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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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Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10 μ 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.
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fig7: Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10 μ 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.

Mentions: 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 10 μ 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 30 min and 1 h of treatment.


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)

Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10 μ 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743360&req=5

fig7: Signalling mechanisms activated by JWH-015 in prostate PC-3 cells. Cells were incubated with 10 μ 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.
Mentions: 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 10 μ 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 30 min and 1 h of treatment.

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