The aim of the study was to assess the effect of TNFα treatment upon AEA turnover in DU145 cells, since little is known about the regulatory effects of TNFα upon the eCB system, in contrast to the large body of data concerning effects of eCBs, synthetic, and plant-derived cannabinoids upon the TNFα and other cytokines [25,26] (review see [49]). There were two main findings that are discussed below:

TNFα treatment affects the balance of AEA catabolic enzymes in DU145 cells. Consistent with the literature [30], TNFα treatment of the cells increased the expression of PTGS2. Levels of PTGS2 expression were low, and we could not detect COX-2 in Western blot experiments. However, Subbarayan et al. [30] were able to detect the protein in both Western blot and immunofluorescence experiments. They found increased expression of COX-2 as early as 0.5 h after TNFα treatment in the cells, and also reported an increased expression of COX-2 in androgen-insensitive PC3 cells, but not in the androgen-sensitive LNCaP cells. In contrast to the increase in PTGS2 expression, we found that FAAH and NAAA expression at the mRNA level were not affected by the TNFα treatment. The functional assay of [Et-³H]AEA hydrolysis also failed to show significant changes following TNFα treatment. Importantly, the [Et-³H]AEA hydrolysis was completely inhibited by the selective FAAH inhibitor URB597, confirming that FAAH was the primary enzyme responsible for the hydrolysis of externally administered AEA in these cells.

In theory, the net result of the increased COX-2 expression relative to the FAAH (and NAAA) expression following TNFα treatment should be a greater production of PG-EAs. However, the low levels of COX-2 in the cells meant that PG-EAs (and PGs themselves) were only detected in the lipidomics experiments when the cells had been incubated for 30 min with 100 nM AEA. In their experiment, Subbarayan et al. [30] detected PGE₂ using an enzyme immunoassay kit and reported a 1.8-fold increase in levels following TNFα treatment. It is possible that the basal expression of COX-2 was higher in their DU145 cells than in our cells. These authors did not look for PG-EAs (although they may inadvertently have measured it together with PGE₂ in their immunoassay [36]). We identified the PG-EA that was detected in our experiments as PGE₂-EA rather than PGF_2α-EA. This is an interesting finding given that PGE₂-EA reduces LPS-induced TNFα production by monocytes [50] whereas PGF_2α-EA is pro-inflammatory in nature [51]. It sounds counter-intuitive from the tumour cells point of view that they should produce anti- rather than pro-inflammatory compounds, but the levels were low and the reduction in NAPE-PLD expression, by reduction of the synthesis of the AEA precursor, would work against a PGE₂-EA → TNFα feedback loop in the cells.

Although not part of the main aim of the study, we also investigated the effect of TNFα treatment on the other endocannabinoid, 2-AG. At the level of mRNA, TNFα reduced the levels of the synthetic enzymes DAGL-α and –β, but this was not sufficient to affect the observed levels of 2-AG in either the medium or the cell lysates. More pronounced pharmacological inhibition of 2-AG synthesis, however, does reduce 2-AG levels in these cells, and this is accompanied by an increased invasivity of the cells across matrigel-coated transwells [14].

COX-2 does not gate the uptake of AEA in LPS+IFNγ-treated RAW264.7 cells. The manner in which AEA is accumulated in cells has been a matter of considerable debate, in particular with respect to whether or not there is a plasma membrane bidirectional transporter protein [52,53]. It is clear, however, that in cells expressing FAAH, this enzyme gates the accumulation by reducing the intracellular free AEA concentration and hence preserving the gradient across the plasma membrane [8]. Very little work has been undertaken on the uptake of AEA into prostate cancer cells, but PC3 cells, which express low levels of FAAH [54], unsurprisingly do not shown this phenomenon, with uptake in the absence and presence of FAAH inhibitors being very similar [53]. In contrast, rat Dunning AT1 prostate cancer cells express FAAH, and AEA uptake into these cells is reduced upon FAAH inhibition [55]. The DU145 cells resemble more the AT1 cells than the PC3 cells in this respect.

Given that FAAH, by removing intracellular AEA, can maintain AEA uptake, a similar property might be expected for other enzymes using AEA as a substrate, such as COX-2, since COX-2-catalysed cyclooxygenation of AEA would deplete local AEA levels. This phenomenon should be expected to be extra prominent in cases of increased COX2 expression. The COX-2 levels were too low in the DU145 cells to test this hypothesis, but in the LPS+IFNγ-treated RAW264.7 cells, which were included in the study as a positive control for COX-2 (albeit with the limitation that they are from mouse, rather than man), the expression levels are very high. As a rough guide, the k_cat and K_m values for human COX-2 towards AEA are 4 s^-1 and 62 μM respectively, at pH 8 [56], whereas the corresponding values for rat FAAH towards this substrate are 4.8 s^-1 and 17 μM, respectively [57]. The very much higher expression of COX-2 than FAAH in the LPS+IFNγ-treated RAW264.7 cells, at least at the mRNA level, would suggest that COX-2 will play a significant role in AEA metabolism. Indeed, we were able to detect a peak eluting in the same region as the PG-EA analogue bimatoprost following incubation of the LPS+IFNγ-treated (but not control) RAW264.7 cells with [Ara-³H]AEA. If COX-2 gated AEA uptake in these cells in the same way as FAAH, then its induction by LPS+IFNγ treatment should increase the rate of uptake, while pharmacological inhibition of the enzyme by flurbiprofen should reduce the uptake back to the level seen for the control RAW264.7 cells. Neither event occurred, in contrast to the clear effect of the FAAH inhibitor URB597 in the cells. It is at first sight surprising that one catabolic enzyme can gate AEA uptake in this way whereas another one cannot. However, for 2-AG, a different pattern is seen, where the hydrolytic enzymes do not regulate the uptake, whereas down-stream processes (incorporation of the arachidonic acid into the phospholipids) are involved [58,59]. Thus, the intracellular processes regulating AEA and 2-AG uptake are complex and different for the two endocannabinoids despite the fact that they are both arachidonoyl derivatives.

In conclusion, the present study has provided novel data on the effects of TNFα treatment on AEA catabolic pathways in DU145 prostate cancer cells and suggest that the balance of catabolism is tilted towards COX-2 by this treatment, although the low expression of this enzyme in the cells limits the functional consequence of such a tilt. Further studies in other prostate cancer cells, ideally with different basal levels of the synthetic and degradative enzymes and with other inflammatory mediators, are warranted in order to shed more light upon the relationship between inflammation and the disturbed eCB system in prostate cancer and the functional consequence of increased COX-2 levels upon the levels and metabolism of AEA.