Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8.

Wu HY, Chu RM, Wang CC, Lee CY, Lin SH, Jan TR.

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Department of Veterinary Medicine, National Taiwan University, No.1, Sec. 4, Roosevelt Road, Taipei, Taiwan 106, ROC.

Abstract

We recently reported that cannabidiol (CBD) exhibited a generalized suppressive effect on T-cell functional activities in splenocytes directly exposed to CBD in vitro or isolated from CBD-administered mice. To investigate the potential mechanisms of CBD effects on T cells, we characterized the pro-apoptotic effect of CBD on primary lymphocytes. The apoptosis of splenocytes was markedly enhanced following CBD exposure in a time- and concentration-dependent manner, as evidenced by nuclear hypodiploidity and DNA strand breaks. Exposure of splenocytes to CBD elicited an early production of reactive oxygen species (ROS) with the peak response at 1 h post CBD treatment. In parallel with the ROS production, a gradual diminishment in the cellular glutathione (GSH) content was detected in CBD-treated splenocytes. Both CBD-mediated ROS production and GSH diminishment were remarkably attenuated by the presence of N-acetyl-L-cysteine (NAC), a thiol antioxidant. In addition, CBD treatment significantly stimulated the activation of caspase-8, which was abrogated in the presence of NAC or GSH. Pretreatment of splenocytes with a cell-permeable inhibitor for caspase-8 significantly attenuated, in a concentration-dependent manner, CBD-mediated apoptosis, but not ROS production. Collectively, the present study demonstrated that the apoptotic effect of CBD in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8.

PMID:

17950393
[PubMed – indexed for MEDLINE]

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Figures and tables from this article:

: Fig. 1.

Enhancement of apoptosis in splenocytes by cannabidiol (CBD). (A and E) Splenocytes (5 × 10⁶ cells/mL) were either left untreated (NA) or treated with CBD (1–8 μM) and/or VH (0.05% ethanol) for 12 h. (B and F) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 1–12 h. The apoptosis was measured using cell cycle analysis (A–D) or the TUNEL assay (E–G) as described in Materials and methods. Data are expressed as the mean ± SE of 3–7 samples per group. ^⁎, p < 0.05 as compared with the matched VH group. Representative histograms of cell cycle (C and D) and TUNEL (G) analyses performed after 12-h exposure to CBD (8 μM) and/or VH were shown. Results from a representative of 3 independent experiments were shown in panels A, C–E and G, and pooled data were shown in panels B (n = 7) and F (n = 4).; Figure options

: Fig. 2.

Enhancement of apoptosis in B220⁺, CD4⁺ and CD8⁺ splenic lymphocytes by CBD. Splenocytes (5 × 10⁶ cells/mL) were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 12 h. The cells were stained with anti-mouse B220-, CD4- or CD8-PE-Cy5 monoclonal antibody as described in Materials and methods, and the apoptosis was measured using the TUNEL assay. (A) Data are expressed as the mean ± SE of triplicate samples per group. The representative histograms of the TUNEL staining in (B) B220⁺, (C) CD4⁺ or (D) CD8⁺ cells were shown. ^⁎, p < 0.05 as compared with the matched VH group. Results are a representative of 3 independent experiments.; Figure options

: Fig. 3.

The effect of CBD on the production of reactive oxygen species (ROS) in splenocytes. (A) Splenocytes were preloaded with DCF-DA (20 μM) for 15 min. After washing, the splenocytes were either left untreated (NA) or treated with CBD (1–8 μM) and/or VH (0.05% ethanol) for 1 h. (B) Splenocytes preloaded with DCF-DA were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 15 min–6 h. (C) Splenocytes preloaded with DCF-DA and treated with CBD (8 μM) and/or VH for 1 h were stained with anti-mouse B220-, CD4- or CD8-PE-Cy5 monoclonal antibody. The oxidation of DCF-DA was determined by flow cytometry as described in Materials and methods. Data are expressed as the mean ± SE of triplicate samples per group. ^⁎, p < 0.05 as compared with the matched VH group. Results are a representative of 3 independent experiments.; Figure options

: Fig. 4.

The effect of CBD on the intracellular level of total thiols and glutathione (GSH) in splenocytes. (A) Splenocytes were either left untreated (NA) or treated with CBD (1–8 μM) and/or VH (0.05% ethanol) for 12 h. (B) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 1–12 h. The cells were then incubated with CMF-DA (25 μM) for 25 min, and the CMF fluorescence was measured by flow cytometry. (C) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 1–12 h. The intracellular GSH levels were measured by the method of Tietze described inMaterials and methods. Data are expressed as the mean ± SE of triplicate samples per group. ^⁎, p < 0.05 as compared with the matched VH group. Results are a representative of 3 independent experiments.; Figure options

: Fig. 5.

Abrogation of CBD-mediated oxidative stress in splenocytes by N-acetyl-l-cysteine (NAC). (A) Splenocytes (5 × 10⁶cells/mL) preloaded with DCF-DA were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 1 h in the absence (control group) or presence of NAC (0.001–1 mM), and the DCF fluorescence was measured. Changes in ROS levels were expressed as the percentage increase in fluorescence. (B) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 12 h in the absence (control group) or presence of NAC (0.001–1 mM). The cells were then incubated with CMF-DA (25 μM) for 25 min, and the CMF fluorescence was measured. (C) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 12 h in the absence (control group) or presence of NAC (0.001–1 mM), and the hypodiploid apoptosis of the cells was measured. (D and E) NAC (1 mM) were added into splenocytes 0.5 h before or 0.5–6 h after CBD treatment. After 12-h treatment with CBD, the CMF fluorescence (D) and the hypodiploid apoptosis (E) were measured. Data are expressed as the mean ± SE of 3–4 independent experiments. ^⁎, p < 0.05 as compared to the control group treated with CBD.; Figure options

: Fig. 6.

The effect of CBD on the activation of caspase-8 and -3 in splenocytes. (A) Splenocytes (5 × 10⁶ cells/mL) were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 12 h, and the activation of caspase-8 was determined by flow cytometry as described in Materials and methods. A representative histogram of caspase-8 activation was shown. (B and C) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 0.5–12 h, and the activation of caspase-8 and -3 was determined. Data are expressed as the mean ± SE of 8 and 4 independent experiments for caspase-8 and -3, respectively. ^⁎, p < 0.05 as compared to the VH group. (D) Splenocytes were treated with CBD (8 μM) and/or VH (0.05% ethanol) for 12 h in the absence (control group) or presence of NAC or GSH (1 mM of each), and the caspase-8 activation was determined. Data are expressed as the mean ± SE of triplicate cultures. ^⁎, p < 0.05 as compared to the control group treated with CBD. Results are a representative of 3 independent experiments.; Figure options

: Fig. 7.

Attenuation of CBD-induced apoptosis in splenocytes by the caspase-8 inhibitor. Splenocytes (5 × 10⁶ cells/mL) were treated with CBD (1–8 μM) and/or VH (0.05% ethanol) in the absence (control group) or presence of the caspase-8 inhibitor (5 and 10 μM) for 12 h. The hypodiploid apoptosis of the cells was determined by cell cycle analysis. Data are expressed as the mean ± SE of triplicate cultures. ^⁎, p < 0.05 as compared to the matched control group. Results are a representative of 3 independent experiments.; Figure options

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Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8.

Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8.

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