betadex and anandamide

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD's anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed.

Topics: Animals; Arachidonic Acids; beta-Cyclodextrins; Cannabidiol; Cell Death; Cell Line; Cholesterol; Culture Media, Serum-Free; Dronabinol; Endocannabinoids; Gene Expression Regulation; Interleukin-1beta; Lipid Metabolism; Lipopolysaccharides; Mice; Microglia; Oligonucleotide Array Sequence Analysis; Plants; Polyunsaturated Alkamides; RNA, Messenger; Time Factors

To study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer.. Human colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-β-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells.. AEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD.. AEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.

Topics: Antineoplastic Agents; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Caco-2 Cells; Cannabinoid Receptor Modulators; Caspase 3; Cell Proliferation; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Indoles; Membrane Microdomains; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.. Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot.. The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05).. AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.

Topics: Amidohydrolases; Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Cholesterol; Endocannabinoids; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Necrosis; p38 Mitogen-Activated Protein Kinases; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction

Anandamide (AEA) belongs to an emerging class of lipid mediators collectively termed "endocannabinoids". This endogenously synthesized compound has been implicated in multiple processes, mainly related to the regulation of cell growth/death. During pregnancy endometrial fibroblast-like stromal cells proliferate and differentiate into decidual cells, forming the decidua. After reaching its maximum development, the decidua undergoes regression, which appears to be associated with apoptosis. In order to study the role of this endocannabinoid in this process, the effects of AEA upon cell viability and cell death in primary rat decidual cell cultures was investigated. The results obtained demonstrated that AEA induces cell death, in a dose-dependent manner which is associated with morphological alterations, such as nuclear condensation, DNA fragmentation and upregulation of caspase-3/7 activities. Moreover, these effects were attenuated by AM251, a selective antagonist for the cannabinoid receptor CB1. High concentrations induced a dramatic effect in cell viability and morphology, though methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor completely reversed the cytotoxic effect. These findings suggest that AEA in the uterine environment may play an important role in regulating apoptosis through CB1 and thereby modulate decidual stability and regression during pregnancy. However, it cannot be discarded the hypothesis that AEA, in high concentrations, represent a deleterious factor during this complex process.

Topics: Animals; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Cell Death; Cells, Cultured; Cholesterol; Decidua; Dose-Response Relationship, Drug; Endocannabinoids; Female; Membrane Lipids; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Rats; Receptor, Cannabinoid, CB1

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

Topics: Anti-Bacterial Agents; Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Drug Screening Assays, Antitumor; Endocannabinoids; Fas Ligand Protein; fas Receptor; Filipin; Glycerides; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Membrane Microdomains; Membrane Proteins; Neoplasm Proteins; Polyunsaturated Alkamides; src-Family Kinases

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.

Topics: Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Carrier Proteins; Cell Line; Cell Line, Tumor; Endocannabinoids; Glycerides; Humans; Immune System; Lymphocytes; Membrane Microdomains; Metabolism; Neurons; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction; Transfection; U937 Cells

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to approximately 3-fold higher [35S]GTPgammaS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to approximately 50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Topics: Animals; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Biological Transport; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Membrane; Cell Separation; Cholesterol; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Flow Cytometry; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrolysis; Kinetics; Lipid Metabolism; Lipids; Membrane Microdomains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Neurons; Polyunsaturated Alkamides; Protein Binding; Rats; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Rhodopsin; Signal Transduction; Time Factors

