RNA microarray studies of aspirin's effect on MnSOD-deficient mutant [EG110, (MT)] and wild-type [EG103, (WT)] Saccharomyces cerevisiae yeast cells

Non-steroidal anti-inflammatory drugs, principally aspirin (acetylsalicylic acid, ASA), have anti-neoplastic properties, as shown by epidemiological studies on colorectal cancer and many other types of tumours. The chemopreventive and anti-proliferative properties of aspirin towards tumour cells have been shown to be due to the induction of programmed cell death such as apoptosis. Yeast cells are among the experimental models used extensively for the study of oxidative stress and apoptosis in living organisms because yeast, such as S. cerevisiae, retains many of the core eukaryotic cellular processes, including the hallmarks of eukaryotic apoptosis. An important contribution of our previous work has been the clarification of the critical defensive role of the antioxidant mitochondrial enzyme manganese superoxide dismutase (MnSOD) against apoptosis, confirmed to be the attenuation of aspirin-induced superoxide radical accumulation in the yeast mitochondria (Farrugia et al. (2013) FEMS Yeast Res 13, 755-768). To study the possible differential expression of gene transcripts in relation to the induction of apoptosis by aspirin, we used gene expression profiling by means of GeneChip® Microarray Technology (Affymetrix®). The yeast strains considered for this study included (1) the wild type strain S. cerevisiae EG103, which contains both MnSOD and cytosolic copper, zinc superoxide dismutase (CuZnSOD) and (2) the redox-compromised MnSOD-deficient S. cerevisiae EG110 strain. [This work was financed by the Malta Council for Science and Technology through the R&I Technology Development Programme (Project R&I-2015-001)]. 1x108 yeast cells were harvested after 48 hours (h) of aerobic growth in ethanol medium (YPE) in the absence and presence of 15 mM ASA, at 28∘C with constant shaking at 250 rpm. The cells were mechanically lysed using a micro-minibeadbeater (Biospec), in the presence of acid-washed glass beads (500 micrometer diameter) and lysis buffer RLT provided with the Qiagen® RNeasy Mini Kit. Total yeast RNA was extracted and purified using the Qiagen® RNeasy Mini Kit including on-column DNase I digestion, according to the manufacturer’s instructions. The quantity and quality of total RNA were determined using a Thermo Scientific™ Nanodrop spectrophotometer and an Agilent™ 2100 Bioanalyser. A 500 ng quantity of total RNA from each biological replicate of cells, grown in both the absence or presence of aspirin, was analyzed by microarrays. The quality of the total RNA samples and the resulting complimentary RNA (cRNA) was assessed using the Bioanalyzer. Fragmented biotin-labeled cRNA was prepared and 7.5 micrograms was hybridized to GeneChip® Yeast Genome 2.0 Array Chips (Affymetrix®), according to the manufacturer’s protocols (Affymetrix®). The data was normalized using the Robust Multi-Array average (RMA) method, implemented AltAnalyze Version 2.0.8.1 (University of Cincinnati).