Cannabidiol Regulates CD47 Expression and Apoptosis in Jurkat Leukemic Cells Dependent upon VDAC-1 Oligomerization

Decision letter: Cannabidiol activates neuronal Kv7 channels

Editor's evaluation: Cannabidiol activates neuronal Kv7 channels

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Decision letter: Cannabidiol sensitizes TRPV2 channels to activation by 2-APB

Molecular Targets of Cannabidiol in Neurological Disorders.

Cannabidiol (CBD) is the most abundant non-psychotropic cannabinoid constituent of Cannabis sativa. CBD is potentially therapeutic for the eye through antioxidant, anti-inflammatory, and neuroprotective effects. However, the effect of CBD on intraocular pressure (IOP)- the major risk factor for glaucoma- is controversial and large variability exists in the literature. IOP is regulated through aqueous humor (AH) dynamics in trabecular meshwork (TM) tissues. Measurement of CBD in AH and correlation with effects on IOP would clarify what concentrations mediate changes to IOP. Therefore, the overall goal of this dissertation is to measure CBD in ocular tissues and correlate concentrations with ocular pharmacologic effects. Effects of CBD on AH outflow were measured in porcine anterior segment explants. Effects of CBD on TM contractility and Rho/ROCK signaling were assessed in vitro. Porcine AH CBD concentrations were measured following ocular topical application ex vivo. A cyclodextrin solution, cyclodextrin solution containing gellan gum (GG) which forms a hydrogel in situ, and a semifluorinated alkane (SFA) were used to apply CBD topically. Murine AH and serum were measured by LC-MS/MS, and porcine AH and cornea concentrations were measured by HPLC. IOP was assessed following intraperitoneal and topical administration in vivo. Corneal analgesia of topical CBD was assessed using an eye wiping test. CBD increased AH outflow ex vivo, decreased TM cell contractility and inhibited TM Rho/ROCK signaling in vitro. Intraperitoneal administration of CBD reached an AH Cmax of 71.55 ng/mL and decreased IOP lasting up to 4 hours. AH concentration of CBD time dependently increased following topical application ex vivo when applied in cyclodextrin vehicles but was localized to the cornea when applied in SFA. Topical CBD lowered IOP when applied in cyclodextrin containing vehicles and reduced corneal pain when applied in an SFA vehicle. Addition of GG increased AH concentrations (Cmax 1864 ng/mL) and extended IOP reduction (from 5 to 8 hours) relative to cyclodextrin-only formulations. We have determined that low micromolar CBD concentrations alter AH outflow and IOP, and vehicle chosen can localize CBD delivery. The results highlight the importance of not only vehicle but also tissue concentrations for CBD mediated pharmacologic effects.

THC and CBD in blood samples and seizures in Norway: Does CBD affect THC-induced impairment in apprehended subjects?

Caffeine protects against memory loss induced by high and non-anxiolytic dose of cannabidiol in adult zebrafish (Danio rerio)

Reprint of “Caffeine protects against memory loss induced by high and non-anxiolytic dose of cannabidiol in adult zebrafish (Danio rerio)”

We aimed to examine, for the first time, the effect of cannabidiol (CBD) and palmitoylethanolamide (PEA) on the permeability of the human gastrointestinal tract in vitro, ex vivo, and in vivo. Flux measurements of fluorescein-labeled dextrans 10 (FD10) and fluorescein-labeled dextrans 4 (FD4) dextran across Caco-2 cultures treated for 24 hours with interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα) (10 ng·mL-1) were measured, with or without the presence of CBD and PEA. Mechanisms were investigated using cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2), transient receptor potential vanilloid 1 (TRPV1), and proliferator activated receptors (PPAR) antagonists and protein kinase A (PKA), nitric oxide synthase, phosphoinositide 3-kinases, extracellular signal-regulated kinases (MEK/ERK), adenylyl cyclase, and protein kinase C (PKC) inhibitors. Human colonic mucosal samples collected from bowel resections were treated as previously stated. The receptors TRPV1, PPARα, PPARδ, PPARγ, CB1, CB2, G-coupled protein receptor 55 (GPR55), G-coupled protein receptor 119 (GPR119), and claudins-1, -2, -3, -4, -5, -7, and -8 mRNA were measured using multiplex. Aquaporin 3 and 4 were measured using enzyme-linked immunosorbent assay (ELISA). A randomized, double-blind, controlled-trial assessed the effect of PEA or CBD on the absorption of lactulose and mannitol in humans taking 600 mg of aspirin. Urinary concentrations of these sugars were measured using liquid chromatography mass spectrometry. In vitro, PEA, and CBD decreased the inflammation-induced flux of dextrans (P < 0.0001), sensitive to PPARα and CB1 antagonism, respectively. Both PEA and CBD were prevented by PKA, MEK/ERK, and adenylyl cyclase inhibition (P < 0.001). In human mucosa, inflammation decreased claudin-5 mRNA, which was prevented by CBD (P < 0.05). Palmitoylethanolamide and cannabidiol prevented an inflammation-induced fall in TRPV1 and increase in PPARα transcription (P < 0.0001). In vivo, aspirin caused an increase in the absorption of lactulose and mannitol, which were reduced by PEA or CBD (P < 0.001). Cannabidiol and palmitoylethanolamide reduce permeability in the human colon. These findings have implications in disorders associated with increased gut permeability, such as inflammatory bowel disease.

