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* This work was supported in part by Istituto Superiore di Sanità (III AIDS Program), by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome (to A.F.A.), and by Telethon Grant E872 (to G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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” (
). 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 (
). 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 (
). 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 (
). 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.
We have shown that AEA can induce apoptotic body formation and DNA fragmentation, hallmarks of PCD, in human neuronal and immune cells through a pathway involving rise in intracellular calcium, mitochondrial uncoupling, and cytochrome c release. Activation of the arachidonate cascade and of the caspase cascade are critical steps in the death program. The pro-apoptotic activity of AEA was observed at physiological concentrations of this compound (
), were unable to force cells into PCD under the same experimental conditions (TableI), ruling out the possibility that the observed effects of AEA were due to unspecific cell poisoning. Since 2-AG may release arachidonate through FAAH activity faster than AEA (
). Consistently, inhibition of FAAH by ATFMK potentiated, instead of reducing, the apoptotic activity of AEA (Table II). Also inhibition of AEA degradation by blocking its uptake enhanced AEA-induced PCD (TableII). Since a slower degradation leads to an increased concentration of AEA in the extracellular matrix, these findings suggest that the pro-apoptotic activity of AEA is mediated by a target molecule on the cell surface. Indeed, [3H]AEA binds to CHP100 and U937 cell membranes (Fig. 3A). However, in these cell lines a different binding site must be involved because the “classical” CB1 or CB2 receptors are not present (Fig. 2). Previous reports have shown that AEA can bind and modulate receptors other than CB1R and CB2R (
) was ineffective on [3H]AEA binding (Fig. 3B) and on AEA-induced PCD (Table II). On the other hand, it is becoming increasingly evident that AEA behaves as a full agonist at human vanilloid receptors (
) mimicked the pro-apoptotic activity of AEA in these cells. Altogether, these findings suggest that AEA-induced PCD was mediated by vanilloid receptors. It should be stressed that this hypothesis is consistent with the observation that 2-AG and the other endocannabinoids did not promote PCD (Table I), because these compounds do not activate vanilloid receptors (
). In this context, it seems noteworthy that AM404 alone was ineffective on PCD, mitochondrial uncoupling, intracellular calcium concentration, or cytochrome c release from cells, although it did potentiate the effect of AEA (Tables Table II, Table III, Table IV and Fig. 4B). These findings suggest that AM404 was unable to activate directly human VR, at variance with a previous report suggesting that it is an agonist for rat VR (
A major finding of this investigation is that CB1R or CB2R antagonists, SR141716 or SR144528, were ineffective in CHP100 or U937 cells, which lack cannabinoid receptors (Fig. 2), but they did potentiate AEA-induced PCD in C6 or DAUDI cells (Table III). In fact, these cells express functional CB1 or CB2 receptors, respectively (Fig. 2), and were able to bind larger amounts of [3H]AEA than CHP100 or U937 cells. Capsazepine displaced approximately 30% [3H]AEA from C6 or DAUDI cells, suggesting that the remaining 70% was bound to CB receptors. Remarkably, capsazepine prevented AEA-induced PCD in these cells in a way fully analogous to that observed in CHP100 or U937 cells (Table III), suggesting that vanilloid receptors mediate the pro-apoptotic activity of AEA also in C6 and DAUDI cells. As a matter of fact, specific vanilloid responses have been described in C6 cells (
) might be involved. On the other hand, it seems noteworthy that the ability of C6 or DAUDI cells to degrade AEA through intracellular uptake and degradation by FAAH was similar to that of CHP100 or U937 cells, respectively. Therefore, it is tempting to speculate that cells bearing functional CB1 or CB2 receptors on their surface are protected against the toxic effects of physiological concentrations of AEA. In C6 or DAUDI cells, the effects on PCD of co-administration of the transporter inhibitor AM404, which increases extracellular concentration of AEA, or of CBR antagonists SR141716 and SR144528, which prevent CBR activation (Table III), support this concept. These findings can be interpreted by suggesting a regulatory loop between CB receptors and the AEA transporter, which has been recently demonstrated in human endothelial cells (
). In this loop, the binding of AEA to CB receptors triggers the activation of AEA uptake by cells, followed by intracellular degradation of AEA by FAAH. Elimination of AEA from the extracellular space might terminate its activity at vanilloid receptors, thus inhibiting the induction of apoptosis. Scheme FSI summarizes the main features of this model.
