am-356 and dihydroceramide

We have recently shown that cannabinoids induce growth inhibition and apoptosis in mantle cell lymphoma (MCL), a malignant B-cell lymphoma that expresses high levels of cannabinoid receptor types 1 and 2 (CB(1) and CB(2)). In the current study, the role of each receptor and the signal transduction triggered by receptor ligation were investigated. Induction of apoptosis after treatment with the synthetic agonists R(+)-methanandamide [R(+)-MA] and Win55,212-2 (Win55; (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone) was dependent on both cannabinoid receptors, because pretreatment with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) and N-((1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528), specific antagonists to CB(1) and CB(2), respectively, abrogated caspase-3 activity. Preincubation with the inhibitors 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190) showed that phosphorylation of MAPK p38 was implicated in the signal transduction leading to apoptosis. Treatment with R(+)-MA and Win55 was associated with accumulation of ceramide, and pharmacological inhibition of ceramide synthesis de novo prevented both p38 activation and mitochondria depolarization assessed by binding of 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)). In contrast, the pancaspase inhibitor z-Val-Ala-Asp(Ome)-CH(2)F (z-VAD-FMK) did not protect the mitochondrial integrity. Taken together, these results suggest that concurrent ligation of CB(1) and CB(2) with either R(+)-MA or Win55 induces apoptosis via a sequence of events in MCL cells: accumulation of ceramide, phosphorylation of p38, depolarization of the mitochondrial membrane, and caspase activation. Although induction of apoptosis was observed in both MCL cell lines and primary MCL, normal B cells remained unaffected. The present data suggest that targeting CB(1)/CB(2) may have therapeutic potential for the treatment of mantle cell lymphoma.

Topics: Apoptosis; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Caspase 3; Cell Line, Tumor; Ceramides; Enzyme Activation; Humans; Lymphoma, Mantle-Cell; Membrane Potentials; Mitochondrial Membranes; Models, Biological; Morpholines; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Cannabinoid

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(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect. R(+)-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 B1 was associated with a suppression of R(+)-MA-induced 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 C2-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 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(+)-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.

Topics: Arachidonic Acids; Ceramides; Cyclooxygenase 2; Dinoprostone; Gene Expression; Glioma; Humans; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Drug; RNA, Messenger; Sphingomyelin Phosphodiesterase; Time Factors; Tumor Cells, Cultured