Gregory I. Giles

Reactive sulfur species: An emerging concept in oxidative stress

Abstract The ingredients of oxidative stress include a variety of reactive species such as reactive oxygen and reactive nitrogen species (ROS, RNS). While sulfur is usually considered as part of cellular antioxidant systems there is mounting evidence that reactive sulfur species (RSS) with stressor properties similar to the ones found in ROS are formed under conditions of oxidative stress. Thiols as well as disulfides are easily oxidised to sulfur species with sulfur in higher oxidation states. Such agents include thiyl radicals, disulfides, sulfenic acids and disulfideSoxides. They rapidly oxidise and subsequently inhibit thiolproteins and enzymes and can be considered as a separate class of oxidative stressors providing new antioxidant drug targets.

The redox regulation of thiol dependent signaling pathways in cancer

Reactive oxygen species (ROS) play a central role as second messengers in many signal transduction pathways, where they can post-translationally modify proteins via the oxidation of redox sensitive cysteine residues. The range of cellular processes under redox regulation is extensive and includes both the proliferative and apoptotic pathways. Control of the cellular redox environment is therefore essential for normal physiological function and perturbations to this redox balance are characteristic of many pathological states. Oxidative stress is particularly prevalent in cancer, where many malignant cell types possess an abnormal redox metabolism involving down-regulation of antioxidant enzymes and impaired mitochondrial function. This provides a major opportunity to design therapeutic strategies to selectively target cancer cells based on their redox profile. This review will provide a background to this emerging field by summarizing the known redox biochemistry of ROS signaling. The mechanisms of ROS generation by the action of oxidoreductases and nitric oxide synthases will be discussed in conjunction with the cells major antioxidant defenses, with special emphasis placed on the subcellular location of these redox reactions. The effect of ROS on proliferation and apoptosis will be examined by looking at interactions with transcription factors and the Akt, TNF and MAPK signaling pathways. The review will also outline the major differences in redox metabolism between cancer cells and their non-malignant counterparts. Although the full extent of the ROS regulation of signaling pathways is only beginning to be mapped, early indications are that this paradigm will provide new therapeutic targets for cancer therapy.

Fatty Acid Transduction of Nitric Oxide Signaling NITROLINOLEIC ACID IS A HYDROPHOBICALLY STABILIZED NITRIC OXIDE DONOR

The aqueous decay and concomitant release of nitric oxide (·NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ·NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and ·NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ·NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ·NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.

Xanthine Oxidase–Dependent Regulation of Hypoxia-Inducible Factor in Cancer Cells

During chemical hypoxia induced by cobalt chloride (CoCl2), hypoxia-inducible factor 1alpha (HIF1-alpha) mediates the induction of a variety of genes including erythropoietin and vascular endothelial growth factor. We used glioma cells with oxidative phosphorylation-dependent (D54-MG) and glycolytic-dependent (U251-MG) phenotypes to monitor HIF1-alpha regulation in association with redox responsiveness to CoCl2 treatment. We showed that CoCl2 increased xanthine oxidase (XO)-derived reactive oxygen species (ROS), which causes accumulation of HIF1-alpha protein in U251-MG cells. Under these conditions, blockade of XO activity by pharmacologic (N-acetyl-L-cysteine or allopurinol) or molecular (by small interfering RNA) approaches significantly attenuated HIF1-alpha expression. Exogenous H2O2 stabilizes HIF1-alpha protein. XO was present in these cells and was the primary source of free radicals. We also showed higher XO activity in cells exposed to CoCl2 compared with cells grown in normoxia. From the experiments shown here, we concluded that ROS were indeed generated in D54-MG cells exposed to CoCl2 but it was unlikely that ROS participated in the hypoxic signal transduction pathways in this cell type. Possibly, cell type-dependent and stimulus-dependent factors may control ROS dependency or redox sensitivity of HIF1-alpha and thus HIF1-alpha activation either directly or by induction of specific signaling cascades. Our findings reveal that XO-derived ROS is a novel and critical component of HIF1-alpha regulation in U251-MG cells, pointing toward a more general role of this transcription factor in tumor progression.

Electrochemical and in vitro evaluation of the redox-properties of kynurenine species

Kynurenines are formed as part of the tryptophan metabolism and are known to exhibit pro- and anti-oxidant activities in vitro. The mapping of these biological redox-systems and identification of potential in vivo targets are therefore of great interest in cellular physiology. Here the redox-behavior of different kynurenines and anthranilic acids is evaluated electrochemically and compared to that of simple model compounds. Electrochemical results are correlated with the activity of these compounds in redox-bioassays where 3-hydroxyanthranilic acid and 3-hydroxykynurenine have significant redox-activity. The specific electrochemical redox-behavior of these two compounds, indicating a particular redox-mechanism involving the hydroxyl group, can be used to rationalize these findings. The results indicate that tryptophan metabolites can undergo a range of complex redox-reactions in vivo whose precise nature critically depends on structural details. As a consequence, some of the kynurenines have the potential to contribute to neuronal damage in brain disorders and stroke.

