Redox Targets Research Articles

P-62 - SR-B1 as a new redox target of cigarette smoke in human sebocytes

For its critical location, the skin represents the major interface between the body and the environment, therefore is one of the major biological barriers against the outdoor environmental stressors. Among the several oxidative environmental stressors, cigarette smoke (CS) has been associated with the development and worsening of many skin pathologies such as acne, dermatitis, delayed wound healing, aging and skin cancer. In our previous work we have demonstrated that CS is able to affect genes involved in skin cholesterol trafficking, among which SRB1, a receptor involved in the uptake of cholesterol from HDL, seems to be very susceptible to the oxidative stress induced by CS.In the present work we wanted to investigate the presence of SRB1 in human sebocytes and whether CS can affect cholesterol cellular uptake via the redox modulation of SRB1.By using a co-culture system of keratinocytes/sebocytes, we found that CS exposure induced a SRB1 protein loss without affecting sebocytes viability. The decrease of SRB1 levels was a consequence of SRB1/HNE adducts formation that leads to SRB1 ubiquitination and degradation. Moreover, the CS-induced loss of SRB1 induced an alteration of sebocytes lipid content, also demonstrated by cholesterol quantification in SRB1 siRNA experiments.In conclusion, exposure to CS, induced SRB1 post-translational modifications in sebocytes and this might affect sebocytes/skin functionality.

Clinically Evaluated Cancer Drugs Inhibiting Redox Signaling.

There are a number of redox-active anticancer agents currently in development based on the premise that altered redox homeostasis is necessary for cancer cell's survival. Recent Advances: This review focuses on the relatively few agents that target cellular redox homeostasis to have entered clinical trial as anticancer drugs. The success rate of redox anticancer drugs has been disappointing compared to other classes of anticancer agents. This is due, in part, to our incomplete understanding of the functions of the redox targets in normal and cancer tissues, leading to off-target toxicities and low therapeutic indexes of the drugs. The field also lags behind in the use biomarkers and other means to select patients who are most likely to respond to redox-targeted therapy. If we wish to derive clinical benefit from agents that attack redox targets, then the future will require a more sophisticated understanding of the role of redox targets in cancer and the increased application of personalized medicine principles for their use. Antioxid. Redox Signal. 26, 262-273.

Compromised redox homeostasis, altered nitroso–redox balance, and therapeutic possibilities in atrial fibrillation

Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso–redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF.

A novel electrochemical aptasensor for bisphenol A assay based on triple-signaling strategy

Based on a triple-signaling strategy, a novel electrochemical aptasensor has been developed for sensitive and selective detection of bisphenol A (BPA). The thiolated ferrocene (Fc)-modified BPA-binding aptamer probe (Fc-P) was immobilized on the gold electrode and then hybridized with the methylene blue (MB)-modified complementary DNA probe (MB-P) to form a rigid double-stranded DNA (ds-DNA). The specific interaction between BPA and Fc-P led to the release of MB-P from the sensing interface and the conformational change of Fc-P. As a result, the oxidation peak currents of Fc and BPA increased with the increase of the concentration of target (BPA) according to the “signal-on” mode while that of MB decreased with the increase of the BPA concentration according to the “signal-off” mode. By superimposing the triple signal changes, BPA was detected sensitively with a linear range from 1pM to 100pM. The detection limit is 0.19pM, and much lower than that obtained by most of the reported electrochemical methods. The aptasensor also exhibited satisfactory specificity, selectivity, reproducibility and stability. By changing the specific aptamers, this strategy could be easily extended to detect other redox targets, showing promising applications in environmental analysis, food safety monitoring, and bioanalysis.

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.

Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall

Superoxide (O2•−) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide (•NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether •NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether •NO exerts cell-specific, differential modulation of O2•− levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates •NO-dependent inhibition of neointimal hyperplasia. In vivo, •NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, •NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2•− levels. Lastly, to determine the role of SOD-1 in •NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without •NO. Interestingly, •NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2•− by •NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of •NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.

Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury

Emerging lines of evidence indicate that oxidants such as hydrogen peroxide exert specific signaling functions during the processing of chronic pain. However, the mechanisms by which oxidants regulate pain processing in vivo remain poorly understood. Here, we investigated whether cyclic guanosine monophosphate (cGMP)-dependent protein kinase Iα (cGKIα), which can be activated by oxidants independently of cGMP, serves as a primary redox target during pain processing. After peripheral nerve injury, oxidant-induced cGKIα activation is increased in dorsal root ganglia of mice. Knock-in (KI) mice in which cGKIα cannot transduce oxidant signals demonstrated reduced neuropathic pain behaviors after peripheral nerve injury, and reduced pain behaviors after intrathecal delivery of oxidants. In contrast, acute nociceptive, inflammatory, and cGMP-induced pain behaviors were not impaired in these mice. Studying cGKIα KI mice, we provide the first evidence that oxidants activate cGKIα in sensory neurons after peripheral nerve injury in vivo. Our results suggest that oxidant-induced activation of cGKIα specifically contributes to neuropathic pain processing, and that prevention of cGKIα redox activation could be a potential novel strategy to manage neuropathic pain.

