Reagents

R(+)-Metanandamide, JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphtalenylmethanone] and Fumonisin B1 were purchased from Sigma (St Louis, MO, USA). SR 144528 (SR2) was kindly provided by Sanofi Recherche (Montpelier, France). Antibody anti-CB₂ receptor was obtained from Affinity BioReagents (Golden, CO, USA). We purchased anti-tubulin, anti-phospho Akt at Ser473, anti-phospho p38, anti-total p38, anti-phospho JNK, anti-total JNK, anti-phosphor eIF2α, anti-caspase 8 and anti-caspase 9 and anti-citochrome c antibodies from Cell Signaling Technology (St Louis, MO, USA). The inhibitor D609 and the enzyme diacylglycerol kinase were supplied by Calbiochem (La Jolla, CA, USA).

Cell cultures

Human prostate epithelial PC-3, DU-145 and LNCaP cells were purchased from American Type Culture Collection (Rockville, MD, USA) and were cultivated in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100Uml⁻¹ penicillin G, 100μgml⁻¹ streptomycin and 0.25μgml⁻¹amphotericin B (Invitrogen, Paisley, UK). Low cell passages (between 10 and 20) were used for this study. Cells were seeded sub-confluently and, 1 day before the experiment, the serum was removed to work with quiescent cells.

Cell viability assays

Cells were set up 2 × 10⁴cells per well of a 24-well plate and were cultured in the RPMI 1640 medium supplemented with 10% FCS. After treatments according to figure legends, cell viability was assayed by MTT as previously described (Sanchez et al, 2003).

Flow cytometry

Flow cytometry was used to detect apoptotic cells and the distribution of cell cycle. After being cultivated with medium alone or medium containing the indicated stimuli, 10⁵ cells in a 35-mm culture dish were harvested in 0.1% Nonidet P-40 and 0.5mgml⁻¹ Rnase for 30min and stained with 0.05mgml⁻¹ Iodure Propidium (IP) to indicate the relative DNA content. The sub-G1 peak (DNA content <2N) and cell-cycle distribution were measured using FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). To analyse apoptosis by Annexin V staining, the cells were washed twice with PBS and incubated in 0.5ml of binding buffer (10mm HEPES pH 7.4, 150mm NaCl, 2.5mmCaCl₂, 1mm MgCl₂ and 4% BSA), with 4μgml⁻¹ Annexin V-FITC for 15min. The cells were then washed in PBS and re-suspended in the binding buffer with 0.6μgml⁻¹ IP (Calbiochem). In all, 20000 cells of each sample were analysed by flow cytometry in a FACScan (Beckton Dickinson).

Measurement of [³H]-thymidine incorporation into DNA

Cells were treated with different concentrations of MET or JWH-015 according to the experiment. DNA synthesis was determined by pulsing the cells with [³H]-thymidine (1μCi per well) during the last 16h of the culture period as previously described (Sanchez et al, 2003).

siRNA tranfections

Cells were transfected in 1ml OPTIMEN containing 4μg lipofectamine 2000 (Invitrogen Co., Carlsbad, CA, USA), with 100nm human cannabinoid receptor-2-specific small interfering RNA (siRNA) duplexes (5′-GGCCUCUUCCCAAUUUAAAtt-3′, Applied Biosystems, Foster City, CA, USA) or control scrambled RNA for 12h according to the manufacturer’s protocols (Ambion, Applied Biosystems). At 24h after transfection, the medium was removed and replaced with RPMI 1640 with 10% FCS medium. At dedicated time points after transfection, the cells were used for flow cytometry assays or western blot.

Measurement of ceramide levels

Ceramide quantification in cell lipid extracts was performed according to the method described by Perry et al for ceramide quantification in cultured cells (Perry et al, 2000). Briefly, cell pellets were suspended in 0.6ml distilled water, and disrupted at 4°C by sonication. Lipids were extracted with chloroform/methanol, and ceramide content was determined by phosphorylation using Escherichia coli diacylglycerol kinase and [³²P]γ-ATP (6000Cimmol⁻¹; Perkin-Elmer, Barcelona, Spain). Products of this reaction were purified by TLC using chloroform–acetone–methanol–acetic acid–water (50:20:15:10:5, by volume) as the developing solvent. Products of this reaction were quantified and expressed as a percentage of the value observed earlier (Sanchez et al, 2007).

