title: Defining the chemical and molecular mechanisms of cytotoxicity Induced by the endoperoxide class of antimalarials
Creators: name: Firman, James
Publisher: name: University of Liverpool Repository
Year: 2015

description: Artemisinin-derived endoperoxide drugs find widespread employment as frontline treatment against malaria. Although evidence of their potential to express toxicity within a clinical setting remains limited, outcomes derived from animal studies attest their ability to induce neurological and developmental toxicity in mammalian systems. Activity is further demonstrated in vitro within rapidly proliferating human cells – most notably those belonging to immortalised, cancer-derived lines – with significant cytotoxic effects being observed upon drug treatment across a range of settings. It is believed that these find their origin through a mechanism dependent upon Fe(II)-mediated reduction of the endoperoxide bridge functionality, culminating in molecular bioactivation and the subsequent formation of carbon-centred free radical species which in turn, owing to their great reactivity, impart deleterious effects upon cellular functioning. Evidence suggests that dysfunction of the mitochondrion and the formation of reactive oxygen species (ROS) are key stages in the route through which artemisinin derivatives are able to induce death. The characteristics of artesunate-stimulated impact upon mitochondrial functioning are examined. It is demonstrated that culturing of cells in the presence of galactose enhances cytotoxic potential within the HeLa line. The magnitude of this variation in sensitivity is indicative that targeting of the mitochondrion affords a route through which activity is mediated. Falls in cell viability are further preceded by declines in ATP production, providing evidence that disruption of oxidative phosphorylation occurs as an early event in the route towards death. Studies performed on mitochondrial bioenergetic function using the Seahorse XF analyser indicate that artesunate imparts dose-dependent and timedependent decreases in respiratory reserve capacity and oxidative phosphorylation coupling efficiency, whilst stimulating a switch towards glycolytic energy production. Attempts to delineate the root causes of these effects are focused upon examining the relationship between oxidative stress, Fe(II) content and mitochondrial performance. The mitochondrially-localised antioxidant tiron and the lysosomal Fe(II) chelator desferrioxamine are shown to induce substantial cytoprotective effects against artesunate within the HeLa line. Evidence derived from Seahorse XF analysis indicates strongly that these outcomes are related to the capacity of both compounds to abrogate drug impact upon the functions of the mitochondrion. It can thus be posited that mitochondrial damage has its origins in the emergence of oxidative stress, with Fe(II) content acting as key determinant in its progression. The outcomes of further examinations performed within the ρ0 HeLa line suggest an origin for ROS emergence independent of the respiratory chain. In order to test the hypothesis that artemisinin derivatives might induce direct peroxidation of the mitochondrial phospholipid cardiolipin, the impact of a cytochrome c peroxidase inhibitor TPP-IOA is examined on the response of HeLa and HL-60 cells towards artesunate treatment. Results indicate that the inhibitor has variable effects upon cardiolipin oxidation state and subsequent cell survival, leaving doubt as to the true validity of such a connection. As a further study, the cytotoxic capacities of a range of novel artemisininderived anticancer agents and wholly synthetic tetraoxane and trioxolane antimalarials are given assessment. In conclusion, it can be stated that the outcomes of the studies performed in this thesis emphasise the importance of mitochondrial liability towards the progression of artemisinininduced cell death. Further insights into the mechanistic routes through which drug administration contributes, via oxidative stress and free Fe(II) content, to the defective functioning of the organelle have been achieved.