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Cancers 2010, 2(2), 1013-1026; doi:10.3390/cancers2021013
Published: 26 May 2010
Abstract: Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells.
In addition to the well-known psychotropic effects of cannabis and its use as an illicit drug, recent studies have suggested a potential application of cannabinoids as therapeutic agents. In particular, cannabinoids, originally derived from the plant Cannabis sativa, as well as its endogenous and synthetic counterparts, have been reported to exert antiproliferative actions on a wide spectrum of tumor cells [1,2] and, of relevance, they are able to induce inhibition and regression of gliomas, one of the most aggressive forms of cancer. The present paper will summarize the anti-cancer properties of cannabinoid compounds on gliomas, and discuss their potential action mechanisms with particular emphasis on stress-related cellular effects.
2. Endocannabinoid System
The endocannabinoid system is a recently-discovered signaling system present in both the brain and its periphery, comprising cannabinoid CB1 and CB2 receptors, their intrinsic lipid ligands endocannabinoids (ECs) such as N-arachidonoyl ethanolamide (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), and associated proteins (transporters and biosynthetic and degradative enzymes). The cannabinoid CB1 receptor is a pre-synaptic receptor widely expressed throughout the brain. High densities are present in the striatum, hippocampus, and cerebellum, as well as moderate to low densities in the amygdala, midbrain, and cerebral cortex ; it is also present at a lower density in peripheral tissues, including the liver, adipocytes, the exocrine pancreas, the GI tract, skeletal muscle and circulating immune cells .
CB2 receptors were cloned a few years after CB1 , and while they were thought to be predominately located in immune cells in tissues such as the spleen and the liver, there is recent evidence that cannabinoid CB2 receptors exhibit limited neuronal expression [6,7,8,9,10,11].
Both CB1 and CB2 are G-protein coupled receptors. The CB1 receptor couples with both Gi/o proteins which function to inhibit adenylyl cyclase activity, activate potassium channels and inhibit voltage-gated calcium channels, while the CB2 receptor is only known to couple with Gi proteins . The discovery of endogenous ligands for the cannabinoid receptor (endocannabinoids) occurred soon after the characterization of the receptor. The two primary ligands which have been characterized as endocannabinoid are N‑arachidonoylethanolamine, or anandamide (AEA) and 2-arachidonoylglycerol. Both AEA and 2-AG are formed post-synaptically from phospholipid precursors through activity-dependent activation of specific phospholipase enzymes 
Endogenous ligands do not share the same biosynthetic or metabolic pathways, indicating distinct regulation mechanisms. Multiple biochemical pathways may synthesize AEA  and the primary pathway for the production of AEA within the CNS has not been clearly determined yet. 2-AG is mainly synthesized through activation of phospholipase C, and the subsequent production of diacylglycerol, which is rapidly converted to 2-AG by diacylglycerol lipase . AEA is hydrolyzed by the enzyme fatty acid amide hydrolase (FAAH), generating arachidonic acid and ethanolamine, while 2-AG is primarily metabolized by monoacylglycerol lipase (MAG lipase), which results in the formation of arachidonic acid and glycerol . The presence of two endogenous ligands for one receptor has not been fully explained, but differences in pharmacokinetics and in the efficacy of these ligands has been demonstrated , suggesting that they may play distinct physiological roles. Furthermore, endocannabinoid signaling acts differently than most neurotransmitter systems. Specifically, endocannabinoids are released “on demand” by post-synaptic cells, function as retrograde signals and traverse back across the synapse where they bind with pre-synaptically located CB1 receptors and reduce synaptic transmitter release .
Gliomas have been shown to possess one or more components of the endocannabinoid system such as the ability to synthesize endocannabinoids, the presence of CB1/CB2 receptors and the enzyme FAAH, thus suggesting a possible role of this system in regulation of cell growth.
3. Gliomas and Cannabinoids
Malignant gliomas are the most common type of brain tumor in adults, and high-grade gliomas (glioblastomas, GBMs) are among the most rapidly growing and devastating neoplasms. GBMs rarely metastasize out of the central nervous system, but their aggressive invasion of normal tissue surrounding the tumor mass makes surgical removal virtually impossible, and substantially complicates clinical management of the disease [18,19]. Despite surgery and radiotherapy, these tumors invariably recur, and generally lead to death within less than one year of diagnosis.
A hallmark characteristic of gliomas is their molecular and cellular heterogeneity (either in terms of pathology or genetic changes even within a single tumor) which is considered one of the reasons for their malignancy [20,21]. A large number of chemotherapeutic agents (e.g., alkylating agents such as temozolomide, and nitrosureas such as carmustine) have been tested, but no remarkable improvement in patient survival has been achieved so far. Therapeutic adjuvant to surgical resection such as focal radiotherapy and chemotherapy provide only a negligible improvement in the disease’s course and life expectancy with a variable toxicity profile among these treatments, with myelosuppression being the most frequent and limiting factor. Despite this multimodality treatment, clinical recurrence or progression is nearly universal and available systemic chemotherapies offer only modest clinical benefits. Likewise, although immunotherapy strategies appear promising as a new and safe approach to induce antitumor immune response , no immunotherapy or gene therapy trial performed to date has been significantly successful.
