title: The association of N-palmitoylethanolamine with the FAAH inhibitor URB597 impairs melanoma growth through a supra-additive action.
abstract: BACKGROUND
  The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.
  
  
  METHODS
  We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.
  
  
  RESULTS
  The N-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.
  
  
  CONCLUSIONS
  This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.
authors.affiliation: Medicinal Chemistry, Cannabinoid and Endocannabinoid Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium. didier.lambert@uclouvain.be
authors.forename: Laurie
authors.initials: L
authors.lastname: Hamtiaux
authors.forename: Julien
authors.initials: J
authors.lastname: Masquelier
authors.forename: Giulio G
authors.initials: GG
authors.lastname: Muccioli
authors.forename: Caroline
authors.initials: C
authors.lastname: Bouzin
authors.forename: Olivier
authors.initials: O
authors.lastname: Feron
authors.forename: Bernard
authors.initials: B
authors.lastname: Gallez
authors.forename: Didier M
authors.initials: DM
authors.lastname: Lambert
mesh_terms: D000577:Amides; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology*; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism; D002219:Carbamates / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival; D004195:Disease Models, Animal; D004357:Drug Synergism; D063388:Endocannabinoids; D004983:Ethanolamines; D008297:Male; D013058:Mass Spectrometry; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010169:Palmitic Acids / Q000494:pharmacology*; D053284:Polyunsaturated Alkamides; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000473:pathology
chemical_list: D000577:Amides; D001095:Arachidonic Acids; D001549:Benzamides; D063385:Cannabinoid Receptor Modulators; D002219:Carbamates; D063388:Endocannabinoids; D004983:Ethanolamines; D010169:Palmitic Acids; D053284:Polyunsaturated Alkamides; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C005958:palmidrol; C078814:anandamide