Metabolomics can be used as an aid to functional genomics in order to investigate the functions of genes or enzymes. In the current study metabolomics was employed in the study of the response of LNCaP prostate cancer cells to sphingosine kinase inhibitors. Cell culture conditions, metabolite extraction and the LC/MS settings were optimized aiming at a reliable, unbiased, sensitive, and high throughput metabolomic protocol. Three different sphingosine kinase inhibitors were studied and reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for prostate cancer cells metabolites. The method involved optimizing the extraction of LNCaP cells metabolites followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry (HRMS). Extraction repeatability and storage were studied and 480 metabolites were putatively identified. In the study protocol ~ 180 standard compounds from different chemical classes were also run. Five different columns were compared in terms of their performance using these metabolites in combination with MS operated in both positive and negative electrospray ionization modes. The ZIC-pHILIC column showed the best performance and the highest number of metabolites separated. An effect of storage conditions on metabolite profiles was assessed using multivariate statistics (PCA). The treatment of LNCaP and LNCaP-AI cells with 2-(p-hydroxyanilino)- 4-(p-chlorophenyl)thiazole (Ski) modulated the metabolome, with marked changes in glutathione, NADPH, pentose phosphate shunt and glycolytic metabolite levels which were indicative of a pronounced oxidative stress response and modulation of the Warburg effect. Diadenosine triphosphate (Ap3A) was not detected in LNCaP-AI but was present in LNCaP. Ap3A and diadenosine tetraphosphate (Ap4A) are novel apoptotic markers and were quantified by using tandem mass spectrometry.(R)-FTY720 methyl ether (ROME), which is a SK2-selective inhibitor, did not affect produce oxidative stress or affect the pentose phosphate pathway but increased in the levels of several lysophosphatidylinositols (Lyso PI). However, increases in phosphatidylserine (PS), sphingosine and sphinganine, hydroxysphingosine and hydroxysphinganine were marked when the cells treated with (S)-FTY720 Vinylphosphonate. In addition, it caused a fall in hypoxanthine, guanine and uridine which which may be linked with purine nucleoside phosphorylase (PNP). Cell based metabolomics provides a method for exploring the mechanism of drug action.
|Date of Award||10 Sep 2015|
- University Of Strathclyde
|Supervisor||David Watson (Supervisor) & Blair Johnston (Supervisor)|