Abstract
Tumour necrosis factor α (TNFα) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase-2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) analysis. TNFα treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acyl-phosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon γ treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE2-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFα treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.
Highlights
The endocannabinoid system consists of a group of endogenous signaling molecules, primarily the arachidonic acid derivatives anandamide and 2-arachidonoyl glycerol (2-AG), two receptors and the enzymes responsible for the synthesis and breakdown of these ligands
We have investigated the effects of Tumour necrosis factor α (TNFα) treatment upon the enzymes involved in affects anandamide (AEA) synthesis and metabolism in human androgen-independent DU145 prostate cancer cells
Consistent with the literature [30], TNFα treatment of the cells increased the expression of PTGS2, and a significant increase was already seen at 1 h following treatment
Summary
The endocannabinoid (eCB) system consists of a group of endogenous signaling molecules, primarily the arachidonic acid derivatives anandamide (arachidonylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), two receptors (cannabinoid receptors 1 and 2 [CB1 and CB2]) and the enzymes responsible for the synthesis and breakdown of these ligands. AEA belongs to a family of N-acylethanolamine (NAE) lipids, which include the endogenous anti-inflammatory agent palmitoylethanolamide (PEA) and the satiety factor oleoylethanolamide (OEA). These lipids are synthesized on demand from phosphatidylethanolamides by multiple routes [2], an important enzyme in this respect is N-acylphosphatidylethanolamine selective phospholipase D (NAPE-PLD) [3,4]. The relative levels of NAEs reflect the corresponding tissue levels of the phosphatidylethanolamide precursor lipids, and in mammalian brains, for example, AEA comprises only ~1% of the total NAE content (expressed in mol%) in contrast to PEA (~30%) and OEA (~12%) [5]. In human seminal plasma, AEA levels are much higher (12 nM) relative to PEA and OEA levels (32 and 33 nM) [6]
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