anandamide and Brain-Neoplasms

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5μg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Topics: Animals; Arachidonic Acids; Benzamides; Brain Neoplasms; Cannabinoid Receptor Antagonists; Carbamates; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Fibrosarcoma; Fluorescent Dyes; Ganglia, Spinal; Hyperalgesia; Indoles; Lactones; Male; Mice; Mice, Inbred C3H; Pain; Pain Threshold; Polyunsaturated Alkamides; Sensory Receptor Cells; Statistics, Nonparametric; Tritium

Endocannabinoids are neuromodulatory lipids that mediate the central and peripheral neural functions. Endocannabinoids have demonstrated their anti-proliferative, anti-angiogenic and pro-apoptotic properties in a series of studies. In the present study, we investigated the levels of two major endocannabinoids, anandamide and 2-arachidonylglycerol (2-AG), and their receptors, CB1 and CB2, in human low grade glioma (WHO grade I-II) tissues, high grade glioma (WHO grade III-IV) tissues, and non-tumor brain tissue controls. We also measured the expressions and activities of the enzymes responsible for anandamide and 2-AG biosynthesis and degradation, that is, N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), and diacylglycerol lipase-alpha (DGL), in the same samples. Liquid chromatography-mass spectometry analysis showed that the levels of anandamide decreased, whereas the levels of 2-AG increased in glioma tissues, comparing to the non-tumor controls. The expression levels and activities of NAPE-PLD, FAAH and MGL also decreased in glioma tissues. Furthermore, quantitative-PCR analysis and western-blot analysis revealed that the expression levels of cananbinoid receptors, CB1 and CB2, were elevated in human glioma tissues. The changes of anandamide and 2-AG contents in different stages of gliomas may qualify them as the potential endogenous biomarkers for glial tumor malignancy.

Topics: Adolescent; Adult; Aged; Arachidonic Acids; Brain; Brain Neoplasms; Cannabinoid Receptor Modulators; Down-Regulation; Endocannabinoids; Female; Glioma; Glycerides; Humans; Male; Middle Aged; Polyunsaturated Alkamides; Receptors, Cannabinoid; RNA, Messenger; Tritium; Young Adult

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

Topics: Aging; Amides; Amidohydrolases; Animals; Antineoplastic Agents; Apoptosis; Arachidonic Acids; Brain; Brain Neoplasms; Capsaicin; Cell Movement; Culture Media, Conditioned; Dopamine; Endocannabinoids; Ethanolamines; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Neoplasm Proteins; Neural Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; TRPV Cation Channels; Tumor Cells, Cultured

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

The anti-tumor properties of cannabinoids have recently been evidenced, mainly with delta9-tetrahydrocannabinol (THC). However, the clinical application of this drug is limited by possible undesirable side effects due to a broad expression of cannabinoid receptors (CB1 and CB2). An attractive field of research therefore is to identify molecules with more selective tumor targeting. This is particularly important for malignant gliomas, considering their poor prognosis and their location in the brain. Here we investigated whether the most potent endogenous cannabinoid, arachidonylethanolamide (AEA), could be a candidate. We observed that AEA induced apoptosis in long-term and recently established glioma cell lines via aberrantly expressed vanilloid receptor-1 (VR1). In contrast with their role in THC-mediated death, both CB1 and CB2 partially protected glioma against AEA-induced apoptosis. These data show that the selective targeting of VR1 by AEA or more stable analogues is an attractive research area for the treatment of glioma.

Topics: Antineoplastic Agents; Apoptosis; Arachidonic Acids; Brain Neoplasms; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cells, Cultured; Endocannabinoids; Gene Expression Regulation, Neoplastic; Glioma; Humans; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Drug; RNA, Messenger

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca(2+) influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH(2)CH(2)OH group was replaced by N-CH(2)CH(2)Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH(2))(12)CH(3)] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH(2)CH(3))(2) (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 microM AEA to a greater degree than the parent compound, whereas the N-(CH(3))(2) analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca(2+) influx into hVR1-HEK293 cells. EC(50) values for AEA to produce Ca(2+) influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 microM in the presence of 0, 1, 3 and 10 microM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 microM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH(2)CH(3) and N-CH(CH(3))(2) analogues (22 and 23, respectively). 22 and 23 produced a small ( approximately 25%) inhibition of the binding of [(3)H]-CP55,940 and [(3)H]-WIN 55,212-2 to CB(1) and CB(2) receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 microM [(3)H]-AEA by rat brain fatty acid amidohydrolase with IC(50) values of 5.6 and 11 microM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 microM [(3)H]-AEA into C6 glioma cells were seen at 10 microM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Brain Neoplasms; Calcium Channel Blockers; Capsaicin; Endocannabinoids; Ethanolamines; Glioma; Humans; In Vitro Techniques; Kidney; Morpholines; Palmitic Acids; Polyunsaturated Alkamides; Radioligand Assay; Rats; Receptor, Cannabinoid, CB1; Receptors, Drug; Transfection; Tumor Cells, Cultured

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

Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography-electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and 2-arachidonoylglycerol (2-AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid-like compounds N-oleoylethanolamine, N-palmitoylethanolamine and N-stearoylethanolamine. Anandamide and 2-AG (0.16 +/- 0.05 and 0.10 +/- 0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid-like compounds, respectively. N-Palmitoyethanolamine was the most abundant (50%), followed by N-oleoyl (23.6%) and N-stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid-like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six-fold smaller content of all N-acylethanolamines, but not of 2-AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.

Topics: Amides; Animals; Arachidonic Acids; Brain Chemistry; Brain Neoplasms; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Ethanolamines; Gas Chromatography-Mass Spectrometry; Glioblastoma; Glycerides; Humans; Lymphoma; Meningioma; Neuroblastoma; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reference Values; Stearic Acids; Tumor Cells, Cultured; U937 Cells

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Topics: Amides; Animals; Arachidonic Acids; Brain Neoplasms; Cannabinoids; Cell Survival; Endocannabinoids; Ethanol; Ethanolamines; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Mice; Neuroblastoma; Palmitic Acids; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Pronase; Protease Inhibitors; Rats; Tumor Cells, Cultured

N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [(3)H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4-45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3-aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4-hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations <10 microM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain Neoplasms; Cannabinoids; Carrier Proteins; Cell Membrane; Cerebellum; Cyclohexanols; Endocannabinoids; Esterases; Fluorescent Dyes; Glioma; Humans; Lactones; Microscopy, Fluorescence; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.

Topics: Aged; Amidohydrolases; Arachidonic Acids; Biological Transport; Brain; Brain Neoplasms; Cannabinoids; Cell Membrane; Endocannabinoids; Enzyme Inhibitors; Humans; Hydrolysis; Kinetics; Male; Meningeal Neoplasms; Meningioma; Neuroblastoma; Polyunsaturated Alkamides; Tumor Cells, Cultured; U937 Cells