Jeffrey S. Armstrong

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well‐known cytotoxic gas. Recently it has been shown to stimulate N‐methyl‐d‐aspartate (NMDA) receptors to enhance long‐term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 µm. Considerably lower H2S levels are reported in the brains of Alzheimers disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S ‘donor’, sodium hydrogen sulfide (NaSH) on peroxynitrite‐mediated damage to biomolecules and to cultured human SH‐SY5Y cells. H2S significantly inhibited peroxynitrite‐mediated tyrosine nitration and inactivation of α1‐antiproteinase to a similar extent to reduced glutathione at each concentration tested (30–250 µm). H2S also inhibited peroxynitrite‐induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH‐SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite‐mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (γGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24 h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3 h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24 h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24 h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (ΔΨm) remained intact in most of the cells during the 72 h observation period, indicating that ΔΨm dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

Glutathione depletion enforces the mitochondrial permeability transition and causes cell death in Bcl-2 overexpressing HL60 cells.

Bcl‐2, a protein that blocks apoptosis by inhibiting the mitochondrial permeability transition (MPT) and release of cytochrome c appears to affect normal mitochondrial function by altering electron flow and increasing rates of reactive oxygen species (ROS) production. In this study, we show that glutathione (GSH) depletion induces ROS production and selective toxicity in HL60 cells that overexpress Bcl‐2 compared with neomycin vector control cells. Toxicity was mediated by the MPT because it was blocked with the adenine nucleotide translocator (ANT) ligand bongkrekic acid and resulted in mitochondrial cytochrome c release, caspase 3 activation, and DNA fragmentation, indicating the involvement of an apoptotic pathway. Respiratory chain inhibitors stigmatellin and antimycin A, which inhibit Qo and Qi sites of respiratory chain complex III, respectively, blocked ROS production, preserved the redox state of protein thiols, and prevented cell death. These results indicate that in the absence of GSH, endogenous ROS generated at respiratory complex III induce MPT independently of Bcl‐2. The results also suggest a new model for MPT in which the central pore protein ANT is regulated by adenine nucleotide and the activity of mitochondrial respiratory complex III.

Mitochondria: a target for cancer therapy

Mitochondria, the cells powerhouses, are essential for maintaining cell life, and they also play a major role in regulating cell death, which occurs upon permeabilization of their membranes. Once mitochondrial membrane permeabilization (MMP) occurs, cells die either by apoptosis or necrosis. Key factors regulating MMP include calcium, the cellular redox status (including levels of reactive oxygen species) and the mobilization and targeting to mitochondria of Bcl‐2 family members. Contemporary approaches to targeting mitochondria in cancer therapy use strategies that either modulate the action of Bcl‐2 family members at the mitochondrial outer membrane or use specific agents that target the mitochondrial inner membrane and the mitochondrial permeability transition (PT) pore. The aim of this review is to describe the major mechanisms regulating MMP and to discuss, with examples, mitochondrial targeting strategies for potential use in cancer therapy.

Mitochondrial Medicine : Pharmacological targeting of mitochondria in disease

Mitochondria play a central role in cell life and death and are known to be important in a wide range of diseases including the cancer, diabetes, cardiovascular disease, and the age‐related neurodegenerative diseases. The unique structural and functional characteristics of mitochondria enable the selective targeting of drugs designed to modulate the function of this organelle for therapeutic gain. This review discusses mitochondrial drug targeting strategies and a variety of novel mitochondrial drug targets including the electron transport chain, mitochondrial permeability transition, Bcl‐2 family proteins and mitochondrial DNA. Mitochondrial drug‐targeting strategies will open up avenues for manipulating mitochondrial functions and allow for selective protection or eradication of cells for therapeutic gain in a variety of diseases.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3‐nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN‐1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium‐dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium‐dependent process that leads to caspase‐independent apoptosis mediated by calpains.

The Mitochondrial Permeability Transition Regulates Cytochrome c Release for Apoptosis during Endoplasmic Reticulum Stress by Remodeling the Cristae Junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.

Cytochrome bc1 regulates the mitochondrial permeability transition by two distinct pathways

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc1 regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc1. The ROS producing mechanism in cytochrome bc1 involves movement of the “Rieske” iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc1 by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc1. These results indicate that electron flux through cytochrome bc1 regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.

Detection and Measurement of Reactive Oxygen Intermediates in Mitochondria and Cells

Reactive oxygen intermediates (ROIs) play a key role in a number of human diseases either by inducing cell death, cellular proliferation, or by acting as mediators in cellular signaling. Therefore, their measurement in vivo and in cell culture is desirable but technically difficult and often troublesome. To address some of the key methodological issues in examining the formation of ROI in cells and mitochondria, this chapter discusses the following: (a) the cellular sources of ROI and their enzymatic removal, (b) common methods used to determine cellular and mitochondrial ROI such as chemiluminescence, electron paramagnetic resonance spectroscopy, fluorescence, and enzymatic techniques, and (c) some common problems associated with these assays and the interpretation of data. We also provide some simple protocols for the estimation of ROI production in cells and mitochondria, and when measuring ROI in cells and mitochondria, we emphasize the need for thorough understanding of results obtained and their interpretation.