摘要：

Summary The endocannabinoid system, comprising the cannabinoid receptors type 1 (CB1) and type 2 (CB2), their endogenous ligands (endocannabinoids), and the proteins that regulate endocannabinoid biosynthesis and degradation, controls several physiological and pathological functions. Indeed, recent evidence indicates that endocannabinoids influence the intracellular events controlling the proliferation and apoptosis of numerous types of cancer cells, thereby leading to both in vitro and in vivo antitumour effects. We have previously observed that the endogenous ligand arachidonoyl ethanolamide (anandamide; AEA) inhibits the proliferation of human breast cancer cells by blocking the G0/G1-S-phase transition of the cell cycle through interference with cannabinoid CB1 receptor-coupled signal-transducing events. We have also shown that a metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), arrests the growth of K-ras-dependent tumours, induced and/or already established in vivo, and it inhibits the formation of metastatic nodules in the Lewis lung carcinoma model, these effects being largely mediated by cannabinoid CB1 receptors. However, the possibility that stimulation of CB1 receptors interferes with metastatic processes has not been fully explored. Therefore, we hypothesized that CB1 receptor activation could induce a noninvasive phenotype in breast cancer cells. In this study we showed that, in a model of metastatic spreading in vivo, Met-F-AEA significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 receptor 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 in vitro adhesion and migration on type IV collagen through a decrease phosphoylation of Fak and src but without modifying integrin expression. Further, in MDA-MB-231 cell line, we showed the intracellular localization of CB1 receptor and its association with cholesterol- and sphyngolipid-enriched membrane domains (lipid rafts). Cholesterol depletion by methyl-β-cyclodextrin (MCD) treatment strongly reduced the flotation of the protein on the raft-fractions (DRMs) of sucrose density gradients suggesting that CB1 raft-association is cholesterol dependent. Interestingly, binding of Met-F-AEA also impaired 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. Finally, we found that the cholesterol depletion by MCD prevented Met-F-AEA mediated antiproliferative and anti-migratory effects. Taken together, these results suggest that anandamide inhibits human breast cancer cell growth via a lipid rafts mediated mechanism.

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