Open Access Research article

The association of N-palmitoylethanolamine with the FAAH inhibitor URB597 impairs melanoma growth through a supra-additive action

Laurie Hamtiaux1, Julien Masquelier2, Giulio G Muccioli2, Caroline Bouzin3, Olivier Feron3, Bernard Gallez4 and Didier M Lambert1*

Author Affiliations

1 Medicinal Chemistry, Cannabinoid and Endocannabinoid Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

2 Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

3 Pole of Pharmacology and Therapeutics, Institute of Experimental and Clinical Research, Université catholique de Louvain, Brussels, Belgium

4 Biomedical Magnetic Resonance, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

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BMC Cancer 2012, 12:92  doi:10.1186/1471-2407-12-92

Published: 19 March 2012

Additional files

Additional file 1:

Structures of the endocannabinoid metabolism inhibitors used in this study.

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Additional file 2:

Receptor expression in B16 cells. B16 cells express cannabinoid receptor CB1 but not CB2, G-protein coupled receptor GPR55, vanilloid receptor TRPV1 and nuclear receptors PPARα and PPARγ. Detection of mRNA was performed by RT-PCR using mouse brain, spleen and liver as control and RPL19 as house keeping gene. The blots are representative of three.

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Additional file 3:

Investigation of the potential molecular targets of PEA and URB597 in B16 cells. Cytotoxicity of PEA (10 μM), URB597 (10 μM) and PEA + URB597 was not significantly affected by CB1 receptor antagonist (0.1 and 1 μM), TRPV1 receptor antagonist (0.1 and 1 μM), PPAR's receptor antagonists (1 and 5 μM) and GPR55 receptor antagonist (1 and 10 μM). B16 cells were seeded 5 h before treatment (2000 cells/well in microwells) and incubated with PEA alone (10 μM), URB597 alone (10 μM) and combinations of these two molecules. Antagonists were added 1 h prior to the addition of PEA and/or URB597. A MTT test was used to evaluate the percentage of viable cells remaining after 72 h. Data are the mean of three experiments performed in triplicate and are expressed as percentage of the vehicle control.

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Additional file 4:

Cytotoxicity of receptor antagonists. Cytotoxicity of CB1 receptor antagonist (AM251), TRPV1 receptor antagonist (capsazepine), PPARα and PPARγ receptor antagonists (GW6471 and T0070907 respectively) and GPR55 receptor antagonist (cannabidiol, CBD). B16 cells were incubated with the antagonists for 72 h. A MTT test was used to evaluate the percentage of viable cells remaining after treatment. Data are expressed as percentage of the vehicle control and are the mean of three experiments performed in quintuplicate.

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Additional file 5:

Effect of MAFP, CAY10499 and URB597 incubation on PEA and 2-AG levels in B16 cells. (A) MAFP, but not CAY10499, increases intracellular levels of PEA. We found in control cells 25.4 ± 3.8 pmol of PEA/107 cells. (B) MAFP and CAY10499, but not URB597, increase intracellular levels of 2-AG. We found in control cells 29.9 ± 4.8 pmol of 2-AG/107 cells. Levels were measured by HPLC-MS. B16 cells (107 cells) were incubated for 8 h with URB597, CAY10499 or MAFP (1 μM). Data are the mean of three experiments performed in quadruplicate and are expressed as percentage of the vehicle control. Significantly different (*P < 0.05; **P < 0.01; ***P < 0.001) from vehicle incubation.

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