It is estimated that more than 23,000 individuals will be newly diagnosed with central nervous system (CNS) cancers in 2016 (Siegel et al., 2016). There are many distinct neuronal and glial tumor types that develop in the brain. Gliomas are a CNS tumor subtype that are derived from glial tissue and account for approximately 80% of all primary malignant brain tumors (Ostrom et al., 2015). The prognosis for this neoplasm is poor with an average 5-year survival rate of 5.1%. As such, novel strategies are needed to impact current therapeutic approaches. The ECS has been examined in clinical and preclinical glioma models. Several studies show that glioma tissues from rodents and humans express functional ECS components including the CB1 and CB2 receptors (Sánchez et al., 2001; Moreno et al., 2014). Furthermore, CB2 expression was found to be elevated in high grade gliomas (Sánchez et al., 2001; Ellert-Miklaszewska et al., 2007) Hence, a series of informative investigations primarily conducted by the Guzman group, have examined the effects of cannabinoids on glioma cell survival with the goal of assessing the feasibility of developing these agents as novel anti-neoplastics.

Numerous studies indicate that Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is a potent inducer of glioma death in vivo. Utilizing a C6 glioma cell xenograft model, it was demonstrated that tumor growth was significantly reduced by Δ⁹-THC (Galve-Roperh et al., 2000). Subsequent to orthotopic implantation of the C6 cells the overall survival of Δ⁹-THC treated animals was also increased. In vitro studies with the C6 cell line suggested that the Δ⁹-THC-induced reduction in tumor growth occurred as a consequence of increased ceramide synthesis and Erk activation. Another study showed that Δ⁹-THC reduced the growth of U87MG glioma xenografts (Salazar et al., 2009). Experimentation in the U87MG cell line determined that ceramide accumulation and the induction of ER stress proteins p8, CHOP10, and TRB3, were required for Δ⁹-THC-induced death. The authors further demonstrated the importance of p8 in tumor growth by utilizing transformed mouse embryo fibroblasts (MEF) in which tumor development was reduced in p8^+/+ animals treated with Δ⁹-THC compared to vehicle treated animals. In contrast, no significant difference in tumor growth was observed in Δ⁹-THC or vehicle treated p8^−/− mice suggesting that p8 was needed for Δ⁹-THC-mediated tumor death. In a subsequent study by this group, endoplasmic reticulum stress-mediated autophagy and cell death was observed in Δ⁹-THC treated glioma. Specifically, Δ⁹-THC caused an increase in ER stress protein, TRB3, autophagy protein, LC3, and apoptosis as well as a decrease in mTORC protein, p-S6 in U87MG xenograft tumors (Salazar et al., 2009). This pattern of protein expression was also observed in glioma biopsies from 2 patients with glioblastoma multiforme treated via intracranial administration with Δ⁹-THC. In vitro experiments with the U87MG cell line suggested that Δ⁹-THC mediated activation of TRB3 was a critical link between the ER stress and autophagy pathways as TRB3 activation was needed for inactivation of Akt and the mTORC1 complex. Other reports indicated that the administration of Δ⁹-THC improves the therapeutic outcome of the clinically utilized glioma anti-neoplastic, temezolamide (TMZ) (Torres et al., 2011). In this study, Δ⁹-THC significantly reduced the growth of U87MG xenograft tumors treated with TMZ when compared to TMZ or Δ⁹-THC alone. Similarly, administration of Δ⁹-THC + cannabidiol (CBD) or Δ⁹-THC + CBD + TMZ significantly reduced tumorigenesis compared to the individual drugs (Torres et al., 2011). The combined drug regimen was associated with prevalent LC3 expression and cell death in U87MG tumors. The mechanism of cell death was also examined in cultured U87MG cells where inhibition of ceramide synthesis and autophagy prevented cellular cytotoxicity (Torres et al., 2011). Since each of these studies also determined that the cytotoxic effect of Δ⁹-THC was dependent on cannabinoid receptors, the findings of these reports indicate that ceramide synthesis, ER stress, autophagy and cannabinoid receptor activation are likely shared mechanism of Δ⁹-THC-induced glioma death.