4. Δ9-Tetrahydrocannabinol and Glioma Growth
In 1998, Guzman’s group reported the antitumoral effect of Δ9-tetrahydrocannabinol (THC) in C6 murine glioma cells . It was shown that THC-induced glioma cell death was independent of CB1 cannabinoid receptor stimulation and accompanied by a significant breakdown of cellular sphingomyelin pathways . Given the favorable safety profile, in March 2002, the Spanish Ministry of Health approved a Phase I/II clinical trial, carried out in collaboration with Tenerife University Hospital and Guzman’s research group, aimed at investigating the effect of local administration of THC on the growth of recurrent glioblastoma multiforme. The study was the first pilot study that investigated cannabinoid antitumoral action but also the intracranial application of THC through an infusion cannula connected to a subcutaneous reservoir. The nine enrolled patients had previously failed standard therapy (surgery and radiotherapy) and constituted a cohort of terminal patients harboring actively-growing recurrent tumors. The results have been recently published . THC delivery was safe and achieved without overt psychoactive effects. Median survival of the cohort from the beginning of cannabinoid administration was 24 weeks, but two patients survived for approximately one year. Survival for GBM patients following diagnosis is typically six to 12 months. The authors reported that due to the characteristics of the study, the effect of THC on patient survival was unclear, and evaluation of survival would require a larger trial with a different design. However, in placebo-controlled trials for recurrent glioblastoma multiforme with temozolomide, a slight impact has been reported on overall length of survival (median survival: 24 weeks, 6-month survival = 46–60%).
Thus, the possibility to have other drugs such as cannabinoids available to manage these devastating tumors has improved research to establish their action mechanisms on tumor cells and to definitively establish their efficacy. Studies with animal models have shown that local administration of THC or the synthetic cannabinoid WIN-55,212-2 reduced in vivo the size of the tumor generated by intracranial inoculation of C6-derived glioma in Wistar rats  with a concomitant involvement of CB1 and CB2 receptors. Moreover, rats bearing malignant gliomas, when treated intratumorally with cannabinoids, survived significantly longer than untreated animals and 20–35% of treated animals showed a complete eradication of the tumors.
5. CB2 Selective Compounds and Glioma Growth
The unwanted psychotropic effects of marijuana-derived cannabinoids are mediated largely or completely by neuronal CB1 receptors. Thus, great efforts have been made to assess alternative possibilities. One of the most obvious strategies to avoid psychotropic side effects in the management of glioma tumor growth is the administration of CB2-selective compounds. Recent evidence that CB2 receptors are present in both cultured neurons and the nervous system has to be taken into account [27,28]. The co-expression of the CB1 and CB2 cannabinoid receptors has been detected in rat C6 glioma cells and in biopsies from human astrocytomas . Moreover, the extent of CB2 expression was related to tumor grade. Another study that surveyed the level of CB2 receptors in biopsies of human astrocytomas and glioblastomas revealed a high level of this receptor subtype among adult and pediatric tumors  and its amounts appeared to be correlated with tumor malignancy. Calatazzolo et al.  found a higher expression of CB2 receptors in glioblastomas as well as in endothelial cells than in low-grade gliomas. High levels of CB2 expression in either gliomas or in endothelial cells of glioblastoma vessels was also demonstrated by Schley et al. . High levels of CB2 expression suggest that these tumors would be vulnerable to a cannabinoid treatment and indicate a specific CB2 cannabinoid agonist-based strategy. In this context, it has been demonstrated that the local and in vivo daily administration of the selective CB2 agonist JWH-133 in mice bearing subcutaneous glioma causes a considerable regression of malignant tumors, inducing a classic pattern of apoptosis via ceramide de novo synthesis .
The hypothesis of the usage of CB2 selective compounds has prompted further research on the effectiveness of a series of novel CB2 cannabinoid compounds in glioma treatment . The lead compound named KM-233 represents the first generation of synthetic C1’ aryl substitute cannabinoid ligands. This compound exhibits a good lipophilicity and affinity for the CB2 receptor that could predict significant transit across the blood brain barrier and good activity at the CB2 receptor on glioma cells. KM-233 has shown excellent cyotoxicity against U87, U373 and C6 glioma cells and it is also effective in vivo in reducing glioma tumor growth subcutaneously in SCID mice, via both direct intra-tumoral injection and systemic administration. In addition, another series of CB2 selective synthetic compounds has been tested in human glioma cells and found to be highly cytotoxic to cells . Of particular interest, Aguado et al. demonstrated the inhibition of gliomagenesis induced by the selective CB2 compound JWH-133 on glioma stem-like cells .
6. Non Psychotropic Cannabinoids and Glioma Growth
Another strategic approach that has been pursued is to explore the usage of natural, non-psychotropic cannabinoids that bind with very low affinity to cannabinoid receptors, thus excluding either psychotropic and/or immune/peripheral effects. Among the bioactive constituents of marijuana, cannabidiol (CBD), does not have significant intrinsic activity on cannabinoid receptors [28,36] and does not induce psychotropic and adverse side effects. For these reasons, it is one of the natural cannabinoids with the highest potential for therapeutic use. A first study of Massi et al.  reported that CBD was effective in inhibiting U87 and U373 human glioma cell proliferation in an in vitro set of experiments. Additional experiments demonstrated in vivo the antitumor activity of CBD . When tumor xenografts generated by subcutaneous injection of glioma cells in the flank region of immune-deficient mice were treated locally with CBD, there was a significant 60% mean reduction of tumor growth over a 23-day period of observation, although no eradication was described . The antiproliferative effect of CBD was dose-correlated and dependent on its ability to induce apoptotic death. All these effects appeared independent of cannabinoid receptor stimulation . Bisogno et al.  have reported that cannabidiol can recognize the transient receptor potential vanilloid type-1 (TRPV1) as a molecular target, demonstrating that the drug is a full, although weak, agonist on human TRPV1. Ligresti et al.  showed that in addition to cannabidiol, the plant cannabinoids cannabigerol and cannabidiol acid were found to activate TRPV1 receptors. Thus, it can be suggested that there are some alternative ways through which nonpsychotropic cannabinoids can induce apoptosis since it is possible that when TRPV1 is stimulated, apoptosis may be induced by mitochondrial events triggered by TRPV1-mediated calcium influx . Recently, De Petrocellis et al.  have also demonstrated an interaction of phytocannabinoids with ankyrin TRPA1 and melastatin TRPM8 channels, with potential implications for the cancer treatment.