PCD of CHP100 or U937 cells induced by AEA was executed through a series of events common to several types of unrelated apoptotic stimuli (
). It involved the following: (i) rise in cytosolic calcium concentration (within 6 min), (ii) uncoupling of mitochondria (within 6 h), and (iii) release of cytochrome c (within 8 h). These events required gene expression of proteins necessary for apoptosis, as shown by the protective effect of actinomycin D and cycloheximide (Table II). Consistently with the data on apoptotic body formation and [3H]AEA binding to cell membranes, (i) capsazepine inhibited the events triggered by AEA, (ii) AM404 or ATFMK potentiated them, and (iii) SR141716, SR144528, or CBD were ineffective (Table IV and Fig. 4B). At variance with other types of PCD (
), calcium rise induced by AEA was not acting through activation of nitric-oxide synthase, because the nitric-oxide synthase inhibitorl-NAME was ineffective in protecting cells against AEA. Instead, arachidonate degradation by 5-lipoxygenase and cyclooxygenase activities, which might be enhanced as a consequence of a rise in intracellular Ca2+ (
), had a role in the process, because the inhibitors ETYA and MK886 significantly inhibited AEA-induced PCD (Table II). It must be mentioned that MK886 can exert lipoxygenase-unrelated effects on mammalian cells (
). However, the observation that ETYA and MK886 yielded the same inhibition of apoptosis seems to rule out the involvement of lipoxygenase-independent pathways. This seems interesting, because formation of arachidonate products unbalances the intracellular redox level and has been implicated in apoptotic death of several cell types (
). In particular, it should be stressed that a function for lipoxygenase in programmed organelle degradation has been recently demonstrated, showing that the enzyme can make pore-like structures in the lipid bilayer (
) did not contribute to AEA-induced PCD, as suggested by the lack of effect of cyclosporin A (Table II). On the other hand, an unbalanced redox level in the cell has been associated to release of cytochrome c, a converging point in apoptosis induced by different stimuli in various cell types (
). Cytochrome c release was observed also in AEA-induced PCD (Fig. 4B), and it was essential for apoptosis, because sequestering cytochrome c within intact U937 cells by electrotransferred anti-cytochrome cmonoclonal antibodies was able to prevent AEA-induced PCD (
). Caspases are thought to form a proteolytic machinery within the cell, resulting in the breakdown of key enzymes and cellular structures, and to activate DNases responsible for chromatin degradation seen in apoptosis (
). Also AEA-induced PCD seemed to be executed through this series of events, because caspase-3 or caspase-9 inhibitors reduced apoptotic body formation to approximately 20–30% of the controls (Table II). Altogether, these results suggest that PCD induced by AEA occurs through an apoptotic pathway based on calcium rise, mitochondrial uncoupling, and cytochrome c release. Upstream activation of the arachidonate cascade leads to redox unbalance and organelle disruption, which both favor cytochromec release, then caspases act as downstream executioners of the death program. In this context, it seems noteworthy that also capsaicin-induced PCD occurs through intracellular calcium rise, imbalance of the redox level, and drop in mitochondrial membrane potential (
), further strengthening the hypothesis that AEA is acting through vanilloid receptors. Scheme FSI summarizes the series of events responsible for AEA-induced cell death. It seems noteworthy that these findings might be relevant also for neuronal apoptosis induced by alcohols (
). Finally, this study shows that endocannabinoids exert similar actions in neuronal and immune cells, perhaps (and significantly) through common signals.
We thank Dr. Dale G. Deutsch (Department of Biochemistry and Cell Biology, State University of New York, Stony Brook) for the kind gift of C6 glioma cells, Drs. Marco Ranalli and Rita Agostinetto for their skillful assistance with cytofluorimetric analysis and cell culture, and Dr. Francesca Bernassola for helpful discussions.