Clarification of apoptosis pathway in Saccharomyces cerevisiae depending on ketoconazole induced conditions

In the scope of this thesis, it was aimed to clarify the effect of ketoconazole which is an antifungal and promising anticancer drug, on the viability and induced cell death types in Saccharomyces cerevisiae BY4742. Cell viability, phosphatidylserine externalisation, chromatin condensation, DNA fragmentation, mitochondrial membrane potentials were examined in S. cerevisiae to decide the cell death types triggered by ketoconazole in time- and concentration- dependent manner. The apoptotic pathways were investigated through the mutant S. cerevisiae BY4742 cells in which the known most efficient apoptotic genes deleted. Furthermore, alterations in intracellular reactive oxygen species, activities of radical producing enzymes and the response of enzymatic and non-enzymatic antioxidant systems and lipid peroxidation and protein carbonyl levels were investigated in a time and concentration dependent manner. The predominant cell death was found as necrosis at relatively higher ketoconazole concentrations and longer treatment time. In addition, there wasn't any cell death with the treatment of lower concentrations of ketoconazole. On the other hand, after optimising apoptotic conditions, apoptotic cell death occurred in a large amount of cell population. It was found that ketoconazole induced more than one apoptotic pathway in S. cerevisiae and all the investigated proteins participated in this process and the lowest efficient protein was determined as yeast caspase. After the treatment of ketoconazole, although the antioxidant system of S. cerevisiae BY4742 was induced strongly, hydrogen peroxide, hydroxyl radical and total ROS levels were over the control values in all treatments. However, the levels of superoxide anion radical and nitric oxide decreased to below the control level at the end of the investigated incubation period. Additionally, due to the oxidative stress resulting from the imbalance between the ketoconazole-induced ROS formation and the antioxidant defence system, membrane lipid peroxidation and protein carbonyl levels increased depending on ketoconazole concentration and incubation time.