am-404 and Glioma

Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl-based uptake inhibitors [N-(4-hydroxyphenyl)arachidonylamide (AM404), N-(4-hydroxy-2-methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) and (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-octadecen-amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 microm) and VDM11 (10 microm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5,000 per well were used. In contrast, UCM707 (30 microm), OMDM2 (10 microm) and the related compound arvanil (10 microm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6-9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl-based compounds was related primarily to the acyl side-chain rather than the head group. A variety of pre-treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N-acetyl-L-cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl-derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.

Topics: Acylation; Animals; Antineoplastic Agents; Arachidonic Acids; Benzyl Compounds; Brain; Brain Neoplasms; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytotoxins; Drug Screening Assays, Antitumor; Endocannabinoids; Fluoresceins; Furans; Glioma; L-Lactate Dehydrogenase; Neurons; Nucleic Acids; Polyunsaturated Alkamides; Rats; Time Factors; TRPV Cation Channels

AM404 [ N-(4-hydroxyphenyl)arachidonylamide] and VDM 11 [(5 Z,8 Z,11 Z,14 Z)- N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] are commonly used to prevent the cellular accumulation of the endocannabinoid anandamide, and thereby to potentiate its actions. However, it has been reported that AM404 can produce an influx of calcium into cells, which might be expected to have deleterious effects on cell proliferation. In the present study, AM404 and VDM 11 were found to reduce C6 glioma cell proliferation with IC(50) values of 4.9 and 2.7 microM, respectively. The inhibition of cell proliferation following a 96-h exposure was not accompanied by dramatic caspase activation, and was not prevented by either a combination of cannabinoid and vanilloid receptor antagonists, or by the antioxidant alpha-tocopherol, suggestive of a non-specific mode of action. Similar results were seen with palmitoylisopropylamide, although this compound only produced significant inhibition of cell proliferation at 30 microM concentrations. AM404 (1 microM), VDM 11 (1 microM) and palmitoylisopropylamide (3-30 microM), i.e. concentrations producing relatively modest effects on cell proliferation per se, reduced the vanilloid receptor-mediated antiproliferative effects of anandamide, as would be expected for compounds preventing the cellular accumulation of anandamide (and thereby access to its binding site on the vanilloid receptor). It is concluded that concentrations of AM404 and VDM 11 that are generally used to reduce the cellular accumulation of anandamide have deleterious effects upon cell proliferation, and that lower concentrations of these compounds may be more appropriate to use in vitro.

Topics: Animals; Arachidonic Acids; Brain Neoplasms; Cannabinoids; Cell Count; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Endocannabinoids; Glioma; Inhibitory Concentration 50; Polyunsaturated Alkamides; Rats; Receptors, Drug

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.

Topics: Animals; Arachidonic Acids; Biological Transport, Active; Cannabinoid Receptor Modulators; Cell Membrane; Endocannabinoids; Glioma; Glycerides; Kinetics; Models, Chemical; Neurotransmitter Agents; Nitric Oxide; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Tumor Cells, Cultured