Limits...
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.

Show MeSH

Related in: MedlinePlus

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 μ MET or 10 μ 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2743360&req=5

fig1: 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 μ MET or 10 μ 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.

Mentions: We first examined the anti-proliferative effects of the stable anandamide analogue, MET, and the synthetic CB2 ligand, JWH-015, on prostate PC-3 cells. The kinetics of MET and JWH-015 treatment showed that cannabinoid-induced cell death was evident from 12 h, although maximal effect was reached at 48–72 h (Figure 1A). Thus, we decided to follow all the studies at 48 h. Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48 h, 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 5 μ. 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 5 μ. 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).


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)

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 μ MET or 10 μ 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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 μ MET or 10 μ 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.
Mentions: We first examined the anti-proliferative effects of the stable anandamide analogue, MET, and the synthetic CB2 ligand, JWH-015, on prostate PC-3 cells. The kinetics of MET and JWH-015 treatment showed that cannabinoid-induced cell death was evident from 12 h, although maximal effect was reached at 48–72 h (Figure 1A). Thus, we decided to follow all the studies at 48 h. Cells were incubated in the presence of increasing concentrations of MET or JWH-015 for 48 h, 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 5 μ. 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 5 μ. 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).

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