Cannabinoids have been implicated in the reduction of glioma growth. The present study investigated a possible relationship between the recently shown induction of cyclooxygenase (COX)-2 expression by the endocannabinoid analog R(ϩ)-methanandamide [R(ϩ)-MA] and its effect on the viability of H4 human neuroglioma cells. Incubation with R(ϩ)-MA for up to 72 h decreased the cellular viability and enhanced accumulation of cytoplasmic DNA fragments in a time-dependent manner. Suppression of R(ϩ)-MA-induced prostaglandin (PG) E 2 synthesis with the selective COX-2 inhibitor celecoxib (0.01-1 M) or inhibition of COX-2 expression by COX-2-silencing small-interfering RNA was accompanied by inhibition of R(ϩ)-MA-mediated DNA fragmentation and cell death. In contrast, the selective COX-1 inhibitor SC-560 was inactive in this respect. Cells were also protected from apoptotic cell death by other COX-2 inhibitors (NS-398 {[N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]} and diclofenac) and by the ceramide synthase inhibitor fumonisin B 1 , which interferes with COX-2 expression by R(ϩ)-MA. Moreover, the proapoptotic action of R(ϩ)-MA was mimicked by the major COX-2 product PGE 2 . Apoptosis and cell death by R(ϩ)-MA were not affected by antagonists of cannabinoid receptors (CB 1 , CB 2 ) and vanilloid receptor 1. In further experiments, celecoxib was demonstrated to suppress apoptotic cell death elicited by anandamide, which is structurally similar to R(ϩ)-MA. As a whole, this study defines COX-2 as a hitherto unknown target by which a cannabinoid induces apoptotic death of glioma cells. Furthermore, our data show that pharmacological concentrations of celecoxib may interfere with the proapoptotic action of R(ϩ)-MA and anandamide, suggesting that cotreatment with COX-2 inhibitors could diminish glioma regression induced by these compounds.

Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(ϩ)-methanandamide (R(ϩ)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(ϩ)-MA was abolished by fumonisin B 1 , a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (L-cycloserine, ISP-1) were inactive in this respect. R(ϩ)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4-to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B 1 was associated with a suppression of R(ϩ)-MAinduced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C 2 -ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2Ј-amino-3Ј-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C 2 -ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(ϩ)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.

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R(+)-MA-induced apoptosis is mediated via a mitochondrial-dependent pathway that becomes activated, at least in part, through up-regulation of the COX-2 enzyme.

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