Hydrogen sulfide (H 2 S) 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 H 2 S in the brain are reported to range between 10 and 160 lM. Considerably lower H 2 S levels are reported in the brains of Alzheimer's 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 H 2 S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H 2 S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitritemediated damage to biomolecules and to cultured human SH-SY5Y cells. H 2 S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of a 1 -antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 lM). H 2 S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H 2 S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H 2 S deserves further study, given that extracellular GSH levels in the brain are very low.

Although paracrine effects of mesenchymal stem cells (MSCs) have been suggested previously, cardioprotection by human MSC secretions has never been demonstrated. Human MSC-conditioned medium (CM) was collected by following a clinically compliant protocol. In a porcine model of ischemia and reperfusion injury, intravenous and intracoronary MSC-CM treatment significantly reduced myocardial nuclear oxidative stress as determined by immunostaining for 8-hydroxy-2'-deoxyguanosine. In addition, expression levels of phospho-SMAD2 and active caspase 3 were diminished following CM treatment, suggesting that TGF-beta signaling and apoptosis were reduced. This was associated with a 60% reduction in infarct size and marked improvement of systolic and diastolic cardiac performance as assessed with echocardiography and pressure volume loops. Fractionation studies revealed that only the fraction of the CM containing products >1000 kDa (100-220 nm) provided cardioprotection in a mouse model of ischemia and reperfusion injury. This indicates that the responsible paracrine factor of human MSCs is likely a large complex rather than a single small molecule. These data identify human MSC-CM as a promising therapeutic option to reduce myocardial infarct size in patients with acute MI and suggest that the use of stem cell secretions could extend the applicability of stem cells for therapeutic purposes.

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 (gGCS), 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 p21 waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a timedependent 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 pancaspase 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 (DC m ) remained intact in most of the cells during the 72 h observation period, indicating that DC m 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.

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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 are essential for maintaining cell life but they also play a role in regulating cell death, which occurs when their membranes become permeabilized. Mitochondria possess two distinct membrane systems including an outer membrane in close communication with the cytosol and an inner membrane involved in energy transduction. Outer membrane permeabilization is regulated by Bcl-2 family proteins, which control the release of proteins from the mitochondrial intermembrane space; these proteins then activate apoptosis. Inner membrane permeabilization is regulated by the mitochondrial permeability transition (MPT), which is activated by calcium and oxidative stress and leads to bioenergetic failure and necrosis. The purpose of this review is to discuss the biochemical mechanisms regulating mitochondrial membrane permeabilization; this is crucial to our understanding of the role of cell death in diseases such as cancer and the neurodegenerative diseases.

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.