Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). Agonists to CB1 and CB2 promote ceramide de novo synthesis, p38–mitogen-activated protein kinase-dependent activation of caspase-3 and apoptotic cell death in most MCLs. However, in this report we describe that in some MCLs the response to treatment with cannabinoids decreased cell viability as assessed by metabolic activity but did not involve the caspase-3 cascade or loss of plasma membrane integrity. Both primary cells from one MCL patient and the MCL cell line Granta519 responded to treatment with cannabinoids by formation of cycloheximide-sensitive cytoplasmic vacuoles, but did not enter apoptosis. The persistent expression of mammalian homolog of Atg8 with microtubule-associated 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 the 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.

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 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 WIN55,212-2, 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, WIN55,212-2 induced a dose-dependent decreased cell viability, as assessed by flow cytometry, at 48h. Half maximal inhibitory concentration (IC50) values represent WIN55,212-2 concentrations at which the viability reaches 50%. These values were varying between ~1.5 and 5μM for PA, PC, PD, PE and PF primary samples. Lymphoma cells isolated from blood or bone marrow from the patient PB (PB1 and PB2, respectively) were resistant to cannabinoid-induced PI uptake (Figure 1a). Using the same viability assay, the Granta519 cell line, but not other MCL cell lines tested (Rec1, JeKo and JVM2), showed a similar resistance to WIN55,212-2-induced PI uptake (Figure 1b). In contrast, using the XTT viability assay, no such difference between Granta519 and the other MCL cell lines was observed (Figure 1c). This discordance in viability assessment results might indicate that different modes of cannabinoid-mediated toxicity are operational.

Figure 1

Primary MCL cells and cell lines respond differently to WIN55,212-2 treatment. Primary MCL cells (a) and MCL cell lines (b) were treated with WIN55,212-2. Cell viability was assessed by PI staining at 48h; the WIN55,212-2 concentration at which …

Table 1

Characteristics of primary samples from patients with MCL and the expression levels of CNR1 and CNR2 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 10μM WIN55,212-2 (Figure 2a) or 10μM anandamide (AEA, N-arachidonoylethanolamine; Figure 2b). At 24h after adding cannabinoids, all cell lines but Granta519 were dead. Kinetic studies for up to 10h, using western blot, showed that after 5h cannabinoids had activated caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage in Rec1, JeKo and JVM2 but not in Granta519 (Figures 2a and b). Similarly, PARP cleavage was not detected in PB1 cells after treatment with 10μM WIN55,212-2 (Figure 2c). These data indicate that Granta519 cells respond to cannabinoids by decreased metabolic activity, and together with PB1 cells are resistant to apoptosis.

Figure 2

MCL cell lines have variable response to WIN55,212-2 and AEA. MCL cell lines were incubated with cannabinoids and harvested at different time points. After 5h, 10μM WIN55,212-2 (a) or 10μM AEA (b) causes PARP…

Phase-contrast microscopy of Granta519 cell cultures for 48h in the presence of 10μM WIN55,212-2 showed cytoplasmic vacuoles (Figure 3a). Vacuolation is a feature of autophagy, but can also be a feature of other processes such as paraptosis/cytoplasmic vacuolation. In autophagy, autophagosomes are formed, which finally fuse to lysosomes forming autolysosomes. The process also involves the conversion of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 I (LC3 I) to a PE-conjugated form, denoted LC3 II, which localizes in the autophagosomes. The formation of LC3 II is, however, not specific for autophagy but also occurs during cytoplasmic vacuolation.²¹ We investigated the changes in LC3 I and LC3 II expression levels in MCL cell lines up to 10h of treatment with 10μM WIN55,212-2. During this time, we did not observe any enhanced expression of LC3 II in any of MCL cell lines investigated (Figures 2a and b). However, after incubating Granta519 cells with 10μM WIN55,212-2 or AEA for 24 and 48h, LC3 II levels were elevated compared with vehicle-treated cells (Figure 3b).

