331-18A, CBDV and CBD mediate their effect through CB2 and TRPV1.
(A) The mRNA levels of receptors CNR1 (CB1), CNR2 (CB2), GPR55, TRPV1, TRPA1 and TRPM8 were evaluated by qRT-PCR. Gene expression levels were calculated as ΔCT normalized to GUSB housekeeping gene. Results are presented as mean expression of three biological replicates. High ΔCT values indicate low receptor expression. (B) MOLT-4 cells were pretreated with 50 µM antagonist to CB2 (AM630) or TRPV1 (AMG9810) for 30 min, then treated with the combination of the three cannabinoids for 3 h. NICD expression was evaluated by western blot (N=3) with GAPDH as the loading control and statistically analyzed with an unpaired student’s t-test (**p<0.01, ***p<0.001). (C) Representative blots of B. (D) Calcium release by MOLT-4 cells was measured with the Fluo-4 calcium probe immediately following treatment with either vehicle, whole extract, a combination of 331-18A, CBDV and CBD, and each cannabinoid separately. The calcium curves represent an average of three independent experiments. (E) Reduction of each phytocannabinoid-induced calcium release by pretreatment with 50 µM of antagonist to CB2 (AM630) or TRPV1 (AMG9810) for 30 min. The calcium curve for each compound is given again in this graph to compare the effects with the antagonists (N=3, two-way ANOVA, *p<0.05, **p<0.01). (F) MOLT-4 cells were pretreated with 50 µM antagonist to CB2 (SR-144,528) or TRPV1 (AMG9810) or both for 30 min, then treated with the combination of the three cannabinoids or left untreated. Apoptosis was analyzed after 24 h (N=3) by Annexin V/PI. Results are presented as mean ± SEM and statistically analyzed with one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).