Cannabinoids in
Glioblastoma Therapy
2018 May 16
Glioblastoma
(GBM) is the most malignant brain tumor and one of the deadliest types of solid
cancer overall. Despite aggressive therapeutic approaches consisting of maximum
safe surgical resection and radio-chemotherapy, more than 95% of GBM patients
die within 5 years after diagnosis. Thus, there is still an urgent need to
develop novel therapeutic strategies against this disease. Accumulating
evidence indicates that cannabinoids have potent anti-tumor functions and might
be used successfully in the treatment of GBM. This review article summarizes
the latest findings on the molecular effects of cannabinoids on GBM, both in
vitro and in (pre-) clinical studies in animal models and patients.
The therapeutic effect of cannabinoids is based on reduction of tumor growth
via inhibition of tumor proliferation and angiogenesis but also via induction
of tumor cell death. Additionally, cannabinoids were shown to inhibit the
invasiveness and the stem cell-like properties of GBM tumors. Recent phase II
clinical trials indicated positive results regarding the survival of GBM
patients upon cannabinoid treatment. Taken together these findings underline
the importance of elucidating the full pharmacological effectiveness and the
molecular mechanisms of the cannabinoid system in GBM pathophysiology.
Keywords: cannabinoids,
glioblastoma, molecular mechanisms, novel therapeutic strategies, cannabinoid
receptors, cannabidiol, THC
Gliomas are the most common primary tumors of the central
nervous system. Half of the newly-diagnosed gliomas are glioblastomas (GBMs),
with an incidence in adults of 0.59–3.69 cases per 100,000 person life-years
(Ostrom et al., 2014). The vast majority of GBM develop de
novo (primary GBM); however, GBM can also evolve from lower grade
gliomas (secondary GBM). Primary GBM occur more commonly in male patients
whereas the reverse is the case for secondary GBM (Adamson et al., 2009). The mean age of primary and secondary
GBM patients is 62 and 45 years, respectively (Adamson et al., 2009).
GBM is an extremely
aggressive type of cancer. These tumors are characterized by high cellular
proliferation and angiogenesis resulting in rapid tumor growth and,
consequently, necrosis. GBM cells also exhibit high migration and invasive
properties, which allow them to produce metachronous lesions and even to spread
through the brain parenchyma. Furthermore, GBM tumors contain a subpopulation
of glioma stem-like cells (GSCs), which, at least partially, account for the
high resistance to therapy and recurrence rates of these tumors (Louis et al., 2016).
Currently, the
standard of care treatment for GBM consists of maximum safe surgical resection
followed by radiotherapy plus concomitant and adjuvant chemotherapy with temozolomide
(TMZ; Stupp et al., 2005). Despite this aggressive therapeutic
regimen, GBM patients have a poor prognosis, with only 0.05%–4.7% of patients
surviving 5 years past initial diagnosis (Ostrom et al., 2014). Recent advances in molecular pathology
identified various GBM subtypes and thus, paved the way for more individualized
therapeutic strategies. However, GBM remains incurable at present and there is
still an urgent need to further characterize and target the molecular
mechanisms involved in its progression.
The term “cannabinoids” originally described bioactive
constituents of the plant Cannabis sativa. The cannabis
ingredients were used traditionally for their medicinal purpose but also for
their recreational properties. In addition to the psychoactive cannabinoid Δ9-tetrahydrocannabinol
(THC), a number of other phytocannabinoids have been successfully extracted
such as cannabinol, cannabidiol (CBD), cannabigerol or the flavoring agent
beta-caryophyllene (BCP; Mechoulam, 1970; Gertsch et al., 2008). Most of the cannabinoids bind to
G-protein coupled cannabinoid receptors, CB1 and CB2, and act as agonists or
inverse agonists. Of special interest for therapeutic purposes are cannabinoids
that are absent of intoxicating effects such as the CB2-selective BCP and CBD
(Sharma et al., 2016; Russo, 2017). The cannabis constituent CBD has no
significant agonistic activity on cannabinoid receptors (Howlett et al., 2002; Pertwee, 2005) however it targets a number of G-protein
coupled receptors like GPR12, GPR6, GPR3, GPR55 and 5-HT1A and also transient
receptor potential vanilloid TRPV1 and TRPV2 (Espejo-Porras et al., 2013; Nabissi et al., 2013; Hassan et al., 2014; Brown et al., 2017; Kaplan et al., 2017; Laun and Song, 2017). Cannabinoid receptors can also be
selectively activated by pharmacologically efficient synthetic cannabinoids.
