Aspirin-induced apoptosis in yeast

Abstract

In this study, the effect of aspirin on yeast cells with differential protection against reactive oxygen species (ROS), as obtained with wild-type, manganese superoxide dismutase (MnSOD)-deficient and copper, zinc superoxide dismutase (CuZnSOD)deficient Saccharomyces cerevisiae cells was investigated, when grown on fermentable and non-fermentable carbon sources. Aspirin was found to induce apoptosis in MnSODdeficient cells cultivated in ethanol medium, whereas the CuZnSOD-deficient and wildtype yeast strains remained viable. The deleterious effect of aspirin on MnSOD-deficient cells in ethanol medium, was not reversed by the antioxidants N-acetylcysteine and vitamin E, or by the SOD mimetics, TEMPO and TEMPOL. Furthermore, aspirin itself appeared to act as an antioxidant in these cells, until the onset of overt apoptosis, when a moderate increase in the intracellular oxidation level occurred.

The reducing power, as measured by the NADPHlNADP+ concentration ratio in the MnSOD-deficient cells cultivated in ethanol medium was significantly lower than in wild-type cells. In the presence of aspirin, an early shift in the redox balance in MnSOD-deficient cells was detected, due to depletion of NADPH and NADP+, which led to a dramatic drop in the GSH/GSSG ratio. Aspirin did not inhibit glucose-6-phosphate dehydrogenase activity in the MnSOD-deficient cells, and the apoptotic effect of aspirin does not seem to involve enzyme acetylation. However, catalase activity decreased significantly with aspirin, possibly due to decreased protection of the enzyme by NADPH. The decreased reducing power as observed in MnSOD-deficient cells in ethanol medium, may be linked to apoptotic induction by aspirin, independently of the level of ROS. The protective effect of MnSOD, exerted in the isogenic wild-type cells,

appears to be related to the cellular reducing power, which is essential for sustaining growth. Aspirin caused a significant drop in the intracellular pH (PHi) in the MnSOD-deficient cells, growing in ethanol medium. This finding suggests that pHi plays a role in the apoptotic process, however, it does not seem to be the initial trigger committing these cells to aspirin-induced apoptosis. No decrease in the pHi of wild-type cells was detected on treatment with aspirin, indicating that the MnSOD-deficient cells were unable to maintain a homeostatic cytosolic pH.

Mitochondria were found to play an important role in aspirin-induced apoptosis in MnSOD-deficient cells cultivated in ethanol medium. Aspirin triggered an initial release of mitochondrial cytochrome c, and after 96 h of cultivation, most of the mitochondrial cytochrome c was detected in the cytosol. This correlated with a dramatic drop in the ∆Ѱm, which decreased to nearly half that of untreated control cells. Mitochondrial mass in the MnSOD-deficient cells was unaltered on treatment with aspirin, even after cytochrome c release from mitochondria. Hence, mitochondrial mass did not influence the data obtained on the ∆Ѱm of aspirin-treated and untreated cells, using Rhodamine 123. These findings indicate that loss of ∆Ѱm is not required for aspirin induced release of cytochrome c. Indeed, the initial release of cytochrome c occurred prior to the disruption of the ∆Ѱm. It may thus be possible that cytochrome c release does not involve the early onset of the mitochondrial permeability transition but only an alteration of the permeability of the outer mitochondrial membrane.