Dominique Melck

Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action.

The existence of an endogenous cannabinoid system was demonstrated conclusively with the discovery of endogenous brain constituents capable of activating the cannabinoid receptors functionally. These compounds are synthesized by neuronal cells and inactivated through re-uptake and enzymatic hydrolysis by both neurons and astrocytes. In analogy with the endorphins they can be referred to as endocannabinoids. Apart from the identification of their metabolic pathways, research carried out in the past six years has focused on the possible cellular and molecular targets for the actions of endocannabinoids. These studies have confirmed a similarity between the endocannabinoids and the psychoactive substance in marijuana, delta9(-)-tetrahydrocannabinol, and have suggested a role for endocannabinoids in the modulation of neurotransmitter action and release.

Biosynthesis, Uptake, and Degradation of Anandamide and Palmitoylethanolamide in Leukocytes

Anandamide (arachidonoylethanolamide, AnNH) and palmitoylethanolamide (PEA) have been proposed as the physiological ligands, respectively, of central and peripheral cannabinoid receptors. Both of these receptors are expressed in immune cells, including macrophages and mast cells/basophils, where immunomodulatory and/or anti-inflammatory actions of AnNH and PEA have been recently reported. We now provide biochemical grounds to these actions by showing that the biosynthesis, uptake, and degradation of AnNH and PEA occur in leukocytes. On stimulation with ionomycin, J774 macrophages and RBL-2H3 basophils produced AnNH and PEA, probably through the hydrolysis of the corresponding N-acylphosphatidylethanolamines, also found among endogenous phospholipids. Immunological challenge of RBL-2H3 cells also caused AnNH and PEA release. The chemical structure and the amounts of AnNH and PEA produced upon ionomycin stimulation were determined by means of double radiolabeling experiments and isotope dilution gas chromatography/electron impact mass spectrometry. Both cell lines rapidly sequestered the two amides from the culture medium through temperature-dependent, saturable and chemically inactivable mechanisms. Once uptaken by basophils, AnNH and PEA compete for the same inactivating enzyme which catalyzes their hydrolysis to ethanolamine. This enzyme was found in both microsomal and 10,000 × g fractions of RBL cell homogenates, and exhibited similar inhibition and temperature/pH dependence profiles but a significantly higher affinity for PEA with respect to neuronal “anandamide amidohydrolase.” The finding of biosynthetic and inactivating mechanisms for AnNH and PEA in macrophages and basophils supports the previously proposed role as local modulators of immune/inflammatory reactions for these two long chain acylethanolamides.

Suppression of Nerve Growth Factor Trk Receptors and Prolactin Receptors by Endocannabinoids Leads to Inhibition of Human Breast and Prostate Cancer Cell Proliferation

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. β-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50–600 nm) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. Th...

Endocannabinoids and fatty acid amides in cancer, inflammation and related disorders

The long history of the medicinal use of Cannabis sativa and, more recently, of its chemical constituents, the cannabinoids, suggests that also the endogenous ligands of cannabinoid receptors, the endocannabinoids, and, particularly, their derivatives may be used as therapeutic agents. Studies aimed at correlating the tissue and body fluid levels of endogenous cannabinoid-like molecules with pathological conditions have been started and may lead to identify those diseases that can be alleviated by drugs that either mimic or antagonize the action of these substances, or modulate their biosynthesis and degradation. Hints for the therapeutic applications of endocannabinoids, however, can be obtained also from our previous knowledge of marijuana medicinal properties. In this article, we discuss the anti-tumor and anti-inflammatory activity of: (1) the endocannabinoids anandamide (arachidonoylethanolamide) and 2-arachidonoyl glycerol; (2) the bioactive fatty acid amides palmitoylethanolamide and oleamide; and (3) some synthetic derivatives of these compounds, such as the N-acyl-vanillyl-amines. Furthermore, the possible role of cannabimimetic fatty acid derivatives in the pathological consequences of cancer and inflammation, such as cachexia, wasting syndrome, chronic pain and local vasodilation, will be examined.

Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells.

Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.

Interactions between synthetic vanilloids and the endogenous cannabinoid system

The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N‐vanillyl‐cis‐9‐octadecenoamide), and the ‘endocannabinoid’ anandamide (arachidonoyl‐ethanolamide, AEA), suggests possible interactions between the cannabinoid and vanilloid signalling systems. Here we report that olvanil is a stable and potent inhibitor of AEA facilitated transport into rat basophilic leukemia (RBL‐2H3) cells. Olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL‐2H3 cells (IC50=9 μM), while capsaicin and pseudocapsaicin (N‐vanillyl‐nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50=80 μM), AM404 (12.9% inhibition at 10 μM) or oleoylethanolamide (27.5% inhibition at 10 μM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL‐2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (K i=1.64–7.08 μM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin‐induced cAMP formation in intact N18TG2 cells (IC50=1.60 μM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but not capsaicin, also selectively bound to CB1 receptor‐containing membranes. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system, and may lead to the design of a novel class of cannabimimetics with potential therapeutic applications as analgesics.

Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer cells.

Anandamide (ANA) inhibits prolactin‐ and nerve growth factor (NGF)‐induced proliferation of human breast cancer cells by decreasing the levels of the 100 kDa prolactin receptor (PRLr) and the high affinity trk NGF receptor, respectively, and by acting via CB1‐like cannabinoid receptors. However, the intracellular signals that mediate these effects are not known. Here, we show that, in MCF‐7 cells: (i) forskolin and the mitogen‐activated protein kinase (MAPK) kinase inhibitor PD098059 prevent, and the protein kinase A inhibitor RpcAMPs mimics, the inhibitory effects of ANA on cell proliferation and PRLr/trk expression and (ii) ANA inhibits forskolin‐induced cAMP formation and stimulates Raf‐1 translocation and MAPK activity, in a fashion sensitive to the selective CB1 antagonist SR141716A. ANA stimulation of MAPK was enhanced by inhibitors of ANA hydrolysis. Forskolin inhibited MAPK and ANA‐induced Raf‐1 translocation. These findings indicate that, in MCF‐7 cells, ANA inhibits adenylyl cyclase and activates MAPK, thereby exerting a down‐regulation on PRLr and trk levels and a suppression of cell proliferation.

Phosphatidic Acid as the Biosynthetic Precursor of the Endocannabinoid 2-Arachidonoylglycerol in Intact Mouse Neuroblastoma Cells Stimulated with Ionomycin

Abstract: In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2‐arachidonoylglycerol (2‐AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2‐AG. [3H]2‐AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N‐ethylmaleimide, two inhibitors of the Mg2+/Ca2+‐dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin‐induced [3H]2‐AG formation. These data indicate that DAG precursors for 2‐AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl‐CoA acylase inhibitor thimerosal significantly reduced [3H]2‐AG biosynthesis. The implications of these latter findings, and of the PA‐dependent pathways of 2‐AG formation described here, are discussed.

Neurobehavioral activity in mice of N-vanillyl-arachidonyl-amide.

We studied the cannabimimetic properties of N-vanillyl-arachidonoyl-amide (arvanil), a potential agonist of cannabinoid CB(1) and capsaicin VR(1) receptors, and an inhibitor of the facilitated transport of the endocannabinoid anandamide. Arvanil and anandamide exhibited similar affinities for the cannabinoid CB(1) receptor, but arvanil was less efficacious in inducing cannabinoid CB(1) receptor-mediated GTPgammaS binding. The K(i) of arvanil for the vanilloid VR(1) receptor was 0.28 microM. Administered i.v. to mice, arvanil was 100 times more potent than anandamide in producing hypothermia, analgesia, catalepsy and inhibiting spontaneous activity. These effects were not attenuated by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A). Arvanil (i.t. administration) induced analgesia in the tail-flick test that was not blocked by either SR141716A or the vanilloid VR(1) antagonist capsazepine. Conversely, capsaicin was less potent as an analgesic (ED(50) 180 ng/mouse, i.t.) and its effects attenuated by capsazepine. The analgesic effect of anandamide (i.t.) was also unaffected by SR141716A but was 750-fold less potent (ED(50) 20.5 microg/mouse) than capsaicin. These data indicate that the neurobehavioral effects exerted by arvanil are not due to activation of cannabinoid CB(1) or vanilloid VR(1) receptors.

Effect on cancer cell proliferation of palmitoylethanolamide, a fatty acid amide interacting with both the cannabinoid and vanilloid signalling systems

Palmitoylethanolamide (PEA) is a bioactive fatty acid amide belonging to the class of N‐acyl‐ethanolamines (NAEs). This compound has been known since the 1950s for its anti‐inflammatory effects, but was re‐discovered only after the finding that another NAE, arachidonoyl‐ethanolamide (anandamide, AEA), could act as an endogenous ligand of cannabinoid receptors. Although a similar function for PEA has also been proposed, this compound does not activate the two cannabinoid receptor subtypes described to date. PEA and AEA are co‐synthesized by cells, and PEA might act as an ‘entourage’ compound for AEA, i.e. as an endogenous enhancer of AEA biological actions. Indeed, long‐term treatment of human breast cancer cells (HBCCs) with PEA downregulates the expression of the enzyme responsible for AEA degradation, the fatty acid amide hydrolase, thereby leading to an enhancement of AEA‐induced, and cannabinoid CB1 receptor‐mediated, cytostatic effect on HBCCs. AEA is also a full agonist for the receptors of another class of bioactive fatty acid amides, the N‐acyl‐vanillyl‐amines (e.g. capsaicin and olvanil). These sites of action are known as vanilloid receptors of type 1 (VR1). PEA enhances the VR1‐mediated effects of AEA and capsaicin on calcium influx into cells. These ‘entourage’ effects of PEA might be attributable to modulation of VR1 activity, and could underlie the enhancement by PEA, described here for the first time, of the antiproliferative effects of VR1 receptor agonists.