There is considerable controversy at present concerning the mechanisms responsible for the cellular uptake of anandamide. One particular issue concerns whether fatty acid-free bovine serum albumin should be used in the assays, it having been argued that such a presence effectively prevents the specific uptake of anandamide. In the present study, it has been demonstrated that in the presence of a low (0.1%, w/v) concentration of fatty acid-free bovine serum albumin, a temperature-dependent and saturable (K(m) approximately 1 microM) uptake of anandamide into P19 embryonic carcinoma cells can be demonstrated using an incubation time of 4 min. Under these conditions, the uptake of anandamide at 4 degrees C is low at a substrate concentration of 100 nM. The uptake at 37 degrees C was not significantly reduced following treatment of the cells with either methyl-beta-cyclodextrin (50 microM) or mevinolin (1 microM), but was reduced by the FAAH inhibitor URB597 (1 microM) and inhibited by the transport inhibitor cum FAAH substrate AM404 with an IC(50) value of 12 microM. When a 45 s incubation time was used, the uptake of anandamide was not saturable at 37 degrees C over the concentration range tested (0.1-1 microM). Analysis of the data at 37 degrees C obtained with 45 s, 4 min and 15 min incubation times revealed a very rapid (i.e. complete by 45 s) non-saturable component followed by a slower saturable (K(m) approximately 1 microM) component of the uptake. It is concluded that the presence of a low concentration of fatty acid-free bovine serum albumin at a suitable concentration reduces non-specific binding (and release) of anandamide to cell culture wells, greatly reduces the cellular accumulation seen at 4 degrees C, and allows the visualisation of both non-saturable and saturable components of the uptake to be seen at 37 degrees C.

Topics: Amidohydrolases; Animals; Anticholesteremic Agents; Arachidonic Acids; beta-Cyclodextrins; Carcinoma; Cell Line, Tumor; Cholesterol; Embryo, Mammalian; Endocannabinoids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mice; Polyunsaturated Alkamides; Serum Albumin, Bovine

In this report we show, by confocal analysis of indirect immunofluorescence, that the type-1 cannabinoid receptor (CB1R), which belongs to the family of G-protein-coupled receptors, is expressed on the plasma membrane in human breast cancer MDA-MB-231 cells. However, a substantial proportion of the receptor is present in lysosomes. We found that CB1R is associated with cholesterol- and sphyngolipid-enriched membrane domains (rafts). Cholesterol depletion by methyl-beta-cyclodextrin (MCD) treatment strongly reduces the flotation of the protein on the raft-fractions (DRM) of sucrose density gradients suggesting that CB1 raft-association is cholesterol dependent. Interestingly binding of the agonist, anandamide (AEA) also impairs DRM-association of the receptor suggesting that the membrane distribution of the receptor is dependent on rafts and is possibly regulated by the agonist binding. Indeed MCD completely blocked the clustering of CB1R at the plasma membrane. On the contrary the lysosomal localization of CB1R was impaired by this treatment only after AEA binding.

Topics: Arachidonic Acids; beta-Cyclodextrins; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cholesterol; Endocannabinoids; Humans; Lysosomes; Membrane Microdomains; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.

Topics: Animals; Apoptosis; Arachidonic Acids; Base Sequence; beta-Cyclodextrins; Cell Line; CHO Cells; Cricetinae; Cyclodextrins; DNA, Complementary; Endocannabinoids; HL-60 Cells; Humans; Jurkat Cells; Membrane Microdomains; Models, Biological; PC12 Cells; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction

Anandamide (arachidonylethanolamide; AEA) is an endogenous ligand for the cannabinoid receptor and its pharmacological effects are under intensive study. However, AEA has a low aqueous solubility and stability which may restrict its use and may eventually endanger the reliability of the obtained results. In the present study, it was found that AEA forms inclusion complexes with cyclodextrins (CDs), resulting in greater aqueous solubility and stability of AEA as AEA/CD complex. Aqueous solubility increased 1 000 to 30 000-fold, depending on the type of CD (10% solution) used. The half-life of AEA in aqueous hydroxypropyl-beta-cyclodextrin solution (10%) at 50 degree C was 2.9 years. These results suggest that CD-technology will be a very useful method to overcome the solubility and stability problems of AEA.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Arachidonic Acids; beta-Cyclodextrins; Cannabinoids; Chemical Phenomena; Chemistry, Pharmaceutical; Chemistry, Physical; Cyclodextrins; Drug Interactions; Drug Stability; Endocannabinoids; Hydrogen-Ion Concentration; Polyunsaturated Alkamides; Solubility