Metabolomic and Metabolomic Flux Studies of the Effects of Sphingosine kinase Inhibitors on Prostate Cancer Cells Using High-Resolution Mass Spectrometry as an analytical technique

Abstract

There is evidence of the involvement of sphingosine kinases (two isoforms termed SK1 and SK2), which catalyse the formation of the bioactive lipid, sphingosine 1-phosphate (S1P), in various diseases including cancer. Over the last few years, metabolomics has become an increasingly critical part in many fields of research. Therefore, metabolomic and lipidomic analysis was performed to investigate the effect of different sphingosine kinase inhibitors in prostate cancer cells in order to establish the role of SK1 and SK2 in this cancer. Previous studies have shown that the SK1/2 inhibitor, SKi (2-(phydroxyanilino)- 4-(pchlorophenyl)thiazole) inhibits aerobic glycolysis (Warburg effect) and this might contribute to the anti-cancer activity of this compound. This was further investigated in the current study, where 13C6- glucose was employed to determine the flux through different metabolic and lipid pathways, including the glycolysis in androgenindependent LNCaP-AI prostate cancer cells. The LNCaP-AI cell line was treated with SKi in the presence of 13C6-glucose for 24 hours before extraction and global metabolite analysis of cell lysate by LC-MS. The levels of glycolytic metabolites, pentose phosphate pathway, glutathione disulfide (GSSG) and sn-G3P were increased in response to the SKi. On the other hand, NADPH, some metabolites in TCA, nucleotides were found to be lower in the SKi-treated cells. These findings suggest that SKi reprograms cellular metabolism of LNCaP-AI cells, which results in reduced flux through glycolytic and TCA cycles and re-diversion of glucose to produce, sn-G3P, which is capable of increasing ROS production, and which might programme senescent death in these cells. The flux in the v 13C experiment suggests that much of glucose is metabolized through the pentose glucuronate interconversion pathway in LNCaP-AI cells. In order to recapitulate a more aggressive phenotype in prostate cancer cells, SK1b was stably expressed in androgen-sensitive LNCaP cells. These LNCaP-SK1b cells were then treated with SKi and compared with LNCaP cells. The treatment of LNCaP-SK1b cells with SKi increased the levels of sphingomyelins, ceramides, lysoPC and PC and these changes were more substantial than in LNCaP cells. It is proposed that the over-expression of SK1b increases flux through sphingolipid and phospholipid pathways such that inhibition of SK1b with SKi results in a more profound increase in lipid metabolites compared with LNCaP cells. Part of this effect is in blockade of the sphingolipid rheostat and inhibition of phospholipid turnover that is limited by the availability of S1P. The use of the SK2 inhibitor, ROMe in LNCaP-SK1b cells suggests that both SK1 and SK2 regulate the sphingolipid rheostat, but inhibition of these enzymes with SKi results in differential effects on ceramide and phospholipid metabolism. Indeed, the effect of ROMe tends to be more robust in LNCAP cells compared with LNCaP-SK1b cells, with linkage of SK2 with sphinganine and sphinganine-1-phosphate metabolism. Several highly potent selective SK1 and SK2 inhibitors (PLR24, ST55 and ST81) were also used in LNCaP-AI cells and LNCaP-SK1b cells, and shown to disrupt the sphingolipid rheostat and modulate phospholipid turnover. The major conclusion of this thesis is that SK1 and SK2 regulate the metabolome (Warburg effect) and the lipidome to protect prostate cancer cells from apoptosis/senescence and this might contribute to certain hallmarks of cancer including replicative immortality and increased cell survival.