Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in both Human and Canine Glioma Cells

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The use of cannabidiol (CBD), the major non‐psychoactive compound derived from cannabis, as a nutraceutical in both human and veterinary medicine has increased steadily within the past ten years despite a lack of scientific evidence for its effectiveness. Of the cornucopia of ailments that CBD supposedly treats, epilepsy, pain, and cancer remain prominent areas of interest to scientists and the general public. In the world of cancer research, several publications have demonstrated that CBD is cytotoxic against breast, endometrial, glial, and blood cancers, and in all cases it appears that CBD triggers apoptosis through a cannabinoid receptor independent process. A recent publication suggests CBD causes cell death in leukemia cells via calcium dependent mitochondrial membrane disruption, leading to activation of intrinsic apoptosis. Other receptors, such as TRPV1, and pathways, such as autophagy, have been implicated in CBD‐induced cell death but the molecular details remain elusive. Because of their remarkable similarities, from a comparative oncology perspective, canine and human gliomas are of particular interest. Regardless of species, gliomas are aggressive tumors that are notoriously challenging to treat, given their resistance to multimodal treatment, thus creating a need for more effective therapeutics. We sought to characterize the mechanism of CBD‐induced cell death in both human and canine glioma cells. We show that CBD by itself is cytotoxic, anti‐proliferative, and anti‐migratory but at concentrations which may not be pharmacologically attainable. Using resazurin and the IncuCyte live cell imaging platform, we show that metabolic‐based assays artificially increase cell line sensitivity to CBD compared to the IncuCyte, suggesting that non‐cytotoxic concentrations of CBD perturb mitochondrial function. Furthermore, we show that CBD treatment results in the formation of large cellular structures whose existence are RIPK3‐dependent. After treatment with a variety of cell death inhibitors, we demonstrate that CBD‐induced cell death occurs via apoptosis; a classically necroptotic protein, RIPK3, also appears to be involved. Lastly, by combining CBD with the autophagy inhibitor hydroxychloroquine (HCQ) we show that sensitivity to CBD increases with HCQ treatment in canine cells, which suggests involvement of the autophagy pathway for mediating CBD‐induced cell death. These data demonstrate that human and canine glioma cells respond similarly to CBD, indicate a non‐canonical function of RIPK3, and support the involvement of autophagy in CBD‐induced cell death.Support or Funding InformationSupport provided by Extract Labs in Boulder, Colorado.