268 台灣獸醫誌 Taiwan Vet J 36 (4): 268-278, 2010

Hsin-Ying WU et al

Cannabidiol Activated the Intrinsic Apoptotic Pathway in Murine Lymphocytes 1,2

Hsin-Ying WU, 3 Mei-Hsiu LIAO, 1,2 Shu-Ting SU, *1,2 Tong-Rong JAN

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（Received: May 18, 2010. Accepted: July 3, 2010）

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[Wu HY, Liao MH, Su ST, * Jan TR. Cannabidiol Activated the Intrinsic Apoptotic Pathway in Murine Lymphocytes. Taiwan Vet J 36 (4): 268-278, 2010. * Corresponding author TEL: 886-2-3366-1287, FAX: 886-2-3366-3257, E-mail: [email protected]]

INTRODUCTION Apoptosis is a tightly regulated program of cell death critically involved in various physiological and pathological processes, such as the homeostatic balance of the immune system. Two major cellular signaling pathways operate in apoptotic cells, including the intrinsic and the extrinsic pathways. The extrinsic apoptotic pathway is initiated by ligand binding to the

death receptors, which induce the recruitment of the adaptor molecule Fas-associated death domain (FADD) and pro-caspase-8 to form the death-inducing signaling complex resulting in the autocleavage and activation of caspase-8. Caspase-8 subsequently triggers the activation of downstream effector caspases, such as caspase-3 to execute apoptosis program. In addition to the death receptor-initiated signaling, the intrinsic pathway is another crucial mechanism for the signaling

Cannabidiol Activated the Intrinsic Apoptotic Pathway in Murine Lymphocytes

transduction of cell apoptosis. Mitochondria play a pivotal role in the initiation of the intrinsic pathway, in which cytotoxic insults disturb the integrity of mitochondria leading to the loss of the mitochondrial membrane potential. The depolarized mitochondria release cytochrome c to the cytosol that subsequently activates caspase-9, an initiator caspase. Caspase-9 then activates the downstream effector caspases for the execution of cell death [16]. Cannabidiol (CBD) is the major non-psychoactive plant-derived cannabinoid that exhibits a number of promising pharmacological activities, such as antiinflammatory and immunomodulatory actions [13,15, 17,22]. The immunosuppressive effects by CBD have been demonstrated in a variety of experimental models [1,8,11,13,15,17]. For example, we recently demonstrated that in vivo administration of CBD attenuated the serum production of antigen-specific antibodies in ovalbumin-sensitized BALB/c mice, which was closely associated with the suppression of T-cell proliferation and cytokine production ex vivo [17]. Follow up mechanistic studies showed that direct exposure of primary lymphocytes to CBD enhanced apoptosis via an oxidative stress-dependent activation of caspase-8 [25]. These results demonstrate the activation of the extrinsic apoptosis pathway, and suggest that the immunosuppressive effects of CBD may be mediated by the induction of lymphocyte apoptosis. The pro-apoptotic property of plant-derived cannabinoids, in particular 9-tetrahydrocannabinol ( 9THC), has been well documented in a variety of nontransformed and transformed cells, including those of immune origin [4,14,21]. The underlying mechanisms of 9-THC-induced apoptosis involved multiple signaling events; both the intrinsic and extrinsic pathways have been reported to be affected, including the activation of caspase-9, -8, -2 and -10, and the disruption of the mitochondrial function [2,4,7,14]. Notably, CBD has also been demonstrated to induce apoptosis in human C6 glioma and Jurkat T cells via the activation of caspase-8, -9 and -3, and the loss of mitochondrial membrane potential [9,20]. These results suggest that in transformed cells comparable apoptotic signaling events were triggered by CBD and 9-THC, including both the extrinsic and intrinsic pathways. However, it is presently unclear whether CBD activa-

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tes the intrinsic apoptotic pathway in primary lymphocytes. Given the evidence that both the extrinsic and intrinsic apoptotic pathways are triggered by CBD and 9 -THC in transformed leukocytes, and our previous studies showing the activation of the extrinsic pathway in CBD-exposed primary splenocytes, we hypothesized that CBD may also activate the intrinsic pathway in primary lymphocytes. We reported here that CBD-enhanced apoptosis in normal lymphocytes was associated with the activation of the mitochondrionmediated signaling pathway, including the disruption of the mitochondrial membrane potential and the activation of caspase-9.

MATERIALS AND METHODS Reagents All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise stated. Cannabidiol was purchased from Tocris Bioscience (Bristol, UK). CBD was dissolved in absolute ethanol and stored at -20℃. CBD was further diluted to the desired concentrations with Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO BRL, Gaithersburg, MD). Annexin V apoptosis kit was purchased from Strong Biotech Corp. (Taipei, Taiwan). Reagents for RNase protection assays were purchased from BD Biosciences (San Jose, CA).