The capability of cannabidiol to either potentiate or inhibit the actions of THC was recently examined by the McAllister group. In the U251 and SF126 glioblastoma cell lines, THC and CBD acted synergistically to inhibit cell proliferation . The treatment of glioblastoma cells with both compounds led to a significant modulation of the cell cycle, induction of reactive oxygen species and apoptosis as well as specific modulations of extracellular signal-regulated kinase and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. These results suggest that the addition of CBD to THC may improve the overall effectiveness of THC in the treatment of glioblastoma in cancer patients.
Finally, the synthetic derivative of THC, ajulemic acid, has also been reported to inhibit glioma cell growth in vitro and in vivo inducing cytostatic rather than cytotoxic effects , although its pharmacological properties are still controversial and not completely clarified.
7. Endocannabinoids and Glioma Growth
Ongoing research is now evaluating whether endogenous cannabinoids exert tumor-suppressing effects in glioma growth, thus potentially representing an alternative approach to the development of possibly harmless anti-cancer drugs [44,45]. In fact, endogenous cannabinoid agonists or selective inhibitors of endocannabinoid degradation with limited action on CB1 receptors, would exhibit little if any psychotropic activity and be effective only in those tissues where the levels of endocannabinoids were altered. However, the anti-tumor potential of substances that modulate the endocannabinoid system is still largely unexplored.
The use of AEA would have a number of additional advantages over THC: (a) AEA is virtually ineffective on CB2 receptors, which would rule out the immunosuppressive effect described for THC. (b) AEA has been shown to promote the growth of hematopoietic cell lines, an effect which may be particularly attractive if AEA-enhancing strategies were to be included in polychemotherapeutic protocols. By contrast, the poor stability and short half-life of AEA make its use as a therapeutic agent largely impractical. However, since a number of tumor cell lines express one or more components of the endocannabinoid system and since AEA is synthesized on demand at multiple sites throughout the body and because of its lipophilic feature it easily reaches tumor sites as well, including the CNS , novel antiproliferative strategies based on the pharmacological modulation of AEA levels through inhibition of AEA uptake and/or degradation by FAAH (an approach which would interfere with endocannabinoid levels mildly and in a neuronal activity-dependent fashion) may be considered for the clinical management of at least some forms of neoplastic disease.
Finally, the studies on the putative anti-tumor properties of endogenous cannabinoids in human gliomas are only beginning. It has been demonstrated that AEA induces apoptosis in cells derived from the neural crest, such as the CPH100 human neuroblastoma cell line through a pathway involving a rise in intracellular calcium, mitochondrial uncoupling and cytochrome c release . Unlike AEA, other ECs such as 2-AG, linoleoylethanolamide (LEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) were unable to force cells into death . Jacobsson et al.  showed that in rat C6 glioma cells AEA exerts antiproliferative effects associated with a combined activation of cannabinoid and vanilloid receptors but, in contrast with Maccarrone’s data , 2-AG inhibited glioma cell proliferation with a similar potency to that of AEA. Another EC such as stearoylethanolamide (SEA), present in the human brain in amounts comparable to those of AEA, induced pro-apoptotic activity in glioma cell line . Contassot et al.  showed that human glioma cell lines, either established for a very long time (U87 and U251) or derived from a tumor biopsy (Ge227 and Ge258) are efficiently killed by AEA. These cell lines contemporarily express CB1, CB2 and the transient receptor potential vanilloid type-1 (TRPV1), and the authors demonstrated that the antiproliferative effects of AEA were essentially due to its ability to bind to the TRPV1 receptor. The stimulation of the TRPV1 receptor by endocannabinoids could represent an alternative mechanism through which AEA causes apoptosis triggering calcium influx in tumor cells . Despite the scarce data available, the selective targeting of TRPV1 and/or CB1/CB2 receptors by EC system modulation could represent an attractive area of drug development, avoiding CB1-mediated psychotropic side effects and CB2-mediated immunosuppression. In addition, another study has demonstrated that the commonly used acylderived AEA uptake inhibitors AM404, VDM11, UCM707 and OMDM2 rapidly affected C6 glioma cell viability .
8. Cellular Action Mechanisms of Cannabinoids on Gliomas and Cells Derived from the CNS
Natural, synthetic and endogenous cannabinoids have all been found to affect a number of pathways involved in the cell survival/death decision in cell lines derived from the CNS and progress has been made towards understanding the intracellular mechanisms underlying in vivo and in vitro antitumor effects.
The signaling pathways activated by cannabinoids to induce tumor cell death have been studied in primary astrocytes and rat and human glioma cell lines, as well as in CB1-transfected CHO cells and a number of downstream effectors have been identified.
