Expression and functional relevance of cannabinoid receptor 1 in Hodgkin lymphoma

Abstract

Background: Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.

Design and methods: We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated.

Results: A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.

Conclusions: The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Figure 1. Expression of CB₁-receptor in classical Hodgkin lymphoma and reactive non-neoplastic lymphatic tissues.

A) Immunohistochemical staining of CB₁–N in classical Hodgkin lymphoma showing strong expression of CB₁ in HRS cells (arrows). B) Confocal image showing CB₁ (green), CD30 (red) and DAPI-stained nuclei (blue) in cHL. Note the CB₁ negativity in non-neoplastic infiltrate. C) In a case of reactive tonsillitis, CB₁-positive cells (arrow heads) were found in the inter-follicular zone (IFZ) and to a lesser extent in germinal center (GC) and mantle zone (MZ). D) Few disseminated cells were found CB₁-positive (arrow heads) in a case of lymphadenitis. Bars  =  20 µm.

Figure 2. CB₁ in reactive lymphoid tissue.

Immunofluorescence staining and confocal imaging of a tonsil against CB₁–N (green, left column), CD3, CD20, CD138 and CD68 (all in red, second left column). Nuclei were visualized using DAPI (blue, second right column). Right column represents merged images. CB₁ signal was present in CD68⁺ macrophages and CD138⁺ plasma cells, not in CD3⁺ and CD20⁺ lymphocytes. Bars  =  20 µm.

Figure 3. CB₁-expression in B-cell derived cells and reduction of viability of cHL cells with AM251.

A) Extracts of HL cell lines L540, L1236, KMH2, HDLM2, L428, as well as B-NHL cell line Karpas 422 and neuroblastoma derived cell line SHSY were used for mRNA analyses. After reverse transcription, cDNA templates were used to quantify mRNA transcripts of Cnr1, Cnr2, GPR55 and ß-actin. B) Western blot analysis for CB₁–N in cHL (L540, L1236, HDLM2, KM-H2 and L428), B-NHL-derived cell lines (Karpas 422, BJAB, SUDHL8, Farage) and isolated peripheral blood CD19⁺ B-lymphocytes. ß-actin signal served as loading control. C) Cell viability was determined in L428, L540, KM-H2 and Karpas 422 cells treated with the indicated concentrations of AM251 for 120 h using the MTT-assay. Reduced viability of cHL cell lines was observed whereas viability of Karpas 422 cells was not affected. Values represent means ± SD.

Figure 4. Effects of CB₁ inhibition on signal transduction, p65-level, cell cycle profile and apoptotic populations in L428 cells.

L428 cells were treated with 10 µM AM251. A) Western blot analysis of crude cell extracts showing a reduction of p65 whereas P-Erk1/2, P-Akt and P-p38 MAPK were not significantly altered compared to vehicle. B) Cell cycle analysis using EdU/DNA-stain and flow cytometric analysis showed strong decline of cells in S-phase and relative increase of cells in G2M phase. C) AnnexinV/7-AAD staining and subsequent flow-cytometric analysis revealed that after 72 h and 120 h of AM251-treatment, the number of vital cells decreased and apoptotic, necrotic and dead fractions were elevated. D) Processing of caspase-3 in a representative Western blot and its statistical analyses of L428 cells treated with AM251 for 96 h. AM251-treatment resulted in higher amounts of cleaved of caspase-3 (cleaved C-3) accompanied by a decrease of full length caspase-3 (C-3). Values represent means ± SD of 3 independent experiments.