Clovis, EMD Serono. As well as Institutional grant/research support from NewLink Genetics, Pfizer, Exelixis, Eisai. The content is solely the responsibility of the authors and does not represent views of the sponsors nor the National Institutes of Health.

Selenium is a metalloid trace element essential for maintaining the optimal redox environment in cells and tissues. It is structurally incorporated into over 25 selenoproteins and enzymes. The glutathione peroxidase (GPx) family of enzymes has a critical role in human health because of its antioxidant function. The recommended daily allowance (RDA) for selenium intake in humans was established to maximize the activity of GPx in plasma. Suboptimal availability of selenium can limit the expression and activities of GPxs leading to a compromised redox environment. This can cause detrimental oxidative distress that could be prevented by increasing the availability of selenium. In cell culture studies, the medium is typically deficient in selenium; supplementation with selenium can increase selenoenzyme activities. However, the optimal level of supplementation in cell culture media has not been well characterized. We performed dose-response experiments for the activities of GPx1 and GPx4 vs. the level of selenium supplementation in cell culture medium. For this, we advanced an assay to determine the activities of both GPx1 and GPx4 efficiently in a single run. During the optimization process, we found that the observed activities of GPx1 and GPx4 depend greatly on the pH of the assay buffer; the observed activities increase with increasing pH, with pH 8 being optimal. Using the combination assay, we also found that the expression and activities for both GPx1 and GPx4 can be maximized in exponentially growing cells by supplementing cell culture media with ≈ 200 nM seleno-l-methionine, without concerns for toxicity. Optimizing the availability of selenium in cell culture to maximize the expression and activities GPx1 and GPx4 may allow for better translation of information from cell culture work to in vivo settings., Competing Interests: Declaration of competing interest Authors declare no potential conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest Douglas R. Spitz PhD and Bryan G. Allen MD PhD have a sponsored research agreement with Galera Therapeutics, Inc. at the University of Iowa and are unpaid scientific consultants for Galera Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pharmacologic ascorbate treatment (P-AscH - , high-dose, intravenous vitamin C) results in a transient short-term increase in the flux of hydrogen peroxide that is preferentially cytotoxic to cancer cells versus normal cells. This study examines whether an increase in hydrogen peroxide is sustained posttreatment and potential mechanisms involved in this process. Cellular bioenergetic profiling following treatment with P-AscH - was examined in tumorigenic and nontumorigenic cells. P-AscH - resulted in sustained increases in the rate of cellular oxygen consumption (OCR) and reactive oxygen species (ROS) in tumor cells, with no changes in nontumorigenic cells. Sources for this increase in ROS and OCR were DUOX 1 and 2, which are silenced in pancreatic ductal adenocarcinoma, but upregulated with P-AscH - treatment. An inducible catalase system, to test causality for the role of hydrogen peroxide, reversed the P-AscH - -induced increases in DUOX, whereas DUOX inhibition partially rescued P-AscH - -induced toxicity. In addition, DUOX was significantly downregulated in pancreatic cancer specimens compared with normal pancreas tissues. Together, these results suggest that P-AscH - -induced toxicity may be enhanced by late metabolic shifts in tumor cells, resulting in a feed-forward mechanism for generation of hydrogen peroxide and induction of metabolic stress through enhanced DUOX expression and rate of oxygen consumption. SIGNIFICANCE: A high dose of vitamin C, in addition to delivering an acute exposure of H 2 O 2 to tumor cells, activates DUOX in pancreatic cancer cells, which provide sustained production of H 2 O 2 ., (©2020 American Association for Cancer Research.)

This study used a nitroaliphatic chemistry approach to synthesize a novel artemisinin-derived carba-dimer (AG-1) and determined its anti-proliferative effects in human normal and cancer cells. AG-1 treatments selectively inhibit proliferation of cancer cells compared to normal human fibroblasts. Compared to artemisinin, AG-1 is more toxic to human breast, prostate, head-neck, pancreas and skin cancer cells; 50% inhibition (IC 50 ) 123 µM in AG-1 vs. 290 µM in artemisinin-treated breast cancer cells. AG-1 treatment decreased (~ 5 folds) cyclin D1 protein expression that correlated with an increase in the percentage of cells in the G 1 -phase, suggesting a G 1 delay. AG-1-induced toxicity was independent of the DNA damage at 72 h post-treatment, as measured by micronuclei frequency and H2AX protein levels. Results from electron paramagnetic resonance spectroscopy showed Fe-catalyzed formation of AG-1 carbon-centered radicals in a cell-free system. Flow cytometry analysis of H 2 DCF-DA oxidation showed a significant increase in the steady-state levels of reactive oxygen species (ROS) in AG-1-treated cells. Pre-treatment with N -acetyl-l-cysteine and antioxidant enzymes (superoxide dismutase and catalase) significantly suppressed AG-1-induced toxicity, suggesting that superoxide and hydrogen peroxide contribute to AG-1-induced toxicity in human cancer cells. AG-1 represents a novel class of anti-cancer drug that is more potent than its parent compound, artemisinin., Competing Interests: The authors declare no conflict of interest.

