Brian Cunniff

Peroxiredoxin 3 Is a Redox-Dependent Target of Thiostrepton in Malignant Mesothelioma Cells

Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.

Oxidative Processing of Latent Fas in the Endoplasmic Reticulum Controls the Strength of Apoptosis

ABSTRACT We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241–252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.

AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion

Mitochondria infiltrate leading edge lamellipodia, increasing local mitochondrial mass and relative ATP concentration. AMPK regulates infiltration of mitochondria into the leading edge of 2D lamellipodia and 3D invadopodia, coupling local metabolic sensing to subcellular targeting of mitochondria during cell movement.

Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial‐derived oxidants and expression of FOXM1, a redox‐responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito‐carboxy‐proxyl (MCP) and Mito‐TEMPOL (MT) caused dose‐dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox‐responsive green fluorescent protein targeted to mitochondria (mito‐roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi‐1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM. J. Cell. Physiol. 228: 835–845, 2013.

Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine

Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates.

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC-TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC-TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC-TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC-TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.