Animals and cell cultures Male BALB/c mice, 4-5 weeks of age were purchased from the Animal Breeding Center, National Taiwan University Hospital (Taipei, Taiwan). The use of animals was approved by the Institutional Animal Care and Use Committee of the National Taiwan University. On arrival, mice were randomized, transferred to plastic cages containing a sawdust bedding (5 mice per cage) and quarantined for 1-2 weeks. The animal room was maintained at a temperature of 24 ± 2℃ and a relative humidity of 60 ± 20%, with a 12-hr light/ dark cycle. Mice were given standard laboratory food and water ad libitum. Their spleens were isolated aseptically and made into single cell suspensions as described previously [18]. The erythrocytes in spleno-

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cyte cultures were lysed using a hypotonic buffer (150 mM NH4Cl, 10 mM KHCO3 and 100 M Na2EDTA; pH 7.2). The splenocytes were cultured in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, and 5% heat inactivated FBS (referred to as complete RPMI). In all cases, cells were cultured at 37℃ in 5% CO2.

Detection of apoptosis using annexin V and propidium iodide (PI) double staining Splenocytes and thymocytes (5 × 106 cells/mL) were cultured in triplicate in a 24-well plate (0.5 mL/ well) and treated with CBD (8 M) and/or vehicle (VH; 0.05% ethanol) for 1-6 h. At the end of incubation, splenocytes were washed twice with PBS and resuspended in binding buffer. Annexin V-fluorescein isothiocyanate (annexin V-FITC) and PI solutions were added to splenocytes following the manufacturer’s instructions. After incubation at room temperature for 15 min in the dark, the single cell fluorescence of 10,000 cells for each sample was measured using a flow cytometer (Becton Dickinson FACS Calibur, San Jose, CA) at emission of 525 and 670 nm. The data were analyzed using the software Flowjo 5.7.

Detection of mitochondrial membrane potential ( m) using 5,5’,6,6’-tetrachloro-1,1’3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1) staining Splenocytes (5×106 cells/mL) were cultured in triplicate in a 24-well plate (0.5 mL/well) and either left untreated (naive, NA) or treated with CBD (1-8 M) and/ or VH (0.05% ethanol) for 1-12 h at 37℃ in a 5% CO 2 incubator. After washing with PBS, cells were stained with JC-1 (2.5 g/mL) by incubating at room temperature for 20 min in the dark. The single cell fluorescence of 10,000 cells for each sample was measured using a flow cytometer (Becton Dickinson FACS Calibur) at emission of 525 and 575 nm. The data were analyzed using the software Flowjo 5.7.

RNase protection assay (RPA) The steady state mRNA expression of Bcl-2 family members, including Bcl-w, Bfl-1, Bcl-xL, Bak, Bax, Bcl-2 and Bad, was simultaneously measured

using a RPA kit (BD Biosciences) according to the supplier’s instructions. Splenocytes (5×106 cells/mL) were either left untreated (NA) or treated with CBD (1-8 M) and/or VH (0.05% ethanol) for 6 h in the absence or presence of phorbol myristate acetate plus inomycin (PMA/Io; 80 nM/1 M). Following the incubation, splenocytes were lyzed in TRI Reagent® (1 mL/sample; Sigma Chemical), and the total RNA was isolated following the supplier’s instructions. RNA (20 g) was resuspended in hybridization buffer and hybridized with Bcl-2 family specific probes, synthesized using a template set from the RPA kit and labeled using biotin-16-UTP and T7 polymerase, at 56℃ overnight. Single strand RNA and free probes were digested by RNase, and protected probes (double strand RNA) were purified and electrophoresed in a 6% polyacrylamide-tris-borate-EDTA-urea gel. The gel was then semi-dry electrotransferred to the nylon membrane. The protected probes were detected by incubating with horse radish peroxidase (HRP)-conjugated-streptavidin and exposure to enhanced chemiluminescence detection reagents. Quantification was performed by assessing the optical density for the hybridization bands using the alpha imager 1200 imaging system (Alpha Innotech, San Leandro, CA). Unhybridized probes were used as size standards for each mRNA analyzed. Expression of Bcl-w, Bfl-1, Bcl-xL, Bak, Bax, Bcl-2 and Bad was semi-quantitated by normalizing to the expression level of the housekeeping gene L32.

