Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism

Abstract

We evaluated the ability of cannabidiol (CBD) to impair the migration of tumor cells stimulated by conditioned medium. CBD caused concentration-dependent inhibition of the migration of U87 glioma cells, quantified in a Boyden chamber. Since these cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antimigratory effect of CBD. The inhibition of cell was not antagonized either by the selective cannabinoid receptor antagonists SR141716 (CB1) and SR144528 (CB2) or by pretreatment with pertussis toxin, indicating no involvement of classical cannabinoid receptors and/or receptors coupled to Gi/o proteins. These results reinforce the evidence of antitumoral properties of CBD, demonstrating its ability to limit tumor invasion, although the mechanism of its pharmacological effects remains to be clarified.

Figure 1

Concentration-dependent inhibition of U87 migration by CBD. CM was added in the lower compartment of the Boyden chamber and cells were added in the upper part in the presence of CBD, and then U87 cell migration was quantified. Cells were exposed to increasing concentrations of CBD from 0.01 to 9 μM (black column) and migration was evaluated after 6 h of incubation. Results are expressed as percentages of control migration (U87 cell migration in presence of vehicle, open column). Values are mean±s.e.m. of at least three independent experiments. ^*P<0.05, ^**P<0.01 vs vehicle Dunnett's t-test.

Figure 2

CB1 and CB2 expression in U87 human glioma cells. (a) Western immunoblotting of protein homogenates from U87 and U373 cells for CB1. Proteins (40 μg lane⁻¹) from lysates of C6 (as positive control), U87 and U373 cells reacted with anti-CB1 antibody gave an immunoreactive band at 60 kDa corresponding to the native form of the CB1 receptor (black arrow). (b) Western immunoblotting of protein homogenates from U87 and U373 cells for CB2. Proteins (40 μg lane⁻¹) from lysates of splenocytes (as positive control), U87 and U373 cells reacted with anti-CB2 antibody gave an immunoreactive band at 40 kDa corresponding to the native form of the CB2 receptor.

Figure 3

(a) CBD inhibition of cell migration is not prevented by pretreatment with selective antagonists for CB1 and CB2, respectively, SR141716 (diagonal bars) and SR144528 (cross-hatched bars). Cells were preincubated for 30 min with the antagonists, then 30 min with CBD (black bar, CBD alone) and loaded in the upper compartment of the Boyden chamber. (b) CBD inhibition of cell migration is not prevented by the combination of the two cannabinoid receptor antagonists (SR1+SR2) and capsazepine (CPZ). Results are expressed as percentages of control migration (U87 cell migration in presence of vehicle, open column). Values are mean±s.e.m. of at least three independent experiments. ^*P<0.05, ^**P<0.01 vs vehicle Tukey's t-test.

Figure 4

CBD-induced cell migration is not prevented by pretreatment with PTX. Cells were pretreated with 100 ng ml⁻¹ PTX for 4 h, then treated with CBD or vehicle B for 30 min (open columns) and loaded in the upper compartment of the Boyden chamber. Black columns represent cells without PTX treatment, with CBD or vehicle A alone. Results are expressed as percentages of control migration (U87 cell migration in the presence of vehicle). Values are mean±s.e.m. of at least three independent experiments. ^*P<0.05, ^**P<0.01 vs vehicle B; °P<0.05, °°P<0.01 vs vehicle A Tukey's t-test.