The apoptosis induced by THC in C6 cells is accompanied by intracellular accumulation of the ubiquitous lipid second messenger ceramide and by activation of one or more families of mitogen-activated protein kinases (MAPKs) . Following exposure to THC, a biphasic pattern of ceramide accumulation has been observed in C6 rat glioma cells, with an early peak occurring within minutes, followed by a sustained second generation of ceramide lasting several days [51,52]. This delayed peak of ceramide generation has been proposed to be fundamental for apoptosis and depend on de novo ceramide synthesis, rather than on sphingomyelin hydrolysis. The close relationship between ceramide accumulation and THC-induced apoptosis is also supported by the finding that primary neurons are resistant to cannabinoid-induced ceramide accumulation and apoptosis, unless fairly high THC concentrations are used [53,54].
Moreover, Carracedo et al. , using wide array of experimental approaches, identified the stress regulated protein p8 as an essential mediator of cannabinoid antitumoral action and showed that p8 up-regulation is dependent on de novo-synthesized ceramide . The p8 upregulation also takes place in vivo and resistance to cannabinoid treatment is associated with a decreased activation of the p8-regulated proapoptotic pathway .
The p8 target is the pseudokinase tribbles homolog 3 (TRB3) and recently the mechanism that promotes the activation of this signaling route as well as the target downstream of TRB3 that mediates its tumor cell-killing action has been partially elucidated. In fact Salazar et al.  showed that in human glioma cells THC-induced ceramide accumulation and the eukaryotic translation initiation factor 2α (eIF2α) phosphorylation thereby activating an ER stress response that promoted autophagy via tribbles homolog 3–dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis . The autophagy is upstream of apoptosis in THC-induced human and mouse cancer cell death and the activation of this process was necessary for the antitumor effects of cannabinoids in vivo.
THC has been demonstrated to acutely enhance the activity of MAPKs, particularly ERKs and JNKs (Jun N-terminal kinases) in a CB1 receptor-dependent fashion, with a time course which parallels that observed for ceramide accumulation [50,51]. However, the relationship between ceramide accumulation and MAPK activation following CB1 receptor engagement by THC is far from obvious, as an increase in intracellular ceramide levels does not seem to be a prerequisite for cannabinoid-induced activation of the JNK family of MAPKs .
Mechanisms other than ceramide induction can be involved in cannabinoid-induced cell death. Massi et al. demonstrated that there was no involvement of ceramide in CBD-induced apoptosis , thus suggesting that CBD and/or other cannabinoids can exert their antineoplastic effects independently by stimulation of this downstream effector.
A report discloses that WIN inhibits C6 glioma cell proliferation through an inhibition of ERK1/2 kinase and AKT, the key mediator of growth factor-promoted cell survival. A decrease of mitogenic/pro-survival signaling precedes reduction of Bad phosphorylation and the events that follow Bad translocation to the mitochondrial membrane. Bad, a pro-apoptotic Bcl2 family member, may be an important link between the down regulation of the survival pathway and caspase activation evoked by cannabinoid treatment and resulting in glioma cell death .
An involvement of PI3K in the acute effects of CB1 receptor stimulation has also emerged from a study on CB1-transfected CHO cells and CB1-expressing human U373 MG astrocytoma cells, where THC has been shown to enhance the activity of protein kinase B (PKB)/Akt . PKB plays a fundamental role in the regulation of basic cell functions, such as energy metabolism and proliferation.
Finally, evidence has been collected supporting the role of cannabinoids in controlling glioma cell growth through the inhibition of lipoxygenase (LOX)-enzyme. In vivo treatment of nude mice bearing subcutaneous glioma tumors with CBD was found to inhibit the activity and content of 5-LOX enzyme in tumor tissues by a significant 40% .
All this data provides further demonstration of other and/or alternative intracellular targets that can be importantly modulated by cannabinoids contributing to their evident antitumoral effects.
9. Role of Oxidative Stress in the Antiproliferative Effects of Cannabinoids
Under physiological conditions, the maintenance of an appropriate level of intracellular reactive oxygen species (ROS) is important in keeping redox balance and standard signaling proliferation. ROS are essential for many biological functions. They can regulate many signal transduction pathways by directly reacting with and modifying the structure of important protein transcription factors and genes. The modulation of their function can ultimately alter the expression and activities of many transcription factors as well as signaling proteins that are involved in the stress response and cell survival through multiple mechanisms. Moreover, an overproduction of ROS or decreased ability to scavenge would result in a significant increase of intracellular ROS, leading to cellular damage, lipid peroxidation, DNA modifications and enzyme inactivation. If the ROS level is consistent and persistent, all of these damages can cause cell death. It is well-known that cancer cells are characterized, in general, by high levels of ROS and intrinsic oxidative stress. Compared with normal cells, cancer cells seem to possess higher levels of endogenous ROS but events that increase ROS levels above a certain threshold seem to induce an incompatible situation with cellular survival, leading to cell death. This provides the rationale for killing cancer cells inducing ROS accumulation in malignant cells with appropriate agents. Thus, treating cancer cells with compounds that possess pro-oxidant properties and increase ROS level or that abrogate the cellular antioxidant system will shift the redox balance resulting in cancer cell cytotoxicity.
Besides the above-mentioned molecular mechanisms underlying antitumoral action of cannabinoids, evidence has been collected showing that an additional cellular mechanism through which cannabinoids can modulate cell survival/death fate is the induction of oxidative stress in cancer cells. Jacobsson et al.  showed that AEA and 2-AG produce antiproliferative effects on rat C6 glioma cells by a mechanism that involves both cannabinoids and vanilloid receptors and oxidative stress since the anti-oxidant α-tocopherol completely reversed the antitumoral activity of the cannabinoids compounds either at 0.1 μM or 10 μM concentration.
