anandamide and Thyroid-Neoplasms

The active components of Cannabis sativa and their derivatives produce a wide spectrum of effects, some of which may have clinical application. The discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has attracted much attention to cannabinoids as agents capable of controlling the decision of cells to survive or die. We analysed the effects exerted by 2-methyl-2'-F-anandamide (Met-F-AEA), a metabolically stable analogue of anandamide, and observed a growth inhibition in cell lines derived from thyroid carcinomas. Growth inhibition was associated with a high level of CB1 receptor expression, suggesting that the cytotoxic effect is due to interaction with the CB1 receptor. This phenomenon was associated with activation of the protein, p53, an increased apoptotic rate, and expression of p21(CIP1/WAF1). This study provides new insights into the mechanism of Met-F-AEA action, and could have significance in providing a basis for the management of thyroid carcinoma.

Topics: Apoptosis; Arachidonic Acids; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Drug Screening Assays, Antitumor; Endocannabinoids; Humans; Molecular Weight; Piperidines; Poly(ADP-ribose) Polymerases; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Thyroid Neoplasms; Tumor Suppressor Protein p53

Endocannabinoids are now emerging as suppressors of key cell-signaling pathways involved in cancer cell growth, invasion, and metastasis. We have previously observed that the metabolically stable anandamide analog, 2-methyl-2'-F-anandamide (Met-F-AEA) can inhibit the growth of thyroid cancer in vivo. Our hypothesis was that the anti-tumor effect observed could be at least in part ascribed to inhibition of neo-angiogenesis. Therefore, the aim of this study was to assess the anti-angiogenic activity of Met-F-AEA, to investigate the molecular mechanisms underlying this effect and whether Met-F-AEA could antagonize tumor-induced endothelial cell sprouting. We show that Met-F-AEA inhibited bFGF-stimulated endothelial cell proliferation, in a dose-dependent manner, and also induced apoptosis, both effects reliant on cannabinoid CB1 receptor stimulation. Analyzing the signaling pathways implicated in angiogenesis, we observed that the bFGF-induced ERK phosphorylation was antagonized by Met-F-AEA, and we found that p38 MAPK was involved in Met-F-AEA-induced apoptosis. Moreover, Met-F-AEA was able to inhibit bi-dimensional capillary-like tube formation and activity of matrix metalloprotease MMP-2, a major matrix degrading enzyme. Importantly, we demonstrated that Met-F-AEA is also functional in vivo since it inhibited angiogenesis in the chick chorioallantoic neovascularization model. Finally, Met-F-AEA inhibited tumor-induced angiogenesis in a three-dimensional model of endothelial and thyroid tumor cell (KiMol) spheroids co-cultures in different 3-D polymeric matrices that resemble tumor microenvironment and architecture. Thus, our results suggest that anandamide could be involved in the control of cancer growth targeting both tumor cell proliferation and the angiogenic stimulation of the vasculature.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Coculture Techniques; Dose-Response Relationship, Drug; Endocannabinoids; Endothelial Cells; Fibroblast Growth Factor 2; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Neovascularization, Pathologic; Neovascularization, Physiologic; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Spheroids, Cellular; Swine; Thyroid Neoplasms

We investigated the effect of 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), a stable analog of the endocannabinoid anandamide, on a rat thyroid epithelial cell line (FRTL-5) transformed by the K-ras oncogene, and on epithelial tumors derived from these cells. Met-F-AEA effect in vivo was evaluated in a nude mouse xenograft model, where K-ras-transformed (KiMol) cells were implanted subcutaneously. Met-F-AEA (0.5 mg/kg/dose) induced a drastic reduction in tumor volume. This effect was inhibited by the CB1 receptor antagonist SR141716A (0.7 mg/kg/dose) and was accompanied by a strong reduction of K-ras activity. Accordingly, KiMol cells and tumors express CB1 receptors. Met-F-AEA inhibited (IC50 ~5 mM) the proliferation in vitro and the transition to the S phase of KiMol cells and it reduced K-ras activity; these effects were antagonized by SR141716A. Met-F-AEA cytostatic action was significantly smaller in nontransformed FRTL-5 cells than in KiMol cells. Met-F-AEA treatment exerted opposite effects on the expression of CB1 receptors in KiMol and FRTL-5 cells, with a strong up-regulation in the former case and a suppression in nontransformed cells. The data suggest that: 1) Met-F-AEA inhibits ras oncogene-dependent tumor growth in vivo through CB1 cannabinoid receptors; and 2) responsiveness of FRTL-5 cells to endocannabinoids depends on whether or not they are transformed by K-ras.

Topics: Animals; Arachidonic Acids; Blotting, Western; Cannabinoid Receptor Modulators; Cannabinoids; Cell Cycle; Cell Division; Cell Line; Cell Line, Transformed; Dose-Response Relationship, Drug; Endocannabinoids; Genes, ras; Mice; Mice, Nude; Neoplasms, Experimental; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Thyroid Neoplasms; Xenograft Model Antitumor Assays