Cannabinoid Receptor-Mediated Apoptosis Induced by R(+)-Methanandamide and Win55,212-2 Is Associated with Ceramide Accumulation and p38 Activation in Mantle Cell Lymphoma (original) (raw)

Research ArticleArticle

Molecular Pharmacology November 2006, 70 (5) 1612-1620; DOI: https://doi.org/10.1124/mol.106.025981

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 (CB1 and CB2). 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-1_H_-pyrazole-3-carboximide hydrochloride (SR141716A) and N_-((1_S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528), specific antagonists to CB1 and CB2, respectively, abrogated caspase-3 activity. Preincubation with the inhibitors 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1_H_-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1_H_-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 (DiOC6). In contrast, the pancaspase inhibitor z-Val-Ala-Asp(Ome)-CH2F (z-VAD-FMK) did not protect the mitochondrial integrity. Taken together, these results suggest that concurrent ligation of CB1 and CB2 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 CB1/CB2 may have therapeutic potential for the treatment of mantle cell lymphoma.

Footnotes

• This study was supported by grants from the Swedish Cancer Society, The Swedish Research Council, the Cancer Society in Stockholm, the Magnus Bergvall foundation, and the Karolinska Institutet Funds.

• ABBREVIATIONS: MCL, mantle cell lymphoma; CB1 and CB2, cannabinoid receptors type 1 and 2; MAPK, mitogen-activated protein kinase; R(+)-MA, R(+)-methanandamide; Win55, Win55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,_e_]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone]; SR141716A, _N_-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1_H_-pyrazole-3-carboximide hydrochloride; SR144528, N_-((1_S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide); SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1_H_-imidazole; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1_H_-imidazole; PBS, phosphate-buffered saline; PE, phycoerythrin; FCS, fetal calf serum; B-PLL, B-prolymphocytic leukemia; 7AAD, 7-amino-actinomycin D; MS/MS, tandem mass spectrometry; IS, internal standard; SPL, sphingolipid; dh, dihydro; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; z-VAD-FMK, _N_-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; FB1, Fumonisin B1; l-CS, l-cycloserine.

• ↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

• • Received April 23, 2006.
  • Accepted August 25, 2006.

• The American Society for Pharmacology and Experimental Therapeutics

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