Brett A. Wagner

Mechanisms of Ascorbate-Induced Cytotoxicity in Pancreatic Cancer

Purpose: Pharmacologic concentrations of ascorbate may be effective in cancer therapeutics. We hypothesized that ascorbate concentrations achievable with i.v. dosing would be cytotoxic in pancreatic cancer for which the 5-year survival is <3%. Experimental Design: Pancreatic cancer cell lines were treated with ascorbate (0, 5, or 10 mmol/L) for 1 hour, then viability and clonogenic survival were determined. Pancreatic tumor cells were delivered s.c. into the flank region of nude mice and allowed to grow at which time they were randomized to receive either ascorbate (4 g/kg) or osmotically equivalent saline (1 mol/L) i.p. for 2 weeks. Results: There was a time- and dose-dependent increase in measured H2O2 production with increased concentrations of ascorbate. Ascorbate decreased viability in all pancreatic cancer cell lines but had no effect on an immortalized pancreatic ductal epithelial cell line. Ascorbate decreased clonogenic survival of the pancreatic cancer cell lines, which was reversed by treatment of cells with scavengers of H2O2. Treatment with ascorbate induced a caspase-independent cell death that was associated with autophagy. In vivo, treatment with ascorbate inhibited tumor growth and prolonged survival. Conclusions: These results show that pharmacologic doses of ascorbate, easily achievable in humans, may have potential for therapy in pancreatic cancer. Clin Cancer Res; 16(2); 509–20

The Rate of Oxygen Utilization by Cells

The discovery of oxygen is considered by some to be the most important scientific discovery of all time--from both physical-chemical/astrophysics and biology/evolution viewpoints. One of the major developments during evolution is the ability to capture dioxygen in the environment and deliver it to each cell in the multicellular, complex mammalian body-on demand, i.e., just in time. Humans use oxygen to extract approximately 2550 calories (10.4 MJ) from food to meet daily energy requirements. This combustion requires about 22 mol of dioxygen per day, or 2.5×10(-4) mol s(-1). This is an average rate of oxygen utilization of 2.5×10(-18) mol cell(-1) s(-1), i.e., 2.5 amol cell(-1) s(-1). Cells have a wide range of oxygen utilization, depending on cell type, function, and biological status. Measured rates of oxygen utilization by mammalian cells in culture range from <1 to >350 amol cell(-1) s(-1). There is a loose positive linear correlation of the rate of oxygen consumption by mammalian cells in culture with cell volume and cell protein. The use of oxygen by cells and tissues is an essential aspect of the basic redox biology of cells and tissues. This type of quantitative information is fundamental to investigations in quantitative redox biology, especially redox systems biology.

Myeloperoxidase Is Involved in H2O2-induced Apoptosis of HL-60 Human Leukemia Cells

We examined the mechanism of H2O2-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H2O2, and at concentrations up to about 20–25 μm, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H2O2 do not involve hydroxyl radical. When HL-60 cells were exposed to H2O2 in the presence of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN⋅ N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H2O2concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN⋅. Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H2O2-induced apoptosis. Based on this we reasoned that the difference in H2O2-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H2O2-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H2O2-induced apoptosis. Taken together these observations indicate that H2O2-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN⋅.

Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones

The reactions of glutathione (GSH) with polychlorinated biphenyl (PCB) quinones having different degrees of chlorination on the quinone ring were examined. EPR spectroscopy and MS revealed 2 types of reactions yielding different products: (i) a nonenzymatic, nucleophilic displacement of chlorine on the quinone ring yielding a glutathiylated conjugated quinone and (ii) Michael addition of GSH to the quinone, a 2-electron reduction, yielding a glutathiylated conjugated hydroquinone. The pKa of parent hydroquinone decreased by 1 unit as the degree of chlorination increased. This resulted in a corresponding increase in the oxidizability of these chlorinated hydroquinones. The reaction with oxygen appears to be first-order each in ionized hydroquinone and dioxygen, yielding hydrogen peroxide stoichiometrically. The generation of semiquinone radicals, superoxide, and hydroxyl radicals was observed by EPR; however, the mechanisms and yields vary depending on the degree of the chlorination of hydroquinone/quinone and the presence or absence of GSH. Our discovery that chlorinated quinones undergo a rapid, nonenzymatic dechlorination upon reaction with GSH opens a different view on mechanisms of metabolism and the toxicity of this class of compounds.

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.

Free radicals produced by the oxidation of gallic acid: An electron paramagnetic resonance study

BackgroundGallic acid (3,4,5-trihydroxybenzoic acid) is found in a wide variety of plants; it is extensively used in tanning, ink dyes, as well as in the manufacturing of paper. The gallate moiety is a key component of many functional phytochemicals. In this work electron paramagnetic spectroscopy (EPR) was used to detect the free radicals generated by the air-oxidation of gallic acid.ResultsWe found that gallic acid produces two different radicals as a function of pH. In the pH range between 7-10, the spectrum of the gallate free radical is a doublet of triplets (aH = 1.00 G, aH = 0.23 G, aH = 0.28 G). This is consistent with three hydrogens providing hyperfine splitting. However, in a more alkaline environment, pH >10, the hyperfine splitting pattern transforms into a 1:2:1 pattern (aH (2) = 1.07 G). Using D2O as a solvent, we demonstrate that the third hydrogen (i.e. aH = 0.28 G) at lower pH is a slowly exchanging hydron, participating in hydrogen bonding with two oxygens in ortho position on the gallate ring. The pKa of this proton has been determined to be 10.ConclusionsThis simple and novel approach permitted the understanding of the prototropic equilibrium of the semiquinone radicals generated by gallic acid, a ubiquitous compound, allowing new insights into its oxidation and subsequent reactions.

Semiquinone Radicals from Oxygenated Polychlorinated Biphenyls: Electron Paramagnetic Resonance Studies

Polychlorinated biphenyls (PCBs) can be oxygenated to form very reactive hydroquinone and quinone products. A guiding hypothesis in the PCB research community is that some of the detrimental health effects of some PCBs are a consequence of these oxygenated forms undergoing one-electron oxidation or reduction, generating semiquinone radicals (SQ•−). These radicals can enter into a futile redox cycle resulting in the formation of reactive oxygen species, that is, superoxide and hydrogen peroxide. Here, we examine some of the properties and chemistry of these semiquinone free radicals. Using electron paramagnetic resonance (EPR) to detect SQ•− formation, we observed that (i) xanthine oxidase can reduce quinone PCBs to the corresponding SQ•−; (ii) the heme-containing peroxidases (horseradish and lactoperoxidase) can oxidize hydroquinone PCBs to the corresponding SQ•−; (iii) tyrosinase acting on PCB ortho-hydroquinones leads to the formation of SQ•−; (iv) mixtures of PCB quinone and hydroquinone form SQ•− via a comproportionation reaction; (v) SQ•− are formed when hydroquinone-PCBs undergo autoxidation in high pH buffer (≈>pH 8); and, surprisingly, (vi) quinone-PCBs in high pH buffer can also form SQ•−; (vii) these observations along with EPR suggest that hydroxide anion can add to the quinone ring; (viii) H2O2 in basic solution reacts rapidly with PCB-quinones; and (ix) at near-neutral pH SOD can catalyze the oxidization of PCB-hydroquinone to quinone, yielding H2O2. However, using 5,5-dimethylpyrroline-1-oxide (DMPO) as a spin-trapping agent, we did not trap superoxide, indicating that generation of superoxide from SQ•− is not kinetically favorable. These observations demonstrate multiple routes for the formation of SQ•− from PCB-quinones and hydroquinones. Our data also point to futile redox cycling as being one mechanism by which oxygenated PCBs can lead to the formation of reactive oxygen species, but this is most efficient in the presence of SOD.

Lactoferrin in the Preterm Infants' Diet Attenuates Iron-Induced Oxidation Products

Free radical injury is thought to play a significant role in the pathogenesis of several disease processes in low birth weight premature infants including retinopathy of prematurity and necrotizing enterocolitis. Because iron is a known catalyst in free radical–mediated oxidation reactions, the objectives of the present in vitro studies were to determine whether after exposure to air 1) iron present in infant formula, or that added to human milk or formula as medicinal iron or as iron contained in human milk fortifier, increases free radical and lipid peroxidation products; and 2) recombinant human lactoferrin added to formula or human milk attenuates iron-mediated free radical formation and lipid peroxidation. Before adding medicinal iron to formula and human milk, significantly more ascorbate and α-hydroxyethyl radical production and more lipid peroxidation products (i.e. thiobarbituric acid reactive substances, malondialdehyde, and ethane) were observed in formula. After the addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. When iron-containing human milk fortifier was added to human milk, free radicals also increased. In contrast, the addition of apo-recombinant human lactoferrin to formula or human milk decreased the levels of oxidative products when medicinal iron or human milk fortifier was present. We speculate that the presence of greater concentration of iron and the absence of lactoferrin in formula compared with human milk results in greater in vitro generation of free radicals and lipid peroxidation products. Whether iron-containing formula with lactoferrin administered enterally to preterm infants will result in less free radical generation in vivo has yet to be established.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

Significance Mitochondrial Ca2+ is a fundamental signal that allows for adaptation to physiological stress but a liability during ischemia-reperfusion injury in heart. On one hand, mitochondrial Ca2+ entry coordinates energy supply and demand in myocardium by increasing the activity of matrix dehydrogenases to augment ATP production by oxidative phosphorylation. On the other hand, inhibiting mitochondrial Ca2+ overload is promulgated as a therapeutic approach to preserve myocardial tissue following ischemia-reperfusion injury. We developed a new mouse model of myocardial-targeted transgenic dominant-negative mitochondrial Ca2+ uniporter (MCU) expression to test consequences of chronic loss of MCU-mediated Ca2+ entry in heart. Here we show that MCU inhibition has unanticipated consequences on extramitochondrial pathways affecting oxygen utilization, cytoplasmic Ca2+ homeostasis, physiologic responses to stress, and pathologic responses to ischemia-reperfusion injury. Myocardial mitochondrial Ca2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU–permeabilized myocardial fibers or isolated mitochondria in low Ca2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca2+] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca2+ homeostasis. Mitochondrial Ca2+ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.