L-glutamate delays aspirin-induced apoptosis of redox-compromised yeast cells by restoring the GSH/GSSG ratio and the mitochondrial respiratory rate

Abstract

Introduction: We showed that aspirin induces apoptosis in Saccharomyces cerevisiae EG110 cells deficient in manganese superoxide dismutase (MnSOD) cultivated in ethanol medium, but not in wild-type EG103 cells. Our microarray study revealed an aspirin-induced downregulation of SNO1 and SNZ1 in the mutant strain. Together, these genes encode a glutamine amidotransferase complex that produces glutamate: a key metabolite involved in synthesising the antioxidant glutathione (GSH) and the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate. Methods: Upon confirmation of gene expression by RT-qPCR, the growth of aspirin-treated EG110 cells supplemented with L-glutamate was monitored via optical density measurements (OD600). The effect of glutamate was confirmed by viability studies, based on colony forming units (CFUs) and flow-cytometric analysis. Further experiments involved the measurement of intracellular GSH and GSSG levels, as well as respirometry measurements. Results: We observed that aspirin-induced apoptosis of EG110 cells is significantly delayed by the addition of 200mM L-glutamate. Furthermore, we show that this rescuing effect is due to a restored GSH/GSSG ratio, as well as a restored mitochondrial respiratory rate. Conclusion: The influence of aspirin on glutamate metabolism in our redox-compromised cells, suggests that their death is preceded by redox imbalance, as shown by a lowered GSH/GSSG ratio and oxidative stress accompanied by decreased respiratory rate. Rescuing by exogenous glutamate is likely mediated by increased synthesis of cellular GSH and the anaplerotic supply of α -ketoglutarate to the TCA cycle. This aspirin-induced effect on glutamate metabolism may help us better understand the antineoplastic effect of aspirin on early-stage cancer cells which are also redox-compromised. Disclosures: Project “R&I-2015-001” is being financed by the Malta Council for Science and Technology through the R&I Technology Development Programme.