Western blot analysis

Cultured cells were lysed into a lysis buffer (50mm Tris-HCl, pH 7.4, 5mm EDTA, 1mm EGTA, 10mm 2-mercaptoethanol) containing 5μgml⁻¹ leupeptin, 5μgml⁻¹ aprotinin and 1mmphenylmethylsulfonyl fluoride, and were disrupted by sonication. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting was carried out as previously described (Sanchez et al, 2006).

In vivo anti-tumour activity

All animal studies were conducted in accordance with the Spanish institutional regulation for the housing, care and use of experimental animals, have been carried out with ethical committee approval and met the European Community directives regulating animal research. Recommendations made by the UKCCCR have been adhered to carefully. Athymic nude (nu/nu) 6-week-old male mice were purchased from Harlan Iberica (Barcelona, Spain) and were housed in a laminar airflow cabinet under pathogen-free conditions on a 12-h light–dark schedule. Mice were injected subcutaneously (s.c.) in the right flank with 2 × 10⁶ PC-3 cells in 0.2ml of complete culture medium. Two weeks after transplantation, tumours had grown to an average volume of 70mm³. Mice were then divided into three experimental groups of eight animals each, which received the following treatments as s.c. injections: group A, saline (control); group B, 0.15mgkg⁻¹ body weight (b.w.) JWH-015; group C, 0.15mgkg⁻¹ b.w. JWH-015 plus 0.15mgkg⁻¹ b.w SR2. The injection was repeated every day and treatment was continued for 14 days. Tumour volumes were monitored every day using calliper measurements and were calculated using the following formula: (4π/3) × (w/2)² × (l/2), where w=width and l=length. The b.w. of the animals was recorded daily.

Statistical analysis

Data are presented as mean±s.e. of the number of experiments indicated. Statistical comparisons among groups were made with Student’s t-test, and the difference was considered to be statistically significant when the P-value was <0.05.

The cannabinoids, MET and JWH-015, inhibited cell growth of prostate cancer cells

We first examined the anti-proliferative effects of the stable anandamide analogue, MET, and the synthetic CB₂ligand, JWH-015, on prostate PC-3 cells. The kinetics of MET and JWH-015 treatment showed that cannabinoid-induced cell death was evident from 12h, although maximal effect was reached at 48–72h (Figure 1A). Thus, we decided to follow all the studies at 48h. Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48h, after which cell viability was evaluated by MTT assay, [³H]-thymidine incorporation assay or by flow cytometry. As shown in Figure 1B, both MET and JWH-015 caused a dose-dependent decrease in cell viability, which was significantly different from control from doses over 5μm. To assess the suppressive effects of R(+)-Methanandamide and JWH-015 on the proliferation of PC-3 cells, DNA synthesis was measured by [³H]-thymidine incorporation. Results shown in Figure 1C indicate that both cannabinoids inhibited the proliferation of PC-3 cells, which was totally blocked from doses over 5μm. The cell-cycle analysis demonstrated that cannabinoid treatment resulted in a small, although significant, accumulation of cells in the sub-G1 phase of the cell cycle (Figure 1D). These results suggest that the compounds used induced a small percentage of apoptosis and growth arrest in prostate cells. To investigate whether the anti-proliferative effect of cannabinoids on prostate cancer cells was generalised, we used the androgen-refractory prostate cancer DU-145 cells and the less tumourigenic androgen-dependent prostate LNCaP cells. Results shown in Figure 2 showed that both MET and JWH-015 inhibited the growth of the three cancer prostate lines studied, although the effect was less pronounced in the androgen-sensitive LNCaP cells. As shown in Figure 2A, low doses (sub-micromolar) of MET induced a slight increase in LNCaP cell viability, as previously reported by our group (Sanchez et al, 2003).

Figure 1

The anti-proliferative effect of the cannabinoids, R(+)-Methanandamide and JWH-015, on prostate PC-3 cells. (A) Time course of cannabinoid effect on prostate PC-3 cells viability. PC-3 cells were incubated with 10μm MET or 10 …

Figure 2

Anti-proliferative effect of cannabinoids on different prostate cancer cell lines. (A) Prostate cancer LNCaP, PC-3 or DU-145 cells were incubated with different doses of MET for 48h and cell viability was assayed by MTT. (B) Prostate cancer LNCaP, …

To quantify the percentage of apoptotic cells after drug treatments, PC-3 cells were stained with Annexin V-FITC/IP. Results show that the rate of late apoptotic cells (Annexin V-FITC positive/IP positive, upper right quadrant) in MET- and JWH-015-treated cells was statistically increased compared with that in control cells (Figure 3). R(+)Methanandamide and JWH-15 treatments also induced cell necrosis, as inferred from IP-positive cells (upper left quadrant). Early apoptotic cells (Annexin V-FITC positive/IP negative, lower right quadrant) were <5% in all cases. These findings indicate that both MET and JWH-015 promoted a low, although significant, percentage of apoptosis in prostate cancer cells, but other processes such as mitotic catastrophe, cytotoxicity or necrosis could also collaborate in the observed growth inhibition.

