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The expression level of CB1 and CB2 receptors determines their efficacy at inducing apoptosis in astrocytomas.

Cudaback E, Marrs W, Moeller T, Stella N - PLoS ONE (2010)

Bottom Line: Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT.Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.

Principal findings: We generated astrocytoma subclones that express set levels of CB(1) and CB(2), and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

Significance: The high expression level of CB(1) and CB(2) receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB(1) and CB(2) receptors, yet still activate ERK1/2.

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Activation of CB1 and CB2 receptors differentially increases AKT phosphorylation in DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and AKT phosphorylation quantified by Luminex multiplex immunoassay. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in AKT phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6 to 8 independent experiments expressed as % of vehicle (i.e. level of AKT phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Please note that basal AKT phosphorylation did not vary significantly over time (Figure S1b). In (a), (*) = p<0.05 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test. Non-significant (ns).
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pone-0008702-g002: Activation of CB1 and CB2 receptors differentially increases AKT phosphorylation in DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and AKT phosphorylation quantified by Luminex multiplex immunoassay. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in AKT phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6 to 8 independent experiments expressed as % of vehicle (i.e. level of AKT phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Please note that basal AKT phosphorylation did not vary significantly over time (Figure S1b). In (a), (*) = p<0.05 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test. Non-significant (ns).

Mentions: With regard to AKT, three points are noteworthy. First, the CP-55,940-induced increase in AKT phosphorylation correlated with receptor expression levels (Figure 2a). Specifically, CP-55,940 did not significantly increase AKT phosphorylation in the CB1-low subclone, which expresses the lowest level of receptor of all six subclones that we have selected; it induced a small, but significant increase in AKT phosphorylation in CB2-low and CB2-mid, which express slightly more receptors than the CB1-low subclone; and it induced the most pronounced increase in AKT phosphorylation in CB1-mid, CB1-high and CB2-high subclones (see Table 1 for Bmax values). Second, all responses were mediated by cannabinoid receptors since they were antagonized by either SR141716A or SR144528, respectively, and CP-55,940 did not increase AKT in wild type DBT cells (Figures 2a and S1b). Third, there was a clear difference in the kinetics of AKT activation linked to either CB1 or CB2 receptors. Specifically, while the response obtained in the CB1-high subclone peaked at 3 min and returned to the basal level at 5 min, the response measured in the CB2-high clone remained above 250% of basal during the entire 30 min of the incubation period (Figure 2b,c).


The expression level of CB1 and CB2 receptors determines their efficacy at inducing apoptosis in astrocytomas.

Cudaback E, Marrs W, Moeller T, Stella N - PLoS ONE (2010)

Activation of CB1 and CB2 receptors differentially increases AKT phosphorylation in DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and AKT phosphorylation quantified by Luminex multiplex immunoassay. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in AKT phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6 to 8 independent experiments expressed as % of vehicle (i.e. level of AKT phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Please note that basal AKT phosphorylation did not vary significantly over time (Figure S1b). In (a), (*) = p<0.05 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test. Non-significant (ns).
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pone-0008702-g002: Activation of CB1 and CB2 receptors differentially increases AKT phosphorylation in DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and AKT phosphorylation quantified by Luminex multiplex immunoassay. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in AKT phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6 to 8 independent experiments expressed as % of vehicle (i.e. level of AKT phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Please note that basal AKT phosphorylation did not vary significantly over time (Figure S1b). In (a), (*) = p<0.05 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test. Non-significant (ns).
Mentions: With regard to AKT, three points are noteworthy. First, the CP-55,940-induced increase in AKT phosphorylation correlated with receptor expression levels (Figure 2a). Specifically, CP-55,940 did not significantly increase AKT phosphorylation in the CB1-low subclone, which expresses the lowest level of receptor of all six subclones that we have selected; it induced a small, but significant increase in AKT phosphorylation in CB2-low and CB2-mid, which express slightly more receptors than the CB1-low subclone; and it induced the most pronounced increase in AKT phosphorylation in CB1-mid, CB1-high and CB2-high subclones (see Table 1 for Bmax values). Second, all responses were mediated by cannabinoid receptors since they were antagonized by either SR141716A or SR144528, respectively, and CP-55,940 did not increase AKT in wild type DBT cells (Figures 2a and S1b). Third, there was a clear difference in the kinetics of AKT activation linked to either CB1 or CB2 receptors. Specifically, while the response obtained in the CB1-high subclone peaked at 3 min and returned to the basal level at 5 min, the response measured in the CB2-high clone remained above 250% of basal during the entire 30 min of the incubation period (Figure 2b,c).

Bottom Line: Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT.Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.

Principal findings: We generated astrocytoma subclones that express set levels of CB(1) and CB(2), and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

Significance: The high expression level of CB(1) and CB(2) receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB(1) and CB(2) receptors, yet still activate ERK1/2.

Show MeSH
Related in: MedlinePlus