Figure 3

Induction of LC3 II in Granta519 cells in response to WIN55,212-2. (a) Light microscopy images showing cellular morphology of Granta519 cells undergoing cytoplasmic vacuolation after treatment with 10μM WIN55,212-2 for 48h ( …

Cannabinoid-induced vacuolation does not involve lysosomal degradation and is insensitive to MEK-1 and -2 inhibitors

During autophagy, the protein p62 localizes in the autophagosomes and is, together with LC3 II, degraded in autolysosomes. Therefore, changes in levels of LC3 II and p62 proteins may reflect stages of the autophagy.²²

To characterize the cannabinoid-induced LC3 II-positive vacuolation, cells were treated with WIN55,212-2 or AEA for 24 and 48h, and levels of the vacuolar membrane protein p62^22, 23 were analyzed. Cannabinoid treatment induced persistent expression of LC3 II and did not influence the levels of p62 (Figure 3b). The fact that LC3 II and p62 levels did not decrease over time suggests that vacuoles were not efficiently fused with the lysosomes. These findings suggest that the autophagocytic process was not completed, whereas the cellular metabolism, measured by XTT, was reduced as shown in Figure 1c.

To further analyze the vacuoles, we investigated whether changes in the lysosomal pH by chloroquine (CQ) treatment would influence the observed effect of cannabinoids. However, the cannabinoid-induced reduction of metabolism was not affected by CQ treatment, indicating that lysosomal degradation was not involved in the process induced by cannabinoids (Figure 3c).

Other reports suggest involvement of the MAP/ERK kinase (MEK)-1/-2 pathway in vacuolation.^21, 24 Therefore, we investigated whether specific inhibitors of MEK-1 and -2 influenced the fate of Granta519 cells treated with WIN55,212-2. However, the MEK-1- and -2-specific inhibitors, PD98059 and U0126, respectively, were ineffective in protecting from cell death (Figure 3d).

Cannabinoid-mediated decrease of Granta519 cell viability occurs in a cannabinoid receptor-independent manner and requires active protein synthesis

To verify whether the decrease in Granta519 viability is mediated via CB1 and CB2, we pretreated cells with SR141716 and SR144528, selective antagonists for CB1 and CB2, respectively. Cells were pretreated with 10 or 100nM of SR141716 and SR144528 for 30min and then treated with a range of concentrations of WIN55,212-2. Neither SR141716, SR144528 nor a combination of both antagonists inhibited WIN55,212-2-induced cell death or LC3 II lipidation (Figures 4a and b). Altogether, these findings suggest that the cannabinoid-mediated decrease in Granta519 viability and LC3 II-positive vacuolation are not mediated by CB1 and/or CB2 activation.

Figure 4

Effect of CB1 and CB2 antagonists on WIN55,212-2-induced cell membrane integrity and LC3 II formation. (a) Granta519 cell were pretreated with SR141716 (antagonist to CB1) and SR144528 (antagonist to CB2) for 30min before incubation with increasing …

To study whether the cannabinoid-induced cell death is reversible after washing cells off WIN55,212-2, we incubated Granta519 and Rec1 cells with 10μM WIN55,212-2 for 3 or 6h before washing off the drug. The cells were then cultured for another 48h. Our results show that the decrease in metabolic activity, measured by XTT, was significantly reversed by removing the drug both in Granta519 and Rec1 cells (Figure 5a). For Granta519 cells, washing off the drug also diminished LC3 II positivity (Figure 5a). We then studied the effect of longer exposure of WIN55,212-2 on Granta519 cells. For this, we cultured Granta519 with WIN55,212-2 for 24h, then washed cells and recultured for another 24h. Then the decrease in metabolic activity could be significantly reversed (Figure 5b).

Figure 5

Washing off WIN55,212-2 or treatment with CHX protect Granta519 from WIN55,212-2-induced cytoplasmic vacuolation and cell death. (a) Cell viability of Granta519 and Rec1 cells incubated with 10μM WIN55,212-2 for 3 and 6h before …

We further studied whether the decrease in cell viability and induction of vacuolation require active protein synthesis. For this, we blocked the biosynthesis of new proteins with cycloheximide (CHX) and then treated Granta519 cells with WIN55,212-2. The addition of CHX significantly protected cells from WIN55,212-2-induced cell death (Figure 5c), implying the requirement of protein biosynthesis.