Goncharov et al.  reported that the activation of CB1 receptors by THC in C6 cells makes these cells more vulnerable to oxidative damage. The study was performed using a cell permeating Fe (III) chelating quinone that provided more physiological conditions for mimicking naturally occurring oxidative stress within the cell, as a better model for natural ROS formation. In fact, the addition of THC for 10 min prior to the induction of oxidative stress increased subsequent cell damage as demonstrated by LDH and MTT assay. This effect was reversed by the addition of the CB1 but not the CB2 selective antagonist. The authors also reported a parallel decrease in glucose uptake, probably contributing to a dramatic depletion in the energy reserve of the c ells. This event could render the gliomas cells more sensitive to oxidative stress, driving them into apoptosis. Massi et al.  also demonstrated that the pretreatment of glioma cells with α-tocopherol antagonized the anti-proliferative effect of the non psychotropic cannabinoid compound CBD, thus suggesting an involvement of oxidative stress in CBD-antitumoral effects. The authors then investigated the presence of the existence of an oxidative stress state in gliomas cells after CBD exposure . They found that CBD induced significant ROS production, GSH depletion and increase activity of GPox and GRed enzymes, as early as 5–6 h after CBD exposure, with a time course preceding caspases activation. The authors concluded that when the generation of ROS exceeds the scavenging capacity of the cell, and if there is a contemporary decrease in GSH levels counteracted by the increased activity of associated anti-oxidant enzymes, the cell could initiate cell death-linked molecular events, namely the activation of caspase-9 and -8 which, in turn, cleave caspase-3. Ligresti et al.  demonstrated that the antiproliferative action of 10 μM CBD in MDA-MB-231 breast cancer cells was significantly prevented by the antioxidant tocopherol, vitamin C as well as astaxantine. They showed that CBD induced ROS formation and that this effect was Ca2+ dependent because it was erased when cells were preloaded with the Ca2+ chelator BAPTA-AM. The rise of intracellular ROS production can account for the proapoptotic effects of CBD in tumor cells although the phenolic chemical structure would rather favor an antioxidant effect. The oxidant properties of CBD can be dependent from the different biochemical and cellular features of tumor versus non-tumor cells rather than from the molecule itself  and/or by its ability to cause an increase in intracellular Ca2+ depending on the cell culturing conditions .
Marcu et al.  demonstrated on human glioblastoma cells that the combination of THC and CBD produced a significant increase in the formation of ROS. The observed initial increase in ROS was clearly linked to a latter induction of apoptosis. Individually, both THC and CBD could increase apoptosis through the production of ROS, but THC was significantly less efficient at inducing this process as a single agent as compared with when it was used in combination with CBD. Although the concentration of CBD used in the combination treatment did not significantly stimulate ROS, it may have primed this pathway for THC through a convergence on shared signal transduction pathways.
The induction of oxidative stress induced by cannabinoids was also a common mechanism reported in other types of cancer cells since either in EL-4 thymoma , in human colorectal carcinoma Caco-2 , and in PC-12 cells  it was demonstrated that oxidative stress plays a role in the antiproliferative effect of cannabinoids.
The therapy of gliomas, the most frequent class of malignant primary brain tumors and one of the most aggressive forms of cancer characterized by high invasiveness, a high proliferation rate and rich neovascularization, could benefit from the use of cannabinoids, the active compounds of Cannabis sativa, and their synthetic derivatives. They have been shown to mimic the endogenous substances named “endocannabinoids” that activate specific cannabinoid receptors (CB1 and CB2).
Cannabinoids have been proven to inhibit glioma tumor growth in either in vitro or in vivo models through several cellular pathways such as elevating ceramide levels, modulating PI3K/Akt, MAPK kinases, inducing autophagy and oxidative stress state in glioma cells, thus arresting cell proliferation and inducing apoptosis. Since cannabinoids kill tumor cells without toxicity on their non transformed counterparts, probably modulating the cell survival/cell death pathways differently, they can represent a class of new potential anticancer drugs.