Cannabinoid regulation of matrix remodeling proteins may also play a role in its anti-tumor activity. Matrix metalloproteinases (MMP) can promote or prevent tumor invasion and metastasis by degrading extracellular matrix and other cell-associated proteins (Lukaszewicz-Zajac et al., 2014). In addition, the effects of MMPs are counteracted by tissue inhibitors of metalloproteinases (TIMPs). MMPs/TIMPs also directly regulate tumor growth, tumor apoptosis and angiogenesis. An examination of the effect of Δ⁹-THC on C6.9 cell xenograft growth showed that animals treated with Δ⁹-THC contained smaller tumors with reduced MMP-2 expression in contrast control group animals (Blázquez et al., 2008a). Δ⁹-THC also reduced tumor growth and the expression of MMP-2 in tumors of Δ⁹-THC-treated patients. Cell culture studies with C6.9 cells confirmed that MMP-2 expression and cell viability were decreased as a result of Δ⁹-THC exposure (Blázquez et al., 2008a). A different study by this research group also indicated selective participation of TIMP-1 in the anti-neoplastic activity of Δ⁹-THC (Blázquez et al., 2008b). Similar to MMPs, TIMPs have both a positive and negative impact on tumor growth (Lukaszewicz-Zajac et al., 2014). In C6.9 xenografts, Δ⁹-THC decreased tumor development and tumoral TIMP-1 but had little impact on TIMP-2 or TIMP-3 expression. Selective TIMP-1 downregulation was also observed in patient tumor samples subsequent to Δ⁹-THC administration. C6.9 cell culture studies showed that Δ⁹-THC decreased TIMP-1 expression by increasing ER stress and ceramide synthesis and also demonstrated that Δ⁹-THC reduced C6.9 cell migration (Lukaszewicz-Zajac et al., 2014). These studies suggest that downregulation of MMP-2 and TIMP-1 is important for Δ⁹-THC regulation of tumor size, however additional work is needed to reveal whether the reduction in these matrix modulating proteins also alters the metastatic and invasive behavior of tumor cells in vivo.

An investigation of the effects of Δ⁹-THC on human glioblastoma-T98G xenografts demonstrated concentration-dependent effects with low Δ⁹-THC concentrations causing a modest increase in tumor growth and high Δ⁹-THC concentrations promoting tumor cell death (Sánchez et al., 2001). Experiments in cultured T98G cells suggested that this effect was mediated by the formation of a functional dimeric complex between GPR55 and CB2 receptors such that low concentrations of Δ⁹-THC activated the CB2 receptor and Erk expression thereby promoting tumor cell growth. However, in the presence of high Δ⁹-THC concentrations, Δ⁹-THC inhibited GPR55 which blocked the CB2 receptor and Erk activation thereby inhibiting tumor cell growth. This finding is consistent with reports showing that the putative G-protein-coupled cannabinoid receptor, GPR55, is overexpressed in cancers and promotes carcinogenesis (Falasca and Ferro, 2016).

Most clinically available cancer chemotherapeutic agents demonstrate significant toxicity toward cancer and normal cells. This lack of selectivity often produces undesirable side effects that limit its therapeutic utility. Δ⁹-THC preferentially induced cell death in tumorigenic compared to non-tumorigenic astrocytes (Salazar et al., 2009). Moreover, in non-tumor bearing rats, Δ⁹-THC (2500 μg) exhibited limited toxicity and did not affect animal survival, health, behavior or hematopoietic parameters (Galve-Roperh et al., 2000). These findings suggest that Δ⁹-THC will cause limited adverse effects thereby making this compound an attractive candidate for therapeutic use. Furthermore, the anti-emetic, appetite stimulating and analgesic effects of Δ⁹-THC and other cannabinoids can provide relief for important co-morbidities commonly associated with cancer and cancer chemotherapy.

