Apoptosis, redox stress and cancer.

Abstract

Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed.