An Examination of the Anti-Cancer Properties of Plant Cannabinoids in Preclinical Models of Mesothelioma

Abstract

Mesothelioma is an aggressive cancer with limited treatment options and a poor prognosis. Phytocannabinoids possess anti-tumour and palliative properties in multiple cancers, however their effects in mesothelioma are unknown. We investigated the anti-cancer effects and potential mechanisms of action for several phytocannabinoids in mesothelioma cell lines. A panel of 13 phytocannabinoids inhibited growth of human (MSTO and H2452) and rat (II-45) mesothelioma cells in vitro, and cannabidiol (CBD) and cannabigerol (CBG) were the most potent compounds. Treatment with CBD or CBG resulted in G0/G1 arrest, delayed entry into S phase and induced apoptosis. CBD and CBG also significantly reduced mesothelioma cell migration and invasion. These effects were supported by changes in the expression of genes associated with the cell cycle, proliferation, and cell movement following CBD or CBG treatment. Gene expression levels of CNR1, GPR55, and 5HT1A also increased with CBD or CBG treatment. However, treatment with CBD or CBG in a syngeneic orthotopic rat mesothelioma model was unable to increase survival. Our data show that cannabinoids have anti-cancer effects on mesothelioma cells in vitro and alternatives of drug delivery may be needed to enhance their effects in vivo.

Keywords: anti-proliferative; apoptosis; cannabidiol; cannabigerol; cannabinoids; mesothelioma.

Figure 1

Phytocannabinoids reduce the proliferation of mesothelioma cell lines. The drug concentration causing 50% growth inhibition (IC50) of (a) ∆⁹-tetrahydrocannabinol (THC) related, (b) cannabidiol (CBD) related, and (c) cannabichromene (CBC) and cannabigerol (CBG) related phytocannabinoids against rat II-45 and human MSTO and H2452 mesothelioma cell lines were determined. Cell viability was assessed using MTT assays in the presence of phytocannabinoid as indicated. Bars show the mean and standard deviation (SD) from at least three independent experiments.

Figure 2

The phytocannabinoids cannabidiol (CBD) and cannabigerol (CBG) induce cell cycle arrest in mesothelioma cell lines. The effects of CBD and CBG (2xIC50) on cell cycle were assessed in rat II-45 (a,b), human MSTO (c,d) and H2452 (e,f) mesothelioma cell lines. After 24 h of treatment with vehicle (control), CBD or CBG, cells were harvested, and assays performed. Bars show the mean and SD from three independent experiments. p-values were calculated using two-way ANOVA Holm-Sidak’s multiple comparisons. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 relative to vehicle-treated control cells.

Figure 3

The phytocannabinoids cannabidiol (CBD) and cannabigerol (CBG) induced apoptosis in mesothelioma cell lines. The effects of CBD and CBG (2xIC₅₀) on apoptosis were assessed in rat II-45 (a), human MSTO (b), and H2452 (c) mesothelioma cell lines. After 24 h of treatment with vehicle (control), CBD or CBG, cells were harvested, and assays performed. Bars show the mean and SD from three independent experiments. p-values were calculated using two-way ANOVA Holm-Sidak’s multiple comparisons. * p < 0.05 relative to vehicle-treated control cells.

Figure 4

Cannabidiol (CBD) and cannabigerol (CBG) reduced migration and invasion of mesothelioma cell lines. Rat II-45 (a,d) and human MSTO (b) and H2452 (c,e) mesothelioma cell lines were examined with CBD and CBG being tested at sub-cytotoxic concentrations (2 µM CBD for II-45, 6 µM for MSTO and H2452 cells, and 10 µM CBG for all cell lines). The number of migrated cells or cells that had invaded through the Matrigel per 5–10 fields of view was counted and analysed using CellProfiler. Bars show the mean and SD from three independent experiments. p-values were calculated using one-way ANOVA Kruskal-Wallis multiple comparisons. *** p < 0.001 and **** p < 0.0001 relative to vehicle treated control cells.

Figure 5

The phytocannabinoids cannabidiol (CBD) and cannabigerol (CBG) affected mRNA expression of cannabinoid and mesothelioma-related targets in cultured mesothelioma cells. Gene expression was quantified by RT-qPCR in (a) rat II-45 and human (b) MSTO and (c) H2452 mesothelioma cells. FC in gene expression was calculated relative to untreated control cells using the 2^-delta-delta Ct method after normalizing to TBP. Bars show the means and SD in gene expression relative to control (n = 3). Grey bars represent a fold change (FC) of one in control treated cells. p-values were calculated using two-way ANOVA Holm–Sidak’s multiple comparisons. * p < 0.01 relative to vehicle treated control cells and FC < 0.5 or >2.

Figure 6

Differentially expressed genes and pathway analysis associated with cannabidiol (CBD) and cannabigerol (CBG) treatment. The number of genes found to be differentially expressed (a,b) and top shared canonical pathways (c,d) as a result of CBD (a,c) and CBG (b,d) treatment in human MSTO and H2452 mesothelioma cell lines. Analysis was performed relative to vehicle treated control cells and only includes genes with FC > 2. Overlapping areas in Venn diagrams (a,b) indicate the number of shared genes found to be differentially expressed in the same direction in both cell lines. Z-scores (c,d) indicate predicted activation state of the top significantly enriched canonical pathways with values < 0 indicating inhibition and values > 0 indicating activation.

Figure 7

Pemetrexed + cisplatin but not cannabidiol (CBD) or cannabigerol (CBG) treatment prolongs survival in a rat syngeneic orthotopic model of pleural mesothelioma. Rats were pleurally engrafted with mesothelioma cells and then treated with control (vehicle), cisplatin + pemetrexed (chemotherapy), 40 mg/kg CBD or 100 mg/kg CBG for 4 weeks and overall survival was assessed. Data were analysed using Log-rank (Mantel-Cox) test. * p < 0.05 versus vehicle control.