am-356 and Lymphoma--Mantle-Cell

Ceramide levels are elevated in mantle cell lymphoma (MCL) cells following treatment with cannabinoids. Here, we investigated the pathways of ceramide accumulation in the MCL cell line Rec-1 using the stable endocannabinoid analogue R(+)-methanandamide (R-MA). We further interfered with the conversion of ceramide into sphingolipids that promote cell growth. Treatment with R-MA led to increased levels of ceramide species C16, C18, C24, and C(24:1) and transcriptional induction of ceramide synthases (CerS) 3 and 6. The effects were attenuated using SR141716A, which has high affinity to cannabinoid receptor 1 (CB1). The CB1-mediated induction of CerS3 and CerS6 mRNA was confirmed using Win-55,212-2. Simultaneous silencing of CerS3 and CerS6 using small interfering RNA abrogated the R-MA-induced accumulation of C16 and C24. Inhibition of either of the enzymes serine palmitoyl transferase, CerS, and dihydroceramide desaturase within the de novo ceramide pathway reversed ceramide accumulation and cell death induced by R-MA treatment. To enhance the cytotoxic effect R-MA, sphingosine kinase-1 and glucosylceramide synthase, enzymes that convert ceramide to the pro-proliferative sphingolipids sphingosine-1-phospate and glucosylceramide, respectively, were inhibited. Suppression of either enzyme using inhibitors or small interfering RNA potentiated the decreased viability, induction of cell death, and ceramide accumulation induced by R-MA treatment. Our findings suggest that R-MA induces cell death in MCL via CB1-mediated up-regulation of the de novo ceramide synthesis pathway. Furthermore, this is the first study were the cytotoxic effect of a cannabinoid is enhanced by modulation of ceramide metabolism.

Topics: Apoptosis; Arachidonic Acids; Cannabinoids; Cell Line, Tumor; Ceramides; Drug Synergism; Enzyme Inhibitors; Flow Cytometry; Gene Knockdown Techniques; Humans; Lymphoma, Mantle-Cell; RNA, Small Interfering; Statistics, Nonparametric; Up-Regulation

Endogenous and synthetic cannabinoids exert antiproliferative and proapoptotic effects in various types of cancer and in mantle cell lymphoma (MCL). In this study, we evaluated the expression of cannabinoid receptors type 1 and type 2 (CB1 and CB2) in non-Hodgkin lymphomas of B cell type (n = 62). A majority of the lymphomas expressed higher mRNA levels of CB1 and/or CB2 as compared to reactive lymphoid tissue. With the exception of MCL, which uniformly overexpresses both CB1 and CB2, the levels of cannabinoid receptors within other lymphoma entities were highly variable, ranging from 0.1 to 224 times the expression in reactive lymph nodes. Low levels of the splice variant CB1a, previously shown to have a different affinity for cannabinoids than CB1, were detected in 44% of the lymphomas, while CB1b expression was not detected. In functional studies using MCL, Burkitt lymphoma (BL), chronic lymphatic leukemia (CLL) and plasma cell leukemia cell lines, the stable anandamide analog R(+)-methanandamide (R(+)-MA) induced cell death only in MCL and CLL cells, which overexpressed both cannabinoid receptors, but not in BL. In vivo treatment with R(+)-MA caused a significant reduction of tumor size and mitotic index in mice xenografted with human MCL. Together, our results suggest that therapies using cannabinoid receptor ligands will have efficiency in reducing tumor burden in malignant lymphoma overexpressing CB1 and CB2.

Topics: Animals; Antimitotic Agents; Apoptosis; Arachidonic Acids; Blotting, Western; Burkitt Lymphoma; Cell Proliferation; DNA, Complementary; DNA, Neoplasm; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Plasma Cell; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Mice; Mice, Inbred NOD; Mice, SCID; Mitosis; Mitotic Index; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transplantation, Heterologous; Up-Regulation

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