The use of NADH anisotropy to investigate mitochondrial cristae alignment.

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340-360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative-especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

Ultra weak photon emission-a brief review.

Cells emit light at ultra-low intensities: photons which are produced as by-products of cellular metabolism, distinct from other light emission processes such as delayed luminescence, bioluminescence, and chemiluminescence. The phenomenon is known by a large range of names, including, but not limited to, biophotons, biological autoluminescence, metabolic photon emission and ultraweak photon emission (UPE), the latter of which shall be used for the purposes of this review. It is worth noting that the photons when produced are neither 'weak' nor specifically biological in characteristics. Research of UPE has a long yet tattered past, historically hamstrung by a lack of technology sensitive enough to detect it. Today, as technology progresses rapidly, it is becoming easier to detect and image these photons, as well as to describe their function. In this brief review we will examine the history of UPE research, their proposed mechanism, possible biological role, the detection of the phenomenon, and the potential medical applications.

Near-Infrared Light Exposure Triggers ROS to Downregulate Inflammatory Cytokines Induced by SARS-CoV-2 Spike Protein in Human Cell Culture.

The leading cause of mortality from SARS-CoV-2 is an exaggerated host immune response, triggering cytokine storms, multiple organ failure and death. Current drug- and vaccine-based therapies are of limited efficacy against novel viral variants. Infrared therapy is a non-invasive and safe method that has proven effective against inflammatory conditions for over 100 years. However, its mechanism of action is poorly understood and has not received widespread acceptance. We herein investigate whether near-infrared (NIR) light exposure in human primary alveolar and macrophage cells could downregulate inflammatory cytokines triggered by the SARS-CoV-2 spike (S) protein or lipopolysaccharide (LPS), and via what underlying mechanism. Our results showed a dramatic reduction in pro-inflammatory cytokines within days of NIR light treatment, while anti-inflammatory cytokines were upregulated. Mechanistically, NIR light stimulated mitochondrial metabolism, induced transient bursts in reactive oxygen species (ROS) and activated antioxidant gene transcription. These, in turn, downregulated ROS and inflammatory cytokines. A causal relationship was shown between the induction of cellular ROS by NIR light exposure and the downregulation of inflammatory cytokines triggered by SARS-CoV-2 S. If confirmed by clinical trials, this method would provide an immediate defense against novel SARS-CoV-2 variants and other inflammatory infectious diseases.

Non-chemical signalling between mitochondria.

A wide variety of studies have reported some form of non-chemical or non-aqueous communication between physically isolated organisms, eliciting changes in cellular proliferation, morphology, and/or metabolism. The sources and mechanisms of such signalling pathways are still unknown, but have been postulated to involve vibration, volatile transmission, or light through the phenomenon of ultraweak photon emission. Here, we report non-chemical communication between isolated mitochondria from MCF7 (cancer) and MCF10A (non-cancer) cell lines. We found that mitochondria in one cuvette stressed by an electron transport chain inhibitor, antimycin, alters the respiration of mitochondria in an adjacent, but chemically and physically separate cuvette, significantly decreasing the rate of oxygen consumption compared to a control (p = <0.0001 in MCF7 and MCF10A mitochondria). Moreover, the changes in O2-consumption were dependent on the origin of mitochondria (cancer vs. non-cancer) as well as the presence of "ambient" light. Our results support the existence of non-chemical signalling between isolated mitochondria. The experimental design suggests that the non-chemical communication is light-based, although further work is needed to fully elucidate its nature.

Inhibiting CDK4/6 in pancreatic ductal adenocarcinoma via microRNA-21.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a 5-year survival rate of 5-10 %. The high mortality rate is due to the asymptomatic progression of clinical features in metastatic stages of the disease, which renders standard therapeutic options futile. PDAC is characterised by alterations in several genes that drive carcinogenesis and limit therapeutic response. The two most common genetic aberrations in PDAC are the mutational activation of KRAS and loss of the tumour suppressor CDK inhibitor 2A (CDKN2A), which culminate the activation of the cyclin-dependent kinase 4 and 6 (CDK4/6), that promote G1 cell cycle progression. Therapeutic strategies focusing on the CDK4/6 inhibitors such as palbociclib (PD-0332991) may potentially improve outcomes in this malignancy. MicroRNAs (miRs/miRNAs) are small endogenous non-coding RNA molecules associated with cellular proliferation, invasion, apoptosis, and cell cycle. Primarily, miR-21 promotes cell proliferation and a higher proportion of PDAC cells in the S phase, while knockdown of miR-21 has been linked to cell cycle arrest at the G2/M phase and inhibition of cell proliferation. In this study, using a CRISPR/Cas9 loss-of-function screen, we individually silenced the expression of miR-21 in two PDAC cell lines and in combination with PD-0332991 treatment, we examined the synergetic mechanisms of CDK4/6 inhibitors and miR-21 knockouts (KOs) on cell survival and death. This combination reduced cell proliferation, cell viability, increased apoptosis and G1 arrest in vitro. We further analysed the mitochondrial respiration and glycolysis of PDAC cells; then assessed the protein content of these cells and revealed numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with PD-0332991 treatment and miR-21 knocking out. Our results demonstrate that combined targeting of CDK4/6 and silencing of miR-21 represents a novel therapeutic strategy in PDAC.

