Cannabidiol-Induced Apoptosis in Human Leukemia Cells: A Novel Role of Cannabidiol in the Regulation of p22phox and Nox4 Expression
- Robert J. McKallip,
- Wentao Jia,
- Jerome Schlomer,
- James W. Warren,
- Prakash S. Nagarkatti and
- Mitzi Nagarkatti
+ Author Affiliations
- Address correspondence to:
Dr. Robert J. McKallip. Department of Pathology, Microbiology and Immunology. University of South Carolina School of Medicine, 6439 Garner's Ferry Road, Columbia, SC 29209. E-mail: rmckallip@gw.med.sc.edu
Abstract
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).
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