Magnetic Resonance Imaging of Iron Metabolism with T2* Mapping Predicts an Enhanced Clinical Response to Pharmacologic Ascorbate in Patients with GBM.

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.

Depletion of Labile Iron Induces Replication Stress and Enhances Responses to Chemoradiation in Non-Small-Cell Lung Cancer.

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.

Pharmacological ascorbate as a novel therapeutic strategy to enhance cancer immunotherapy.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

Polyoxometalate Nanoparticles as a Potential Glioblastoma Therapeutic via Lipid-Mediated Cell Death.

Polyoxometalate nanoparticles (POMs) are a class of compounds made up of multiple transition metals linked together using oxygen atoms. POMs commonly include group 6 transition metals, with two of the most common forms using molybdenum and tungsten. POMs are suggested to exhibit antimicrobial effects. In this study, we developed two POM preparations to study anti-cancer activity. We found that Mo-POM (NH4)Mo7O24) and W-POM (H3PW12O40) have anti-cancer effects on glioblastoma cells. Both POMs induced morphological changes marked by membrane swelling and the presence of multinucleated cells that may indicate apoptosis induction along with impaired cell division. We also observed significant increases in lipid oxidation events, suggesting that POMs are redox-active and can catalyze detrimental oxidation events in glioblastoma cells. Here, we present preliminary indications that molybdenum polyoxometalate nanoparticles may act like ferrous iron to catalyze the oxidation of phospholipids. These preliminary results suggest that Mo-POMs (NH4)Mo7O24) and W-POMs (H3PW12O40) may warrant further investigation into their utility as adjunct cancer therapies.

Utilization of Pharmacological Ascorbate to Enhance Hydrogen Peroxide-Mediated Radiosensitivity in Cancer Therapy.

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.

Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage.

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.

Comprehensive Subchronic Inhalation Toxicity Assessment of an Indoor School Air Mixture of PCBs.

Few in vivo inhalation studies have explored the toxicity of environmentally relevant mixtures of polychlorinated biphenyls (PCBs). The manufacture of industrial PCBs was banned in 1978, but PCBs continue to be formed in industrial and consumer products. Schools represent a significant source of airborne exposures to legacy and nonlegacy PCBs, placing children at risk. To evaluate the impact of these exposures, we generated an airborne mixture of PCBs, called the School Air Mixture (SAM), to match the profile of an older school from our adolescent cohort study. Female Sprague-Dawley rats were exposed either to SAM or filtered air in nose-only exposure systems, 4 h/day for 4 weeks. Congener-specific air and tissue PCB profiles were assessed using gas chromatography with tandem mass spectrometry (GC-MS/MS). PCB exposures recapitulated the target school air profile with a similarity coefficient, cos θ of 0.83. PCB inhalation yielded µg/g ∑209 PCB levels in tissues. Neurobehavioral testing demonstrated a modest effect on spatial learning and memory in SAM-exposed rats. PCB exposure induced oxidative stress in the liver and lungs, affected the maturational stages of hematopoietic stem cells, reduced telomerase activity in bone marrow cells, and altered the gut microbiota. This is the first study to emulate PCB exposures in a school and comprehensively evaluate toxicity.

Understanding the Redox Biology of Selenium in the Search of Targeted Cancer Therapies.

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.

Simultaneous detection of the enzyme activities of GPx1 and GPx4 guide optimization of selenium in cell biological experiments.

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.

Dual Oxidase-Induced Sustained Generation of Hydrogen Peroxide Contributes to Pharmacologic Ascorbate-Induced Cytotoxicity.

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 H2O2 to tumor cells, activates DUOX in pancreatic cancer cells, which provide sustained production of H2O2.

Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper.

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 CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. 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% O2vs. 21% O2. 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.