Furthermore, cannabinoid receptors are activated by endogenously-produced
arachidonic acid derivatives. The so-called endocannabinoids, anandamide and
2-arachidonoylglycerol (2-AG), are synthesized from cell membrane phospholipids
by specific enzymes. In GBM, increased levels of anandamide and reduced
activity of the synthesizing enzyme N-acylglycerol
phosphatidylethanolamine–phospholipase D (NAPE-PLD) and degrading enzyme fatty
acid amide hydrolase (FAAH) have been identified (Petersen et al., 2005).
The activation of
G-alpha i/o-coupled cannabinoid receptors inhibits adenylate cyclases, signals
via ceramide, and induces kinase phosphorylation of focal adhesion kinase
(FAK), mitogen-activated protein kinase (MAPK), and
phosphatidylinositol-3-kinase (PI3K). Cannabinoid receptors also regulate the
expression of immediate early genes and regulate the production of nitric oxide
(Howlett et al., 2002). Additionally, certain voltage dependent
calcium and inwardly rectifying potassium channels can be modulated via
cannabinoid receptor signaling (Lu and Mackie, 2016). Thus, activation of CB1 or CB2 receptors
exerts diverse consequences on cellular biology and functions (Lu and Mackie, 2016).
GBM tumors are known to express both major
cannabinoid-specific receptors CB1 and CB2. The expression of these receptors
has been detected in GBM cell lines, in ex-vivoprimary tumor
cells derived from GBM patients and in situ, in GBM tissue
biopsies. There is a general consensus that high-grade gliomas, including GBM,
express high levels of CB2. Furthermore, CB2 expression positively correlates
with the malignancy grade (reviewed in Ellert-Miklaszewska et al., 2013). In contrast, the expression of CB1
still requires characterization, as it has been reported to be either unchanged
(Schley et al., 2009), decreased (De Jesús et al., 2010) or even increased (Wu et al., 2012; Ciaglia et al., 2015) in GBM compared to low-grade gliomas or
non-tumor control tissues.
The identification
of altered expression of cannabinoid receptors in gliomas and GBM led to the
hypothesis that cannabinoid receptor agonists might be used as anticancer
agents. Indeed, a pilot clinical study was already developed more than a decade
ago to investigate the anti-tumor activity of THC in patients with glioma. The
study held promising results as it showed a decrease of tumor cell proliferation
upon administration of THC in two of nine patients (Guzmán et al., 2006). Since then, an increasing number of
studies sought to elucidate the molecular mechanisms triggered via the
cannabinoids-cannabinoid receptors axis in gliomas and GBM. The major findings
are described below and a summary is provided in Figure Figure11.
Cannabinoids
and GBM Tumor Growth
The best studied effect of cannabinoids on GBM
pathophysiology is the inhibition of tumor growth. A number of in vivo studies
demonstrated that cannabinoids could significantly reduce tumor volume in
orthotopic and subcutaneous animal models of glioma (for a comprehensive
review, see Rocha et al., 2014). The mechanisms mediating this
phenomenon can be roughly grouped into three categories: (1) cell death-inducing
mechanisms (apoptosis and cytotoxic autophagy); (2) cell
proliferation-inhibiting mechanisms; and (3) anti-angiogenic mechanisms.
Cannabinoid-induced
cell death occurs mainly through the intrinsic (mitochondria-dependent)
apoptotic pathway (reviewed in Ellert-Miklaszewska et al., 2013). Briefly, the pro-apoptotic Bcl-2 family
member Bad is phosphorylated in response to cannabinoid treatment and
translocates to the mitochondria. This results in loss of integrity of the
outer mitochondrial membrane, release of cytochrome c and activation of
apoptosis-executioner caspases. The activation of the intrinsic apoptosis
pathway by cannabinoids is thought to be mediated by an increase in
intracellular ceramide which, in turn, inhibits the pro-survival pathways
PI3K/Akt and Raf1/MEK/ERK thereby allowing Bad to translocate to the
mitochondria. Interestingly, ceramide has been also implicated in
cannabinoid-induced autophagy of glioma cells through the p8/TRB3 pathway and
subsequent inhibition of the Akt/mTORC1 axis (Carracedo et al., 2006; Salazar et al., 2009). Recent studies additionally showed that
THC altered the balance between ceramides and dihydroceramides in
autophagosomes and autolysosomes, which promoted the permeabilization of the
organellar membrane, the release of cathepsins in the cytoplasm and the
subsequent activation of apoptotic cell death (Hernández-Tiedra et al., 2016).
