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

Cooke, Deirdre (1999) Studies on the mode of action of coumarins (coumarin, 6-hydroxycoumarin, 7-hydroxycoumarin & esculetin) at a cellular level. PhD thesis, Dublin City University.

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Abstract

Coumarin, a member of the benzopyrone family of compounds, is a natural substance that has shown anti-tumour activity in vivo, with this effect believed to be due to its metabolites. However, no definitive mode o f action has been identified, and this thesis aimed at gaining further insight into the precise target o f coumarin molecules at a cellular level. A novel biosensing instrument, the Cytosensor Microphysiometer, which detects cellular metabolism, was used throughout, to aid this investigation. The effect o f four coumarin compounds (coumarin, 6-hydroxycoumarin, 7-hydroxycoumarin and esculetin) on the growth, metabolism and metastatic potential of a range o f tumour cell lines was investigated. The toxicity o f these four compounds was examined using a variety of in vitro tests (medium-term growth assays, Lactate dehydrogenase (LDH) assay and a tetrazolium salt-based (MTT) assay). A superior method to the MTT assay for examining the effect of compounds on metabolism was achieved using the Cytosensor Microphysiometer. The effect of coumarins on the metastatic potential of tumour cells (in terms o f their protease secretion) was also explored. The effect o f 7-hydroxycoumarin and esculetin on growth signalling pathways within tumour cells was probed. Using the A431 cell line (which over-expresses the EGF-Receptor) and EGF as a model growth factor signalling mechanism, the effect of the two coumarins on tyrosine phosphorylation events in cells was explored. Direct in vitro tyrosine kinase assays with purified EGFreceptor, and ELISA, Western Blotting and Cytosensor studies in intact cells, were used to achieve this. The involvement o f coumarin compounds in protein kinase C signalling was also examined. A “model” monocyte system was developed and used in a preliminary assessment of the immunomodulatory role of coumarins. The activation of two “monocytic” cell lines was assessed using the Cytosensor Microphysiometer. Subsequently, the effect o f coumarins on the release of reactive oxygen species, reactive nitrogen intermediates and proteases from activated immune cells, was accomplished using luminometric, colourimetric and substrate gel analyses, respectively.

Item Type:Thesis (PhD)
Date of Award:1999
Refereed:No
Supervisor(s):O'Kennedy, Richard
Uncontrolled Keywords:Coumarins; anti-tumour activity; metabolites
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:18438
Deposited On:18 Jul 2013 14:02 by Celine Campbell. Last Modified 18 Jul 2013 14:02

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