The literature widely reports on the regulatory actions of the endocannabinoid system in health and disease, including cancer. Endocannabinoids and synthetic cannabinoids are essentially described as protective factors limiting cell proliferation, differentiation and survival as well as tumor development. In this study, we aimed at investigating the possibility of enhancing endocannabinoid cytotoxicity using inhibitors of their hydrolysis in a melanoma model.
After looking for the presence of enzymatic activity for AEA, 2-AG and PEA hydrolysis and elucidating which enzymes were present in our melanoma model, we showed a time-dependent effect of these three endocannabinoids on B16 cell viability. As frequently described for many cancer cell lines like colon cancer cells [17, 21], glioma cells  breast cancer cells [19, 25, 27] or prostate cancer cells , AEA and 2-AG reduced B16 cell viability. Surprisingly, at 10 μM, we found PEA to decrease cell viability. Indeed, this endocannabinoid was reported to act as an "entourage" agent able to increase AEA antiproliferative effects but not to induce those when incubated alone, even at concentrations up to 10 μM [29, 30]. However, here PEA could clearly reduce B16 cell viability at 10 μM but also at lower concentrations. We also confirmed that PEA degradation into palmitic acid was not responsible for the effects observed with PEA .
We then sought to increase PEA levels to investigate if this could affect B16 melanoma cell viability by potentiating PEA cytotoxicity. Some reports indicate that the use of inhibitors of endocannabinoid hydrolysis can be of interest in the development of anticancer therapies. For example, elevation of endocannabinoid concentrations by inhibitors of their re-uptake and degradation produced a decrease in thyroid transformed cells growth . In a colorectal cancer cell line, inhibitors of endocannabinoid inactivation increased their levels and reduced cell proliferation . Other experiments performed on prostate cancer cells also testified of the benefits of inhibiting 2-AG hydrolysis to block cell growth and invasion [28, 41–43]. Therefore, we assayed five inhibitors of either FAAH or NAAA, the two main enzymes known to hydrolyze PEA and that we found to be expressed in B16 melanoma cells. The most cytotoxic treatment was obtained by the co-incubation of 10 μM of PEA with the irreversible FAAH inhibitor, URB597 at 10 μM. Using a human melanoma cell line, we also evidenced a significant cytotoxicity of this treatment. Interestingly, among the inhibitors tested, the highest inhibition of PEA hydrolysis was obtained with URB597. This compound already exerted a significant decrease in cell viability when used alone. Of note, the other selective FAAH inhibitor CAY10402 was also able to potentiate PEA cytotoxicity without inducing any decrease in cell viability by itself. This last observation suggested that the PEA-URB597 cytotoxicity might be partly due to elevation of endocannabinoid levels. Indeed we found that incubation of B16 cells with URB597 could raise PEA levels up to 163%, indicating that the cytotoxicity of this inhibitor could be partly assigned to modulation of PEA levels. Actually, even though the concentrations obtained when inhibiting FAAH were lower than those required to reduce cell viability by adding PEA exogenously, we considered that locally available PEA levels might be high enough to produce pharmacological effects when inhibiting FAAH. The higher effects, when looking at PEA hydrolysis or at cell viability, of URB597 compared to CAY10402 could be related to a reversible FAAH inhibition by CAY10402, while URB597 was characterized as an irreversible inhibitor. In the liver, URB597 was previously shown to enhance AEA-induced cell death via FAAH inhibition . Surprisingly, the two dual FAAH/MAGL inhibitors MAFP and CAY10499 only slightly accentuated PEA effects on cells viability even though they were effective at inhibiting PEA hydrolysis and exhibited cytotoxic effects by themselves. Nevertheless, in comparison to URB597, the effect of MAFP on intracellular PEA levels was less pronounced and CAY10499 did not significantly affect PEA concentration even though it tended to increase it (see Additional file 5A). It is therefore supposed that the action of these two dual inhibitors is not sufficient to increase PEA levels and, consequently, its cytotoxicity upon melanoma cells. In addition, as mentioned above these compounds are also irreversible inhibitors of MAGL and consequently can influence 2-AG levels as well (see Additional file 5B). The inhibition of 2-AG degradation was frequently evidenced to result in antitumor effects, either by making profit of 2-AG antiproliferative and anti-invasive properties [40, 41, 43] or by limiting the production of arachidonic acid known to be associated to aggressiveness of cancer cells [45, 46]. Since this endocannabinoid also exhibited cytotoxic properties in B16 cells, we would have assumed that the concomitant inhibition of the FAAH and the MAGL should have produced an enlarged diminution of cell viability. Astonishingly, we only observed that inhibition of 2-AG hydrolysis produced a small decrease in cell viability and poorly contributed to induction of cytotoxicity when combining to PEA. This suggests a minor role of 2-AG in cell viability in our model as compared to PEA. Finally, although the poor inhibition of PEA hydrolysis by the NAAA inhibitor is puzzling at a first glance, the almost full inhibition of PEA hydrolysis by URB597 suggests that FAAH is likely to account for most of PEA degradation in our cellular model. Thus, even if CCP inhibits the NAAA-mediated PEA hydrolysis, FAAH can largely compensate for the decreased NAAA activity .
