Biosynthesis and degradation of bioactive fatty acid amides in human breast cancer and rat pheochromocytoma cells - Implications for cell proliferation and differentiation

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [C-14]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 mu M for oleamide and 2.1 mu M for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB 1 receptor selective antagonist SR 141716A (0.5 mu M) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [C-14]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [C-14]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first rime that: (a) induction by nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on arachidonoylethanolamide/oleamide metabolism, (b) arachidonoylethanolamide and oleamide are autacoid suppressors of human breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that oleamide may act as an enhancer of arachidonoylethanolamide actions through competitive inhibition of its degradation.