anandamide and Lung-Neoplasms

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and 2-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for 2-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between 2 and 17 pmol mg(-1) protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.

Topics: Amides; Arachidonic Acids; Cell Line, Tumor; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Glycerides; Humans; Limit of Detection; Lung Neoplasms; Mass Spectrometry; Mesenchymal Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results

The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

Topics: Adenocarcinoma; Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Calcium Channel Blockers; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Chemotaxis; Drug Resistance, Neoplasm; Endocannabinoids; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Nude; NF-kappa B; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tissue Array Analysis; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Delta-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 microM) and significant decreases at 100 microM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.

Topics: Apoptosis; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoids; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dronabinol; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Endocannabinoids; Humans; Hydrogen Peroxide; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Models, Biological; Oxygen Consumption; Polyunsaturated Alkamides

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB1 receptors could induce a non-invasive phenotype in breast metastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.

Topics: Animals; Apoptosis; Arachidonic Acids; Breast Neoplasms; Cannabinoid Receptor Modulators; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Endocannabinoids; Female; Focal Adhesion Kinase 1; Humans; Integrins; Lung Neoplasms; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; src-Family Kinases; Time Factors