title: Cannabinoid receptor-mediated apoptosis induced by R(+)-methanandamide and Win55,212-2 is associated with ceramide accumulation and p38 activation in mantle cell lymphoma.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital Huddinge, F-46, SE-14186 Stockholm, Sweden.
authors.forename: Kristin
authors.initials: K
authors.lastname: Gustafsson
authors.forename: Birger
authors.initials: B
authors.lastname: Christensson
authors.forename: Birgitta
authors.initials: B
authors.lastname: Sander
authors.forename: Jenny
authors.initials: J
authors.lastname: Flygare
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001095:Arachidonic Acids / Q000494:pharmacology*; D048588:Benzoxazines; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002186:Cannabinoids / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D020522:Lymphoma, Mantle-Cell / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology*; D009281:Naphthalenes / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D043882:Receptors, Cannabinoid / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D001095:Arachidonic Acids; D048588:Benzoxazines; D002121:Calcium Channel Blockers; D002186:Cannabinoids; D002518:Ceramides; D009025:Morpholines; D009281:Naphthalenes; D043882:Receptors, Cannabinoid; C109343:dihydroceramide; C088155:methanandamide; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3