Increasing Antiproliferative Properties of Endocannabinoids in N1E-115 Neuroblastoma Cells through Inhibition of Their Metabolism

The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines – N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) - which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis

Palmitoylethanolamide inhibits rMCP-5 expression by regulating MITF activation in rat chronic granulomatous inflammation

Chronic inflammation, a condition frequently associated with several pathologies, is characterized by angiogenic and fibrogenic responses that may account for the development of granulomatous tissue. We previously demonstrated that the chymase, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties in a model of chronic inflammation sustained by mast cells (MCs), granuloma induced by the subcutaneous carrageenan-soaked sponge implant in rat. In this study, we investigated the effects of palmitoylethanolamide (PEA), an anti-inflammatory and analgesic endogenous compound, on rMCP-5 mRNA expression and Microphtalmia-associated Transcription Factor (MITF) activation in the same model of chronic inflammation. The levels of rMCP-5 mRNA were detected using semi-quantitative RT-PCR; the protein expression of chymase and extracellular signal-regulated kinases (ERK) were analyzed by western blot; MITF/DNA binding activity and MITF phosphorylation were assessed by electrophoretic mobility shift assay (EMSA) and immunoprecipitation, respectively. The administration of PEA (200, 400 and 800 µg/ml) significantly decreased rMCP-5 mRNA and chymase protein expression induced by λ-carrageenan. These effects were associated with a significant decrease of MITF/DNA binding activity and phosphorylated MITF as well as phosphorylated ERK levels. In conclusion, our results, showing the ability of PEA to inhibit MITF activation and chymase expression in granulomatous tissue, may yield new insights into the understanding of the signaling pathways leading to MITF activation controlled by PEA

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>The <it>N</it>-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and <it>N</it>- 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.</p> <p>Conclusions</p> <p>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.</p

Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms

Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets

Numerical simulation of natural convection in mixed porous/pure-fluid domains

This thesis is dedicated to the exploration of natural convection phenomena within domains characterized by a heat-generating porous matrix immersed in a pure-fluid region. Such flows hold particular significance in the context of Loss-of-Cooling Accidents (LOCA) occurring within Spent Fuel Pools (SFP) in nuclear power plants. In this application, the porous medium models the fuel assemblies placed in storage racks at the pool's base. During a LOCA event, the cooling circuit, which maintains low temperatures in the pool, becomes inoperative, leading to a free convection regime within the pool. The Fukushima accident in 2011 exposed our limited comprehension of the intricate processes that take place during LOCA. This work aims to address this problem by producing accurate data on the fundamental physics governing the flow dynamics and heat transfer in this context. This objective is of great interest to the research community involved in industrial numerical simulations of SFP during LOCA scenarios. To achieve these goals, we have developed a numerical tool for conducting Direct Numerical Simulations of three-dimensional natural convection within porous/pure-fluid domains. The tool has been validated and verified against a large set of cases, and its numerical properties have been studied. We perform an analysis on the parameters that drive the physical modeling of the porous medium, providing valuable insights into the flow dynamics and heat transfer in such a configuration. The investigation extends to exploring the effects of altering rack heights relative to the bottom wall as well as the consequences of uneven heat load distribution within the racks. This examination provides valuable insights and paves the way for an enhanced understanding of LOCA phenomena.(FSA - Sciences de l'ingénieur) -- UCL, 202

Potentiation of endocannabinoid antitumoral properties by inhibitors of their hydrolysis

