beta-Phenylethyl isothiocyanate mediated apoptosis: a proteomic investigation of early apoptotic protein changes

beta-Phenylethyl isothiocyanate (PEITC) is a promising chemopreventative agent found in abundance in watercress (Rorripa nasturtium aquaticum) as its glucosinolate precursor. In the present investigation, we sought to determine the early changes in protein expression that contribute to the mechanism(s) of PEITC-mediated apoptosis in the human hepatoma HepG2 cell line. Such data may invariably identify new molecular targets of PEITC, contributing to a greater understanding of the mechanism(s) by which isothiocyanates mediate apoptotic cascades. Using two-dimensional difference gel electrophoresis we determined the changes in global protein expression between control (0.01% dimethyl sulfoxide) and PEITC (IC50 approximately 20 microM) treated cells after 3 and 6 h, such time points being used to circumvent the effects of caspase mediate proteolysis. Comparison between PEITC treated cells with their respective controls showed that 17 protein spots were differentially expressed. Fourteen of these spots, representing 9 unique proteins, were successfully identified using matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) and MALDI tandem time of flight (TOF/TOF) mass spectrometry. We observed significant shifts in isoelectric points on two-dimensional electrophoresis gels in heat shock 27 kDa protein (HSP27), macrophage migration inhibition factor and heterogeneous nuclear ribonucleoprotein K (hnRNP K) indicating that these proteins are probably involved in protein phosphorylation. Indeed, hnRNP K was determined to be phosphorylated on key tyrosine residues as assessed by using antiphosphotyrosine antibodies. In separate experiments we also showed that c-myc is up-regulated in PEITC treated cells, and since hnRNP K is reported to induce overexpression of c-myc, we proposed that PEITC-induced apoptosis may involve a c-myc dependent apoptotic pathway in HepG2 cells.     

Tolerance to oxidative stress is associated with both oxidative stress response and inherent growth in a fungal wheat pathogen

Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen–plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxidative stress tolerance, and we identified two uncharacterized genes in a major QTL associated with this trait. Our data suggest that melanization does not affect tolerance to oxidative stress, which differs from what was found for animal pathogens. This study provides a whole-genome perspective on the genetic basis of oxidative stress tolerance in a plant pathogen.     

The ARG tyrosine kinase interacts with Siva-1 in the apoptotic response to oxidative stress

The Abl family of mammalian nonreceptor tyrosine kinases consists of c-Abl and ARG (Abl-related gene). Certain insights are available regarding the involvement c-Abl in the response of cells to stress. ARG, however, has no known function in cell signaling. The present studies demonstrate that ARG associates with the proapoptotic Siva-1 protein. The functional significance of the ARG-Siva-1 interaction is supported by the finding that ARG is activated by oxidative stress and that this response involves ARG-mediated phosphorylation of Siva-1 on Tyr(48). The proapoptotic effects of Siva-1 are accentuated in cells stably expressing ARG and are inhibited in ARG-deficient cells. Moreover, the proapoptotic effects of Siva-1 are abrogated by mutation of the Tyr(48) site. We also show that the apoptotic response to oxidative stress is attenuated in ARG-deficient cells and that this defect is corrected by reconstituting ARG expression. These findings support a model in which the activation of ARG by oxidative stress induces apoptosis by a Siva-1-dependent mechanism.     

A novel proteomic approach reveals a role for Mg-protoporphyrin IX in response to oxidative stress

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression. In order to investigate whether the tetrapyrrole Mg-ProtoIX itself is an important part of plastid-to-nucleus communication, we used an affinity column containing Mg-ProtoIX covalently linked to an Affi-Gel matrix. The proteins that bound to Mg-ProtoIX were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with nano liquid chromatography-mass spectrometry (MS)/MS. Thus, we present a novel proteomic approach to address the mechanisms involved in cellular signaling and we identified interactions between Mg-ProtoIX and a large number of proteins associated with oxidative stress responses. Our approach revealed an interaction between Mg-ProtoIX and the heat shock protein 90-type protein, HSP81-2 suggesting that a regulatory complex including HSP90 proteins and tetrapyrroles controlling gene expression is evolutionarily conserved between yeast and plants. In addition, our list of putative Mg-ProtoIX-binding proteins demonstrated that binding of tetrapyrroles does not depend on a specific amino acid motif but possibly on a specific fold of the protein.     

Human polynucleotide phosphorylase protein in response to oxidative stress

8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in their precursors due to the actions of oxygen radicals produced through a normal cellular metabolism. Since oxidized guanine can pair with both cytosine and adenine, it causes alterations in the phenotypic expression when it is present in RNA. To prevent such an outcome, organisms must have some mechanism for eliminating such oxidized guanine nucleotides from RNA and its precursors. In mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to 8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has been suggested to be a good candidate for such a role. The human PNP protein has an ability to bind specifically to RNA containing 8-oxoGua. When human cells are exposed to agents that induce oxidative stress, such as hydrogen peroxide and menadion, the amounts of PNP protein decrease rapidly while amounts of other proteins in the cells do not change after such treatments. No specific decrease in the PNP protein level is observed when cells are treated with ACNU and cycloheximide at doses sufficient to provide the same degree of growth suppression. These results imply that the PNP protein might thus play a role in excluding oxidized forms of RNA from the translation mechanism.     

Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat

Stroke is a life-threatening disease with major cause of mortality and morbidity worldwide. The neuronal damage following cerebral ischemia is a serious risk to stroke patients. Oxidative stress and apoptotic damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The objective of this study was to test the hypothesis that administration of edaravone (Edv) maintains antioxidant status in brain, improves the cholinergic dysfunction and suppresses the progression of apoptosis response in rat. To test this hypothesis, male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) of 2 h followed by reperfusion for 22 h. Edv was administered (10 mg/kg bwt) intraperitoneally 30 min before the onset of ischemia and 1 h after reperfusion. After reperfusion, rats were tested for neurobehavioral activities and were sacrificed for the infarct volume, estimation of oxidative damage markers. Edv treatment significantly reduced ischemic lesion volume, improved neurological deficits, contended oxidative loads, and suppressed apoptotic damage. In conclusion, treatment with Edv ameliorated the neurological and histological outcomes with elevated endogenous anti-oxidants status as well as reduced induction of apoptotic responses in MCA occluded rat. We theorized that Edv is among the pharmacological agents that reduce free radicals and its associated cholinergic dysfunction and apoptotic damage and have been found to limit the extent of brain damage following stroke.     

Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.     

Diva reduces cell death in response to oxidative stress and cytotoxicity

Diva is a member of the Bcl2 family but its function in apoptosis remains largely unclear because of its specific expression found within limited adult tissues. Previous overexpression studies done on various cell lines yielded conflicting conclusions pertaining to its apoptotic function. Here, we discovered the expression of endogenous Diva in PC12 neuronal-like cell line and rat bone marrow mesenchymal stem cells (BMSCs), leading to their utilisation for the functional study of Diva. Through usage of recombinant Fas ligand, hydrogen peroxide, overexpression and knock down experiments, we discovered that Diva plays a crucial pro-survival role via the mitochondrial death pathway. In addition, immunoprecipitation studies also noted a decrease in Diva's interaction with Bcl2 and Bax following apoptosis induced by oxidative stress. By overexpressing Diva in BMSCs, we had observed an increase in the cells' capacity to survive under oxidative stress and microglial toxicity. The result obtained from our study gives us reason to believe that Diva plays an important role in controlling the survival of BMSCs. Through overexpression of Diva, the viability of these BMSCs may be boosted under adverse conditions.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Differential carbonylation of proteins as a function of in vivo oxidative stress

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the 35 proteins identified and quantified, Apo AII, clusterin, hemopexin precursor, and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, 11 of which changed more than 2-fold in oxidation state. Three types of carbonylation were identified at these sites: direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homologue were the most heavily oxidized proteins.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

Role of zinc in pulmonary endothelial cell response to oxidative stress

Although zinc is a well-known inhibitor of apoptosis, it may contribute to oxidative stress-induced necrosis. We noted that N,N,N',N'- tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; >10 microM), a zinc chelator, quenched fluorescence of the zinc-specific fluorophore Zinquin and resulted in an increase in spontaneous apoptosis in cultured sheep pulmonary artery endothelial cells (SPAECs). Addition of exogenous zinc (in the presence of pyrithione, a zinc ionophore) to the medium of SPAECs caused an increase in Zinquin fluorescence and was associated with a concentration-dependent increase in necrotic cell death. Exposure of SPAECs to TPEN (10 microM) resulted in enhanced apoptosis after lipopolysaccharide or complete inhibition of t-butyl hydroperoxide (tBH)-induced necrosis. We further investigated the role of two zinc-dependent enzymes, poly(ADP-ribose) polymerase (PARP) and protein kinase (PK) C, in tBH toxicity. tBH toxicity was only affected by the PARP inhibitors 4-amino-1,8-naphthalimide or 3-aminobenzamide over a narrow range, whereas the PKC inhibitors bisindolylmaleimide and staurosporine significantly reduced tBH toxicity. tBH caused translocation of PKC to the plasma membrane of SPAECs that was partially inhibited by TPEN. Thus pulmonary endothelial cell zinc inhibits spontaneous and lipopolysaccharide-dependent apoptosis but contributes to tBH-induced necrosis, in part, via a PKC-dependent pathway.     

Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.     

Role of oxidative carbonylation in protein quality control and senescence

Proteins can become modified by a large number of reactions involving reactive oxygen species. Among these reactions, carbonylation has attracted a great deal of attention due to its irreversible and unrepairable nature. Carbonylated proteins are marked for proteolysis by the proteasome and the Lon protease but can escape degradation and form high-molecular-weight aggregates that accumulate with age. Such carbonylated aggregates can become cytotoxic and have been associated with a large number of age-related disorders, including Parkinson's disease, Alzheimer's disease, and cancer. This review focuses on the generation of and defence against protein carbonyls and speculates on the potential role of carbonylation in protein quality control, cellular deterioration, and senescence.     

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from −218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.