- Sarfaraz, S.; Adhami, V.M.; Syed, D.N.; Afaq., F.; Mukhtar, H. Cannabinoids for cancer treatment: progress and promise. Cancer Res. 2008, 68, 339–342. [Google Scholar] [CrossRef]
- Bifulco, M.; Laezza, C.; Pisanti, S.; Gazzerro, P. Cannabinoids and cancer: pros and cons of an antitumour strategy. Br. J. Pharmacol. 2006, 148, 123–135. [Google Scholar] [CrossRef]
- Herkenham, M.; Lynn, A.B.; Johnson, M.R.; Melvin, L.S.; de Costa, B.R.; Rice, K.C. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J. Neurosci. 1991, 11, 563–583. [Google Scholar]
- Matias, I.; Bisogno, T.; Di Marzo, V. Endogenous cannabinoids in the brain and peripheral tissues: regulation of their levels and control of food intake. Int. J. Obes. 2006, 30 (Suppl. 1), S7–S12. [Google Scholar] [CrossRef]
- Munro, S.; Thomas, K.L.; Abu-Shaar, M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993, 365, 61–65. [Google Scholar] [CrossRef]
- Beltramo, M.; Bernardini, N.; Bertorelli, R.; Campanella, M.; Nicolussi, E.; Fredduzzi, S.; Reggiani, A. CB2 receptor-mediated antihyperalgesia: possible direct involvement of neural mechanisms. Eur. J. Neurosci. 2006, 23, 1530–1538. [Google Scholar] [CrossRef]
- Brusco, A.; Tagliaferro, P.A.; Saez, T.; Onaivi, E.S. Ultrastructural localization of neuronal brain CB2 cannabinoid receptors. Ann. N. Y. Acad. Sci. 2008, 1139, 450–457. [Google Scholar] [CrossRef]
- Gong, J.P.; Onaivi, E.S.; Ishiguro, H.; Liu, Q.R.; Tagliaferro, P.A.; Brusco, A.; Uhl, G.R. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res. 2006, 3, 10–23. [Google Scholar]
- Ross, R.A.; Coutts, A.A.; McFarlane, S.M.; Anavi-Goffer, S.; Irving, A.J.; Pertwee, R.G.; MacEwan, D.J.; Scott, R.H. Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception. Neuropharmacology 2001, 40, 221–232. [Google Scholar] [CrossRef]
- Van Sickle, M.D.; Duncan, M.; Kingsley, P.J.; Mouihate, A.; Urbani, P.; Mackie, K.; Stella, N.; Makriyannis, A.; Piomelli, D.; Davison, J.S.; Marnett, L.J.; Di Marzo, V.; Pittman, Q.J.; Patel, K.D.; Sharkey, K.A. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science 2005, 14, 329–332. [Google Scholar]
- Wotherspoon, G.; Fox, A.; McIntyre, P.; Colley, S.; Bevan, S.; Winter, J. Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons. Neuroscience 2005, 135, 235–245. [Google Scholar] [CrossRef]
- Dalton, G.D; Bass, C.E.; Van Horn, C.G.; Howlett, A.C. Signal transduction via cannabinoid receptors. CNS Neurol. Disord. Drug Targets 2009, 8, 422–431. [Google Scholar] [CrossRef]
- Piomelli, D. The endocannabinoid system: a drug discovery perspective. Curr. Opin. Investig. Drugs 2005, 6, 672–679. [Google Scholar]
- Ahn, K.; McKinney, M.K.; Cravatt, B.F. Enzymatic pathways that regulate endocannabinoid signalling in the nervous system. Chem. Rev. 2008, 108, 1687–1707. [Google Scholar] [CrossRef]
- Bisogno, T.; Howell, F.; Williams, G.; Minassi, A.; Cascio, M.G.; Ligresti, A.; Matias, I.; Schiano-Moriello, A.; Paul, P.; Williams, E.J.; Gangadharan, U.; Hobbs, C.; Di Marzo, V.; Doherty, P. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 2003, 163, 463–468. [Google Scholar] [CrossRef]
- Freund, T.F.; Katona, I.; Piomelli, D. Role of endogenous cannabinoids in synaptic signaling. Physiol. Rev. 2003, 83, 1017–1066. [Google Scholar]
- Hillard, C.J. Biochemistry and pharmacology of the endocannabinoids arachidonylethanolamide and 2-arachidonylglycerol. Prostaglandins Other. Lipid. Mediat. 2000, 61, 3–18. [Google Scholar] [CrossRef]
- Franceschi, E.; Tosoni, A.; Bartolini, S.; Mazzocchi, V.; Fioravanti, A.; Brandes, A.A. Treatment options for recurrent glioblastoma: pitfalls and future trends. Expert Rev. Anticancer Ther. 2009, 5, 613–619. [Google Scholar]
- Chi, A; Norden, AD; Wen, PY. Inhibition of angiogenesis and invasion in malignant gliomas. Expert Rev. Anticancer Ther. 2007, 7, 1537–1560. [Google Scholar] [CrossRef]
- Sathornsumetee, S.; Reardon, D.; Desjardins, A.; Quinn, J.A.; Vredenburgh, J.J.; Rich, J.N. Molecularly targeted therapy for malignant glioma. Cancer 2007, 110, 13–24. [Google Scholar] [CrossRef]
- Sanai, N.; Alvarez-Buylla, A.; Berger, M.S. Neural stem cells and the origin of gliomas. N. Engl. J. Med. 2005, 353, 811–822. [Google Scholar] [CrossRef]
- Yamanaka, R. Novel immunotherapeutic approaches to glioma. Curr. Opin. Mol. Ther. 2006, 8, 46–51. [Google Scholar]
- Sánchez, C.; Galve-Roperh, I.; Canova, C.; Brachet, P.; Guzmán, M. Delta9-tetrahydrocannabinol induces apoptosis in C6 glioma cells. FEBS Lett. 1998, 436, 6–10. [Google Scholar] [CrossRef]
- Carracedo, A.; Lorente, M.; Egia, A.; Blázquez, C.; García, S.; Giroux, V.; Malicet, C.; Villuendas, R.; Gironella, M.; González-Feria, L.; Piris, M.A.; Iovanna, J.L.; Guzmán, M.; Velasco, G. The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells. Cancer Cell 2006, 9, 301–312. [Google Scholar] [CrossRef]