Since the identification of the ∆9-tetrahydrocannabinol as the main psychoactive component of the plant Cannabis sativa, a notable amount of studies have been carried out in order to elucidate the implication of the so-called endocannabinoid system in health and various diseases. This system is composed of endogenous bioactive lipids, principally anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which activate the CB1 and CB2 cannabinoid receptors. Additionally, these endocannabinoids have also been described to activate other plasma membrane proteins like the vanilloid type 1 receptor (TRPV1), two G protein-coupled receptors – GPR55 and GPR119 – as well as peroxisome proliferator-activated receptors (PPAR’s). Along with the endocannabinoids, other endogenous molecules belonging to the N-acylethanolamine family, including N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA), are known to exert cannabimimetic actions without binding the cannabinoid receptors. These “endocannabinoid-like” share with AEA common metabolic pathways as well as some molecular targets. Endocannabinoid levels are tightly regulated by enzymes responsible for their degradation. The fatty acid amide hydrolase (FAAH) and the N-acylethanolamine-hydrolyzing acid amidase (NAAA) are in charge of the hydrolysis the N-acylethanolamines AEA, PEA and OEA, while the major enzyme regulating 2-AG levels is the monoacylglycerol lipase (MAGL). Cancer is one of the main causes of death worldwide and a major health care challenge. During the last few years, the involvement of the endocannabinoids in antitumoral processes received a particular attention and evidence has accumulated to demonstrate the ability of cannabinoids to decrease proliferation and viability in numerous cancer cell lines both in vitro and in vivo. The present work intends to investigate the influence of the endocannabinoids AEA and 2-AG, as well as PEA and OEA, on tumor cell proliferation and survival. Selective endocannabinoid hydrolyzing enzyme inhibitors served as useful probes to explore the function and potentiate the action of these lipid mediators. We focused on two mouse cancer models: N1E-115 neuroblastoma and B16 melanoma. We further highlighted the interest of targeting the endocannabinoid system in the management of cancer and evidenced the cytotoxic effects of endocannabinoids in both cell lines. In neuroblastoma cells, the most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597. This compound drastically reduced cell viability, partly due to the inhibition of AEA hydrolysis and the resulting increase in AEA levels. We found that this combination decreased cell proliferation and slowed cell cycle progression without inducing cell death. In contrast, the association of PEA with URB597 synergistically induced melanoma cell death. The effect was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. This antitumor action was associated with an elevation of PEA levels and larger necrotic regions in the tumor. The targets mediating the antiproliferative effects of AEA and URB597 on neuroblastoma cells and those responsible for the decrease of cell survival induced by PEA and URB597 on melanoma cells are still conflicting and remain to be further investigated. Though our results suggest a mechanism independent of the activation of cannabinoid, TRPV1, PPARs and GPR55 receptors in both cancer models, it seems to occur through a lipid raft-dependent pathway in the case of neuroblastoma. There is still a long way to go until the introduction of endocannabinoids in the therapeutic arsenal against cancer progression. However, this work emphasizes the great potential benefit of associating endocannabinoids with enzymatic hydrolysis inhibitors for the development of novel anticancer therapies.(FARM 3) -- UCL, 201

Numerical Simulations of turbulent thermal convection in a domain composed of a pure-fluid region and an immersed heated porous medium

Heat transfer and fluid flow in domains that include a pure- fluid region and porous region are encountered in many natural phenomena as well as in technological applications. In this work, we are concerned with turbulent thermal convection above and in a heated porous medium. The main motivation for this study comes from the need to better understand the phenomena occurring during loss of cooling accidents in spent fuel pools of nuclear power stations. To that end, the fuel racks that transfer heat to the surrounding water are macroscopically modeled as an internally heated porous medium. In the first part of the talk, we briefly present the mathematical model which is based on the single-domain approach. In this model, the porosity is introduced as a field variable to formulate one single set of governing equations that is valid both in the pure-fluid as well as the porous region. Also, in this context, thermal non-equilibrium is assumed between the solid and the fluid inside the porous region. Hence, two distinct energy equations are used, one for the solid and one for the fluid. In the second part, we discuss and analyze the results of Direct Numerical Simulations of turbulent convection in a cubical cavity with a uniformly heated porous region that is placed at the bottom of the domain and away from the side walls. We consider a free slip at the top boundary kept at a constant temperature. All other boundaries are adiabatically isolated walls. The convection is thereby only driven by the heat flux originating from the solid phase of the porous medium. In a third part, we vary the coverage of the porous medium in the horizontal direction and the heat-source distribution profile while keeping the total power constant in all cases and investigate their effects on the flow structures, the turbulence statistics, etc... In particular, we pay interest to the differences between the area- averaged temperature profiles along the vertical direction and the obtained Nusselt numbers in the three studied cases and explain the difference

Modelling and simulation of natural convection in cavities with immersed internally heated porous structures

In this talk we are concerned with three-dimensional natural convection in domains that contain immersed porous structures heated internally. The formulation of the problem is based on the single-domain approach which incorporates porosity as a field variable. Also, the solid matrix is not in thermal equilibrium with the fluid inside the porous structure. A homogeneous boiling model is also implemented to take account of the phase change that may occur when water reaches the saturation temperature. The numerical scheme is based on a fractional-step time-marching scheme coupled with a projection method for the computation of the pressure. First, we present briefly the formulation of the mathematical model and describe the basic aspects of the proposed numerical algorithm. Then, we present and discuss numerical results from a series of test cases for the problem in hand. Emphasis is placed on the effect of thermal non-equilibrium between the solid matrix and the fluid inside the porous medium