Cannabidiol (CBD) is a non-psychotropic phytocannabinoid whose effect on tumor growth is being extensively explored. CBD significantly reduced U87MG xenograft growth when compared to vehicle control animals (Massi et al., 2004). An in vitro examination of the mechanism of action suggested that the induction of reactive oxygen species (ROS) and activation of the CB2 receptor was involved in the anti-tumor effect. However, unlike Δ⁹-THC, CBD toxicity did not appear to be regulated by CB1 or ceramide. Furthermore, in an orthotopic and subcutaneous tumor model, CBD significantly decreased U251 cell tumor growth (Soroceanu et al., 2013). In tumors generated by both methods, the expression of the proliferation marker, Ki67, and inhibitor of DNA binding (Id-1) protein were reduced. Id-1 is a transcription factor that prevents tumor cell differentiation and its expression is elevated in highly aggressive tumors (Ling et al., 2006). Cell culture studies showed that CBD inhibited Id-1 expression which occurred coincident with a reduction in U251 cell invasiveness (Soroceanu et al., 2013). A follow up study by this group demonstrated that CBD inhibited tumor growth and increased the survival of animals orthotopically implanted with primary 3832 and 387 glioblastoma cells (Singer et al., 2015). The reduction in tumor size was accompanied by decreased expression of Id-1 and Sox-2 indicating a reduction in tumor aggressiveness. Continued administration of CBD in this model resulted in reduced responsiveness to CBD, increased antioxidant response gene expression and upregulated expression of mesenchymal markers in the tumors. Investigation of the in vitro mechanism suggested that resistance to CBD was primarily caused by activation of SCL7A11 (a transcriptional target of Nrf2) antioxidant signaling as inhibition of this target decreased tumor cell survival and augmented the antitumor activity of CBD. A different study revealed that CBD potentiated the killing effect of ionizing radiation (IR), a common clinical therapy for glioblastoma. Using an orthotopic xenograft model, CBD + Δ⁹-THC + IR significantly decreased the size of GL261 gliomas compared to CBD + Δ⁹-THC or IR alone (Scott et al., 2014). Increased TUNEL positive cells in tumors isolated from animals exposed to CBD + Δ⁹-THC + IR implicated cell death as the mechanism of tumor growth inhibition. Thus, the antitumor activity of Δ⁹-THC and CBD has been demonstrated in different in vivo tumor models and the diverse pathways by which these agents elicit death appear to favor co-administration with other chemotherapeutic agents.

Numerous studies suggest that synthetic cannabinoids such as JWH-133, WIN 55,212-2 and HU-210 are promising chemotherapeutic agents for treatment of gliomas. In murine C6 xenografts, the growth of glioma solid tumors was significantly inhibited by JWH-133 compared to control group animals (Sánchez et al., 2001). This effect was found to be mediated by the CB2 but not the CB1 receptor. In the same animal model, JWH-133 also inhibited the growth of human glioma cells. Examination of the mechanism of cytotoxicity in cultured C6 cells showed that ceramide synthesis was needed for JWH-133-mediated tumor cell death. Later studies by this group validated the in vitro mechanism by demonstrating that blockade of ceramide synthesis in vivo decreased the anti-tumor activity of JWH-133 (Blázquez et al., 2008a,b). These studies also revealed that ceramide was involved in JWH-133-induced suppression of the invasion associated proteins, MMP-2 and TIMP-1. The combined reports suggest that the induction of ceramide synthesis by JWH-133 causes a reduction tumor cell survival and invasion.