- Guzmán, M.; Duarte, M.J.; Blázquez, C.; Ravina, J.; Rosa, M.C.; Galve-Roperh, I.; Sánchez, C.; Velasco, G.; González-Feria, L. A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme. Br. J. Cancer 2006, 95, 197–203. [Google Scholar] [CrossRef]
- Galve-Roperh, I.; Sánchez, C.; Cortés, M.L.; Gomez del Pulgar, T.G.; Izquierdo, M.; Guzmán, M. Anti-tumoural action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nat. Med. 2000, 6, 313–319. [Google Scholar] [CrossRef]
- Howlett, A.C.; Breivogel, C.S.; Childers, S.R.; Deadwyler, S.A.; Hampson, R.E.; Porrino, L.J. Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology 2004, 47, 345–358. [Google Scholar] [CrossRef]
- Pertwee, R. Pharmacological actions of cannabinoids. Handb. Exp. Pharmacol. 2005, 168, 1–51. [Google Scholar] [CrossRef]
- Sánchez, C.; de Ceballos, M.L.; del Pulgar, T.G.; Rueda, D.; Corbacho, C.; Velasco, G.; Galve-Roperh, I.; Huffman, J.W.; Ramón y Cajal, S.; Guzmán, M. Inhibition of glioma growth in vivo by selective activation of the CB(2) cannabinoid receptor. Cancer Res. 2001, 61, 5784–5789. [Google Scholar]
- Ellert-Miklaszewska, A.; Grajkowska, W.; Gabrusiewicz, K.; Kaminska, B.; Konarska, L. Distinctive pattern of cannabinoid receptor type II (CB2) expression in adult and pediatric brain tumours. Brain Res. 2007, 1137, 161–169. [Google Scholar] [CrossRef]
- Calatozzolo, C.; Salmaggi, A.; Pollo, B.; Sciacca, F.L.; Lorenzetti, M.; Franzini, A.; Boiardi, A.; Brogli, G.; Marras, C. Expression of cannabinoid receptors and neurotrophins in human gliomas. Neurol. Sci. 2007, 28, 304–310. [Google Scholar] [CrossRef]
- Schley, M.; Ständer, S.; Kerner, J.; Vajkoczy, P.; Schüpfer, G.; Dusch, M.; Schmelz, M.; Konrad, C. Predominant CB2 receptor expression in endothelial cells of glioblastoma in humans. Brain Res. Bull. 2009, 79, 333–337. [Google Scholar] [CrossRef]
- Duntsch, C.; Divi, M.K.; Jones, T.; Zhou, Q.; Krishnamurthy, M.; Boehm, P.; Wood, G.; Sills, A.; Moore, B.M. Safety and efficacy of a novel cannabinoid chemotherapeutic, KM-233, for the treatment of high-grade glioma. J. Neurooncol. 2006, 77, 143–52. [Google Scholar] [CrossRef]
- Krishnamurthy, M.; Gurley, S.; Moore, B.M., II. Exploring the substituent effects on a novel series of C1'-dimethyl-aryl Delta8-tetrahydrocannabinol analogs. Bioorg. Med. Chem. 2008, 16, 6489–6500. [Google Scholar] [CrossRef]
- Aguado, T.; Carracedo, A.; Julien, B.; Velasco, G.; Milman, G.; Mechoulam, R.; Alvarez, L.; Guzmán, M.; Galve-Roperh, I. Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis. J. Biol. Chem. 2007, 282, 6854–6862. [Google Scholar]
- Howlett, A.C.; Barth, F.; Bonner, T.I.; Cabral, G.; Casellas, P.; Devane, W.A.; Felder, C.C.; Herkenham, M.; Mackie, K.; Martin, B.R.; Mechoulam, R.; Pertwee, R.G. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol. Rev. 2002, 54, 161–202. [Google Scholar] [CrossRef]
- Massi, P.; Vaccani, A.; Ceruti, S.; Colombo, A.; Abbracchio, M.P.; Parolaro, D. Antitumour effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. J. Pharmacol. Exp. Ther. 2004, 308, 838–845. [Google Scholar]
- Bisogno, T.; Hanus, L.; De Petrocellis, L.; Tchilibon, S.; Ponde, D.E.; Brandi, I.; Moriello, A.S.; Davis, J.B.; Mechoulam, R.; Di Marzo, V. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br. J. Pharmacol. 2001, 134, 845–852. [Google Scholar] [CrossRef]
- Ligresti, A.; Morello, A.S.; Starowicz, K.; Matias, I.; Pisanti, S.; De Petrocellis, L.; Laezza, C.; Portella, G.; Bifulco, M.; Di Marzo, V. Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. J. Pharmacol. Exp. Ther. 2006, 318, 375–387. [Google Scholar]
- Maccarrone, M.; Lorenzon, T.; Bari, M.; Melino, G.; Finazzi-Agrò, A. Anandamide induces apoptosis in human cells via vanilloid receptors. Evidence for a protective role of cannabinoid receptors. J. Biol. Chem. 2000, 275, 31938–31945. [Google Scholar] [CrossRef]
- De Petrocellis, L.; Vellani, V.; Schiano-Moriello, A.; Marini, P.; Magherini, P.C.; Orlando, P.; Di Marzo, V. Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8. J. Pharmacol. Exp. Ther. 2008, 325, 1007–1015. [Google Scholar] [CrossRef]
- Marcu, J.P.; Christian, R.T.; Lau, D.; Zielinski, A.J.; Horowitz, M.P.; Lee, J.; Pakdel, A.; Allison, J.; Limbad, C.; Moore, D.H.; Yount, G.L.; Desprez, P.Y.; McAllister, S.D. Cannabidiol enhances the inhibitory effects of delta9-tetrahydrocannabinol on human glioblastoma cell proliferation and survival. Mol. Cancer Ther. 2010, 9, 180–189. [Google Scholar]
