Figure 6.

Synthetic cannabinoids induce autophagy associated with inhibition of mTOR pathway. (a) Representative microphotographs of LN18 cells transfected with GFP-LC3 and treated with DMSO (vehicle), JWH133, WIN55,212-2 or its inactive enantiomer WIN55,212-3 (SWIN), JWH133 for 24 h show the increase of LC3 punctation in cannabinoid-treated cells. Increase of GFP-LC3–positive cells with GFP-LC3 dots in cells exposed to JWH133 or WIN55,212-2 was reduced following 3-methyladenine (3MA, 2 mM for 24 h). A scale bar corresponds to 10 µm (b) and by pre-treatment with antagonists of CB1 receptor (AM251) and/or CB2 receptor (AM630) in WIN55,212-2-treated cells. Statistical significance of changes was determined using one-way ANOVA and is indicated as follows: * p < 0.05, *** p < 0.001. (c). Cells were scored for the presence of GFP-LC3 puncta (more than five dots per cell) among GFP-LC3-transfected cells. A minimum of 100 cells per sample were counted (mean ± SEM, three independent experiments). One-way ANOVA followed by Fisher post-hoc test; * p < 0.05, *** p < 0.001. (d,e) LN18 glioma cells were exposed to synthetic cannabinoids JWH133 (JWH) or WIN55,212-2 (WIN) and control treatments with DMSO or WIN-55,212-3 (S-WIN) for 12–48 h. Representative immunoblots show the effects on LC3-I and LC3-II levels (d) and on the phosphorylation level of mTORC1 downstream substrate: p70 S6 kinase and its target S6 ribosomal protein (e). (f) The graphs show the relative intensity of the bands on immunoblots (LC3-II, ph-p70S6K, ph-S6Rb) as compared to the control (DMSO-treated cells) and normalized to total p70S6K levels; based on the densitometric evaluation (the mean ± SD of at least two independent experiments). Statistical significance of changes was determined using one-way ANOVA and is indicated as follows: * p < 0.05, ** p < 0.01 (vs. DMSO) and ^# p < 0.05, ^### p < 0.001 (SWIN vs. WIN).