Detection of caspase-9 activation The activation of caspase-9 was measured using a cell-permeable specific substrate of the enzyme (FAM-LEHD-FMK; CaspaTag Assay Kit, Chemicon International Inc., Temecula, CA). The reagent is carboxyfluorescein-labeled fluromethyl ketone-peptide-inhibiting substrate that covalently binds only to the target caspase. When added to cells, the substrate binds to the active caspase, and the green fluorescent signal is a direct measure of the amount of active caspase present in the cell. In brief, splenocytes were treated with CBD (8 M) and/or VH (0.05% ethanol) for 0.5-12 h. The cells were washed with PBS, and incubated with FAM-LEHD-FMK for 1 h at 37℃ in the dark, and then washed again. The single cell fluor-

Cannabidiol Activated the Intrinsic Apoptotic Pathway in Murine Lymphocytes

escence of 10,000 cells for each sample was measured using a flow cytometer at emission of 525 nm. The data were analyzed using the software Flowjo 5.7.

Statistical analysis The mean ± standard error (SE) was determined for each treatment group in the individual experiments. Dunnett’s two-tailed t-test was used to compare treatment groups to the control group. P value < 0.05 was defined as statistical significance.

RESULTS Cannabidiol enhanced apoptosis in murine splenocytes We first confirmed the apoptotic effect of CBD on murine splenocyes [25]. Freshly isolated splenocytes were exposed to CBD (8 M) and/or VH (0.05% ethanol), and the apoptosis of splenocytes was determined using annexin V and propidium iodide (PI) double staining. Annexin V binds to phophatidylserine present on the external surface of apoptotic cells, an indicator widely used to measure apoptosis. Positioning of quadrants on the dot plots of annexin V/PI staining was performed, and the proportion of live cells (annexin V PI ), early apoptotic cells (annexin V+PI ), late apoptotic cells (annexin V+PI+) and necrotic cells (annexin V PI+) were determined. As shown in Fig. 1A, splenocytes treated with CBD (8 M) markedly enhanced the proportion of late apoptotic cells in a time-dependent manner (3-6 h), as evidenced by the increased proportion of annexin V+PI+ cells. Representative dot plots of the cells exposed to CBD and/ or VH for 6 h are shown in Fig. 1B and C.

CBD induced depolarization of the mitochondrial membrane potential ( m) The effect of CBD on m was measured by flow cytometry using JC-1 staining. JC-1 is a lipophilic cation that forms aggregates in the inner mitochondrial membrane of normal viable cells, which emit orange fluorescence. Upon depolarization of m, JC-1 aggregates reversibly convert to monomers that emit green fluorescence [24]. The cells emitting

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green fluorescence were defined as depolarized cells. As shown in Fig. 2A, exposure of splenocytes to CBD (8 M) significantly induced the depolarization of mitochondria in a time-dependent manner (6-12 h). The effect of CBD was further demonstrated to be concentration (4-8 M)-dependent (Fig. 2B). Representative dot plots of the cells exposed to CBD and/or VH for 12 h are shown in Fig. 2C and D.

CBD increased the mRNA expression of Bak and Bax Bcl-2 family members play an important role in mitochondrion-mediated apoptotic pathway. Pro-apoptotic Bcl-2 family proteins, such as Bak and Bax, can translocate to mitochondrial outer membrane and form pores that alter mitochondrial membrane permeability (MMP), leading to the release of cytochrome c and several apoptogenic factors. In contrast, anti-apoptotic Bcl-2 family proteins, such as Bcl-2 and BclxL, can maintain the integrity of mitochondrial membrane by inhibiting the activation and translocation of pro-apoptotic proteins [23]. In light of the JC-1 staining showing the depolarization of the mitochondrial membrane potential, we examined the mRNA expression of the Bcl-2 family members using RPA. Splenocytes were either left untreated (naive, NA) or treated with CBD (1-8 M) and/or VH (0.05% ethanol) for 6 h in the absence or the presence of phorbol myristate acetate/inomycin (PMA/Io; 80 nM/1 M). As shown in Fig. 3, treatment of unstimulated splenocytes with CBD (4-8 M) increased the mRNA levels of Bak and Bax. Exposure of PMA/Io-stimulated splenocytes to CBD also enhanced the expression of bax mRNA. The mRNA expression of other investigated genes was not altered by CBD treatment.

