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. 2010 Jan 14;5(1):e8702.
doi: 10.1371/journal.pone.0008702.

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

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Free PMC article

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Eiron Cudaback et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activation of CB1 and CB2 receptors increases ERK1/2 phosphorylation in all DBT subclones.
DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and ERK1/2 phosphorylation quantified by Western blot analysis. (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 ERK1/2 phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6–8 independent experiments expressed as % of vehicle (i.e. level of ERK1/2 phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Note that basal ERK1/2 phosphorylation did not vary significantly over time (Figure S1a). In (a), (*) = p<0.05 and (**) = p<0.01 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.
Figure 2
Figure 2. 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).
Figure 3
Figure 3. CP-55,940 kills DBT cells via cannabinoid receptor-dependent and -independent mechanisms.
Wild-type (wt) DBT cells and DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP) at either 1 µM (receptor mediated) or 10 µM (receptor independent), and cell viability was assessed by quantifying WST-1 conversion. (a) Five days after treatment, only CB1-low DBT cells showed significant sensitivity to CP at 1 µM, whereas all subclones were sensitive to CP at 10 µM. Data represent means±s.e.m. of 5–9 independent experiments in triplicate. (b) Only the pretreatment of CB1-low DBT cells with SR141617A (5 µM) prevented the CP-induced killing, whereas pretreatment with PD 98059 (PD, 10 µM) significantly reduced both the CP 1 µM and CP 10 µM induced toxicity in CB1-low and wild-type, respectively. Data represent means±s.e.m. of 3–5 independent experiments in triplicate. (c) Dose-response and (d) kinetics of CP-induced killing of wt and CB1-low DBT cells. Data represent means±s.e.m. of 3–9 independent experiments in triplicate. All results are expressed as % of WST-1 measurements in corresponding cells treated with vehicle (0.1% DMSO) for the indicated time point. In (a), (*) = p<0.05 and (**) = p<0.01 significantly different from the viability in the presence of vehicle only, ANOVA followed by Bonferroni's post-test. In (b), (**) = p<0.01 significantly different the viability after treatment with either 1 or 10 µM CP, ANOVA followed by Bonferroni's post-test. In (c), (**) and (##) = p<0.01 significantly different from the viability in the presence of vehicle only, ANOVA followed by Bonferroni's post-test. In (d), (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from the viability in the presence of vehicle only at corresponding time point, ANOVA followed by Bonferroni's post-test.
Figure 4
Figure 4. Time course of the apoptotic response induced by CP-55,940 in wild-type and CB1-low DBT cells.
(a,b) Wild-type (wt) DBT cells and (c,d) CB1-low DBT cells were expanded in 6-well plates, incubated with CP-55,940 (CP) at 10 and 1 µM, respectively, and apoptosis assessed by quantifying propidium iodine (PI) and annexin V fluorescence by FACS after 1, 3, and 5 days. Shown are representative results, with values within each quadrant = mean of 3–5 independent experiments.
Figure 5
Figure 5. Inhibition of AKT signaling confers sensitivity of cannabinoid receptor-expressing DBT cells to cannabinoid-dependent cytotoxicity.
(a) Wild-type (wt) DBT cells and DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP) at 1 µM (receptor-mediated) without or with Inhibitor X (5 µM added 10 min before CP), and cell viability was assessed after 5 days by quantifying WST-1 conversion. Data represent means±s.e.m. of 3–9 independent experiments in triplicate. (*) = p<0.05 and (**) = p<0.01 significantly different from the viability in the presence of vehicle only, ANOVA followed by Bonferroni's post-test. (b) wt and CB2-low DBT cells were expanded in 6-well plates, incubated with CP at 1 µM (receptor-mediated) without or with Inhibitor X (5 µM added 10 min before CP), and the apoptotic response assessed after 5 days by propidium iodine (PI) and annexin V staining by FACS after 1, 3, and 5 days. Shown are representative results, with values within each quadrant = mean of 3–5 independent experiments.

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