- Recht, L.D.; Salmonsen, R.; Rosetti, R.; Jang, T.; Pipia, G.; Kubiatowski, T.; Karim, P.; Ross, A.H.; Zurier, R.; Litofsky, N.S.; Burstein, S. Antitumour effects of ajulemic acid (CT3), a synthetic non-psychoactive cannabinoid. Biochem. Pharmacol. 2001, 62, 755–763. [Google Scholar] [CrossRef]
- Pacher, P.; Bátkai, S.; Kunos, G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol. Rev. 2006, 58, 389–462. [Google Scholar] [CrossRef]
- Di Marzo, V. The endocannabinoid system: its general strategy of action, tools for its pharmacological manipulation and potential therapeutic exploitation. Pharmacol. Res. 2009, 60, 77–84. [Google Scholar] [CrossRef]
- Jacobsson, S.O.; Wallin, T.; Fowler, C.J. Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors. J. Pharmacol. Exp. Ther. 2001, 299, 951–959. [Google Scholar]
- Maccarrone, M.; Pauselli, R.; Di Rienzo, M.; Finazzi-Agrò, A. Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells. Biochem. J. 2002, 366, 137–144. [Google Scholar]
- Contassot, E.; Wilmotte, R.; Tenan, M.; Belkouch, M.C.; Schnüriger, V.; de Tribolet, N.; Burkhardt, K.; Dietrich, P.Y. Arachidonylethanolamide induces apoptosis of human glioma cells through vanilloid receptor-1. J. Neuropathol. Exp. Neurol. 2004, 63, 956–963. [Google Scholar]
- De Lago, E.; Gustafsson, S.B.; Fernández-Ruiz, J.; Nilsson, J.; Jacobsson, S.O.; Fowler, C.J. Acyl-based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations. J. Neurochem. 2006, 99, 677–688. [Google Scholar] [CrossRef]
- Guzman, M.; Galve-Roperh, I.; Sanchez, C. Ceramide: a new second messenger of cannabinoid action. Trends Pharmacol. Sci. 2001, 22, 19–22. [Google Scholar]
- Galve-Roperh, I.; Sanchez, C.; Cortes, M. L.; Gomez del Pulgar, T.; Izquierdo, M.; Guzman, M. Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nat. Med. 2000, 6, 313–319. [Google Scholar] [CrossRef]
- Rueda, D.; Galve-Roperh, I.; Haro, A.; Guzman, M. The CB1 cannabinoid receptor is coupled to the activation of c-Jun N-terminal kinase. Mol. Pharmacol. 2000, 58, 814–820. [Google Scholar]
- Campbell, V. A. Tetrahydrocannabinol-induced apoptosis of cultured cortical neurones is associated with cytochrome c release and caspase-3 activation. Neuropharmacology 2001, 40, 702–709. [Google Scholar] [CrossRef]
- Chan, G.C.; Hinds, T.R.; Impey, S.; Storm, D.R. Hippocampal neurotoxicity of Delta9-tetrahydrocannabinol. J. Neurosci. 1998, 18, 5322–5332. [Google Scholar]
- Salazar, M.; Carracedo, A.; Salanueva, I.J.; Hernández-Tiedra, S.; Lorente, M.; Egia, A.; Vázquez, P.; Blázquez, C.; Torres, S.; García, S.; Nowak, J.; Fimia, G.M.; Piacentini, M.; Cecconi, F.; Pandolfi, P.P.; González-Feria, L.; Iovanna, J.L.; Guzmán, M.; Boya, P.; Velasco, G. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J. Clin. Invest. 2009, 119, 1359–1372. [Google Scholar] [CrossRef]
- Vaccani, A.; Ceruti, S.; Colombo, A.; Abbracchio, M.P.; Parolaro, D. Antitumour effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. J. Pharmacol. Exp. Ther. 2004, 308, 838–845. [Google Scholar]
- Ellert-Miklaszewska, A.; Kaminska, B.; Konarska, L. Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad protein. Cell Signal. 2005, 17, 25–37. [Google Scholar] [CrossRef]
- Gomez del Pulgar, T.; Velasco, G.; Guzman, M. The CB1 cannabinoid receptor is coupled to the activation of proteinkinase B/Akt. Biochem. J. 2000, 347, 369–373. [Google Scholar] [CrossRef]
- Massi, P.; Valenti, M.; Vaccani, A:; Gasperi, V:; Marras, E.; Fezza, F.; Maccarrone, M.; Parolaro, D. 5-Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non-psychoactive cannabinoid. J. Neurochem. 2008, 104, 1091–1100. [Google Scholar] [CrossRef]
- Goncharov, I.; Weiner, L.; Vogel, Z. Delta9-tetrahydrocannabinol increases C6 glioma cell death produced by oxidative stress. Neuroscience 2005, 134, 567–574. [Google Scholar] [CrossRef]
- Massi, P.; Vaccani, A.; Bianchessi, S.; Costa, B.; Macchi, P.; Parolaro, D. The non-psychoactive cannabidiol triggers caspase activation and oxidative stress in human glioma cells. Cell Mol. Life Sci. 2006, 63, 2057–2066. [Google Scholar] [CrossRef]
- Lee, C.Y.; Wey, S.P.; Liao, M.H.; Hsu, W.L.; Wu, H.Y.; Jan, T.R. A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells. Int. Immunopharmacol. 2008, 8, 732–740. [Google Scholar] [CrossRef]
- Gustafsson, K.; Sander, B.; Bielawski, J.; Hannun, Y.A.; Flygare, J. Potentiation of cannabinoid-induced cytotoxicity in mantle cell lymphoma through modulation of ceramide metabolism. Mol. Cancer Res. 2009, 7, 1086–1098. [Google Scholar] [CrossRef]
- Sarker, K.P.; Obara, S.; Nakata, M.; Kitajima, I.; Maruyama, I. Anandamide induces apoptosis of PC-12 cells: involvement of superoxide and caspase-3. FEBS Lett. 2000, 472, 39–44. [Google Scholar] [CrossRef]
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