 but additional administration of submaximal doses of CBD induced glioma cell death through an altogether different mechanism. However, these authors have used botanical drug cannabinoids, and not purified molecules in vivo after human U87 and T98 subcutaneous injections to nude mice; therefore, the two studies are not comparable.
Finally, we explored whether combined treatment affects glioma cellular invasion, which is a highly relevant hallmark of GB progression. Successful anti-invasive therapy would not only prevent primary tumour spread that would better target GB cells, but may also prevent relapse following surgical resection. The anti-invasive effects of CBD on cancer cells have been demonstrated in numerous publications [15,65]. Here, we have demonstrated for the first time that subtoxic concentrations of CBG inhibit the invasion of GB and GSC spheroids (Figure 7). Different GB cell types (U87, T98, and U373) significantly differ in their response to the anti-invasive properties of CBG [30]. These differences may be due to the different genetic/molecular subtypes that we demonstrated to exist between U87 and U373 cells [38], and/or different levels of cannabinoid receptors in these lines [26,66]. Screening for the presence of the two major cannabinoid-selective receptors CB1 and CB2 in a plethora of GB lines and the two GSCs by semiquantitative evaluations using immunolabelling of the respective primary in vitro cell cultures, we observed that both types of the cells express both kinds of receptors, justifying patient treatment with cannabinoids. Secondly, as CB1 and CB2 differ in their expressions among patients, different responses may be expected; what explains the observed variability is their primary glioblastoma cells. Noteworthily, other non-specific ionotropic receptors may also mediate cannabinoid signaling, as reviewed by De Petrocellis et al. [26]. The mechanism of CBG inhibition of invasion may be similar to results from Soroceanu et al. [59], attributing this to the inhibition of transcription factor Id-1 expression, regulating GB invasion by CBD, and was observed in several GB cell lines, in the ex-vivo primary GB cells and in an orthotopic xenograft murine model. Alternatively, CBD treatment of GB cells may significantly downregulate metalloproteases-associated proteolytic systems. This needs to be confirmed in CBG treatment, as in our case the invasion inhibition seems to be similar as observed with CBD [30]. Additionally, upon cannabinoid binding, CB1 and CB2 may interact with GB receptors that are related to their invasion activity. For example, Nabissi et al. [62] have demonstrated that the CBD:THC combination reduced myeloma cell migration by down-regulating the expression of the important chemokine receptor CXCR4 that, along with its ligand SDF-1α (CXCL12), regulates in particular GSC homing to their niches [67] and critically affects GB progression. Coke et al. [68] reported that a CB2 agonist specifically reduced CXCR4-mediated migration. Clinically, CXCR4 expression in tumours is used to predict cancer aggressiveness [43], and the CXCR4 blocking ligand plerixafor successfully inhibited the progression of acute myeloid leukaemia (Aml I-a) in a clinical setting [69].
Interestingly, we found that the chemotherapeutic drug TMZ at 400 µM reduced the invasion of U87 cells by approximately 50%, U373 cell invasion by 70 %, and T98 cell invasion by 80%. Conversely, in the GSC line, NCH421k TMZ treatment highly increased GSC invasion (Figure 7D). These preliminary, though consistent, data are difficult to interpret. TMZ is known to methylate nuclear DNA guanine residues at several positions via complex mechanisms, resulting in cytostatic G2/M cell cycle arrest and autophagy-induced apoptosis, depending on the cell context [70], which may not be detected by MTT assays alone. Of note, TMZ also potentially alkylates mitochondrial DNA and RNA, as well as proteins and lipids carrying nucleophilic groups, acting at post-transcriptional/post-translational levels. Although TMZ is a standard chemotherapeutic, used in the treatment of GB patients at much lower concentrations (14.95–34.54 μM) [70], highly different concentrations (100 and 4000 μM) were required to be effective in the in vitro models. Altogether there is still much debate surrounding TMZ’s mechanisms and its efficacy in clinical use [9], taken together, our data corroborate the statement by Deng et al. [41] on the dichotomy of the antiproliferative and apoptotic effects of TMZ in GB.