The effect of synthetic cannabinoids on angiogenesis was also examined. JWH-133 reduced the growth of C6 gliomas that was accompanied by a decrease in tumoral, pro-angiogenic VEGF and VEGFR (Blázquez et al., 2004). It was determined that VEGFR levels were also downregulated in glioma biopsies from patients treated with Δ⁹-THC. In vitro studies with C6 cells demonstrated that both JWH-133 and WIN 55,212-2 decreased VEGFR2 phosphorylation and VEGF levels by increasing ceramide production. The reduction in VEGF signaling by the cannabinoids was also dependent on CB1 and CB2 receptors. A more detailed examination of the effect of cannabinoids on angiogenesis showed that JWH-133 decreased the permeability of tumor localized blood vessels as well as the growth of C6 and patient-derived glioma cells in xenograft tumor studies (Blázquez et al., 2003). Consistent with these results, the mRNA expression of the angiogenesis and metastasis-promoting proteins VEGF, Ang2 and MMP-2 were decreased by JWH-133. Furthermore, in vitro studies using HUVEC cells demonstrated that JWH-133 inhibited endothelial cell migration and initiated death that was reversed by antagonism of CB1 and CB2 receptors. The findings of these reports indicate that cannabinoid prevention of angiogenesis is reliant on ceramide production and cannabinoid receptor activation.

Accumulating evidence suggests that a subpopulation of brain tumors originate from pluripotent stem-like cells (Singh et al., 2004). These undifferentiated cells are often associated with poor prognosis and highly aggressive tumor phenotypes whereas well-differentiated glial cells are typically less aggressive (Wan et al., 2011). As such, the stem-like population of U87MG cells and human gliomas were treated with JWH-133 or HU-210 and then subcutaneously implanted to examine the effect of cannabinoids on this tumor cell subtype (Aguado et al., 2007). Cannabinoid treated cells formed smaller tumors which contained low levels of stem-like stage proteins such as nestin and high levels of differentiation-associated proteins including β-tubulin III and S-100β. Consistent with these findings, JWH-133 and HU-210 decreased the expression of nestin and increased the expression of GFAP, S-100β and β-tubulin III in cultured stem-like glioma cells. Hence it appears that the anti-neoplastic activity of the synthetic cannabinoids may be attributed to their ability to prevent tumor cell survival, invasion, and angiogenesis while promoting tumor cell differentiation.

KM-233 is a synthetic cannabinoid that signals through CB1 and CB2 receptors and is a derivative of Δ⁸-THC (Krishnamurthy et al., 2008). The anti-neoplastic activity of KM-233 was examined in an orthotopic U87MG cell tumor protocol where a significant reduction in tumor size occurred (Gurley et al., 2012). These results were verified in a xenograft tumor study using patient-derived glioblastoma multiforme tissue. In vitro studies showed that KM-233 induced U87MG death was prevented by antagonizing the CB1 receptor. A subsequent study by this group confirmed the anti-tumor activity of KM-233 against U87MG xenograft tumors and also demonstrated that KM-233 produced minimal damage to neuronal tissue within the therapeutic dosage range (Duntsch et al., 2006). As such, KM-233 may be an effective agent against glioma with an acceptable safety profile similar to other cannabinoids.

The bulk of our knowledge about the effect of cannabinoids on cancer growth in in vivo models is derived from the aforementioned glioma studies. Much of this work demonstrated potent antitumor activity, confirmed in vitro mechanisms of cannabinoid antitumor action and provided evidence that these molecules were minimally toxic and therefore paved the way for execution of a clinical study with a small number of glioma patients (Clinical trial ID# NCT01812603). However, additional experimentation in animals is needed to define the pharmacokinetic properties of cannabinoids before large scale human studies are conducted. It will be particularly important to examine drug accumulation in tumors and drug disposition in body compartments subsequent to drug administration via different routes as most of the glioma studies conducted thus far employ intra/peri-tumor drug delivery. Such investigations may identify more clinically convenient routes of administration, establish the extent of drug stability and metabolism while providing clues about potential adverse effects of these agents.