Studies on the mode of action of coumarins (coumarin, 6-hydroxycoumarin, 7-hydroxycoumarin esculetin) at a cellular level

摘要

Coumarin, a member of the benzopyrone family of compounds, is a natural substance that hasudshown anti-tumour activity in vivo, with this effect believed to be due to its metabolites. However, nouddefinitive mode o f action has been identified, and this thesis aimed at gaining further insight into theudprecise target o f coumarin molecules at a cellular level. A novel biosensing instrument, the CytosensorudMicrophysiometer, which detects cellular metabolism, was used throughout, to aid this investigation.ududThe effect o f four coumarin compounds (coumarin, 6-hydroxycoumarin, 7-hydroxycoumarinudand esculetin) on the growth, metabolism and metastatic potential of a range o f tumour cell lines wasudinvestigated. The toxicity o f these four compounds was examined using a variety of in vitro testsud(medium-term growth assays, Lactate dehydrogenase (LDH) assay and a tetrazolium salt-basedud(MTT) assay). A superior method to the MTT assay for examining the effect of compounds onudmetabolism was achieved using the Cytosensor Microphysiometer. The effect of coumarins on theudmetastatic potential of tumour cells (in terms o f their protease secretion) was also explored.ududThe effect o f 7-hydroxycoumarin and esculetin on growth signalling pathways within tumourudcells was probed. Using the A431 cell line (which over-expresses the EGF-Receptor) and EGF as audmodel growth factor signalling mechanism, the effect of the two coumarins on tyrosineudphosphorylation events in cells was explored. Direct in vitro tyrosine kinase assays with purified EGFreceptor,udand ELISA, Western Blotting and Cytosensor studies in intact cells, were used to achieveudthis. The involvement o f coumarin compounds in protein kinase C signalling was also examined.ududA “model” monocyte system was developed and used in a preliminary assessment of theudimmunomodulatory role of coumarins. The activation of two “monocytic” cell lines was assessedudusing the Cytosensor Microphysiometer. Subsequently, the effect o f coumarins on the release ofudreactive oxygen species, reactive nitrogen intermediates and proteases from activated immune cells,udwas accomplished using luminometric, colourimetric and substrate gel analyses, respectively.