This is the first report to demonstrate that the non-intoxicating cannabinoid CBG alone and in combination with CBD efficiently targets two key elements that otherwise prevent the successful treatment of GB patients with current therapeutics: Firstly, to overcome GSC resistance to cytotoxic agents and to induce apoptosis, and secondly, to inhibit GB cell invasion. Our findings on the effects of cannabinoids on GSCs are consistent with previous observations that demonstrate that cannabinoid receptor agonists promote glial differentiation by altering the expression of genes involved in the regulation of stemness. As such, differentiated GB cells were also exposed to the cytotoxic, proapoptotic, and anti-invasive effects of CBG. We have demonstrated that the combination of CBG and CBD, each at sub-cytotoxic concentrations, results in additive effects on reduced cell viability and induced apoptosis, which are sufficient to replace the application of THC. Formulations containing THC are thus not needed and could be avoided due to the psychoactive activity of THC that is particularly harmful to GB patients with neurological distortions associated with tumour progression. Furthermore, besides exerting clear antitumour effects, CBG and CBD also exert known palliative off-target effects, such as analgesia, increased appetite, and the prevention of chemotherapy-induced cachexia and nausea.
The following are available online at https://www.mdpi.com/2073-4409/10/2/340/s1, Figure S1: Biosynthesis and metabolism of cannabigerol (CBG), Figure S2: Cell death determination by flow cytometry using Annexin V-FITC and propidium iodide staining, Table S1: High-performance liquid chromatography purity results for the CBG, CBD, and THC solutions used in this study.
Conceptualization, T.T.L., M.N. (Metka Novak), M.M. and M.N. (Massimo Nabissi); Data curation, M.N. (Metka Novak), B.B. and M.N. (Massimo Nabissi); Formal analysis, M.N. (Metka Novak), M.A.P.A., O.M., B.Ž.B., B.M., B.B. and M.N. (Massimo Nabissi); Investigation, B.Ž.B. and M.N. (Massimo Nabissi); Methodology, M.N. (Metka Novak), M.A.P.A., O.M., B.Ž.B., B.M., M.M. and M.N. (Massimo Nabissi); Resources, Roman Bošnjak and Roby Zomer; Supervision, T.T.L., M.N. (Metka Novak) and B.B.; Visualization, B.M.; Writing—original draft, T.T.L., M.N. (Metka Novak) and B.B.; Writing—review & editing, T.T.L. and M.N. (Metka Novak). All authors have read and agreed to the published version of the manuscript.
This work was supported by the Slovenian Research Agency (Grant Program P1-0245 to T.T.L., post-doctoral project Z3-1870 to B.B.) and by the European Program of Cross-Border Cooperation for Slovenia-Italy Interreg TRANS-GLIOMA (to T.T.L). This work was also supported by MGC Pharmaceuticals.
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the the National Medical Ethics Committee of the Republic of Slovenia (approval No. 0120-179 190/2018/4).
Informed consent was obtained from all patients, whose tumours were involved in the study.
We thank Eva Lasič and Stephen Minger for carefully editing this manuscript. We thank Andrej Porčnik from the Department of Neurosurgery, University Medical Centre, Ljubljana, for providing the tumour samples. We also highly appreciate Jernej Mlakar from the Institute of Pathology, Medical Faculty, University of Ljubljana, for providing glioblastoma tissue sections.
The author T.T.L. received research grants from MGC Pharmaceuticals; however, the sponsors had no decisive role in the execution, interpretation, or writing of the study.
Figure 1. Cannabinoids reduce viability of GB and GSC cells. CBG (A,D), CBD (B,E), and THC (C,F) impaired the viability of differentiated GB cells and GSCs. Dose responses of cell viability measured by MTT assay (y-axis) and different CBG, CBD and THC concentrations increasing in the range of 0.1–50 µM (x-axis log scale) on established GB cells (U87 and U373) (A–C; red lines) in comparison to primary patient-derived GB cells (A–C; black lines). Established GSC lines NCH421k and NCH644 (D–F; red lines) are compared to primary patient-