Redox catalysts as sensitisers towards oxidative stress

The predominance of oxidative stress in many tumour cell environments provides a means to selectively target these cells via protein oxidation. The zinc fingers of transcription factors utilise cysteine thiols for structural zinc coordination. Redox control of DNA binding regulates transcription and therefore the overall rates of proliferation, apoptosis and necrosis in the carcinoma. We report here the adverse effects of glutathione peroxidase (GPx) mimics towards zinc finger motifs and PC12 cell survival. Nanomolar catalyst concentrations facilitated H2O2‐induced oxidation of an Sp1 transcription factor fragment. In PC12 cells GPx catalysis triggered a significant increase in cell death, correlating with severity of oxidative stress. As a consequence, we conclude that GPx mimics are potential chemotherapeutic agents.

Novel Method for Measuring S-Nitrosothiols Using Hydrogen Sulfide

Recent advances in techniques that allow sensitive and specific measurement of S-nitrosothiols (RSNOs) have provided evidence for a role for these compounds in various aspects of nitric oxide (NO) biology. The most widely used approach is to couple reaction chemistry that selectively reduces RSNOs by one electron to produce NO, with the sensitive detection of the latter under anaerobic conditions using ozone based chemiluminescence in NO analyzers. Herein, we report a novel reaction that is readily adaptable for commercial NO analyzers that utilizes hydrogen sulfide (H2S), a gas that can reduce RSNO to NO and, analogous to NO, is produced by endogenous metabolism and has effects on diverse biological functions. We discuss factors that affect H2S based methods for RSNO measurement and discuss the potential of H2S as an experimental tool to measure RSNO.

Reactive sulphur species in oxidative signal transduction

Intense interest has been generated by the discovery that reactive oxygen species can function as intracellular second messengers. Reactive oxygen species have been implicated in diverse cellular processes, including growth factor signal transduction, gene expression and apoptosis. Additionally, there is evidence for proteins that are regulated by redox environment through the reversible oxidation of their cysteine residues. However, the direct reaction of reactive oxygen species with cysteine at physiological concentrations is generally a slow process, suggesting that intermediates are required to convey efficiently the oxidative stimulus. Here, we discuss the evidence that DSOs (disulphide-S-oxides) are formed from glutathione under oxidizing conditions and specifically modulate the redox status of thiols, indicating the existence of specialized cellular oxidative pathways. DSO inactivated glyceraldehyde 3-phosphate and alcohol dehydrogenases and released zinc from metallothionein and a zinc finger domain. In contrast, equivalent concentrations of H(2)O(2) showed minimal effect. The antioxidants ascorbate, NADH, trolox and melatonin were unable to quench DSO-induced oxidation. These findings support the paradigm of oxidative signal transduction and provide a general pathway whereby reactive oxygen species can convert thiols into disulphides.

Targeting oxidative stress-related diseases: organochalcogen catalysts as redox sensitizers.

Tumor cells proliferate under conditions of oxidative stress. A novel therapeutic approach would be to enhance the cellular effects of the reactive oxygen species formed under these conditions by supplementation with a redox catalyst. This provides a means to target and specifically destroy cancer cells via oxidation of redox-sensitive proteins, such as transcription factors, while leaving cells with a normal redox balance largely unaffected. We have previously reported a preliminary observation on the effects of pro-oxidant catalysts that enhance cancer cell death. This paper presents a detailed in vitro investigation into the mechanism of action of synthetic glutathione peroxidase mimics on a model Sp1 transcription factor peptide. The structure and redox potential of these mimics correlate with their ability to catalyze the oxidation of this zinc-binding motif by H(2)O(2) and these compounds promise therapeutic potential by promoting H(2)O(2)-induced PC12 cell death.

Intracellular targeting and pharmacological activity of the superoxide dismutase mimics MnTE-2-PyP5+ and MnTnHex-2-PyP5+ regulated by their porphyrin ring substituents.

Manganese porphyrin-based drugs are potent mimics of the enzyme superoxide dismutase. They exert remarkable efficacy in disease models and are entering clinical trials. Two lead compounds, MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), have similar catalytic rates, but differ in their alkyl chain substituents (ethyl vs n-hexyl). Herein we demonstrate that these changes in ring substitution impact upon drug intracellular distribution and pharmacological mechanism, with MnTnHex-2-PyP(5+) superior in augmenting menadione toxicity. These findings establish that both catalytic activity and intracellular distribution determine drug action.