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

Olea-Herrero N, Vara D, Malagarie-Cazenave S, Díaz-Laviada I - Br. J. Cancer (2009)

Bottom Line: 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.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.In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

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.

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Inhibition of cannabinoid-induced cell death by the CB2 antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10 μ MET or 10 μ JWH-015 for 48 h in the presence or absence of 0.5 μ Rimonabant (SR1) or 2 μ 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.
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fig4: Inhibition of cannabinoid-induced cell death by the CB2 antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10 μ MET or 10 μ JWH-015 for 48 h in the presence or absence of 0.5 μ Rimonabant (SR1) or 2 μ 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.

Mentions: 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.


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

Olea-Herrero N, Vara D, Malagarie-Cazenave S, Díaz-Laviada I - Br. J. Cancer (2009)

Inhibition of cannabinoid-induced cell death by the CB2 antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10 μ MET or 10 μ JWH-015 for 48 h in the presence or absence of 0.5 μ Rimonabant (SR1) or 2 μ 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743360&req=5

fig4: Inhibition of cannabinoid-induced cell death by the CB2 antagonist, SR 144528 (SR2). PC-3 cells were incubated with 10 μ MET or 10 μ JWH-015 for 48 h in the presence or absence of 0.5 μ Rimonabant (SR1) or 2 μ 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.
Mentions: 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.

Bottom Line: 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.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.In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus