Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702720
Title: Liquid chromatography and mass spectrometry based metabolomic investigations of sulphur containing metabolites in human prostate cancer
Author: Childs, Stephen Andrew
ISNI:       0000 0004 6058 9670
Awarding Body: University of Sunderland
Current Institution: University of Sunderland
Date of Award: 2015
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Abstract:
Low molecular weight thiols constitute a biologically important class of metabolites, some of which play a principal role in cellular defence against oxidative stress. The aetiology of cancer is generally linked with DNA mutation; often as a result of oxidative damage when antioxidant defences are dysregulated. Accordingly, the investigation of redox metabolites within cancer models is relevant to better understand the initiation and development of the disease. Specifically, when detected at an early stage, prostate cancer treatment by androgen ablation often carries a high rate of success. After a period of 18-24 months however, the disease is characterised by a shift to androgen insensitivity, and mortality increases significantly in advanced states. Hence, early detection and improved understanding of the changes in metabolism which accommodate a shift to androgen insensitivity and increased rate of proliferation are also relevant. Metabolomic profiling is a rapidly expanding field of systems biology which combines sensitive, high resolution equipment with powerful chemometric data processing to determine alterations in metabolic pathways in response to stress factors, including internal and external stimuli; thus providing valuable insight into the mechanisms involved in disease development. Whilst this approach has been applied to cancer research in the past to discover new drug targets and putative biomarkers for early detection, the complex metabolic pathways involved in cancer progression are not fully understood. Moreover, recently reported dysregulation of redox status and glutathione content in prostate cell models suggested significantly altered metabolism in some cancers. In order to better understand the metabolic events occurring, the aim of this study was to detect, and quantify where possible the sulphur-containing metabolites in prostate cancer cell models. Targeted metabolomic based methods using derivatisation with a specific reagent (DTNB) were developed and validated to provide comprehensive quantitative measurements of reduced thiols in cell models representing androgen sensitive (LNCaP) and androgen insensitive (DU145) disease, in addition to control cells representing healthy prostate epithelium (PZ-HPV-7). Furthermore, metabolomic profiling was performed using these cell lines to identify up and down regulation of key sulphur containing metabolites including disulphides and thioethers. Measurements of glutathione and the oxidised form indicated increased oxidative stress in LNCaP cells, whilst DU145 exhibited signs of adaptation to oxidative stress by up-regulation of glutathione biosynthesis. Investigation of the metastatic, androgen insensitive cell line, LNCaP, revealed a significant disparity in total thiol content and glutathione, suggesting the presence of additional thiol metabolites. Methods were developed and refined to determine the presence of cysteine, cysteinylglycine and an additional previously unidentified thiol species in LNCaP cells. Quantitative HPLC methods were validated and used to determine the concentration of individual thiol components in each cell line, successfully accounting for the total thiol content for the first time. The control cell contained 0.5 (± 0.03) and 6.3 (± 0.14) femtomoles per cell of cysteine and glutathione respectively, DU145 cells contained 0.3 (± 0.1) and 32.3 (± 2.3) femtomoles per cell of cysteine and glutathione respectively, and LNCaP cells contained 2.7 (± 0.05), and 8.3 (± 0.73) femtomoles per cell of cysteine and glutathione respectively. LNCaP cells additionally contained 0.8 (± 0.1) femtomoles per cell of cysteinylglycine. Further investigations proved that the unknown thiol (compound x) was a molecule of cysteine and glycerate linked by a peptide bond. Through examination of metabolite databases and chemical literature it was determined that the molecule had not previously been reported. Profiling of the cells highlighted this metabolite as a key component of the LNCaP metabolic fingerprint, in addition to other metabolites with roles in cell energy production. The developed methods stand as potentially useful tools for the sensitive detection and quantitation of thiols and for metabolomic investigations in various cell lines. Detection of a new thiol, cysteinyl-glycerate, in LNCaP cells warrants further investigations into the biological role of this metabolite and the potential as a putative biomarker.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.702720  DOI: Not available
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