Julie Gardener
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Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors
EVIDENCE FOR A PROTECTIVE ROLE OF CANNABINOID RECEPTORS
Mauro Maccarrone, Tatiana Lorenzon, Monica Bari, Gerry Melino and Alessandro Finazzi-Agrò
The Journal of Biological Chemistry, October 13, 2000
Abstract
The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-L-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.
Anandamide (arachidonoylethanolamide, AEA)1 belongs to an emerging class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids and collectively indicated as “endocannabinoids” (1). In fact, AEA has been isolated and characterized as an endogenous ligand for cannabinoid receptors in the central nervous system (CB1 subtype) and peripheral immune cells (CB2 subtype). AEA is released from depolarized neurons, endothelial cells and macrophages (2), and mimics the pharmacological effects of Δ9-tetrahydrocannabinol, the active principle of hashish and marijuana (3). Recently, attention has been focused on the possible role of AEA and other endocannabinoids in regulating cell growth and differentiation, which might account for some pathophysiological effects of these lipids. An anti-proliferative action of AEA has been reported in human breast carcinoma cells, due to a CB1-like receptor-mediated inhibition of the action of endogenous prolactin at its receptor (4). An activation of cell proliferation by AEA has been reported instead in hematopoietic cell lines (5). Moreover, preliminary evidence that the immunosuppressive effects of AEA might be associated with inhibition of lymphocyte proliferation and induction of programmed cell death (PCD or apoptosis) has been reported (6), and growing evidence is being collected that suggests that AEA might have pro-apoptotic activity, both in vitro (7) and in vivo (8). This would extend to endocannabinoids previous observations on Δ9-tetrahydrocannabinol, shown to induce PCD in glioma tumors (8), glioma cells (9), primary neurons (10), hippocampal slices (10), and prostate cells (11). However, the mechanism of AEA-induced PCD is unknown. The various effects of AEA in the central nervous system and in immune system (reviewed in Refs.1-3), as well as its ability to reduce the emerging pain signals at sites of tissue injury (12), are terminated by a rapid and selective carrier-mediated uptake of AEA into cells (13), followed by its degradation to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH) (14). Recently, we showed that human neuroblastoma CHP100 cells and human lymphoma U937 cells do have these tools to eliminate AEA (15). Therefore, these cell lines were chosen to investigate how AEA and related endocannabinoids induce apoptosis and how the removal and degradation of AEA are related to this process. The existence of a neuroimmune axis appears to be confirmed by the finding that endocannabinoids elicit common responses in these two cell types.
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