Abstract C91: Targeting enzalutamide resistance in prostate cancer.

Abstract Prostate cancer initially responses to androgen ablation or castration, but progression to incurable castrate-resistant prostate cancer often occurs within three years. Androgen receptor pathway inhibitor Enzalutamide (ENZ) has produced promising survival gains of patients with CRPC. However, resistance to this novel therapy also rapidly occurs and novel treatment strategies are needed. We have recently developed a unique ENZ and castrate-resistant LNCaP xenograft model. To identify key regulators and active pathways in ENZ resistant cells, genome-wide gene expression analysis was performed and analyzed using systems biological approaches. We identified that forkhead box M1 (FOXM1) is one of the key upstream regulators activated in ENZ resistant prostate cancer cells. FOXM1 is overexpressed in several cancers and it is an important regulator of cancer characteristics such as cell cycle, proliferation, invasion, metastases and cancer stem cells. FOXM1 is also a critical regulator of oxidative stress and its depletion sensitises cells to oxidative stress induced apoptosis. Interestingly, we further found out that the antibiotic ionophore and oxidative stress inducer monensin that was previously identified as a cancer-specific prostate cancer inhibitor in high-throughput screening, reduces FOXM1 pathway activity and viability of ENZ resistant cells. Thus, the results suggest that inhibition of FOXM1 is a potent novel mean to impair redox control and target ENZ resistant prostate cancer cells. Taken together, our results indicated that FOMX1 activity is increased in ENZ resistant prostate cancer cells and that targeting FOXM1 is a potential means to overcome ENZ resistance in prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C91. Citation Format: Kirsi Ketola, Jennifer Bishop, Martin Gleave, Amina Zoubeidi. Targeting enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C91.

KEAP1 Is a Redox Sensitive Target That Arbitrates the Opposing Radiosensitive Effects of Parthenolide in Normal and Cancer Cells

Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells toward death, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. We previously showed that parthenolide enhances ROS production in prostate cancer cells through activation of NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes to parthenolide's radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury. Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Furthermore, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective properties in normal cells.

Electrochemical sensor based on direct electron transfer of HIV-1 Virus at Au nanoparticle modified ITO electrode

In this study, for the first time, an electrochemical detection method was proposed to detect direct electron transfer signal from HIV-1 Virus. Au nanoparticles were fabricated on the Indium Tin Oxide coated glass (ITO) electrode by electrochemical deposition to improve the surface area to provide better electron-transfer kinetics, and higher background charging current. On the Au nanoparticle modified ITO electrode, antibody fragment was immobilized by self-assembly method with gold–thiol interaction and different concentrations of HIV-1 Virus like particles (VLPs) were applied for the direct determination. Due to the advantage of fabricated Au nanoparticle's excellent electrical and mechanical properties, HIV-1 VLPs were successfully detected from 600fg/mL to 375pg/mL. Furthermore, since the proposed electrochemical virus sensor is designed for direct determination without any labeling structure, the electrochemical communication between redox target and electrode surface will reduce interference reactions and simplifies the detection system with small amount of reagent and better stability. Therefore, highly sensitive virus sensor was developed to detect HIV-1 Virus in a label free system.

Investigating electron-transfer processes using a biomimetic hybrid bilayer membrane system

Here we report a protocol to investigate the electron-transfer processes of redox-active biomolecules in biological membranes by electrochemistry using biomimetic hybrid bilayer membranes (HBMs) assembled on gold electrodes. Redox-active head groups, such as the ubiquinone moiety, are embedded in HBMs that contain target molecules, e.g., nicotinamide adenine dinucleotide (NADH). By using this approach, the electron-transfer processes between redox molecules and target biomolecules are mediated by mimicking the redox cycling processes in a natural membrane. Also included is a procedure for in situ surface-enhanced Raman scattering (SERS) to confirm the electrochemically induced conformational changes of the target biomolecules in the HBMs. In addition, each step in constructing the HBMs is characterized by electrochemical impedance spectroscopy (EIS), high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The time required for the entire protocol is ∼12 h, whereas the electrochemical measurement of electron-transfer processes takes less than 1 h to complete.

