Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). protein-1 light chain-3 II (LC3 II) and p62 as well as the lack of protection from chloroquine indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process distinguishing from classical autophagy. Transmission electron microscopy images and immunofluorescence staining of endoplasmic reticulum (ER) chaperone calreticulin showed that this vacuoles were of ER origin and that chromatin remained normal. These features resemble paraptosis-like cell death-a third type of a programmed cell death not previously described in response to cannabinoids. synthesis of ceramides followed by p38-mitogen-activated protein kinase (MAPK)-dependent apoptosis of lymphoma cells.19 20 Furthermore the cannabinoid methanandamide reduced tumor growth in MCL in a xenograft mouse model.4 Intriguingly high expression of cannabinoid receptors did P005672 HCl not always result in caspase-3-mediated cell death in B-cell lymphomas treated with cannabinoids4 but still as we show in the current study decreased the mitochondrial activity. We therefore hypothesized that cannabinoids may induce other types of programmed cell death (PCD) than apoptosis (PCD I). Here we show that cannabinoids may induce non-apoptotic PCD in MCL widening their therapeutic potential. Results Cannabinoid-mediated cell death of primary MCL cells and MCL cell lines Primary MCL lymphoma cells were obtained from six patients. From two patients – PA and PB – two different tissues were obtained. The expression levels of gene encoding cannabinoid receptor 1 (CNR1) and cannabinoid Gdf11 receptor 2 (CNR2) was determined by quantitative PCR (Table 1). The expression levels of the cannabinoid receptors were normalized to B cells purified from a buffy coat from a healthy donor. The effect of the synthetic cannabinoid WIN55 212 a potent agonist to CB1 and CB2 receptors on cell viability was assessed by two principally different methods. The integrity of the plasma cell membrane was analyzed by flow cytometry for the uptake of the DNA stain propidium iodide (PI) which cannot pass through intact cell membranes. In addition the XTT viability assay which is based on detecting mitochondrial activity was used for viability assessment. In five out of six primary MCLs WIN55 212 induced a dose-dependent decreased cell viability as assessed by flow P005672 HCl cytometry at P005672 HCl 48?h. Half maximal inhibitory concentration (IC50) values represent WIN55 212 concentrations at which the viability reaches 50%. These values were varying between ～1.5 and 5?and in primary MCL cells and in MCL cell lines Granta519 and PB1 cells are resistant to cannabinoid-induced apoptosis We have further analyzed the possible role of caspase-3-dependent effector mechanism as a factor underlying the observed differences in cell death. The response of Granta519 was compared with the other MCL cell lines Rec1 JeKo and JVM2 to incubation with 10?do not proliferate and the vacuolation process requires new protein synthesis. XTT assay on P005672 HCl PB1 cells treated with WIN55 212 for 48?h did not show any changes in mitochondrial activity upon treatment (Figure 7b). Figure 7 WIN55 212 induced vacuolation in PB1 cells. (a) Normal ultrastructure morphology of primary PB1 cells was predominately found in cells treated with the vehicle for 24?h. These cells had a well-defined plasma membrane and uniformly distributed … WIN55 212 induces ER stress in MCL The morphological changes of ER in WIN55 212 Granta519 and PB1 cells prompted further investigation on expression of ER stress-associated proteins: the ER chaperone binding immunoglobulin protein (BiP) that binds to the misfolded proteins and helps them to refold properly and the transcription factor C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP) that participates in the pro-apoptotic pathway of the unfolded protein response (UPR). The analysis of BiP and CHOP by western blot revealed that WIN55 212 treatment upregulates BiP and CHOP proteins in all MCL cell lines studied up to 10?h of treatment with WIN55 212 (Figure 8a). This suggests that WIN55 212 activates ER stress in MCL cells but as this response is similar in all investigated cell lines the ER stress does not discriminate between LC3-II-positive vacuolation or apoptotic cell death. The levels of P005672 HCl BiP and CHOP in Granta519 cells remained high also after 24 and 48?h of treatment (Figure 8b). Figure 8 Expression of ER stress markers in MCL cell lines treated with 10?and tumor growth.