Mechanisms of lipid metabolism in a prostate cancer cell model

The work explores mechanisms underlying the altered phospholipid metabolism that is characteristic of many human tumors and animal tumor models. Two main areas are investigated: (1) the regulation of enzymatic pathways involving choline and ethanolamine kinase that lead to the synthesis and sequestering of the phosphomonoesters phosphocholine and phosphoethanolamine during tumor cell development, and (2) the accumulations of phospholipid catabolic byproducts, neutral lipids and glycerophosphocholine, that occur during drug-induced apoptosis with a differentiating agent. Using a perfused DU145 prostate cancer cell line as a paradigm, changes in phospholipid metabolites are explored non-invasively and in real time using 31P and 1H magnetic resonance spectroscopy. DU145 cells perfused in the presence of choline or ethanolamine exhibit time dependent increases in their corresponding metabolites, namely phosphocholine and phosphoethanolamine, due to uptake and phosphorylation by kinases. The uptake and/or phosphorylation of choline can be inhibited in the presence of the choline kinase inhibitor MN58b, but not by the classic choline transport inhibitor HC-3. In contrast, the uptake and phosphorylation of ethanolamine is not affected by either of these inhibitors, indicating distinct metabolic pathways for these two metabolites. The increased mobile lipids, total choline, and glycerophosphocholine resonances induced in DU145 cells by differentiation therapy was attenuated by pretreatment with AACOCF3, an inhibitor of the phospholipase A2 isoforms cPLA2 and iPLA2. Pretreatment with BEL, a specific inhibitor of iPLA2, caused a reduction in glycerophosphocholine and to a lesser extent in total choline but not mobile lipids. This provides evidence that functional activation of phospholipase(s) A2 are responsible for increased total choline metabolites and the associated accumulation of mobile lipids. Levels of cPLA2, phosphorylated cPLA2, iPLA2, and sPLA2 X expression were not changed by treatment with differentiating agents which indicates that the changes in phospholipase activity are regulated by alternate mechanisms. The role of external contributions of lipids was explored using fluorescent fatty acid uptake in the differentiation therapy model as a proof-of-concept. Increased fatty acid uptake was determined to occur within 2 hours of drug treatment and that it was independent of caspase regulation. In addition, a screen of oncology drugs identified and validated the necessity and universality of fatty acid transport in environments of cellular stress. Fatty acid uptake leading to mobile lipid production appears to be an early stress response in the apoptotic process. Collectively, these studies establish the importance of the analogous yet independent pathways of choline and ethanolamine regulation, the critical role of phospholipase A2 in phosphatidylcholine cycling during drug treatment, and the external recruitment of lipids during cellular stress. It is anticipated that further refinement and advances will add to the framework of phospholipid homeostasis disruption in cancer and therapeutic application and ultimately to our understanding of cancer metabolism