Fig. 6: CBD directly induces mitochondrial Ca2+ overload and mPTP opening. | Cell Death & Disease

Fig. 6: CBD directly induces mitochondrial Ca2+ overload and mPTP opening.

From: Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia

Fig. 6

a CEPIA3mt fluorescence is colocalized with mitochondrial marker TMRE in Jurkat cells. Scale bar: 10 μm. bd Concurrent monitoring of [Ca2+]i and [Ca2+]m in Jurkat cells. [Ca2+]i and [Ca2+]m changes were evaluated with Fura-2 (2 μM) and CEPIA3mt, respectively. Note that cells were loaded either with Fura-2 or with CEPIA3mt; individual time courses for Fura-2 and CEPIA3mt, were synchronized with respect to the timepoint of CBD addition and averaged [Ca2+]i and [Ca2+]m responses were plotted at the upper and lower panels, respectively. CBD (30 μM), histamine (10 μM) and membrane-permeable IP3R blocker 2 APB (50 μM μM) were added as indicated. Traces are mean ± SD of at least six samples from independent experiments. e Peak values for [Ca2+]m changes, induced by CBD, from the experiments shown in bd, with a variable level of [Ca2+]i due to manipulations with Ca2+ release from the ER. Bars represent mean ± SD of at least six samples from independent experiments. One-way ANOVA test (*p < 0.05). f [Ca2+]i monitoring in Jurkat cells, loaded with Fura-2 (2 μM). ER Ca2+ was depleted by thapsigargin (1 μM). Experiments were performed in Ca2+-free medium (HBSS). Addition of CBD causes an abrupt decrease of [Ca2+]i (cf with c). Traces are mean ± SD of at least three samples from independent experiments. g, h [Ca2+]m monitoring in Jurkat cells, transfected with CEPIA3mt. CBD (30 μM) was added as indicated. When indicated, cells were preincubated over 20 min with either MCU blocker RU360 (1 μM), mPTP inhibitor CsA (10 μM), or inhibitor of mitochondrial Na+/Ca2+ exchanger NCLX CGP37157 (1 μM). Traces are mean ± SD of at least six samples from independent experiments. i [Ca2+]i monitoring in Jurkat cells, loaded with Fura-2 (2 μM). CBD (30 μM) was added as indicated. Cells were preincubated during 20 min with vehicle or CsA (10 μM), specific inhibitor of the mPTP. Values Δ [Ca2+]i were obtained by subtracting the [Ca2+]i baseline level from the peak [Ca2+]i. Traces are ±SD of at least six samples from independent experiments. j Cytosolic Ca2+ response to CBD (30 μM) in Jurkat cells was abolished by a preincubation with the MCU blocker Ru360 (1 μM) over 20 min. Values Δ [Ca2+]i were obtained by subtracting the [Ca2+]i baseline level from peak [Ca2+]i. Data are mean ± SD of a minimum of six independent experiments (**p < 0.01; ***p < 0.001; Student’s t-test). k Representative images of Jurkat cells, transfected with EYFP-Cyt-C, pretreated with RU360 (1 μM, 20 min), and subsequently treated with CBD (30 μM, 1 h). Discrete green fluorescent puncta (pseudocolor) represent Cyt-C localization in intact mitochondria whereas Cyt-C release from mitochondria is evidenced by a more diffuse EYFP-Cyt-C distribution. Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of RU360. Scale bar: 10 μm. l MCU blocker RU360 effectively prevents CBD-induced cell death in Jurkat cells. Cell death was evaluated by flow cytometry, using Annexin V-AF488/PI double staining. Cells were preincubated with vehicle or RU360 (1 μM, 2 min), and then treated with CBD (30 μM, 6 h). Data of three independent experiments are present (**p < 0.01, one-way ANOVA test). m ROS levels were evaluated by DCF fluorescence intensity. Cells were either only treated with CBD (10 or 30 μM, 1 h, light and dark green bars, respectively) or additionally pretreated with RU360 (1 μM, 20 min). In all, 50 cells from at least three independent experiments were analyzed for each condition. Data are mean of ±SD. Statistic comparisons between control and CBD-treated samples, or between RU360-pretreated and non-pretreated samples were performed. ****p < 0.0001, one-way ANOVA. n Effects of the CB2 inverse agonist, AM630 (n = 8), mPTP inhibitor CsA (n = 6) and membrane-permeable IP3R blocker 2APB (n = 6) on the viability of Jurkat cells, treated with CBD. Cell viability was evaluated by resazurin-based metabolic assay (24 h). Data are mean ± SD. Statistical comparison was made in relation to CBD-treated samples; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, one-way ANOVA

Back to article page