Acetate Induces Growth Arrest in Colon Cancer Cells Through Modulation of Mitochondrial Function.

Acetate is one of the main short chain fatty acids produced in the colon when fermentable carbohydrates are digested. It has been shown to affect normal metabolism, modulating mitochondrial function, and fatty acid oxidation. Currently, there is no clear consensus regarding the effects of acetate on tumorigenesis and cancer metabolism. Here, we investigate the metabolic effects of acetate on colon cancer. HT29 and HCT116 colon cancer cell lines were treated with acetate and its effect on mitochondrial proliferation, reactive oxygen species, density, permeability transition pore, cellular bioenergetics, gene expression of acetyl-CoA synthetase 1 (ACSS1) and 2 (ACSS2), and lipid levels were investigated. Acetate was found to reduce proliferation of both cell lines under normoxia as well as reducing glycolysis; it was also found to increase both oxygen consumption and ROS levels. Cell death observed was independent of ACSS1/2 expression. Under hypoxic conditions, reduced proliferation was maintained in the HT29 cell line but no longer observed in the HCT116 cell line. ACSS2 expression together with cellular lipid levels was increased in both cell lines under hypoxia which may partly protect cells from the anti-proliferative effects of reversed Warburg effect caused by acetate. The findings from this study suggest that effect of acetate on proliferation is a consequence of its impact on mitochondrial metabolism and during normoxia is independent of ACCS1/2 expression.

Cannabidiol Modulates Mitochondrial Redox and Dynamics in MCF7 Cancer Cells: A Study Using Fluorescence Lifetime Imaging Microscopy of NAD(P)H.

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.

Mitochondrial Function as a Potential Tool for Assessing Function, Quality and Adulteration in Medicinal Herbal Teas.

Quality control has been a significant issue in herbal medicine since herbs became widely used to heal. Modern technologies have improved the methods of evaluating the quality of medicinal herbs but the methods of adulterating them have also grown in sophistication. In this paper we undertook a comprehensive literature search to identify the key analytical techniques used in the quality control of herbal medicine, reviewing their uses and limitations. We also present a new tool, based on mitochondrial profiling, that can be used to measure medicinal herbal quality. Besides being fundamental to the energy metabolism required for most cellular activities, mitochondria play a direct role in cellular signalling, apoptosis, stress responses, inflammation, cancer, ageing, and neurological function, mirroring some of the most common reasons people take herbal medicines. A fingerprint of the specific mitochondrial effects of medicinal herbs can be documented in order to assess their potential efficacy, detect adulterations that modulate these effects and determine the relative potency of batches. Furthermore, through this method it will be possible to assess whole herbs or complex formulas thus avoiding the issues inherent in identifying active ingredients which may be complex or unknown. Thus, while current analytical methods focus on determining the chemical quality of herbal medicines, including adulteration and contamination, mitochondrial functional analysis offers a new way of determining the quality of plant derived products that is more closely linked to the biological activity of a product and its potential clinical effectiveness.

Cannabidiol Affects Extracellular Vesicle Release, miR21 and miR126, and Reduces Prohibitin Protein in Glioblastoma Multiforme Cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.

Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer.

Exosomes and microvesicles (EMV) are lipid bilayer-enclosed structures, released by cells and involved in intercellular communication through transfer of proteins and genetic material. EMV release is also associated with various pathologies, including cancer, where increased EMV release is amongst other associated with chemo-resistance and active transfer of pro-oncogenic factors. Recent studies show that EMV-inhibiting agents can sensitize cancer cells to chemotherapeutic agents and reduce cancer growth in vivo. Cannabidiol (CBD), a phytocannabinoid derived from Cannabis sativa, has anti-inflammatory and anti-oxidant properties, and displays anti-proliferative activity. Here we report a novel role for CBD as a potent inhibitor of EMV release from three cancer cell lines: prostate cancer (PC3), hepatocellular carcinoma (HEPG2) and breast adenocarcinoma (MDA-MB-231). CBD significantly reduced exosome release in all three cancer cell lines, and also significantly, albeit more variably, inhibited microvesicle release. The EMV modulating effects of CBD were found to be dose dependent (1 and 5 µM) and cancer cell type specific. Moreover, we provide evidence that this may be associated with changes in mitochondrial function, including modulation of STAT3 and prohibitin expression, and that CBD can be used to sensitize cancer cells to chemotherapy. We suggest that the known anti-cancer effects of CBD may partly be due to the regulatory effects on EMV biogenesis, and thus CBD poses as a novel and safe modulator of EMV-mediated pathological events.