Vacuoles in cannabinoid-treated Granta519 cells are of ER origin

Light and transmission electron microscopy (TEM) analysis of Granta519 cells at different time points after treatment with 10μM WIN55,212-2 was used to further characterize the vacuole formation. Cells incubated with WIN55,212-2 for 24h showed large cytoplasmic vacuoles, whereas the plasma membrane was unaffected. We also observed that the nuclei were intact and noted the occasional appearance of mitotic figures (Figure 6a), whereas vehicle-treated Granta519 cells retained a normal ultrastructural morphology, with well defined nuclei containing uniformly distributed chromatin and well preserved organelles (Figures 6bi and ii). In Granta519 cells treated with 10μM WIN55,212-2, small amounts of vacuoles in the cytoplasm were seen already at 2h of incubation, which increased in number and size with time (Figures 6biii–viii). The vacuoles were surrounded by membranes and some of these membranes were decorated by ribosomes (Figure 6biv), indicating that the intracellular vacuoles were derived from the rough endoplasmic reticulum (ER) and possibly also smooth ER. Most mitochondria remained unchanged up to 6–12h of WIN55,212-2 treatment. Then, we observed dilation of the cristae membranes and denser mitochondria matrix. However, in some cells the mitochondrial morphology remained normal even after 24h of treatment.

Figure 6

Granta519 cells form vacuoles in response to WIN55,212-2 treatment. (a) Light microscopy pictures of 0.5μm sections prepared as for TEM of Granta519 cells treated with 10μM WIN55,212-2 or vehicle for 24h. Note …

The nuclear membrane was also affected, displaying a widening of the membrane space but with intact nuclear pore complexes (Figure 6bvi). Notably, the outer plasma membranes were not affected, and the general cell morphology did not display any pronounced signs of apoptosis or necrosis. Furthermore, the vacuoles were often empty, but membrane structures could occasionally be found (Figure 6bv).

Immunofluorescence staining showed altered distribution of the ER chaperone calreticulin (Figure 6c). The ER formed large vacuoles in WIN55,212-2-treated cells, confirming that the empty vacuoles within the cytoplasm are indeed of ER origin.

Similar vacuoles, although smaller in size, were formed in PB1 primary cells treated with WIN55,212-2 (Figures 7aiii and iv) compared with PB1 treated with vehicle (Figures 7ai and ii) for 24h. The smaller size of the vacuoles in primary cells might be due to the fact that lymphoma cells ex vivo do not proliferate and the vacuolation process requires new protein synthesis. XTT assay on PB1 cells treated with WIN55,212-2 for 48h did not show any changes in mitochondrial activity upon treatment (Figure 7b).

Figure 7

WIN55,212-2 induced vacuolation in PB1 cells. (a) Normal ultrastructure morphology of primary PB1 cells was predominately found in cells treated with the vehicle for 24h. These cells had a well-defined plasma membrane and uniformly distributed …

WIN55,212-2 induces ER stress in MCL

The morphological changes of ER in WIN55,212-2-treated 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 WIN55,212-2 treatment upregulates BiP and CHOP proteins in all MCL cell lines studied up to 10h of treatment with WIN55,212-2 (Figure 8a). This suggests that WIN55,212-2 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 BiP and CHOP in Granta519 cells remained high also after 24 and 48h of treatment (Figure 8b).

Figure 8

Expression of ER stress markers in MCL cell lines treated with 10μM WIN55,212-2. (a) Western blots depicting expression levels of proteins, BiP, CHOP and β-actin, in Granta519, Rec1, JeKo and JVM2 cell lines treated with 10 …

Reagents

WIN55,212-2 and AEA were from Tocris (Bristol, UK). CQ, CHX and PI were from Sigma-Aldrich (Dorset, UK). PD98059 and U0126 were from Calbiochem (Merck, Darmstadt, Germany). SR141716 and SR144528 were from Cayman Chemical (Ann Arbor, MI, USA).