In addition to
ceramide-mediated cell death, cannabinoids were also shown to trigger apoptosis
via oxidative stress (reviewed in Massi et al., 2010). Specifically, glioma cells treated with
CBD responded with reactive oxygen species (ROS) production, GSH depletion and
caspase-9, -8 and -3 activation. Furthermore, combined treatment of GBM cells
with THC and CBD induced a significant increase in the formation of ROS, which
was linked to a later induction of apoptosis (Marcu et al., 2010). Recently however, Scott et al. (2015) showed that, while CBD treatment of
glioma cells did induce a significant increase in ROS production, this
phenomenon was accompanied by an upregulation of a large number of genes
belonging to the heat-shock protein (HSP) super-family. As the subsequent
upregulation of HSP client proteins diminished the cytotoxic effect of CBD, the
authors proposed that the inclusion of HSP inhibitors might enhance the
anti-tumor effects of cannabinoids in glioma/GBM treatment regimens (Scott et
al., 2015).
Apart from a direct
killing effect on tumor cells, cannabinoids can also induce cell cycle arrest
thereby inhibiting tumor cell proliferation. For instance, treatment of GBM
cells with THC and/or CBD increases the population of cells in the G0-G1 phase
and G2-GMphase
while decreasing the number of cells in the S-phase (Marcu et al., 2010). Similarly, Galanti et al. (2008) found that administration of THC to
human GBM cell lines induced G0-G1 phase arrest. The authors also
characterized some of the molecular mechanisms involved in cannabinoid-induced
cell cycle arrest and found that THC decreased the levels of E2F1 and Cyclin A
(two proteins that promote cell cycle progression) while upregulating the level
of the cell cycle inhibitor p16INK4A (Galanti et al., 2008).
The inhibitory
effects of cannabinoids on GBM growth are, however, not restricted to the
direct modulation of tumor cell death/survival or proliferation pathways.
Several studies showed that cannabinoids were also able to inhibit tumor
angiogenesis. For instance, Blázquez et al. (2003) found that local administration of the
nonpsychotic cannabinoid JWH-133 to mice inhibited angiogenesis of malignant
gliomas, since the cannabinoid-treated tumors had a small, differentiated and
impermeable vasculature while the vasculature of the control tumors was large,
plastic and leaky (Blázquez et al., 2003). The same group later demonstrated that
local administration of THC resulted in a decrease of pro-angiogenic VEGF
levels in two patients with recurrent GBM (Blázquez et al., 2004). In vitro, cannabinoids
inhibited endothelial cell migration via the ERK pathway and endothelial cell
survival via protein kinase C (PKC) and p38-MAPK pathways (Blázquez et al., 2003). Similarly, Solinas et al. (2012) demonstrated that CBD induced
endothelial cell cytostasis, inhibited endothelial cell migration and
sprouting in vitro and inhibited angiogenesis in
vivo. These effects were accompanied by a downregulation of pro-angiogenic
factors such as matrix metalloprotease-2 and -9 (MMP2 and MMP9), urokinase-type
plasminogen activator (uPA), endothelin-1 (ET-1), platelet-derived growth
factor-AA (PDGF-AA) and chemokine (c-x-c motif) ligand 16 (CXCL16; Solinas et
al., 2012).
