Chracterization of the mechanisms of action of anticancer agents in vitro and monitoring their effects in vivo

Sammanfattning: Chemotherapeutic drug resistance is a major clinical problem and cause for failure in the therapy of human cancer. Mutations in apoptotic programs may promote tumour progression and reduce the efficacy of cancer therapy. Mutations within the p53 gene are found in about half of all human cancers and loss of p53 function correlates with multi-drug resistance in many tumour types. Anti-cancer therapy is generally believed to induce tumour apoptosis and apoptosis is an attractive clinical endpoint for evaluation of treatment efficiency. The aims of this thesis were to identify agents that induce p53-independent apoptosis and to characterize the mechanisms of action such drugs and to develop a new approach for monitor treatment responses. We have developed a simple assay system for detection of apoptosis and also necrosis both in vivo and in vitro. The apoptosis assay is based on measurement of caspase-cleaved cytokeratin-1 8 and detected by the monoclonal antibody M30. The M30-ELISA is a novel high-throughput assay for screening and characterization of compounds that induce apoptosis Another assay, the M65-ELISA, was also developed that measures full-length soluble CK18 and CK18 COOH terminal fragments. Our results showed large differences in the ratios of caspase-cleaved to total CK 18 in the sera of different patients. Our results suggest that apoptosis is not the main mechanisms of spontaneous or therapy associated cell death in the many tumours. We identified agents that induce p53-independent apoptosis and characterized their mechanisms of action by screening a drug library from the National Cancer Institute (NCI). The result shows that a large number of potential anti-cancer drugs induce p53- independent apoptosis and lysosomal membrane potential (LMP) is a mediator of many such responses. We identified two different mechanisms of LMP induction. One group of compounds induce reactive oxygen species (ROS) whereas the second group did not. Evidence suggesting a Baxregulated pathway of LMP induction was obtained. These results establish a unique role of LMP in the pathways of cell death and may have implications for cancer chemoprevention strategies. Enhancing the lysosomal cell death pathway may be a therapeutic strategy to overcome the chemoresistance. Due to the high frequency of p53 mutations in human cancer cells, the demonstration that LMP is potentially a common mechanism of action of p53-independent drugs and the association of ROS with induction of LMP are interesting in terms of future drug development.