Patient samples

Tumour cells were isolated as described previously,²⁰ viability frozen in freezing medium containing 10% DMSO (Merck, Darmstadt, Germany), 40% fetal bovine serum (FBS; Invitrogen, Paisley, UK) and 50% RPMI+GlutaMax (Invitrogen) according to the Biobank procedures at Karolinska University Hospital, Huddinge, and stored at −135°C

Cell culture of primary cells and cell lines

Granta519, Rec1, JeKo, JVM2 cell lines and primary cells were obtained and cultured as previously described.⁴ Before setting up experiments, cells were cultured for 4h (primary cells) or overnight (cell lines) in RPMI+GlutaMax (Invitrogen) with 1% FBS (Invitrogen). It is common to use 1% serum in media when performing experiments with cannabinoids, as many cannabinoids bind to serum components (for a review, see Flygare and Sander¹⁸).

Cell viability

Viability was assessed by (1) the XTT assay (measuring the (primarily) mitochondrial, metabolic activity) according to the manufacturer’s instructions (Roche, Mannheim, Germany) or by (2) flow cytometric analysis (FACSCalibur, BD Biosciences, Bedford, MA, USA) of PI (Sigma-Aldrich; final concentration 1μg/ml) uptake by cells lacking plasma membrane integrity. Results were analyzed using FlowJo 7 (Tree Star Inc., Ashland, OR, USA) for Windows, and pharmacological interpretation was done using a four-parameter logistic equation with iterative fitting (dose–response fitting) using OriginPro 8 (OriginLab, Northampton, MA, USA).

Western blotting

Cells were pelleted and frozen at −20°C or used immediately after pelleting for preparing cell lysates using RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Proteins were separated on 12% NuPAGE gels from Invitrogen and then transferred using semidry transfer system onto PVDF membrane (Millipore, Solna, Sweden). Membranes were blocked with 10% dry milk in TBS solution for 2h at room temperature (RT) and then probed overnight at 4°C or for 2h at RT with primary antibodies: anti-PARP, anti-caspase-3, anti-LC3, anti-BiP or anti-Akt (Cell Signaling, Danvers, MA, USA) or anti-p62, anti-β-actin (Sigma-Aldrich) in 5% dry milk or 5% BSA for anti-CHOP (Cell Signaling) in TBS. Membranes were then washed in TBS and probed with secondary antibodies (anti-rabbit or anti-mouse; GE Healthcare Bio-Sciences Corp., Uppsala, Sweden). Blots were developed using Supersignal West Pico (Pierce, Biotechnology, Shelton, CT, USA) and wet-developing method (developer and fixer solutions were from Kodak (Sigma-Aldrich) and films were from GE Healthcare). For reprobing membranes, HRP was blocked using the SG substrate kit (Vector Labs, Peterborough, UK).

RNA isolation and cDNA synthesis

RNA was isolated using the RNeasy Plus mini kit (Qiagen, Sollentuna, Sweden) according to the manufacturer’s protocol. Quantification and quality of the RNA preparations were measured using the NanoDrop ND-1000 spectrophotometer (Saveen Werner, Stockholm, Sweden). Complementary DNA (cDNA) was synthesized using the Omniscript Reverse Transcription kit (Qiagen) according to manufacturer’s protocol. RNaseOut recombinant ribonuclease inhibitor and the oligo dT primers used were purchased from Invitrogen.

