2.1 Reagents

THC, the CB-receptor antagonists (SR141716 and SR144528) and JWH-133 were kind gifts from Dr. Javier Fernández-Ruiz (Complutense University, Madrid), Sanofi-Aventis (Montpellier, France) and Dr. John W. Huffman (Clemson University, SC), respectively. The fluorescent probes Hoechst 33258 and tetramethylrhodamine metyl ester (TMRM) as well as annexin V-fluorescein isothiocyanate (FITC) were from Molecular Probes (Leiden, The Netherlands). Anti-p38 MAPK, anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-JNK, anti-caspase 3 and anti-poly(ADP-ribose) polymerase (PARP) antibodies were from Cell Signalling (Berverly, MA). Anti-α-tubulin was from Sigma Chemical Co. (St. Louis, MO), anti-BH3 interacting-domain death-agonist (Bid) antibody was from R&D Systems (Minneapolis, MN), and anti-cytochrome c and anti-Bcl-x antibodies were from BD Pharmingen (San Diego, CA). PD169316 was from Calbiochem (Darmstadt, Germany).

2.2 Cell culture and viability

Exponentially growing Jurkat cells were cultured in RPMI 1640 medium containing 10% heat-inactivated foetal bovine serum, 10 mM HEPES, 5 U/mL penicillin and 5 mg/mL streptomycin. Jurkat cells stably transfected with Bcl-x_L expression vector [17] were maintained in the same medium supplemented with 1 mg/mL G418. Cells were transferred to a serum-free medium 30 min before performing the different treatments.

2.3 Nuclear DNA content

Cells were collected by centrifugation at 1500 × g for 5 min, washed once, and incubated for 30 min at room temperature in PBS containing 1% (w/v) BSA, 30% ethanol and 5 μg/mL Hoechst 33342. Fluorescence intensity was analyzed using a LSR flow cytometer (Becton Dickinson, San Jose, CA).

2.4 Quantitative real time PCR

Total RNA was isolated using the RNeasy Protect kit (Qiagen, Hilden, Germany) including a DNase digestion step using the RNase-free DNase kit (Qiagen). cDNA was obtained using the Transcriptor (Roche, Basel, Switzerland). Taqman probes for human CB₁ and CB₂ were obtained from Applied Biosystems (Foster City, California). Experiments were routinely performed including a negative control (no cDNA) as well as a positive control for CB₁ receptor mRNA expression (cDNA obtained from RNA of the human astrocytoma cell line U373MG).

2.5 Apoptosis

Cells (approximately 0.55 × 10⁶ cells per assay) were collected by centrifugation at 1500 × g for 5 min, washed once, and incubated for 30 min at room temperature in 50 μL of binding buffer (10 mM HEPES, pH 7.4, 2.5 mM CaCl₂, 140 mM NaCl) supplemented with 3 μL annexin V-FITC. After incubation for 1 additional minute with the same medium containing 4.5 μg/mL propidium iodide, fluorescence intensity was analyzed using a FACSCalibur flow cytometer. Ten thousand cells were recorded in each analysis.

2.6 Mitochondrial transmembrane potential

Cells (approximately 0.55 × 10⁶ cells per assay) were collected by centrifugation at 1500 × g for 5 min, washed once, and incubated for 30 min at room temperature with binding buffer (see above) plus 0.25 μM TMRM and 3 μL annexin V-FITC. After incubation for 1 additional minute with 1 μM Hoechst 33258 – in order to discriminate living cells – fluorescence intensity was analyzed using a LSR flow cytometer. Ten thousand cells were recorded in each analysis.

2.7 Mitochondria- and cytosol-enriched fractions

They were isolated as described in [18].

2.8 Western blot

Samples (15–45 μg protein/condition) were subjected to SDS/PAGE in 10% or 15% polyacrylamide gels and transferred to PVDF membranes. Western blot analysis was performed using the corresponding antibodies.

2.9 Caspase activity

Caspase 3/7 activity (DVEDase activity) and caspase 8 (LETDase activity) were determined according to manufacturer instructions using a luminogenic substrate (Caspase Glo, Promega, Madison, WI). Luminiscence was determined in a Microplate Fluorescence Reader FLUOstar Optima (BMG Labtech, Offernburg, Germany).

2.10 Statistics

Results shown represent means ± S.D. In statistical analyses of Western blot bands, S.D. values are omitted for clarity. Statistical analysis was performed by ANOVA with a post hoc analysis by the Student–Neuman–Keuls test.

