Dataset Information

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

ABSTRACT: Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

SUBMITTER: Booty LM

PROVIDER: S-EPMC6436940 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Booty Lee M LM Gawel Justyna M JM Cvetko Filip F Caldwell Stuart T ST Hall Andrew R AR Mulvey John F JF James Andrew M AM Hinchy Elizabeth C EC Prime Tracy A TA Arndt Sabine S Beninca Cristiane C Bright Thomas P TP Clatworthy Menna R MR Ferdinand John R JR Prag Hiran A HA Logan Angela A Prudent Julien J Krieg Thomas T Hartley Richard C RC Murphy Michael P MP

Cell chemical biology 20190131 3

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjug ...[more]

PMID: 30713096

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Project description:Glioblastoma is one of the most aggressive brain tumors, characterized by a pronounced redox imbalance, expressed in a high oxidative capacity of cancer cells due to their elevated glycolytic and mitochondrial oxidative metabolism. The assessment and modulation of the redox state of glioblastoma are crucial factors that can provide highly specific targeting and treatment. Our study describes a pharmacological strategy for targeting glioblastoma using a redox-active combination drug. The experiments were conducted in vivo on glioblastoma mice (intracranial model) and in vitro on cell lines (cancer and normal) treated with the redox cycling pair menadione/ascorbate (M/A). The following parameters were analyzed in vivo using MRI or ex vivo on tissue and blood specimens: tumor growth, survival, cerebral perfusion, cellular density, tissue redox state, expression of tumor-associated NADH oxidase (tNOX) and transforming growth factor-beta 1 (TGF-β1). Dose-dependent effects of M/A on cell viability, mitochondrial functionality, and redox homeostasis were evaluated in vitro. M/A treatment suppressed tumor growth and significantly increased survival without adverse side effects. This was accompanied by increased oxidative stress, decreased reducing capacity, and decreased cellular density in the tumor only, as well as increased cerebral perfusion and down-regulation of tNOX and TGF-β1. M/A induced selective cytotoxicity and overproduction of mitochondrial superoxide in isolated glioblastoma cells, but not in normal microglial cells. This was accompanied by a significant decrease in the over-reduced state of cancer cells and impairment of their "pro-oncogenic" functionality, assessed by dose-dependent decreases in: NADH, NAD+, succinate, glutathione, cellular reducing capacity, mitochondrial potential, steady-state ATP, and tNOX expression. The safety of M/A on normal cells was compromised by treatment with cerivastatin, a non-specific prenyltransferase inhibitor. In conclusion, M/A differentiates glioblastoma cells and tissues from normal cells and tissues by redox targeting, causing severe oxidative stress only in the tumor. The mechanism is complex and most likely involves prenylation of menadione in normal cells, but not in cancer cells, modulation of the immune response, a decrease in drug resistance, and a potential role in sensitizing glioblastoma to conventional chemotherapy.

Project description:The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and pathophysiological roles of reactive oxygen species. The relative contributions of the thioredoxin (Trx) and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on endothelial nitric oxide (NO) synthase (eNOS) and NO-dependent pathways in the endothelium. We designed duplex small interfering RNA (siRNA) constructs to specifically "knock down" the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione-to-oxidized glutathione ratio. siRNA-mediated knockdown of either GR, TrxR1, or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) and also blocked eNOS enzyme activity (using the [(3)H]arginine/[(3)H]citrulline assay). Pretreatment of endothelial cells with N,N'-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production. siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide (H(2)O(2)) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased H(2)O(2) production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in H(2)O(2) generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall.

Dataset Information

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Publications

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

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