While most studies
found that the agonistic stimulation via CB receptors is responsible for the
anti-tumor effects of cannabinoids, recent evidence suggests that CB1
antagonists might also be useful in glioma therapy. Specifically, Ciaglia et
al. (2015) found that the pharmacological
inactivation of CB1 by SR141716 inhibited glioma cell growth through cell cycle
arrest and induction of caspase-dependent apoptosis. Interestingly however,
SR141716 additionally upregulated the expression of NKG2D ligands (MICA and
MICB) on the surface of glioma cells via STAT3 inactivation. The increase of
MICA/B levels subsequently enhanced the recognition and killing of glioma cells
by NK-cells. Notably, SR141716-induced MICA/B upregulation directly correlated
with the degree of CB1 expression and occurred only in malignant glioma cells
but not in normal human astrocytes (Ciaglia et al., 2015). Taken together these findings suggest
that CB1 specific antagonists might be useful in multimodal therapeutic
strategies, at least for certain subsets of GBM with high expression of CB1.
Cannabinoids
and GBM Invasion
Although gliomas and GBM rarely metastasize, these tumor
cells are very adept at infiltrating the surrounding healthy brain tissue and
spreading through the brain parenchyma (reviewed in Manini et al., 2018). Therefore, therapeutic strategies aimed
at inhibiting the migration and invasion of GBM cells are of great clinical
relevance in the management of this disease.
The role of
cannabinoids in GBM migration and invasion is still poorly characterized.
Nevertheless, accumulating evidence suggests that cannabinoids have potent
anti-invasive effects on glioma cells both in vitro and in
vivo. For instance, Soroceanu et al. (2013) showed that CBD inhibited the invasion
of GBM cells through organotypic brain slices. This anti-invasive effect was
attributed to the inhibition of Id-1 expression by CBD and was observed in
several GBM cell lines, in ex-vivo primary GBM cells and in
an orthotopic xenograft murine model (Soroceanu et al., 2013). Solinas et al. found that CBD
significantly inhibited GBM invasion even at low concentrations, which were
otherwise not sufficient to induce tumor cell death (Solinas et al., 2013). The authors further demonstrated that
CBD treatment of GBM cells significantly downregulated major proteins
associated with tumor invasion, in particular MMP-9 and TIMP-4 (Solinas et al., 2013). Moreover additional MMPs and TIMPs have
been linked to the anti-invasive effects of cannabinoids in glioma.
Specifically, both TIMP-1 and MMP-2 were downregulated by THC treatment of
glioma cells. These effects were mediated via ceramide accumulation and
activation of p8 stress protein and, interestingly, were observed in glioma
bearing mice as well as in two patients with recurrent GBM who had received
intra-tumor injections with THC (Blázquez et al., 2008a,b).
Cannabinoids
and Glioma Stem-Like Cells (GSCs)
A major challenge for GBM treatment is the resistance of
the recurrent tumor to therapy. Accumulating evidence indicates that a
subpopulation of GSCs contributes to this phenomenon through multiple
mechanisms, such as alteration of DNA damage response, hypoxic
microenvironment, Notch signaling pathway or multidrug resistance (reviewed in
Liebelt et al., 2016).
GSCs express both
major cannabinoid receptors, CB1 and CB2, as well as other components of the
endocannabinoid system (Aguado et al., 2007). Exploratory gene array studies found
that cannabinoid agonists altered the expression of genes involved in stem cell
proliferation and differentiation. Cannabinoid-treated GSCs responded with
increased S-100ß and GFAP expression and with simultaneous downregulation of
the neuroepithelial progenitor marker nestin. Furthermore, cannabinoid
challenge reduced the efficiency of GSCs to initiate glioma formation in
vivo, as indicated by decreased neurosphere formation and cell
proliferation in secondary xenografts (Aguado et al., 2007). The differentiation of GSCs was
recently linked to the expression levels of the transcription factor Aml-1a.
Nabissi et al. (2015) found that Aml-1a was upregulated during
GSCs differentiation while Aml-1a knock-down restored a stem-cell phenotype in
differentiated GSCs. Interestingly, treatment of GSCs with CBD upregulated the
expression of Aml-1a in a TRPV2- and PI3K/Akt-dependent manner thereby inducing
autophagy and abrogating the chemoresistance of GSCs to BCNU therapy (Nabissi
et al., 2015).