CBD induced the activation of caspase-9 Caspase-9 is a key caspase mediating the apoptotic signaling in the intrinsic pathway. To further examine the effect of CBD on the intrinsic apoptotic pathway, the activation of caspase-9 was measured. The results showed that CBD (8 M) treatment significantly activated caspase-9 in a time-dependent manner (6-12 h; Fig. 4A). Moreover, the presence of N-acetylL-cysteine (NAC; 1 mM) almost completely attenu-

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CBD-mediated enhancement of apoptosis in splenocytes. (A) Splenocytes (5×106 cells/mL) were treated with CBD (8 M) and/or VH (0.05% ethanol) for 1-6 h. The apoptosis of splenocytes was measured using annexin V-FITC and propidium iodide (PI) double staining. Data are expressed as the mean ± SE of triplicate samples per group. *, < 0.05 compared to the matched VH group. Results are a representative of three independent experiments. (B and C) Representative dot plots of the cells exposed to CBD and/or VH for 6 h are shown. The results showed that CBD markedly increased the proportion of late apoptotic (annexin V+PI+) cells.

ated the activation of caspase-9 induced by CBD (Fig. 4B).

DISCUSSION To date, the majority of literature reports investigating the apoptotic effect of CBD and its underlying mechanisms primarily focused on transformed or immortalized cells, in which CBD induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways [2,4,6,7,9,14,20,25]. Our previously studies in-

vestigating the effect of CBD on nontransformed lymphocytes have demonstrated that the apoptosis induced by CBD is closely associated with an oxidative stress-dependent activation of caspase-8, a key caspase involved in the extrinsic pathway [25]. In the present study, we further showed that the intrinsic pathway is activated in splenocytes exposed to CBD, as evidenced by the observed depolarization of the mitochondrial membrane potential and the activation of caspase-9. These results suggest that CBD triggers similar apoptotic signaling pathways in both trans-

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6 CBD-induced depolarization of the mitochondrial membrane potential ( m). (A) Splenocytes (5×10 cells/mL) were treated with CBD (8 M) and/or VH (0.05% ethanol) for 1-12 h. (B) Splenocytes (5×106 cells/mL) were either left untreated (naive, NA) or treated with CBD (1-8 M) and/or VH (0.05% ethanol) for 12 h. m was determined using JC-1 staining. Data are expressed as the mean ± SE of triplicate samples per group. *, < 0.05 compared to the matched VH group. (A and B) Results are a representative of three independent experiments. (C and D) Representative dot plots of the cells exposed to CBD and/or VH for 12 h are shown.

formed and primary lymphocytes, in which both the intrinsic and the extrinsic pathways are involved. The objective of the present study was to investigate whether the intrinsic death pathway is activated in CBD-induced apoptosis in primary lymphocytes. A

hallmark of the activation of the intrinsic pathway is the disruption of the mitochondrial membrane integrity [23]. We therefore examined the mitochondrial membrane potential ( m) in CBD-induced apoptosis in splenocytes. Our flow cytometric analysis employ-

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The effects of CBD on the mRNA expression of family genes. Splenocytes (5×106 cells/ mL) were either left untreated (naive, NA) or treated with CBD (1-8 M) and/or VH (0.05% ethanol) for 6 h in the absence or the presence of PMA/Io (80 nM/1 M). The family mRNA expression of genes was determined using RNase protection assay (RPA) as described in Material and methods. (A) Results are a representative of two independent experiments. (B) Quantitative results are shown: the levels of bak and bax mRNA were normalized to the house keeping gene L32. The results showed that CBD enhanced the mRNA expression of bax and bak.

Cannabidiol Activated the Intrinsic Apoptotic Pathway in Murine Lymphocytes

CBD-induced activation of the caspase-9. (A) Splenocytes (5×106 cells/mL) were treated with CBD (8 M) and/or VH (0.05% ethanol) for 1-12 h. (B) Splenocytes were treated with CBD (8 M) and/or VH (0.05% ethanol) for 12 h in the absence (control group) or presence of NAC (1 mM). The activation of caspase-9 was determined by flow cytometry. Data are expressed as the mean ± SE of triplicate samples per group. *, < 0.05 compared to the VH group (A) or the matched control (B). Results are a representative of three independent experiments.

ing JC-1 staining clearly showed that CBD treatment induced a marked depolarization of the m in a timeand concentration-dependent manner (Fig 2). Similar results have been reported in CBD-treated leukemia cells, in which CBD interfered with the mitochondrial membrane integrity and induced the release of cytochrome c [20]. Based on these results, it is apparent that mitochondria may be a common intracellular target involved in CBD-mediated apoptosis in both normal and immortalized lymphocytes. It has been well established that Bcl-2 family