Tetrahydrobiopterin: A Vascular Redox Target to Improve Endothelial Function

Loss of normal endothelial function and bioactivity of nitric oxide (NO), associated with increased production of reactive oxygen species (ROS), are characteristics of cardiovascular disease states. There is good experimental evidence that these abnormalities are causally related to cardiovascular disease pathogeneses, and are amenable to therapeutic intervention. However, simple attempts to increase NO levels or reduce "oxidative stress", for example using nonselective anti-oxidant drugs, have shown no benefit as treatments of cardiovascular disease. Increasing evidence highlights the need to better understand NO and ROS mediated signaling mechanisms in endothelial function, in order to identify more rational and selective therapeutic targets. The NO synthase co-factor, tetrahydrobiopterin (BH4) is a redox active molecule which regulates NO and ROS production by NO synthase and provides an exemplar of redox dependent signaling in the endothelium, with relevance to cardiovascular disease. Loss of endothelial cell BH4 is observed in cardiovascular disease states and results in loss of NO, but increased ROS production by endothelial NO synthase. Genetic mouse models of augmented endothelial cell BH4 synthesis have shown proof of concept that endothelial cell BH4 can alter cardiovascular disease pathogenesis, but clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial cell uptake of BH4. In contrast, some existing therapies such as statins appear to exert favourable effects on endothelial cell BH4 and endothelial NO synthase function. Identifying specific redox mechanisms and targets in the endothelium will provide new potential targets for future drug treatments.

A Novel Strategy for Global Analysis of the Dynamic Thiol Redox Proteome

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

Redox Regulation of Actin by Thioredoxin-1 Is Mediated by the Interaction of the ProteinsviaCysteine 62

Actin is a highly conserved protein in eukaryotic cells, and has been identified as one of the main redox targets by redox proteomics under oxidative stress. However, little is known about the mechanisms of regulation of the redox state of actin. In this study, we investigated how thioredoxin-1 (Trx1) affected the redox state of actin and its polymerization under oxidative stress in SH-SY5Y cells. Trx1 decreased the levels of reactive oxygen species (ROS) in the cells, and cysteine residues at positions 32, 35, and 69 of the Trx1 protein were active sites for redox regulation. Actin could be kept in a reduced state by Trx1 under H(2)O(2) stimulation. A physical interaction was found to exist between actin and Trx1. Cysteine 62 in Trx1 was the key site that interacted with actin, and it was required to maintain cellular viability and anti-apoptotic function. Taken together, these results suggested that Trx1 could protect cells from apoptosis under oxidative stress not only by increasing the total antioxidant capability and decreasing the ROS levels, but also by stabilizing the actin cytoskeletal system, which cooperatively contributed to the enhancement of cell viability and worked against apoptosis.

Protein deacetylation by sirtuins: delineating a post-translational regulatory program responsive to nutrient and redox stressors

Lysine acetylation/deacetylation is increasingly being recognized as common post-translational modification that appears to be broadly operational throughout the cell. The functional roles of these modifications, outside of the nucleus, have not been extensively studied. Moreover, as acetyl-CoA donates the acetyl group for acetylation, nutrient availability and energetic status may be pivotal in this modification. Similarly, nutrient limitation is associated with the deacetylation reaction. This modification is orchestrated by a novel family of sirtuin deacetylases that function in a nutrient and redox dependent manner and targets non-histone protein deacetylation. In compartment-specific locations, candidate target proteins undergoing lysine-residue deacetylation are being identified. Through these investigations, the functional role of this post-translational modification is being delineated. We review the sirtuin family proteins, discuss their functional effects on target proteins, and postulate on potential biological programs and disease processes that may be modified by sirtuin-mediated deacetylation of target proteins.

Role of Hydrogen Peroxide in NF-κB Activation: From Inducer to Modulator

Hydrogen peroxide (H2O2) has been implicated in the regulation of the transcription factor NF-kappaB, a key regulator of the inflammatory process and adaptive immunity. However, no consensus exists regarding the regulatory role played by H2O2. We discuss how the experimental methodologies used to expose cells to H2O2 produce inconsistent results that are difficult to compare, and how the steady-state titration with H2O2 emerges as an adequate tool to overcome these problems. The redox targets of H2O2 in the NF-kappaB pathway--from the membrane to the post-translational modifications in both NF-kappaB and histones in the nucleus--are described. We also review how H2O2 acts as a specific regulator at the level of the single gene, and briefly discuss the implications of this regulation for human health in the context of kappaB polymorphisms. In conclusion, after near 30 years of research, H2O2 emerges not as an inducer of NF-kappaB, but as an agent able to modulate the activation of the NF-kappaB pathway by other agents. This modulation is generic at the level of the whole pathway but specific at the level of the single gene. Therefore, H2O2 is a fine-tuning regulator of NF-kappaB-dependent processes, as exemplified by its dual regulation of inflammation.

