Glutathione depletion causes cytochrome c release even in the absence of cell commitment to apoptosis

Abstract

We demonstrate here that the release of mature cytochrome c from mitochondria is a cellular response to the depletion of glutathione, the main intracellular antioxidant, independently from the destiny of the cells, i.e., apoptosis or survival. On the one hand, cytosolic cytochrome c was detected in cells where the inhibition of glutathione synthesis led to glutathione depletion without impairing viability or in tight concomitance with glutathione depletion prior to puromycin-induced apoptosis. Removal of the apoptogenic agent prior to apoptosis, but after glutathione extrusion and cytochrome c release, led to recovery of preapoptotic cells, which resume healthy features, i.e., restoration of normal glutathione levels and disappearance of cytosolic cytochrome c. On the other hand, in an example of apoptosis occurring without glutathione depletion, no translocation of cytochrome c from mitochondria to cytosol was detected. Unlike the other instances of apoptosis, in this case caspase 3 was not activated, thus suggesting the following oxidant-related apoptotic pathway: glutathione depletion, cytochrome c release, and caspase 3 activation. These results show that cytochrome c release is not a terminal event leading cells to apoptosis, but rather is the consequence of a redox disequilibrium that, under some circumstances, may be associated with apoptosis.

Superoxide Dismutase Inactivation in Pathophysiology of Asthmatic Airway Remodeling and Reactivity

“…Previous work has shown that oxidative stress decreases intracellular GSH through efflux, and GSH efflux has been identified as a proximal mediator of apoptosis through induction and activation of BAX. 32,33 Here, inhibition of SOD activity also caused rapid depletion of intracellular GSH, consistent with increased intracellular oxidant stress ( Figure 6B). Thus, our results also link BAX activation to oxidative stress and decreased intracellular GSH in vitro.…”

“…22 Interestingly, efflux of glutathione reproducibly activates BAX and cytochrome c release in epithelial cells in vitro and is one established mechanism for induction of apoptosis. 32,33,46 In support of increased transcellular glutathione fluctuation in the upstream events leading to epithelial cell apoptosis in asthma, glutathione levels are higher than normal in the asthmatic airway lining fluid, indicating increased efflux from epithelial cells in vivo. 42,[47][48][49] An overwhelming number of studies support oxidative and nitrative processes in the pathogenesis of asthma.…”

Multistep and multitask Bax activation

“…It was shown that after treatment with H 2 O 2 at low concentrations, or after glutathione depletion in U937 and HepG2 cells, cytochrome c is released in the absence of apoptosis (Ghibelli et al, 1999); at the same time, Bax translocates to mitochondria, and undergo dimerization, as detected in non-reducing but denaturing electrophoresis (SDS-PAGE) (D'Alessio et al, 2005). Cell-free oxidation of cytosolic extracts with H 2 O 2 leads to disulfide dimerization.…”

Disruption of Redox Homeostasis in Tumor Necrosis Factor-Induced Apoptosis in a Murine Hepatocyte Cell Line

“…Immunoblot analysis of nuclear protein was performed using 10 g of protein isolated as described above. Release of cytochrome C into the cytosol was determined by immunoblot as described by Ghibelli et al 29 using a mouse monoclonal anti-cytochrome C antibody (PharMingen, La Jolla, CA). Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% milk (blotting grade; BioRad, Hercules, CA) at 4°C and incubated with primary antibodies at the following dilutions: p65, 1:2,000; Mch5, 1:1,000; caspase-3, 1:1,000; and cytochrome C, 1:1,000, in 0.5% milk in Tris-buffered saline with 0.1% Tween 20 for 1 to 2 hours.…”