The Cytotoxic Effects of Cannabidiol and Cannabigerol on Glioblastoma Stem Cells May Mostly Involve GPR55 and TRPV1 Signalling

Abstract

Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60-70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.

Keywords: cannabidiol; cannabigerol; cannabinoid receptors; glioblastoma; glioma; stem cells.

Figure 1

Receptor mRNA levels in different types and grades of glioma samples. The expression of (A) CNR1 (CB1), (B) GPR55, and (C) TRPV1 in glioma, non-cancerous brain tissues, and GBM cells. mRNA values were normalized to the housekeeping genes HPRT1 and GAPDH and analysed with quantGenius software (developed at NIB). n: number of samples; N: non-cancerous brain tissues; LGG: grade I or II gliomas (pilocytic astrocytoma, astrocytoma, oligodendroglioma); GBM: glioblastoma; GBM rec: recurrent glioblastoma; GBM cells: primary glioblastoma cells; GSCs: glioblastoma stem cells isolated from patient tumour samples; NA: normal astrocytes versus N: the non-cancerous brain tissues (* p < 0.05).

Figure 2

Correlation of cannabinoid receptors with glioma patient survival. (A) Graphical representation of the correlation matrix for the correlations between the expression of the receptors GPR55 (n = 113), CNR1 (n = 129), and TRPV1 (n= 116) and glioma sample type and overall glioma patient survival (months). The correlations range from −1 (red dots) to 1 (blue dots). The closer the values are to 0, the more uncorrelated the variables are. (B) Estimation of the regression model between patient survival and the expression of three cannabinoid receptors: GPR55 (a.; n = 106.), CNR1 (b.; n = 120), and TRPV1 (c.; n = 109). R: the correlation coefficient between the analysed variables; p: statistical p-value (considered significant when p < 0.05).

Figure 3

Correlation matrix and overall survival curves for cannabinoid receptor expressions in GBMs. (A) Correlation matrix data show positive significance between GBM patient (n = 89) survival and GPR55 (p = 0.042) and TRPV1 (p = 0.049) receptor gene expressions. (B,C) Kaplan–Meier survival curves of GBM patients (n = 89) stratified by three cut-off levels of GPR55 (B) and TRPV1 (C) mRNA expression. (D) Kaplan–Meier survival curves of GBM patients, as analysed from the TCGA data bank GLIOVIS with GBM patients (n = 152) with GPR55 mRNA expression level data. Survival at median value was not significant. GBM: glioblastoma. * p < 0.05.

Figure 4

Correlations between the expressions of cannabinoid receptors and GSC markers in glioblastoma. Stemness genes are framed in red below. Correlations with cannabinoid receptors are framed in red above. GPR55 significantly correlated with the GSC markers SOX2, CD15, ID1, and OCT4. TRPV1 significantly correlated with NOTCH; OLIG2; and the putative markers CD9, TRIM28, and TUFM (the latter three of which are potential candidates to be classified as established stem cell markers [32,33,34]). CNR1 only correlated (poorly) with TRIM28. * p < 0.05.

Figure 5

Correlation matrix of GPR55, CNR1, and TRPV1 with MES, CL, and PN markers. Behnan et al. [38] suggested the following markers: (A) MES (COL1A2, COL1A, TGFBI, THBS1, DAB2, and S100A4), (B) CL (ACSBG1 and KCNF), and (C) PN (P2RX7, STMN4, SOX10, and ERBB3). GPR55 correlated with MES markers COL1A2 (r = 0.24) and S100A4 (r = 0.55), very strongly with CL marker KCNF1 (r = 0.96), and poorly with PN marker ERBB3 (r = 0.33). CNR1 did not markedly correlate with any of these markers. TRPV1 correlated with MES markers DAB2 (r = 0.46) and TGBF1 (r = 0.35), CL marker ASCBG1 (r = 0.37), and most highly with PN markers P2RX7 (r = 050) and STMN4 (r = 045). The star under the correlation number (r) between the two genes in the boxes, means that the correlation was significant ( p < =0.05) n ^® (* p < 0.05).

Figure 6

The cannabidiol (CBD) and cannabigerol (CBG) reduce viability of glioblastoma (GBM) and GBM stem cells (GSCs). CBD (A,C) and CBG (B,D) decreased the viability of ten differentiated GBM cells and eight GSCs. Dose responses of cell viability measured by MTT assay (y-axis) and different CBD and CBG concentrations increasing in the range of 0.32–320 µM (x-axis log scale) on established GBM cells (U373 line) (A, B; red line) in comparison with patient-derived primary GBM cells (A, B; black lines). Established GSC lines NCH644k (C, D; red line) are compared with patient-derived primary GSCs (C, D; black lines) after 48 h of single treatment with cannabinoids. The solvents base emulsion or DMSO (≤0.4%, v/v) were used as controls. Data are expressed as mean ± SE (n = 3–5 independent biological experiments, each with technical triplicates). Vehicles comprised ≤0.1% base emulsion.

Figure 7

Cytotoxicity of cannabidiol (CBD) and cannabigerol (CBG) mixtures on glioblastoma (GBM) cells and GBM stem cells (GSCs). Mean IC50 values for GSCs (n = 8) and differentiated GBM cell lines (n = 10) treated with CBD and CBG (at a molar ratio of 3:1) taken from Table 2 are presented as bars of the mean cytotoxic effects, expressed in mean CBD and CBG absolute concentrations. The paired t-test was used to evaluate statistical difference (* p < 0.0105, ** p < 0.0094).

Scheme 1

The cannabinoids cannabidiol (CBD) and cannabigerol (CBG) affect glioblastoma tumour differentiated d-glioblastoma cells and glioblastoma stem cells (GSCs).