H2O2-mediated modulation of cytosolic signaling and organelle function in rat hippocampus

Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox targets involved in such signaling, we exposed hippocampal neurons to hydrogen peroxide (H2O2). Redox-sensitive dyes indicated that externally applied H2O2 may oxidize intracellular targets in cell cultures and acute tissue slices. In cultured neurons, H2O2 (EC50 118 µM) induced an intracellular Ca2+ rise which could still be evoked upon Ca2+ withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, dantrolene, 2-aminoethoxydiphenyl borate, and high levels of ryanodine, which identifies the endoplasmic reticulum (ER) as the intracellular Ca2+ store involved. Intracellular accumulation of endogenously generated H2O2—provoked by inhibiting glutathione peroxidase—also released Ca2+ from the ER, as did extracellular generation of superoxide. Phospholipase C (PLC)-mediated metabotropic signaling was depressed in the presence of H2O2, but cytosolic cyclic adenosine-5′-monophosphate (cAMP) levels were not affected. H2O2 (0.2–5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a Ca2+- and cAMP-independent manner, and directly oxidized cellular nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). In part, the mitochondrial depolarization reflects uptake of Ca2+ previously released from the ER. We conclude that H2O2 releases Ca2+ from the ER via both ryanodine and inositol trisphosphate receptors. Mitochondrial function is not markedly impaired even by millimolar concentrations of H2O2. Such modulation of Ca2+ signaling and organelle interaction by ROS affects the efficacy of PLC-mediated metabotropic signaling and may contribute to the adjustment of neuronal function to redox conditions and metabolic supply.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-009-0672-0) contains supplementary material, which is available to authorized users.

Blockade of the erbB2 Receptor Induces Cardiomyocyte Death through Mitochondrial and Reactive Oxygen Species-dependent Pathways

Overexpression of the receptor tyrosine kinase erbB2 (Her2 in humans) is correlated with a poor prognosis in breast and ovarian cancers. Treatment with trastuzumab (a monoclonal antibody against erbB2) improves survival; however, it also causes cardiomyopathy. We hypothesized that blockade of the erbB2 receptor induces cardiomyocyte death through a mitochondrial pathway that is dependent on the production of reactive oxygen species (ROS). We first showed that levels of erbB2 receptor are significantly decreased in an animal model of ischemic heart disease and in human ischemic cardiomyopathy. We treated neonatal rat cardiomyocytes with an inhibitory erbB2 antibody to study the mechanism behind the deleterious effects of erbB2 blockade. These cells displayed a dose-dependent increase in ROS production and cell death compared with control IgG-treated cells; these processes were reversed by the antioxidant, N-acetylcysteine. The effects of erbB2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively. Furthermore, mouse embryonic fibroblasts lacking Bax and Bak (proteins that mediate cell death through a mitochondrial pathway) were resistant to the deleterious effects of erbB2 antibody. These effects of erbB2 blockade appear to occur through a pathway involving AKT and PKC-alpha. Our results suggest that erbB2 plays a role in cardiomyocyte survival, and that the deleterious effects of trastuzumab on the heart occur through a mitochondrial pathway and is mediated by ROS production. Manipulation of redox signaling may be beneficial in cancer patients receiving trastuzumab.

Effects of oxidative stress on protein thiols and disulphides in Mytilus edulis revealed by proteomics: Actin and protein disulphide isomerase are redox targets

Many proteins contain cysteines which are sensitive to oxidation. This is sometimes reversible through interaction with glutathione, glutaredoxin or thioredoxin systems making these proteins potential sensors of oxidative stress. In this study we analysed whether there was an increase in mixed disulphide bond (–S–S–) formation in the blue mussel Mytilus edulis in response to menadione. This was achieved by initially blocking reduced thiols with N-ethylmaleimide, –S–S– were then reduced with dithiothreitol (DTT) and labelled with 5-iodoacetamidofluorescein (5-IAF). Free –SHs were also labelled directly with 5-IAF. Separations were performed on 1D or 2D SDS PAGE and images analysed. There was an increase in –S–S– in response to menadione and detection of changes in oxidised proteins was easier than that of changes in the amount of reduced proteins. Protein disulphide isomerase (PDI) was labelled both as –SH and –S–S–, underlining its involvement in the redox status of the animal. A glutathione transferase (GST P1-1) forms an inter-chain disulphide bridge in response to menadione.