We observed that plant derived cannabinoids inhibit the proliferation of human glioblastoma cell lines. Compared to Δ9
-THC, CBD was significantly more potent than Δ9
-THC at inhibiting cancer cell growth. This finding is in agreement with studies using models of aggressive breast cancers (26
-THC is currently being used in a clinical trial for treatment of recurrent glioblastoma (5
). Past studies have suggested that non-psychoactive cannabinoids can modulate the actions of Δ9
). We hypothesized that the cannabinoid therapy utilizing Δ9
-THC alone could be improved using a strategy of combination treatments.
We discovered that CBD enhanced the ability of Δ9-THC to inhibit cell proliferation and induce cell cycle arrest and apoptosis. This activity occurred in two of three glioblastoma cell lines tested. Treatment of U251 cells with the combination led to a substantial down-regulation of ERK activity, but not p38 MAPK and JNK1/2. The reduction in ERK activity was specific for the combination treatment and occurred in more than one glioblastoma cell line. Importantly, continuing to increase the concentration of Δ9-THC alone did not result in inhibition of ERK activity. This data indicates that the enhanced effects observed were not solely due to an increase in potency of Δ9-THC in U251 cells upon co-application with CBD. Further support for this conclusion was observed when studying the activity of Δ9-THC and CBD on U251 cell invasiveness. Both compounds were effective at inhibiting the invasiveness of U251 cells, however, there was no evidence that CBD improved the activity of Δ9-THC upon co-application.
In human glioblastoma cells, the ability of Δ9
-THC to inhibit growth and induce apoptosis has been linked to the initial activation of CB1
). Similar effects produced by CBD have been linked in part to CB2
receptor activation, but the initial interaction site for the additional activity of CBD remains to be clarified (7
). We observed that increases in apoptosis produced by Δ9
-THC alone, or the combination of Δ9
-THC and CBD, were partially dependent on CB2
receptor activation. Apoptosis produced by CBD alone was not dependent on CB2
receptor activation. Importantly, the induction of apoptosis in the presence of the combination treatment was significantly greater than that observed with Δ9
-THC alone. Apoptosis produced by the combination of Δ9
-THC and CBD was dependent on the production of oxidative stress and resulted in a unique activation of both intrinsic and extrinsic caspases.
Studies have shown that the inhibitory activity of cannabinoids in glioblastoma is dependent on activation of CB1
receptors, modulation of MAPKs and induction of multiple types of cellular stresses leading to apoptosis (32
). In the case of Δ9
-THC, up-regulation of p8 appears to be a specific event which leads to apoptosis in multiple types of cancers (35
). Treatment of U251 cells with the combination of Δ9
-THC and CBD led to an up-regulation of p8 expression, but similar activity was seen with Δ9
-THC alone. This is in contrast to what we observed when studying modulation of caspase activity, and suggests that the enhanced apoptotic activity produced by the combination treatment was not the results of an interaction with the p8 pathway.
The ability of CBD to inhibit growth and induce apoptosis in glioblastoma and additional cancers has been primarily associated with the up-regulation of ROS and multiple caspases, and has been linked to alterations in NADPH oxidases (9
). A link between ROS production and modulation of the LOX pathway has been hypothesized as a potential mechanism of antitumor activity of CBD in glioblastoma (16
). In this study, an initial increase in ROS was clearly linked to a latter induction of apoptosis. Individually, both Δ9
-THC and CBD could increase apoptosis through the production of ROS, however, Δ9
-THC was significantly less efficient at inducing this process as a single agent as compared to when it was used in combination with CBD. Even though the concentration of CBD used in the combination treatment did not significantly stimulate ROS, it may have primed this pathway for Δ9
-THC through a convergence on shared signal transduction pathways. A similar hypothesis could explain the unique down-regulation of phosphorylated ERK that was produced by the combination treatment. Alternatively, CBD may have potentiated the activity of Δ9
-THC by inhibiting pathways that impart drug resistance in glioblastoma. For instance, a recent study showed that amphiregulin expression was associated with increased ERK activation, which mediated resistance to THC-induced apoptosis in gliomas (40
). Therefore, CBD may have potentiated the activity of THC-induced apoptosis by inhibiting amphiregulin regulated increases in ERK activation. Future studies will be needed in order to elucidate the detailed mechanism associated with the unique effects of the Δ9
-THC and CBD combination treatment.
-THC and CBD can activate distinct pathways in glioblastoma cells that ultimately culminate in inhibition of cancer cell growth and invasion as well as induction of cell death (2
). We hypothesized that, if the individual agents were combined, a convergence on shared pathways may ensue leading to an enhanced ability of the combination treatment to inhibit certain cancer cell phenotypes. We found this to be true in this investigation. Cannabidiol significantly improved the inhibitory effects of Δ9
-tetrahydrocannabinol on glioblastoma cell proliferation and survival, but not on cell invasiveness. The data suggests that the improved activity observed with the combination treatment is the result of a specific modulation of ERK and ROS activity leading to inhibition of cell cycle and induction of apoptosis.
Combinations, compared to individual drug treatments with specific cannabinoid-based compounds, may represent an improvement for the treatment of patients with glioblastoma and perhaps additional cancers. It is also possible that other constituents of Cannabis sativa which are not structurally related to cannabinoids could improve antitumor activity when combined. An important next step will be to perform studies testing for synergistic antitumor activity of cannabinoids in additional preclinical models of glioblastoma. Even if synergism is not evident, combination treatments may allow for increased dosing due to non-overlapping toxicities and decrease development of resistance to the activity of Δ9-THC or CBD when administered alone. With the growing evidence demonstrating cannabinoids are effective inhibitors of multiple types of cancer, it is likely that additional clinical trials will be carried out. Combination treatments with cannabinoids may improve overall efficacy in these future clinical trials.