The receptor mediating the cytotoxic effects of PEA and URB597 could not be identified as one of the classical molecular targets mediating endocannabinoid action. However, though pharmacological blockade of receptors constitutes a reliable and widely used method, silencing of these receptors may constitute a matter of interesting perspective to completely rule out their implication in the cytotoxic effects produced by the treatments.
Co-treatment of PEA and URB597 induced cell death in cultured B16 melanoma cells, while PEA and URB597 incubated alone only slightly increased the number of apoptotic and necrotic cells. This drug activity reinforcement was confirmed in vivo where tumor volume and tumor weight were decreased after 6 days of treatment only when melanoma-bearing mice were treated with both PEA and URB597. When looking at the endocannabinoid levels in the tumor after treatment, the growth delay induced by PEA-URB597 treatment appears to be related to an elevation of PEA levels within the tumor. Conversely, AEA and 2-AG levels were not significantly affected by treatments even though AEA levels tended to increase following URB597 injections. These results contrast with the observations made by Bifulco et al. with rat thyroid transformed cells, in which tumor levels of AEA, 2-AG and PEA were all three augmented after an intratumor treatment with the FAAH inhibitor arachidonoyl-serotonin . This difference may arise from variations in the experimental conditions, such as the injection modalities, resulting in variable availabilities of the inhibitor, or the timing at which tumors were resected. Since our results show a tendency to increase for N-acylethanolamine concentrations in URB597-treated mice and since only the co-incubation of PEA and URB597 increased PEA levels, we may think that the elevation of AEA and PEA levels are transitory and that these molecules are rapidly degraded. Along this line, we support the hypothesis that an earlier excision of the tumors after the last injection could have revealed a significant increase in AEA and PEA concentrations. Nevertheless, in our melanoma system, only the co-injection of PEA and URB597 is able to sufficiently increase the concentrations in order to reduce tumor growth. In addition, some reports have shown that FAAH inhibition induces an increase in 2-AG levels [39, 41]. However, in our melanoma cells, 2-AG levels were not influenced at all by URB597 treatments. This may be attributed to the fact that FAAH is weakly responsible for 2-AG hydrolysis in B16 cells.
We could also evidence that the decrease in tumor growth observed with PEA-URB597 treatment was the result of increased necrotic events in the tumor. Although tumor growth delay obtained with PEA and URB597 may look marginal, the extent of necrosis observed in this very aggressive tumor model indicates that measurements of tumor volume/weight certainly underestimate the real impact of the co-treatment. Furthermore, because neither PEA nor URB597 or the association of both molecules produced antiangiogenic effects, a reduced oxygen and nutrient supply is unlikely to account for the increased necrosis induced by the treatment. It seemed of interest to investigate this point because PEA and analogues have already been described as owning antiangiogenic effects in a model of chronic inflammation [48, 49]. Likewise, AEA was reported to influence cancer growth via inhibition of angiogenesis  and synthetic cannabinoids WIN-55.212-2 and JWH-133 were shown to decrease melanoma vascularization .
A large number of reports suggest the therapeutic interest of using PEA in medicine. This lipid mediator has been emerging as a potent antinociceptive molecule [50, 51] and exhibits anti-inflammatory properties . Of note, PEA is already used as the active molecule of anti-inflammatory and analgesic preparations (e.g. Normast®, Pelvilen®) [53, 54]. These advantageous effects associated with the present observations put into light the possibility of emerging therapies implicating PEA for pathological conditions including cancer.