Cannabidiol and Cannabigerol Inhibit Cholangiocarcinoma Growth In Vitro via Divergent Cell Death Pathways

Abstract

Cholangiocarcinoma (CCA) is a rare and highly lethal disease with few effective treatment options. Cannabinoids, cannabidiol (CBD) and cannabigerol (CBG) are non-psychedelic components extracted from cannabis. These non-psychoactive compounds have shown anti-proliferative potential in other tumor models; however, the efficacy of CBD and CBG in CCA is unknown. Furthermore, two cell death pathways are implicated with CBD resulting in autophagic degeneration and CBG in apoptosis. HuCC-T1 cells, Mz-ChA-1 cells (CCA cell lines) and H69 cells (immortalized cholangiocytes), were treated with CBD and CBG for 24 to 48 h. The influence of these cannabinoids on proliferation was assessed via MTT assay. Apoptosis and cell cycle were evaluated via Annexin-V apoptosis assay and propidium iodide, respectively. The expression of proliferation biomarker Ki-67, apoptosis biomarker BAX, and autophagic flux biomarkers LC3b and LAMP1 were evaluated via immunofluorescence. Cell migration and invasion were evaluated via wound healing assay and trans-well migration invasion assays, respectively. The colony formation was evaluated via colony formation assay. In addition, the expression of autophagy gene LC3b and apoptosis genes BAX, Bcl-2, and cleaved caspase-3 were evaluated via Western blot. CBD and CBG are non-selective anti-proliferative agents yielding similar growth curves in CCA; both cannabinoids are effective, yet CBG is more active at lower doses. Low doses of CBD and CBG enhanced immortalized cholangiocyte activity. The reduction in proliferation begins immediately and occurs maximally within 24 h of treatment. Moreover, a significant increase in the late-stage apoptosis and a reduction in the number of cells in S stage of the cell cycle indicates both CBD and CBG treatment could promote apoptosis and inhibit mitosis in CCA cells. The fluorescent expression of BAX and LC3b was significantly enhanced with CBD treatment when compared to control. LAMP1 and LC3b colocalization could also be observed with CBD and CBG treatment indicating changes in autophagic flux. A significant inhibition of migration, invasion and colony formation ability was shown in both CBD and CBG treatment in CCA. Western blot showed an overall decrease in the ratio of anti-apoptotic protein Bcl-2 with respect to pro-apoptotic protein BAX with CBG treatment. Furthermore, CBD treatment enhanced the expression of Type II cell death (autophagic degeneration) protein LC3b, which was reduced in CBG-treated CCA cells. Meanwhile, CBG treatment upregulated Type I cell death (programmed apoptosis) protein cleaved caspase-3. CBD and CBG are effective anti-cancer agents against CCA, capable of inhibiting the classic hallmarks of cancer, with a divergent mechanism of action (Type II or Type I respectively) in inducing these effects.

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

“…Based on our previous work [ 34 ], we propose a mechanism in which adding CBG arrests the cell cycle in the G1/S1 and G2/M phases, facilitating apoptosis via cytostatic effects to a greater extent than CBD ( Scheme 1 ). Similar has been recently reported in cholangiocarcinoma [ 46 ]. A decrease in the number of cholangiocarcinoma cells in the S stage of the cell cycle and a significant increase in late-stage apoptosis were observed after combined CBD:CBG vs. single cannabinoid treatment.…”

“…(III) The role of TRPV1 at plasma and ER membranes is to regulate Ca 2+ influx/efflux. Additionally, when TRPV1 is desensitised by CBD/CBG binding, it also induces intrinsic pro-apoptotic mechanisms [ 46 ] via Ca 2+ depletion, triggering ER stress. We found significantly higher expression of TRPV1 in GSCs as in GBM cells ( Figure 1 C), and a correlation between TRPV1 and NOTCH and OLIG2 and other GSC stemness and epithelial-to-mesenchymal transition markers.…”

Characterization of the Chemopreventive Properties of Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy

“…Notably, the inflorescence of Felina 32 and Fedora crops collected in September contained higher levels of cannabidiol than the corresponding samples collected in June as well as the samples collected from other crops. Cannabidiol has been reported to induce cytotoxicity in other noncancerous cell models and to act in a nonspecific manner upon cancer cells [80][81][82], although opposite evidence was reported, too [83,84]. Our samples were less toxic in H69 cells than in HepG2 ones, suggesting that the potential cytotoxicity of cannabidiol may be modulated by other compounds in the hemp phytocomplex, such as β-caryophyllene, leading to an increased tolerability in noncancerous cells.…”

Cannabigerol Induces Autophagic Cell Death by Inhibiting EGFR-RAS Pathways in Human Pancreatic Ductal Adenocarcinoma Cell Lines

“…Herein, the combination of CBG with GEM or PTX increased the cytotoxicity compared to the administration of the drug alone, also showing a synergistic effect for some combinations. In GBM cells, CBD and CBG plus temozolomide did not show an additive effect, but in cholangiocarcinoma cells, CBG synergized with GEM and cisplatin [13,29]. Moreover, in PDAC, CBD showed the ability to increase GEM and PTX efficacy in in vitro tests and KPC mice treated with CBD and GEM showed a survival three times longer than mice treated with GEM [18,19].…”