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|Title:||Mechanisms of aspirin-induced apoptosis in redox-compromised yeast cells : the effect of aspirin on the cell cycle and glutamate metabolism|
|Authors:||Azzopardi, Maria (2020)|
|Keywords:||Nonsteroidal anti-inflammatory agents|
|Citation:||Azzopardi, M. (2020). Mechanisms of aspirin-induced apoptosis in redox-compromised yeast cells : the effect of aspirin on the cell cycle and glutamate metabolism (Doctoral dissertation).|
|Abstract:||Various non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin (acetylsalicylic acid, ASA), are deemed chemopreventive. This is due to their anti-proliferative effects particularly on early-stage cancer cells, which are redox-compromised due to their reduced expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). The mechanisms by which ASA induces cell death in redox-compromised cells are not yet fully elucidated. In this study, the redox-compromised MnSOD-deficient Saccharomyces cerevisiae EG110 cells and the wild-type MnSOD-proficient EG103 cells, grown aerobically in ethanol medium, were used as suitable models to study the effect of ASA on the cell cycle and glutamate metabolism in redox-compromised . The significant ASA-induced downregulation of the genes SNO1 and SNZ1 in EG110, but not in EG103 yeast cells, which was previously determined by microarray analysis at 48 hours of cultivation, was validated by qRT-PCR analyses. Since the proteins encoded by these two genes together catalyse the deamination of glutamine into glutamate, this study confirmed that ASA-treated EG110 yeast cells, but not their untreated counterparts, cultivated for 96 hours in ethanol medium, suffer a highly significant decline in the intracellular levels of glutamate and of its immediate metabolic products α-ketoglutarate and reduced glutathione (GSH), which are involved in mitochondrial function and redox homeostasis, respectively. Also, at 96 hours of cultivation, ASA-treated EG110 cells were observed to die, as determined by growth curves and viability assays. Flow-cytometric DNA content analysis performed in this work revealed that ASA induces a G0/G1 cell-cycle arrest in EG110 cells, whereas their untreated counterparts accumulate at the G2/M phase. Notably, the deleterious effects of ASA on EG110 yeast cells were reversed by exogenous L-glutamate. In fact, the addition of 200 mM L-glutamate to ASA-treated EG110 yeast cells iv (i) restored their level of α-ketoglutarate, (ii) increased their level of GSH such that their redox balance was restored, as determined by the GSH/GSSG ratio, and (iii) protected the cells against cell cycle arrest and apoptotic cell death. These results indicated that glutamate depletion is a critical event in ASA-induced apoptosis in redox-compromised yeast cells. Since early-stage cancer cells are also considered to be redox-compromised and heavily rely on glutamate metabolism to proliferate rapidly and uncontrollably, these findings may (i) throw light on a potential chemopreventive mechanism by which aspirin selectively commits cancer cells to apoptosis, and (ii) contribute to the development of ASA-like drugs for chemoprevention.|
|Appears in Collections:||Dissertations - FacM&S - 2020|
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