Abstract
The cannabinoid, cannabidiol (CBD), is part of the plant's natural defense system that when given to animals has many useful medicinal properties, including activity against cancer cells, modulation of the immune system, and efficacy in epilepsy. Although there is no consensus on its precise mode of action as it affects many cellular targets, CBD does appear to influence mitochondrial function. This would suggest that there is a cross-kingdom ability to modulate stress resistance systems that enhance homeostasis. As NAD(P)H autofluorescence can be used as both a metabolic sensor and mitochondrial imaging modality, we assessed the potential of this technique to study the in vitro effects of CBD using 2-photon excitation and fluorescence lifetime imaging microscopy (2P-FLIM) of NAD(P)H against more traditional markers of mitochondrial morphology and cellular stress in MCF7 breast cancer cells. 2P-FLIM analysis revealed that the addition of CBD induced a dose-dependent decrease in bound NAD(P)H, with 20 µM treatments significantly decreased the contribution of bound NAD(P)H by 14.6% relative to the control (p < 0.001). CBD also increased mitochondrial concentrations of reactive oxygen species (ROS) (160 ± 53 vs. 97.6 ± 4.8%, 20 µM CBD vs. control, respectively, p < 0.001) and Ca2+ (187 ± 78 vs. 105 ± 10%, 20 µM CBD vs. the control, respectively, p < 0.001); this was associated with a significantly decreased mitochondrial branch length and increased fission. These are all suggestive of mitochondrial stress. Our results support the use of NAD(P)H autofluorescence as an investigative tool and provide further evidence that CBD can modulate mitochondrial function and morphology in a dose-dependent manner, with clear evidence of it inducing oxidative stress at higher concentrations. This continues to support emerging data in the literature and may provide further insight into its overall mode of action, not only in cancer, but potentially its function in the plant and why it can act as a medicine.
Highlights
Uncontrolled cell growth, or cancer, is frequently associated with increased aerobic glycolysis and alterations in mitochondrial function (Trigos et al, 2018)
CBD had no significant effect on the fluorescence lifetimes of mitochondrial free NAD(P)H and bound NAD(P)H, shown in Figures 1C and D, respectively
In this study we used FLIM, fluorescence microscopy, and conventional reactive oxygen species (ROS) and calcium detecting stains to investigate the effects of CBD on NAD(P)H lifetime, calcium and ROS levels, and mitochondrial morphology in a breast cancer cell line
Summary
Uncontrolled cell growth, or cancer, is frequently associated with increased aerobic glycolysis (the Warburg effect) and alterations in mitochondrial function (Trigos et al, 2018). Mitochondria are central in managing oxidative stress via thermodynamic buffering, involving enhancement of antioxidant capacity and uncoupling, as well as detoxification and induction of programmed cell death (Popov, 2020; Gandin et al, 2021) This is supported by evidence that some bioactive plant compounds can protect plant mitochondria (Laus and Soccio, 2020). The stress response system has to be adaptive and protect cells and induce cell death when necessary, suggesting a biphasic or hormetic dose–response curve One such compound, cannabidiol (CBD), has demonstrated a plethora of pharmacological effects ranging from antiinflammatory to anxiolytic, antiepileptic, anticancer, and even antibacterial (Mechoulam et al, 2002; Gray et al, 2020; Kis et al, 2019). Rather than focusing on individual receptors and channels, here we investigate how it might modulate a key nexus in cell function, the mitochondrion
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