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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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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 μ JWH-015in the presence or absence of 2 μ SR2, 50 μ Fumonisin B1 (Fumo) or 5 μ D609 for 48 h and intracellular ceramide was assayed as above. (C) Cell cycle of PC-3 cells incubated with 10 μ JWH-015±50 μ 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.
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fig6: 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 μ JWH-015in the presence or absence of 2 μ SR2, 50 μ Fumonisin B1 (Fumo) or 5 μ D609 for 48 h and intracellular ceramide was assayed as above. (C) Cell cycle of PC-3 cells incubated with 10 μ JWH-015±50 μ 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.

Mentions: 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 48 h 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 10 μ Fumonisin B1, but not with D609, prevented the increase in ceramide concentration, a fact that suggests that ceramide came from de novo biosynthesis.


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)

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 μ JWH-015in the presence or absence of 2 μ SR2, 50 μ Fumonisin B1 (Fumo) or 5 μ D609 for 48 h and intracellular ceramide was assayed as above. (C) Cell cycle of PC-3 cells incubated with 10 μ JWH-015±50 μ 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743360&req=5

fig6: 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 μ JWH-015in the presence or absence of 2 μ SR2, 50 μ Fumonisin B1 (Fumo) or 5 μ D609 for 48 h and intracellular ceramide was assayed as above. (C) Cell cycle of PC-3 cells incubated with 10 μ JWH-015±50 μ 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.
Mentions: 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 48 h 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 10 μ Fumonisin B1, but not with D609, prevented the increase in ceramide concentration, a fact that suggests that ceramide came from de novo biosynthesis.

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