3.1 p38 MAPK is involved in CB₂ receptor-induced apoptosis

THC has been shown to induce apoptosis of human leukaemia cells via the CB₂ receptor [12]. We confirmed by real time quantitative PCR that CB₂ was the unique cannabinoid receptor expressed by these cells (CB₂ amplification was evident after 22 cycles whereas no amplification of CB₁ receptor mRNA was detected after 45 cycles) and that THC-induced cell death was prevented by the CB₂-selective antagonist SR144528 but not by the CB₁-selective antagonist SR141716 (Fig. 1A ). Here, we investigated whether coupling of the CB₂ receptor to MAPKs participates in this effect. As shown in Fig. 1B, incubation with THC led to activation of ERK, JNK and p38 MAPK. Pharmacological inhibition of p38 MAPK but not of ERK or JNK significantly prevented THC-induced apoptosis of Jurkat cells (Fig. 1C, E and F). In line with that observation, THC treatment activated p38 MAPK and SR144528 but not SR141716 prevented this effect (Fig. 1D). The CB₂-selective agonist JWH-133 [19] at 10 μM reduced cell viability by 39 ± 10% (6 h, n = 4) and slightly activated p38 MAPK (Fig. 1D), but for unknown reasons its potency of action was notably lower than that of THC.

In order to study the functional role of p38 MAPK in THC-induced apoptosis, we sought for a system in which this process was blunted. For this purpose, we used Jurkat cells overexpressing the pro-survival protein Bcl-x_L [17]. As shown in Fig. 2A , Bcl-x_L overexpressing Jurkat cells were resistant to THC-induced apoptosis. Moreover, THC treatment did not stimulate p38 MAPK in Bcl-x_L overexpressing cells and the amount of phosphorylated p38 MAPK was much lower in Bcl-x_L overexpressing than in control cells (Fig. 2B). Taken together, these results indicate that activation of p38 MAPK kinase plays a significant role in CB₂ receptor-induced apoptosis of Jurkat cells.

3.2 p38 MAPK is involved in CB₂ receptor-induced caspases 3 and 8 activation

We have recently observed that THC treatment of Jurkat cells leads to a CB₂ receptor-dependent loss of mitochondrial-inner-membrane transmembrane potential (Δψ _m) and cytochrome c release that lead in turn to caspases 3 and 8 activation and apoptosis (manuscript in preparation). We therefore investigated the involvement of p38 MAPK in these events. Pharmacological inhibition of p38 MAPK did not prevent THC-induced loss of Δψ _m (Fig. 3A ) and only slightly attenuated mitochondrial cytochrome c release [as a control, pharmacological inhibition of the mitochondrial respiratory chain with rotenone completely prevented mitochondrial cytochrome c release (Fig. 3B) – but did not affect p38 MAPK phosphorylation (Fig. 3C)]. In contrast, incubation with PD169316 significantly prevented caspases 3 (Fig. 4A and B ) and 8 (Fig. 4D and E) activation as well as cleavage of the caspase 3 selective substrate PARP (Fig. 4C) and the caspase 8 substrate Bid (Fig. 4F). The notion that p38 MAPK may play a role in caspase activation is in line with previous observations in other cellular systems [20] and suggest that, upon cannabinoid challenge, p38 MAPK is involved in regulating the activation of caspases 3 and 8 rather than in acting as a primary regulator of the mitochondrial events that trigger cytochrome c release.

3.3 Concluding remarks

Activation of cannabinoid receptors has been shown to induce apoptosis in different tumour cells. Nevertheless, the molecular mechanisms involved in this effect are not completely understood yet. It has been recently reported that activation of the CB₂ receptor induces apoptosis in the human leukaemia cell line Jurkat [12]. Here, we show that activation of p38 MAPK participates in this effect. Cannabinoid receptors induce apoptosis via sustained ERK activation [5] and Akt inhibition [21]. JNK has also been implicated in the pro-apoptotic effect of cannabinoids [22]. However, although p38 MAPK is coupled to CB₁ [14, 15] and CB₂ [23] receptors, and this kinase has been repeatedly shown to participate in programmed cell death [24], this is the first time in that p38 MAPK is shown to participate in cannabinoid receptor-induced apoptosis. This effect could be cell-specific since cannabinoid-induced apoptosis of glioma [5] and dendritic [25] cells does not depend on p38 MAPK activation. In contrast, ERK and JNK are not apparently involved in THC-induced apoptosis of Jurkat cells. Nevertheless, further research is still necessary to elucidate the molecular bases of the activation of the different MAPK cascades in the context of CB₂ receptor-induced apoptosis.