Targeting cannabinoid receptors to treat leukemia: Role of cross-talk between extrinsic and intrinsic pathways in Δ9-tetrahydrocannabinol (THC)-induced apoptosis of Jurkat cells
Introduction
Although marijuana is popularly known as one of the oldest and most widely used drugs of abuse worldwide, its medicinal properties have been exploited for centuries [1]. Marijuana's major psychoactive component, Δ9-tetrahydrocannabinol (THC), is now believed to act through two main receptors: cannabinoid receptor (CB) 1, expressed mainly on cells of the central nervous system, and CB2, expressed primarily on cells of the immune system [1], [2], [3]. THC and other synthetic cannabinoids have been proposed as potential therapeutic agents in the treatment of pain, cachexia and nausea in AIDS and cancer therapy patients, as well as intraocular pressure due to glaucoma [1], [4]. Recently, the use of THC has also been suggested in the treatment of gliomas and prostate cancer [5], [6]. In addition, studies conducted in our laboratory have shown that various cannabinoids, both plant-derived and synthetic, can induce apoptosis in transformed immune cells, suggesting the potential use of cannabinoids in the treatment of cancers of immune origin [7]. We have shown that apoptosis in normal and transformed immune cells is mediated through CB2 [7], [8], although the precise signaling mechanism still remains to be established.
Apoptosis is primarily mediated through two pathways: the death receptor pathway and the mitochondrial pathway [9], [10], [11]. In the death receptor pathway, a death receptor ligand, such as Fas ligand, binds to its receptor, such as Fas, triggering aggregation of the death receptor, recruitment of an adaptor molecule, such as FADD, as well as pro-caspase-8 or -10 forming a complex named the death inducing signaling complex (DISC) [12]. This results in the autocatalytic cleavage and activation of caspase-8 or caspase-10, leading to activation of caspase-3 or -7 and induction of apoptosis [9]. In the mitochondrial pathway, cytochrome c is released from the mitochondria to form a complex called the apoptosome with apoptosis protease activator factor-1 (Apaf-1), dATP, and pro-caspase-9 [13], [14], [15], [16]. The apoptosome then activates caspase-9, which in turn activates caspase-3, thereby inducing apoptosis. In cells referred to as type I cells, the death receptor pathway and the mitochondrial pathway are distinct [17]. In type II cells, such as the widely studied transformed human Jurkat T cell line, the amount of caspase-8 or -10 activated by death receptor ligation is insufficient to activate caspase-3 [17]. Instead, caspase-8 or -10 cleaves Bid, a pro-apoptotic Bcl-2 family member, which then translocates to the mitochondria, triggering release of cytochrome c and recruitment of the mitochondrial pathway [18], [19].
In the current study, we used Jurkat cells to further investigate the pathways of apoptosis induced by THC. While the mitochondrial pathway was sufficient to induce apoptosis, it was facilitated by activation of the death receptor pathway through cross-talk and, interestingly, THC-induced apoptosis was regulated at least in part by caspase-2 and caspase-10.
Section snippets
Reagents
THC was obtained from the National Institute of Drug Abuse (Rockville, MD) and was initially dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) to a concentration of 20 mM and stored at −20 °C. For experiments, THC was further diluted in RPMI 1640 (Invitrogen, Carlsbad, CA) medium free of fetal calf serum.
Cell lines
Wild-type (WT) Jurkat is an acute T-lymphoblastic leukemia cell line generated from a 14-year-old male. In addition, we used Jurkat cell lines that were deficient in signaling pathways
Effect of THC on wild-type Jurkat cells and those deficient in FADD, caspase-8 and caspase-9
In order to characterize the respective roles of the death receptor and the mitochondrial pathways in THC-induced apoptosis, we examined the effect of THC on wild-type, FADD-deficient, caspase-8 deficient, and caspase-9 dominant negative Jurkat cells. As shown in Fig. 1a, after 3 h of incubation with THC at a concentration of 10 μM, there was 61.3% apoptosis in wild-type Jurkat cells, which was similar to the amount of apoptosis found in wild-type Jurkat cells incubated with membrane bound FasL,
Discussion
Recently, THC has been shown to induce apoptosis in a variety of cells [5], [6], [7], [8], although the precise mechanisms remain unclear. In the current study, we used Jurkat cells, which were shown to undergo apoptosis following exposure to THC [7], to further investigate the pathways of apoptosis. Treatment of wild-type Jurkat cells with THC triggered the extrinsic (death receptor) pathway of apoptosis, which was evidenced by the fact that THC treatment led to activation of caspase-8 and
Acknowledgment
This work was supported in part by National Institutes of Health grants.
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