Purpose: Standard treatment for glioblastoma (GBM) includes surgery, radiation therapy (RT), and temozolomide (TMZ), yielding a median overall survival (OS) of approximately 14 months. Preclinical models suggest that pharmacologic ascorbate (P-AscH - ) enhances RT/TMZ antitumor effect in GBM. We evaluated the safety of adding P-AscH - to standard RT/TMZ therapy., Patients and Methods: This first-in-human trial was divided into an RT phase (concurrent RT/TMZ/P-AscH - ) and an adjuvant (ADJ) phase (post RT/TMZ/P-AscH - phase). Eight P-AscH - dose cohorts were evaluated in the RT phase until targeted plasma ascorbate levels were achieved (≥20 mmol/L). In the ADJ phase, P-AscH - doses were escalated in each subject at each cycle until plasma concentrations were ≥20 mmol/L. P-AscH - was infused 3 times weekly during the RT phase and 2 times weekly during the ADJ phase continuing for six cycles or until disease progression. Adverse events were quantified by CTCAE (v4.03)., Results: Eleven subjects were evaluable. No dose-limiting toxicities occurred. Observed toxicities were consistent with historical controls. Adverse events related to study drug were dry mouth and chills. Targeted ascorbate plasma levels of 20 mmol/L were achieved in the 87.5 g cohort; diminishing returns were realized in higher dose cohorts. Median progression-free survival (PFS) was 9.4 months and median OS was 18 months. In subjects with undetectable MGMT promoter methylation ( n = 8 ), median PFS was 10 months and median OS was 23 months., Conclusions: P-AscH - /RT/TMZ is safe with promising clinical outcomes warranting further investigation., (©2019 American Association for Cancer Research.)

Background: High-dose, pharmacological ascorbate (P-AscH - ) is preferentially cytotoxic to human cancer cells in vitro . Investigations on the efficacy of P-AscH - as an adjuvant treatment for multiple human cancers are on-going, but has yet to be formally investigated in dogs. The primary objectives of this study were to determine the pharmacokinetic (PK) profile of P-AscH - in healthy Beagle dogs and the effects of P-AscH - on canine osteosarcoma cells in vitro . Methods: Eight purpose-bred, healthy, spayed female Beagle dogs, between 20 and 21 months old, and 8-10 kg were administered two doses of P-AscH - (550 or 2,200 mg/kg) via intravenous infusion over 6 h, on separate days. Plasma ascorbate concentrations were measured at 12 time points during and after infusion for PK analysis using nonlinear mixed-effects (NLME) and non-compartmental analysis (NCA). Clonogenic assays were performed on 2 canine osteosarcoma cell lines (D-17 and OSCA-8) and canine primary dermal fibroblasts after exposure to high concentrations of ascorbate (75 pmoles/cell). Results: Plasma ascorbate levels in the dogs peaked at approximately 10 mM following 2,200 mg/kg and returned to baseline 6-8 h after dosing. Minor adverse effects were seen in two dogs. Ascorbate (75 pmoles/cell) significantly decreased survival in both the osteosarcoma cell lines (D-17 63% SD 0.010, P = 0.005; OSCA-8 50% SD 0.086, P = 0.026), compared to normal fibroblasts (27% SD 0.056). Conclusions: Pharmacological ascorbate is preferentially cytotoxic to canine-derived cancer cells. High levels of ascorbate can be safely administered to dogs. Further studies are needed to determine the effects of P-AscH - on canine patients., (Copyright © 2019 Musser, Mahaffey, Fath, Buettner, Wagner, Schneider, Seo, Mochel and Johannes.)

The clinical potential of pharmacologic ascorbate (P-AscH - ; intravenous delivery achieving mmol/L concentrations in blood) as an adjuvant in cancer therapy is being reevaluated. At mmol/L concentrations, P-AscH - is thought to exhibit anticancer activity via generation of a flux of H 2 O 2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer to examine the effects of P-AscH - on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H 2 O 2 produced by P-AscH - induces DNA damage. In response to this DNA damage, we observed that PARP1 is hyperactivated. Using our unique absolute quantitation, we found that P-AscH - mediated the overactivation of PARP1, which results in consumption of NAD + , and subsequently depletion of ATP leading to mitotic cell death. We have also found that Chk1 plays a major role in the maintenance of genomic integrity following treatment with P-AscH - . Hyperactivation of PARP1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 analyzer, we demonstrated that the severe decrease in ATP after challenging with P-AscH - is because of increased demand, not changes in the rate of production. Genetic deletion and pharmacologic inhibition of PARP1 preserved both NAD + and ATP; however, the toxicity of P-AscH - remained. These data indicate that disruption of bioenergetics is a secondary factor in the toxicity of P-AscH - ; damage to DNA appears to be the primary factor. IMPLICATIONS: Efforts to leverage P-AscH - in cancer therapy should first focus on DNA damage., (©2019 American Association for Cancer Research.)

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lipid hydroperoxides within biological membranes. Although therapy-resistant mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially phospholipid glutathione peroxidase 4 (GPx4) inhibitors, the underlying mechanism is yet to be deciphered. As such, the full application of GPx4 inhibitors in cancer therapy remains challenging. Here we demonstrate that metadherin (MTDH) confers a therapy-resistant mesenchymal-high cell state and enhanced sensitivity to inducers of ferroptosis. Mechanistically, MTDH inhibited GPx4, as well as the solute carrier family 3 member 2 (SLC3A2, a system X c - heterodimerization partner), at both the messenger RNA and protein levels. Our metabolomic studies demonstrated that MTDH reduced intracellular cysteine, but increased glutamate levels, ultimately decreasing levels of glutathione and setting the stage for increased vulnerability to ferroptosis. Finally, we observed an enhanced antitumor effect when we combined various ferroptosis inducers both in vitro and in vivo; the level of MTDH correlated with the ferroptotic effect. We have demonstrated for the first time that MTDH enhances the vulnerability of cancer cells to ferroptosis and may serve as a therapeutic biomarker for future ferroptosis-centered cancer therapy.