Tumor Cell Responses to a Novel Glutathione<i>S</i>-Transferase–Activated Nitric Oxide-Releasing Prodrug

Findlay, Victoria J.; Townsend, Danyelle M.; Saavedra, Joseph E.; Buzard, Gregory S.; Citro, Michael L.; Keefer, Larry K.; Ji, Xinhua; Tew, Kenneth D.

doi:10.1124/mol.65.5.1070

Cited by 115 publications

(125 citation statements)

References 39 publications

(39 reference statements)

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“…Previous studies by our group observed an in vivo induction of protein nitration by PABA/NO (30). Furthermore, a critical ratio of GSH:PABA/NO is necessary to induce glutathionylation versus nitration in vitro.…”

Section: Discussionmentioning

confidence: 97%

A Novel Role for Human Sulfiredoxin in the Reversal of Glutathionylation

Findlay

Townsend²,

Morris³

et al. 2006

Cancer Research

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181

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Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys 99 ) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS À moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.

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Section: Discussionmentioning

confidence: 97%

A Novel Role for Human Sulfiredoxin in the Reversal of Glutathionylation

Findlay

Townsend²,

Morris³

et al. 2006

Cancer Research

Self Cite

181

140

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“…In previous studies, we showed the toxicity of PABA/NO to be significantly diminished in NIH/3T3 cells overexpressing MRP1, with an IC 50 value of 50 versus 11 M in wild-type (Findlay, 2004). We used these cells to address whether PABA/NO-induced S-glutathionylation is independent of toxicity (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Glutathione S-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with S-Glutathionylation of Proteins

Townsend

Findlay²,

Fazilev³

et al. 2005

Mol Pharmacol

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Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione, liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (Ͻ5 min) and is sustained for ϳ7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including ␤-lactate dehydrogenase, Rho GDP dissociation inhibitor ␤, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces Sglutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.The human genome is composed of Ͻ30,000 genes, yet complexity of protein structure/function seems distinctly more layered. In the proteomics era, it has become clear that the dogma of genetic determinism is distinctly influenced by a number of processes, including polymorphic variation, gene splicing events, exon shuffling, protein domain rearrangements, and a number of post-translational modifications that contribute to alterations in tertiary and quaternary protein structure. Among these, phosphorylation, glycosylation, methylation, and acetylation can account for a large proportion of modifications. Cellular homeostasis is an intricate balance of survival and death signals, the control of which can occur through post-translational modification of target proteins. Protein phosphorylation is regulated by kinase/ This research was funded through the National Cancer Institute grant CA53783, the Intramural Research Program of the National Institutes of Health/National Cancer Institute Center for Cancer Research, and National Cancer Institute c...

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“…Although NOV-002 treatment did not alter the expression levels of actin, S-glutathionylation modification of the protein increased in a dose-and timedependent manner. S-glutathionylation of actin alters the ratio of F ( filamentous) and G (globular) actin, leading to marked changes in the overall cytoskeletal architecture and concomitant changes in cellular trafficking (25). Using confocal microscopy, we evaluated the cytoskeletal architecture and stress fibers with phalloidin staining (which binds specifically to actin; Fig.…”

Section: Resultsmentioning

confidence: 99%

NOV-002, a Glutathione Disulfide Mimetic, as a Modulator of Cellular Redox Balance

Townsend

Hutchens

et al. 2008

Cancer Research

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NOV-002 is a novel glutathione disulfide mimetic that when administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance to chemotherapy (e.g., hematologic recovery) in advanced non-small cell lung cancer patients. We show that NOV-002, which is not cytotoxic as a single agent, generated time-and concentration-dependent oxidative signals at the cell surface (reduction in protein thiols) and intracellularly [altered oxidized glutathione (GSSG) and reduced glutathione levels and ratio; increased reactive oxygen species] in the premyeloid HL-60 cell line and that this was associated with an increase in S-glutathionylation of cell proteins, particularly actin. Commensurate with these effects, NOV-002 activated p38, c-Jun-NH 2 -kinase, and extracellular signal-regulated kinase and caused a dosedependent increase in phosphorylation of three proteins that have previously been linked with hematopoiesis, AKT, JAK2, and STAT5. The effect of NOV-002 on enzymes involved in glutathione metabolism was evaluated. Relative to oxidized glutathione, NOV-002 was an equivalent substrate for glutathione reductase and was an inhibitor of protein disulfide isomerase, one of the components of the redox-sensitive unfolded protein response pathway. These redox-stimulated cell signaling actions occurred in the context of increased HL-60 cell proliferation after treatment with NOV-002. Overall, the pleiotropic pharmacologic effects of NOV-002 can be attributed to the GSSG component of the drug, and modulation of cellular redox balance is a feature central to the mechanism of action of NOV-002. Such modulation may underlie its clinical actions, including hematologic recovery and immunostimulation in the face of chemosuppression.

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Tumor Cell Responses to a Novel GlutathioneS-Transferase–Activated Nitric Oxide-Releasing Prodrug

Cited by 115 publications

References 39 publications

A Novel Role for Human Sulfiredoxin in the Reversal of Glutathionylation

A Novel Role for Human Sulfiredoxin in the Reversal of Glutathionylation

A Glutathione S-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with S-Glutathionylation of Proteins

NOV-002, a Glutathione Disulfide Mimetic, as a Modulator of Cellular Redox Balance

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