Restrictive threshold-dose effect of cannabidiol on cellular oxygen consumption rate

TL Macpherson1, J Westwick0,2, KL Wright1. 1Lancaster University, Lancaster, UK, 2Novartis, Horsham, West Sussex, UK

Introduction: Cannabidiol (CBD), a phytocannabinoid from the Cannabis sativa plant, is believed to have therapeutic potential for a range of ailments. It acts across a range of concentrations; at high micromolar concentrations CBD has an anti-proliferative effect on proliferating cancer cells (1). In leukemic cells, exposure with CBD at >2.5µM lead to a loss of mitochondrial membrane potential and a release of cytochrome C (2). The aim of this study was to identify how CBD affects oxygen consumption at the mitochondrial level.

Methods: Proliferating Caco-2 colon cancer cells were plated at 5x104 cells/well and maintained overnight in medium supplemented with FBS and NEAA at 37ºC. Cells were then washed twice with PBS and changed into unbuffered serum free DMEM medium and placed in a CO2 free 37ºC incubator before the experiment. An XF Flux analyser from Seahorse Bioscience was used to assess the metabolic action of CBD on proliferating Caco-2 cells over 2-12 hours. The analyser measures the oxygen consumption rate (OCR) of cells simultaneously with the extracellular acidification rate (ECAR), a measure of glycolysis.

Results: A dose dependent study of CBD at 1x10-5 – 1x10-8 M, showed that 1x10-5 M CBD decreased the OCR, with a 2.19-fold decrease (± 1.88) compared to the baseline OCR after two hours incubation and a 3.06-fold decrease (±1.96) by the third hour of incubation, with complete blockage of OCR by 8 hours (AUC p<0.001, n=3). This reduction in OCR is accompanied by an increase in ECAR at 2 hours, although this subsequently results in complete inhibition of ECAR by 11 hours in the presence of CBD. FCCP (0.5x10-5 M) increases OCR by 2.1-fold (±0.1, p<0.05), an effect reversed by CBD, with accelerated complete cessation of OCR by 3 hours. Pre-treatment of cells with 1x10-5 M CBD prevents increased OCR by FCCP (n=4). In addition to this, treatment with α-tocopherol (1x10-7 -1x10-5 M) a potent antioxidant, inhibited 1x10-5 M CBD-induced reduction of OCR by 54.9% (±11.4, p<0.01).

Conclusions: CBD acts on cellular bioenergetic processes limiting the respiratory capacity of proliferating cancer cells, culminating in cell death. This effect is not dose-dependent and acts within a small therapeutic window. These effects are not recapitulated in fully differentiated Caco-2 cells, a model of normal enterocytic epithelium, which may explain the well-documented anti-inflammatory effects of CBD (3).

References

1. Massi, P., M. Solinas, et al. (2012). Cannabidiol as a potential anticancer drug. British Journal of Clinical Pharmacology doi: 10.1111/j.1365-2125.2012.04298.x. [Epub ahead of print]

2. McKallip, R. J., W. Jia, et al. (2006) Cannabidiol-induced apoptosis in human leukemia cells: a novel role of cannabidiol in the regulation of p22phox and Nox4 expression. Molecular Pharmacology. 70(3): 897

3. Alhamoruni, A., K. L. Wright, et al. (2012). Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability. B.J.Pharmacol. 165(8): 2598-2610

Acknowledgements: Dr Jane Armstrong and Dr Dave Criddle, Liverpool NIHR Pancreas Biomedical Research Unit (PBRU), Royal Liverpool University Hospital, UK.