Figure 3

Evaluation of apoptosis by Annexin V-FITC/IP staining, followed by flow cytometry analysis. Representative plots of Annexin V-FITC/IP staining of PC-3 cells cultured in the presence of increasing concentrations of MET or JWH-015 are shown. Data showing …

Involvement of CB₂ in the anti-proliferative effect of cannabinoids

As we have previously shown, prostate PC-3 cells express both CB₁ and CB₂ cannabinoid receptors (Sanchez et al, 2003). We then investigated the role of CB₁ and CB₂ in cannabinoid-induced prostate cell death. Pharmacological blockage of CB₁ with its antagonist Rimonabant (SR1) did not reduce the effect of MET on cell cycle or apoptosis (Figure 4A). However, the CB₂ antagonist, SR 144528 (SR2), reduced the number of apoptotic cells and the number of sub-G1 cells induced by MET treatment (Figure 4A). As MET is a weak ligand for CB₂, we confirmed this result with the CB₂-selective agonist JWH-015. The JWH-015-induced cell death effect was reverted by SR2, suggesting a role for CB₂ in the apoptotic mechanisms of cannabinoids in PC-3 cells.

Figure 4

Inhibition of cannabinoid-induced cell death by the CB₂ antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10μm MET or 10μm JWH-015 for 48h in the presence or absence of 0.5μmRimonabant …

To confirm the involvement of CB_2, we silenced its expression with siRNA. PC-3 cells were transfected with CB₂-selective siRNA or control scrambled RNA for 48h, after which the expression of CB₂ was notably reduced as it was corroborated by western blotting (Figure 5). Under these conditions, apoptosis induced by 10μm JWH-015 was almost totally blocked in cells transfected with CB₂ siRNA when compared with scrambled siRNA-transfected cells (Figure 5). These results confirm the involvement of CB₂ receptor in the pro-apoptotic effect of cannabinoids in prostate cells. Therefore, we conducted the rest of the experiments with JWH-015, which is a potent and selective ligand for CB₂ and which exhibits more efficacy than MET for CB₂ activation.

Figure 5

CB₂ is involved in the anti-proliferative effect of JWH-015 in PC-3 cells. Cells were transfected with CB₂-specific small interfering RNA (siRNA) or with control scrambled RNA for 48h and then treated with 10μm JWH-015 for an …

Ceramide synthesis mediates the anti-proliferative effect induced by CB₂ activation

Ceramide is a sphingolipid messenger that has a relevant role in the regulation of tumour cell fate (Velasco et al, 2005;Carpinteiro et al, 2008). Recent studies have suggested that apoptosis induced by cannabinoids can be preceded by ceramide accumulation (Gomez del Pulgar et al, 2002; Gustafsson et al, 2006). To further analyse the apoptotic mechanism of JWH-015, we measured intracellular ceramide concentration in PC-3 cells. As shown in Figure 6A, incubation with JWH-015 for 48h led to a dose-dependent increase in intracellular ceramide accumulation. This effect was prevented by the CB₂ antagonist SR2, which indicates that ceramide accumulation was mediated by the activation of CB₂ (Figure 6B). Ceramide is formed in cellular membranes by de novo synthesis through a pathway involving the ceramide synthase or by hydrolysis of sphingomyelin catalysed by acid, neutral and alkaline sphingomyelinases. To gain insight into the origin of JWH-015-induced ceramide increase, cells were incubated in the presence of the ceramide synthase inhibitor, Fumonisin B1, or the sphingomyelinase inhibitor, D609. As shown in Figure 6B, treatment of cells with 10μm Fumonisin B1, but not with D609, prevented the increase in ceramide concentration, a fact that suggests that ceramide came from de novo biosynthesis.

Figure 6

Involvement of ceramide synthesis in JWH-015-induced cell growth inhibition. (A) PC-3 cells were incubated in the presence of increasing concentrations of JWH-015 for 48h and intracellular ceramide was measured by the DAG kinase method as indicated …

Moreover, treatment with Fumonisin B1 prevented the inhibition of cell growth induced by JWH-015 (Figure 6C), indicating that ceramide biosynthesis was involved in the action of the cannabinoid.

