Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: involvement of CB2

Abstract

Background: We have previously shown that cannabinoids induce growth inhibition and apoptosis in prostate cancer PC-3 cells, which express high levels of cannabinoid receptor types 1 and 2 (CB(1) and CB(2)). In this study, we investigated the role of CB(2) receptor in the anti-proliferative action of cannabinoids and the signal transduction triggered by receptor ligation.

Methods: The human prostate cancer cell lines, namely PC-3, DU-145 and LNCaP, were used for this study. Cell proliferation was measured using MTT proliferation assay, [(3)H]-thymidine incorporation assay and cell-cycle study by flow cytometry. Ceramide quantification was performed using the DAG kinase method. The CB(2) receptor was silenced with specific small interfering RNA, and was blocked pharmacologically with SR 144528. In vivo studies were conducted by the induction of prostate xenograft tumours in nude mice.

Results: We found that the anandamide analogue, R(+)-Methanandamide (MET), as well as JWH-015, a synthetic CB(2) agonist, exerted anti-proliferative effects in PC-3 cells. R(+)-Methanandamide- and JWH-015-induced cell death was rescued by treatment with the CB(2) receptor antagonist, SR 144528. Downregulation of CB(2) expression reversed the effects of JWH-015, confirming the involvement of CB(2) in the pro-apoptotic effect of cannabinoids. Further analysing the mechanism of JWH-015-induced cell growth inhibition, we found that JWH-015 triggered a de novo synthesis of ceramide, which was involved in cannabinoid-induced cell death, insofar as blocking ceramide synthesis with Fumonisin B1 reduced cell death. Signalling pathways activated by JWH-015 included JNK (c-Jun N-terminal kinase) activation and Akt inhibition. In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.

Conclusions: This study defines the involvement of CB(2)-mediated signalling in the in vivo and in vitro growth inhibition of prostate cancer cells and suggests that CB(2) agonists have potential therapeutic interest and deserve to be explored in the management of prostate cancer.

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 10 μM MET or 10 μM JWH-015 for different times and cell viability was assayed by MTT. (B) Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48 h and cell viability was assayed by MTT. (C) Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48 h and cell proliferation was measured by [³H]-thymidine incorporation. (D) Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48 h and cell cycle was assayed by flow cytometry. Data are the means±s.e. of three different experiments, each performed in triplicate. ^*P<0.05 and ^**P<0.01 using Student's t-test for the comparison between vehicle-treated and cannabinoid-treated cells.

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 48 h and cell viability was assayed by MTT. (B) Prostate cancer LNCaP, PC-3 or DU-145 cells were incubated with different doses of JWH-015 for 48 h and cell viability was assayed by MTT. Data are means±s.e. of two different experiments, each performed in triplicate. ^*P<0.05 and ^**P<0.01 using Student's t-test for the comparison between vehicle-treated and cannabinoid-treated cells.

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 the percentage of late apoptotic cells (upper right quadrant) are the mean±s.e. of three different experiments, each performed in duplicate. ^*P<0.05 and ^**P<0.01 using Student's t-test for the comparison between vehicle-treated and cannabinoid-treated cells.

Figure 4

Inhibition of cannabinoid-induced cell death by the CB₂ antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10 μM MET or 10 μM JWH-015 for 48 h in the presence or absence of 0.5 μM Rimonabant (SR1) or 2 μM SR2. Apoptosis was assayed by Annexin V-FITC/IP staining (panels A and C) and cell cycle was measured by IP staining (panels B and D). Representative plots are shown in the figure and data are the mean±s.e. of three different experiments, each performed in duplicate. ^*P<0.05 and ^**P<0.01 using Student's t-test for the comparison between vehicle-treated and cannabinoid-treated cells, and ^#P<0.05 and ^##P<0.01 for the comparison between cannabinoid-treated and antagonist-treated cells.

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 48 h and then treated with 10 μM JWH-015 for an additional 48 h. Cell apoptosis or cell cycle was assayed by flow cytometry. A representative plot is shown. Data are the mean±s.e. of two different experiments performed in duplicate. ^*P<0.05 using Student's t-test for the comparison between control and JWH-015-treated cells. Upper panel, western blot for CB₂ in control (scrambled) and CB₂ siRNA-transfected cells.

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 48 h and intracellular ceramide was measured by the DAG kinase method as indicated in the Methods section. (B) PC-3 cells were incubated with 10 μM JWH-015in the presence or absence of 2 μM SR2, 50 μM Fumonisin B1 (Fumo) or 5 μM D609 for 48 h and intracellular ceramide was assayed as above. (C) Cell cycle of PC-3 cells incubated with 10 μM JWH-015±50 μM Fumo for 48 h. Data are the mean±s.e. of three different experiments performed in duplicate. ^*P<0.05 and ^**P<0.01 using Student's t-test for the comparison between vehicle-treated and JWH-015-treated cells, and ^#P<0.01 for the comparison between JWH-015-treated and inhibitor-treated cells.

Figure 7

Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10 μM JWH-015 for different times. (A) Phosphorylation levels of p38, JNK, Akt and eIF2α were measured by western blot. (B) Levels of pro-caspase 8, pro-caspase 9 and cytochrome c in the cell cytosol were detected by western blot. Figure shows a representative image of the other three experiments. Tubulin levels are shown as loading control.

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), 1.5 mg kg⁻¹ JWH-015 or 1.5 mg kg⁻¹ JWH-015 plus 1.5 mg kg⁻¹ SR2. Treatments were carried out by injections in the peritumoural area every day. Tumour volumes were measured daily. (A) The dorsal side of representative mice and dissected tumours after treatment. (B) Tumour growth curve after administration of vehicle (diamonds), JWH-015 (squares) or JWH-015+SR2 (triangles). Results represent the mean±s.e. of eight mice in each group. ^*P<0.01 vs control and ^#P<0.01 vs JWH-015, compared by Student's t-test.