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members, including the anti-apoptotic and pro-apoptotic proteins play a pivotal role in regulating the mitochondrial membrane integrity. Translocation of the pro-apoptotic proteins, including Bak, Bax and Bad, from cytosol to mitochondria forms pores on the membrane resulting in the release of cytochrome c and other apoptogenic factors. In contrast, anti-apoptotic proteins, such as Bcl-2, Bcl-XL and Bcl-w, inhibit the activation and translocation of Bax and Bak, thereby maintaining the mitochondrial membrane integrity. Interestingly, the results from our RPA demonstrated that CBD treatment upregulated the steady state mRNA expression of pro-apoptotic Bax and Bak (Fig 3). In 9-THC-treated human Jurkat T cells, the inhibition of Bcl-2 and translocation of Bad were detected [14]. In the present studies, we did not observe marked influence by CBD on the mRNA expression of Bcl-2 and Bad. Further investigation on the protein level of the Bcl-2 family members is needed to more comprehensively elucidate the influence of CBD on Bcl-2 expression in primary lymphocytes. Oxidative stress is known to trigger apoptosis [3]. Many cytotoxic agents induce apoptosis through the induction of reactive oxygen species (ROS) in various models of apoptotic cell death, including T cells [12]. Consistent with the role of ROS in apoptosis, antioxidants such as NAC and glutathione have been shown to prevent lymphocyte apoptosis by scavenging ROS [5,10]. Of relevant to the current study, CBD-mediated apoptosis has been reported to be associated with the induction of ROS in leukemia and glioma, as well as in splenocytes and thymocytes [9,19,20,26]. Consistent with these reports, we demonstrated that CBD-induced activation of caspase-9 was abrogated in the presence of NAC, confirming a crucial role of oxidative stress in the activation of the intrinsic pathway induced by CBD. In summary, the present study demonstrated that CBD exposure resulted in a remarkable induction of apoptosis in splenocytes, which was associated with the depolarization of the mitochondrial membrane potential, the up-regulation of bak and bax mRNA expression, and the activation of caspase-9. These data suggest that the intrinsic apoptotic pathway is activated in CBD-mediated apoptosis in splenocytes. Based on the results from the present and our previous stu-

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dies, we conclude that both intrinsic and extrinsic apoptotic pathways are involved in CBD-mediated apoptosis in primary lymphocytes.

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Acknowledgments

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This work was supported by grants 99AS-9.2.5BQ-B1(2) and 99AS-9.2.3-BQ-B2(1) from Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan, ROC. HY Wu is recipient of graduate fellowship awarded by the Institute of Nuclear Energy Research, Atomic Energy Council, Executive Yuan, Taiwan, ROC.

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大麻二酚活化小鼠淋巴球之內在凋亡路徑 1,2

吳欣穎

3

廖美秀

1,2

蘇淑婷

*1,2 詹東榮

國立臺灣大學生物資源暨農學院 動物癌症中心 10617 台北市羅斯福路四段 1 號 國立臺灣大學獸醫專業學院 獸醫學系暨研究所 10617 台北市羅斯福路四段 1 號 3 行政院原子能委員會核能研究所 同位素應用組 32546 桃園縣龍潭鄉佳安村文化路 1000 號 1 2

（收稿日期：99 年 05 月 18 日。接受日期：99 年 07 月 03 日）

摘要

大麻二酚 (cannabidiol; CBD) 是大麻成分中主要不具中樞活性的大麻生物鹼，已被報告經由活化外在及內在

凋亡路徑誘發神經膠質瘤及血癌細胞之凋亡。我們先前指出 CBD 在小鼠初代淋巴球細胞所誘發的凋亡和氧化性壓力 引起的半胱氨酸蛋白-8 (caspase-8) 之活化有關，顯示 CBD 會活化外在凋亡路徑。本研究進一步探討 CBD 誘發小 鼠脾臟細胞之凋亡是否涉及粒腺體媒介之凋亡路徑。利用流式細胞儀分析粒腺體之膜電位，結果顯示脾臟細胞給予 CBD 會造成粒腺體膜電位之去極化，此作用具有時間和濃度相關性。給予 CBD 處理之脾臟細胞，其 Bax 和 Bak mRNA 的表現量增加。此外，隨著 CBD 處理時間的增加，脾臟細胞之半胱氨酸蛋白-9 明顯地被活化；給予抗氧化 劑 -乙醯-L-半胱胺酸 (N-acetyl-L-cysteine) 可有效地降低 CBD 所誘發之半胱氨酸蛋白-9 的活化。綜合上述，本研 究顯示 CBD 在初代淋巴球細胞會經由引發氧化性壓力之機制活化內在凋亡路徑。[吳欣穎、廖美秀、蘇淑婷、* 詹東 榮。大麻二酚活化小鼠淋巴球之內在凋亡路徑。台灣獸醫誌 36 (4): 268-278，2010。* 通訊作者 TEL: 886-2-3366-1287， FAX: 886-2-3366-3257，E-mail: [email protected]]