Quantitative PCR

mRNA expression levels of CNR1 and CNR2 were assessed by real-time PCR using Platinum SYBR Green qPCR Supermix-UDG (Invitrogen) according to the manufacturer’s protocol. Primers sequences (Eurogentec, Seraing, Belgium) for the selected genes were as follows: CNR1 forward: 5′-CATTAAGACGGTGTTTGCATTCT-3′ and reverse: 5′-CGTGTCGCAGGTCCTTACTC-3′, amplifying 1185–1282bp (transcript variant 1) and 1018–1115bp (transcript variant 2). CNR2 primers were 5′-GACACGGACCCCTTTTTGCT-3′ (forward primer) and 5′-CCTCGTGGCCCTACCTATCC-3′ (reverse primer), amplifying 1321–1423bp. β-Actin primers were 5′-AAAGACCTGTACGCCAACACA-3′ (forward primer) and 5′-AGTACTTGCGCTCAGGAGGA-3′ (reverse primer), amplifying 955–1096bp. Each sample was prepared in triplicates in a 96-well plate (Bio-Rad Laboratories, Sundyberg, Sweden) and the reactions were performed with the C1000 Thermal cycler (Bio-Rad). An initial step was performed at 95°C for 2min, followed by 40 cycles at 95°C for 15s and finished by 57°C for 30s. The results were analyzed and cycle threshold (Ct) values of transcripts were quantified using CFX manager software (Bio-Rad). The ΔCt values were calculated using β-actin as reference. cDNA from buffy coat B cells was used as control for CNR1 and CNR2 to calculate the ΔΔCt value and to determine the relative fold increase.

Immunofluorescence microscopy

Cells were grown on poly–lysine-coated coverslips, then spun down, washed and incubated with 4% PFA at RT. After washing, cells were incubated with primary mouse anti-calreticulin (Abcam, Cambridge, UK) antibody at 4°C overnight. After washing secondary anti-mouse, Alexa488-conjugated antibody was applied for 1h at RT. Cells were washed and mounted onto slide using mounting media with DAPI (Vector Labs). Staining was examined on Leica DM RXA fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using SlideBook 5.0 software (Intelligent Imaging Innovations, Göttingen, Germany). The images were analyzed using software ImageJ for Macintosh (US National Institutes of Health, Bethesda, MD, USA).

Transmission electron microscopy

Cells were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4, at RT for 30min. Next, cells were transferred to Eppendorf tubes (Hamburg, Germany) and further fixed overnight in the refrigerator. After fixation, cells were rinsed in 0.1M phosphate buffer and centrifuged. The pellets were then postfixed in 2% osmium tetroxide in 0.1M phosphate buffer, pH 7.4, at 4°C for 2h, dehydrated in ethanol, followed by acetone and embedded in LX-112 (Ladd, Burlington, VT, USA). Sections were contrasted with uranyl acetate, followed by lead citrate and examined in a Tecnai 12 TEM (Fei Company, Eindhoven, The Netherlands) at 80kV. Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Munster, Germany).

Ethical permission

This study was approved by the Ethics Committee at Karolinska Institutet.

We are grateful to the personnel at the Flow Cytometry Unit at Karolinska University Hospital Huddinge for help with collection of patient samples and flow cytometry. This study was financially supported by grants from the Swedish Cancer Society, the Swedish Research Council, the Cancer Society in Stockholm, the Karolinska Institutet Funds and the Stockholm County Council.

CB1

cannabinoid receptor 1

CB2

cannabinoid receptor 2

CNR1

gene encoding cannabinoid receptor 1

CNR2

gene encoding cannabinoid receptor 2

MCL

mantle cell lymphoma

PCD

programmed cell death

PI

propidium iodide

IC50

half maximal inhibitory concentration

AEA

anandamide

PARP

poly (ADP-ribose) polymerase

LC3

mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3

ER

endoplasmic reticulum

CHOP

(CCAAT/enhancer-binding protein) homologous protein

BiP

binding immunoglobulin protein

UPR

unfolded protein response

IGF1R

insulin-like growth factor-1 receptor

EGF

epidermal growth factor

MAPK

mitogen-activated protein kinase

Bcl2

B-cell lymphoma 2

Bax

Bcl2-associated X protein

Bcl-x_L

B-cell lymphoma-extra large

EBV

Epstein–Barr virus

EBER

EBV-encoded RNA

TEM

transmission electron microscopy

FBS

fetal bovine serum

CHX

cycloheximide

CQ

chloroquine

Edited by A Stephanou

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