Cannabidiol-Induced Apoptosis in Human Leukemia Cells: A Novel Role of Cannabidiol in the Regulation of p22phox and Nox4 ExpressionRobert J. McKallip, Wentao Jia, Jerome Schlomer, James W. Warren, Prakash S. Nagarkatti and Mitzi Nagarkatti
Molecular Pharmacology September 2006
In the current study, we examined the effects of the nonpsychoactive cannabinoid, cannabidiol, on the induction of apoptosis in leukemia cells. Exposure of leukemia cells to cannabidiol led to cannabinoid receptor 2 (CB2)-mediated reduction in cell viability and induction in apoptosis. Furthermore, cannabidiol treatment led to a significant decrease in tumor burden and an increase in apoptotic tumors in vivo. From a mechanistic standpoint, cannabidiol exposure resulted in activation of caspase-8, caspase-9, and caspase-3, cleavage of poly(ADP-ribose) polymerase, and a decrease in full-length Bid, suggesting possible cross-talk between the intrinsic and extrinsic apoptotic pathways. The role of the mitochondria was further suggested as exposure to cannabidiol led to loss of mitochondrial membrane potential and release of cytochrome c. It is noteworthy that cannabidiol exposure led to an increase in reactive oxygen species (ROS) production as well as an increase in the expression of the NAD(P)H oxidases Nox4 and p22phox. Furthermore, cannabidiol-induced apoptosis and reactive oxygen species (ROS) levels could be blocked by treatment with the ROS scavengers or the NAD(P)H oxidase inhibitors. Finally, cannabidiol exposure led to a decrease in the levels of p-p38 mitogen-activated protein kinase, which could be blocked by treatment with a CB2-selective antagonist or ROS scavenger. Together, the results from this study reveal that cannabidiol, acting through CB2 and regulation of Nox4 and p22phox expression, may be a novel and highly selective treatment for leukemia.
Marijuana has been suggested as a potent therapeutic agent alleviating such complications as intraocular pressure in glaucoma and cachexia, nausea, and pain in AIDS and cancer patients. A number of recent studies now suggest the possible use of these compounds for the treatment of cannabinoid receptor-expressing tumors. For example, anandamide was shown to inhibit the proliferation of the human breast cancer cell lines MCF-7 and EFM-19 in vitro (De Petrocellis et al., 1998). In addition, THC was shown to induce apoptosis in human prostate PC-3 cells and in C6 glioma cells in culture (Sanchez et al., 1998; Ruiz et al., 1999; Galve-Roperh et al., 2000). THC-induced apoptosis involved cannabinoid receptor-dependent (Sanchez et al., 1998; Galve-Roperh et al., 2000) or -independent pathways (Ruiz et al., 1999). Such studies have triggered interest in targeting cannabinoid receptors in vivo to induce apoptosis in transformed cells. To this end, cannabinoids were shown to inhibit the growth of C6 glioma cells in vivo (Sanchez et al., 2001). Furthermore, recent studies from our laboratory demonstrated that targeting cannabinoid receptors may be a novel approach to treating lymphoblastic disease (McKallip et al., 2002).
A significant limitation to the use of a number of these compounds is their unwanted psychotropic activity. Cannabidiol (CBD) is a nonpsychoactive derivative of marijuana that is currently being examined for its use in the treatment of cancer. For example, Massi et al. (2004) demonstrated that cannabidiol was capable of suppressing the proliferation of human glioma cell lines. In addition, the HL-60 myeloblastic cell line was shown to be sensitive to CBD-induced apoptosis, whereas monocytes from healthy subjects were relatively resistant to CBD-induced apoptosis, suggesting that CBD may be effective at treating acute myelogenous leukemia (Gallily et al., 2003).
Although a number of reports demonstrate the ability of CBD to induce apoptosis in tumor cells, little work has been done demonstrating CBD mechanism of action. Massi et al. (2004) found that apoptosis in human glioma cell lines after exposure to CBD was mediated through CB2 receptor and the generation of reactive oxygen species (ROS). The generation of ROS can play an important role in the induction of apoptosis in T cells undergoing either activation-induced cell death or activated T cell autonomous cell death (Hildeman et al., 2003). Furthermore, the regulation of ROS generation can be significantly affected by NAD(P)H oxidases (Suzuki et al., 1998; Lee et al., 2000). Numerous studies have been carried out examining the ability of compounds to induce apoptosis in tumor cells by increasing ROS production (Kang et al., 2004; Kim et al., 2004; Chang et al., 2005; Hu and Brindle, 2005; Lebedeva et al., 2005). However, little is known about the ability of cannabinoids or signaling through cannabinoid receptors to regulate the expression or activity of NAD(P)H oxidases and/or to control of ROS generation in leukemia. However in a recent study, the NAD(P)H oxidase Nox5, which plays a significant role in mediating Ca2+-dependent ROS generation, was shown to be expressed in lymph nodes and the spleen, suggesting a possible role of NAD(P)H oxidases in the regulation of ROS production in cells of the immune system (Banfi et al., 2001).
The observation that CBD can mediate apoptosis through cannabinoid receptor 2 (CB2) combined with results from our previous study, where we demonstrated that, compared with a human glioma cell line, a number of human leukemias and lymphomas expressed significantly higher levels of CB2, suggests the possibility that human leukemias and lymphomas may be highly sensitive to the CB2-mediated effects of cannabidiol (McKallip et al., 2002; Massi et al., 2004). Therefore, in the current study, we examined the potential use of cannabidiol in the treatment of lymphoblastic disease. Using the murine EL-4 leukemia and the human Jurkat and Molt-4 leukemia cell lines, we demonstrated that exposure to cannabidiol led to tumor killing by induction of apoptosis. Examination of the mechanism of cannabidiol-induced apoptosis revealed that cannabidiol was acting through CB2. Furthermore, we demonstrated the involvement of the NAD(P)H oxidases p22phox and Nox4 and the subsequent generation of reactive oxygen species in CBD-induced apoptosis. Together, these studies suggest that cannabidiol, acting through CB2, may be a novel regulator of NAD(P)H oxidase expression and that CBD may prove to be a potent nonpsychoactive and specific treatment of leukemia.
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