Another potential
mechanism regulating the “stemness” of GSCs upon cannabinoid treatment involves
the intracellular increase of ROS. Specifically, CBD was shown to inhibit the
self-renewal of GSCs via activation of the p38-MAPK pathway and downregulation
of key stem cell mediators such as Sox2, Id1 and p-STAT3, while co-treatment
with antioxidants abrogated these effects. In vivo, treatment of
intracranial GSCs-derived tumors with CBD inhibited tumor cell proliferation,
activated the pro-apoptotic caspase-3 and significantly prolonged the survival
of tumor-bearing mice. Even though a subset of GSCs adapted to CBD treatment
and led to tumor regrowth, this phenomenon could be abrogated by combined
therapy with CBD and small molecule modulators of ROS (Singer et al., 2015).
Clinical
Relevance and Future Perspective of Cannabinoids in GBM Therapy
The antineoplastic effects of cannabinoids have been
investigated in a number of in vitroand in vivo studies
(reviewed in Ladin et al., 2016). A pilot phase I clinical trial for the
treatment of GBM patients indicated a good safety profile for THC (Velasco et
al., 2007). The
intra-tumor administration of THC in nine patients with actively growing
recurrent GBM decreased tumor cell proliferation (Guzmán et al., 2006) and induced apoptosis (Carracedo et al., 2006). In contrast, cannabinoids promoted the
survival of healthy oligodendrocytes (Molina-Holgado et al., 2002), astrocytes (Gómez Del Pulgar et al., 2002), and neurons (Howlett et al., 2002; Mechoulam, 2002). A tumor-specific cytostatic/cytotoxic
effect of cannabinoids would, therefore, have great relevance for the treatment
of GBM.
Pre-clinical studies
have also investigated the anti-tumor effects of cannabinoid combinations (in
particular THC:CBD) and found that the anti-neoplastic effect of THC was
enhanced when combined with CBD (reviewed in Ladin et al., 2016). The therapeutic potential of THC:CBD
combinations was, furthermore, tested in combination with standard GBM
chemotherapy, such as the alkylating anti-neoplastic drug TMZ or with ionizing
radiotherapy. In a GBM xenograft model in nude mice, the reduction of tumor
size could be enhanced by co-administration of THC with CBD and TMZ in
comparison to the effects of THC, CBD and TMZ alone (Torres et al., 2011). In a further study, THC:CBD
co-treatment of orthotopic GBM tumors in C57BL/6 mice enhanced the killing
effect of ionizing radiation (Scott et al., 2014; Ladin et al., 2016).
These beneficial
effects of THC:CBD preparations in pre-clinical models have led to a
placebo-controlled phase II clinical trial investigating a THC:CBD mixture in
combination with dose-intense TMZ in GBM patients (clinical trial NCT01812603). The company GW Pharmaceuticals
reported in their orphan drug-designated study positive results in the
treatment of GBM (Schultz and Beyer, 2017;
Schultz, 2018).
This study included 21 adult patients with histopathologically-confirmed GBM
and with a Karnofsky performance scale of 60% or greater (clinical trial NCT01812603; Schultz and Beyer, 2017).
Patients received orally a maximum of 12 sprays per day delivering 100 μl of a
solution containing 27 mg/ml THC and 25 mg/ml CBD. The control group received
TMZ only and had a 44% 1-year survival rate. In contrast the THC:CBD plus TMZ
group showed a 83% 1-year survival rate with a median survival over 662 days
compared with 369 days in the control group. (Schultz and Beyer, 2017;
Schultz, 2018).
These first results of clinical investigations are promising and point to the
importance of cannabinoid translational research leading to clinically relevant
studies. In the future, endocannabinoid-degrading MAGL enzyme might also be an
interesting target since it changes the fatty acid network of cancer cells
modulating their pathogenicity (Nomura et al., 2010).
In conclusion,
cannabinoids show promising anti-neoplastic functions in GBM by targeting
multiple cancer hallmarks such as resistance to programmed cell death,
neoangiogenesis, tissue invasion or stem cell-induced replicative immortality.
The effects of cannabinoids can be potentially enhanced by combination of
different cannabinoids with each other or with chemotherapeutic agents. This
requires, however, a detailed understanding of cannabinoid-induced molecular
mechanisms and pharmacological effects. Ultimately, these findings might foster
the development of improved therapeutic strategies against GBM and, perhaps,
other diseases of the nervous system as well.
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