Signal mechanisms involved in JWH-015-induced prostate PC-3 cell death

To further explore the signalling pathways in which the CB₂ agonist exerted its effect in prostate PC-3 cells, we studied stress-related MAP kinase cascades activation by western blot. PC-3 cells were treated for different times with 10μm JWH-015 and then phosphorylated forms of JNK and p-38 kinases, indicative for activated kinases, were detected by western blot. Results in Figure 7A show that JWH-015 activates the stress-signal-related kinase JNK as phosphorylated JNK is increased at 30min and 1h of treatment.

Figure 7

Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10μm JWH-015 for different times. (A) Phosphorylation levels of p38, JNK, Akt and eIF2α were measured by western blot. (B) Levels of …

Eukaryotic cells respond to stress in their endoplasmic reticulum by phosphorylating the α-subunit of translation initiation factor 2 (eIF2α). This adaptation inhibits general protein synthesis while promoting translation and expression transcriptional regulators that induce gene expression important for cellular remediation and apoptosis. It has recently been described that cannabinoids promote endoplasmic reticulum stress and autophagy-mediated cell death in glioma cells through the Akt/mammalian target of rapamycin (mTOR) pathway inhibition and eIF2α activation (Salazar et al, 2009b). To investigate whether JWH-015 regulated this pathway in prostate cells, we treated the cells with the CB₂ agonist and measured the phosphorylation of Akt in Ser473, which is negatively involved in the autophagy pathway (Todde et al, 2009). PC-3 treatment with 10μm JWH-015 resulted in a long-term AKT phosphorylation decrease that was evident from 6h of treatment (Figure 7A). This was in concordance with the increase in phosphorylated eIF2α (Figure 7A), suggesting that endoplasmic reticulum stress signals were activated by JWH-015 in prostate cells. We further investigated whether JWH-015 induced caspase activation, which can be detected after the decrease in the pro-caspase form and the increase in the proteolytic active form by western blot (Hoffmann et al, 2009). Results in Figure 7 show that JWH-015 induced activation of caspase 9, which has been described as an initiator caspase essential in the intrinsic or mitochondrially gated pathway of apoptosis (Johnson and Jarvis, 2004; Denault and Salvesen, 2008). To further confirm the activation of the intrinsic apoptotic pathway, we determined the cytosolic levels of cytochrome c, which is released into the cytosol when the cell receives an apoptotic stimulus to trigger programmed death through caspase 9 activation (Ow et al, 2008).

JWH-015 caused regression of prostate cancer xenografts in nude mice

The above observations of the anti-proliferative effects of JWH-015 were examined in a xenograft model of prostate cancer. Xenograft human prostate tumours were established in nu/nu mice by a s.c. injection. Tumour-bearing animals (~70mm²) were treated daily with vehicle, 1.5mgml⁻¹ JWH-015 or 1.5mgml⁻¹ JWH-015 plus 1.5mgkg⁻¹ SR2. Animals were treated for 15 days and tumour volume was calculated every day. At the end of the experiment, tumours were dissected and weighed. As shown in Figure 8, JWH-015-treated animals had a rapid and dramatic reduction in tumour growth, whereas uncontrolled growth was observed in the control group. The final tumour volume as well as the final tumour weight was significantly lower in the JWH-015-treated group compared with that in the control group (Table 1). Treatment with JWH-015 plus SR2 resulted in a similar growth compared with that in the control group, suggesting that thein vivo effect of JWH-015 is also mediated through CB₂ activation (Figure 8 and Table 1).

Figure 8

In vivo anti-tumoural properties of JWH-015. Athymic nude mice were injected s.c. in the right flank with PC-3 cells and 4 weeks later (day 0) were treated for 15 days with vehicle (control), 1.5mgkg⁻¹JWH-015 or 1.5mgkg …

Table 1

Effect of JWH-015 on PC-3 xenograft tumour growth

This work was supported by Ministerio de Ciencia e Innovación (Grant SAF2008-03220), Comunidad de Madrid (Grants CAM/UAH CCG08-UAH/BIO-3914 and CAM S-SAL-0261-2006) and Comunidad Castilla-LaMancha (Grant PII1/09-0165-0822). SM-C receives a fellowship from the Juan de la Cierva programme from the Spanish MEC. NO-H and DV receive fellowships from the University of Alcalá.

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