P01.17 * ROLE OF PHOSPHATYDILCHOLINE-SPECIFIC PHOSPHOLIPASE C IN MODULATION OF CXCL12/CXCR4 AXIS IN A HUMAN GLIOMA CELL LINE

Abstract

BACKGROUND: The CXCR4 chemokine receptor and its ligand CXCL12 are one of the most well studied chemokine systems in tumor biology. In gliomas CXCL12/CXCR4 axis regulates multiple mechanisms that sustain tumor growth as migration, proliferation, angiogenesis and is emerging as a novel biologically relevant target for brain tumor therapy. Previous studies, in our laboratory highlights a role of the phosphatydilcholine-specific phospholipase C (PC-PLC) in the regulation and expression of growth factor membrane receptors, suggesting a relationship between the metabolism of phosphatydilcholine (PC) and cell receptor status. AIM: Investigate the crosstalk between the chemokine receptor CXCR4 and PC-PLC and its implications in glioma cells growth. MATERIALS AND METHODS: U87MG, a glioblastoma cell line was treated with a conventional CXCR4 antagonist, AMD3100 (10 µM), or with a PC-PLC inhibitor, D609 (Xantate) (100 µM). Confocal Laser Scanning Microscopy (CLSM) and Western Blotting (WB) experiments were performed on cells to investigate protein expression and intracellular localization. Cell metabolism on both treated or untreated U87MG cells was evaluated by in vitro Nuclear Magnetic Resonance Spectroscopy (MRS). RESULTS: CLSM analyses highlighted for the first time an extensive co-localization of CXCR4 and PC-PLC on the plasma membrane of U87MG cells. Furthermore we observed a down-modulation of CXCR4 from cell membrane in response to both inhibitors, AMD3100 and D609, however D609 only induced a decrease in CXCR4 total protein content. Moreover, D609 treatment reduced PC-PLC expression, induced a reduction of pAKT and exerted a significative anti-proliferative effect on U87MG cells (49% inhibition). MRS analyses revealed alterations on U87MG metabolism, showing in D609-treated cells an increase of choline-containing metabolites (Glycerophosphocholine, GPCho; Choline, Cho) (increase of 77%) that could be correlated with activation of lipases and phospholipases that can induce an accumulation of neutral lipids associated with the inhibition of cell proliferation. D609 treatment also induced a significative decrease of intracellular concentration of Lactate (38%), and preliminary analyses on lactate deydrogenase (LDH) showed a decrement of the enzyme activity (30%) suggesting that inactivation of PC-PLC could impaired the glycolysis pathway in U87MG. CONCLUSIONS: In this study we identified for the first time an interaction between the chemokine receptor CXCR4 and the enzyme PC-PLC on a glioma cell line. The specific inhibition of PC-PLC modulates CXCR4 receptor localization on cellular membrane and protein expression. Our findings suggest that the inhibition of PC-PLC could represent a new molecular approach in CXCR4-overexpressing glioma cells.