In this study, the pharmacokinetic characteristics and tissue distribution of cannabidiol(CBD)/γ-polyglutamic acid-g-cholesterol(γ-PGA-g-CHOL) nanomicelles [CBD/(γ-PGA-g-CHOL)NMs] were investigated by pharmacokinetic experiments, and the effect of CBD/(γ-PGA-g-CHOL)NMs on the lipopolysaccharide(LPS)-induced inflammatory damage of cells was evaluated by cell experiments. CBD/(γ-PGA-g-CHOL)NMs were prepared by dialysis. The CBD concentrations in the plasma samples of male SD rats treated with CBD and CBD/(γ-PGA-g-CHOL)NMs were investigated, and the pharmacokinetic parameters were calculated and compared. UPLC-MS/MS was employed to determine the concentration of CBD in tissue samples. The heart, liver, spleen, lung, kidney, and muscle samples were collected at different time points to explore the tissue distribution of CBD and CBD/(γ-PGA-g-CHOL)NMs. The Caco-2 cell model of LPS-induced inflammation was established, and the cell viability, transepithelial electrical resistance(TEER), and secretion levels of inflammatory cytokines were determined to compare the anti-inflammatory activity between the two groups. The results showed that CBD/(γ-PGA-g-CHOL)NMs had the average particle size of(163.1±2.3)nm, drug loading of 8.78%±0.28%, and encapsulation rate of 84.46%±0.35%. Compared with CBD, CBD/(γ-PGA-g-CHOL)NMs showed increased peak concentration(C_(max)) and prolonged peak time(t_(max)) and mean residence time(MRT_(0-t)). Within 24 h, the tissue distribution concentration of CBD/(γ-PGA-g-CHOL)NMs was higher than that of CBD. In addition, both CBD and CBD/(γ-PGA-g-CHOL)NMs significantly enhanced Caco-2 cell viability and TEER, lowered the secretion levels of inflammatory cytokines, and alleviated inflammation. Moreover, CBD/(γ-PGA-g-CHOL)NMs demonstrated stronger anti-inflammatory effect. It can be inferred that γ-PGA-g-CHOL blank nanomicelles are good carriers of CBD, being capable of prolonging the circulation time of CBD in the blood, improving the bioavailability and tissue distribution concentration of CBD, and protecting against LPS-induced inflammatory injury. The findings can provide an experimental basis for the development and clinical application of oral CBD preparations.

Background: Cannabis extract has a long history of being used in the treatment and prevention of several medical conditions. The utilization of cannabis extracts, whether for medical or localized purposes, is widely observed. In cannabis extract, cannabidiol (CBD) is one of the most important non-psychoactive compounds. Several studies have demonstrated that CBD has several benefits in the treatment of various medical conditions. Nevertheless, CBD has also been demonstrated to suppress both innate and adaptive immune responses. Despite CBD has claimed to have many benefits, the toxicity of CBD is often pointed out and discussed. Nonetheless, the data on the toxicity effects of CBD on immune cells are limited. Objectives: In this study, we aimed to investigate the toxicity effects of various concentrations of CBD on immune cells, including CD4 T cells, CD8 T cells, B cells, NK cells, and monocytes. Materials and methods: Various concentrations of peripheral blood mononuclear cells (PBMCs) were treated with various concentrations of CBD or relative concentrations of methanol as a diluent control for 12, 24, and 48 hrs. Cell morphology was observed using flow cytometry. The percentage of cell death in the treated cells was determined by cell viability assay. In addition, the toxic effects of CBD on PBMC sub-populations were determined by staining with fluorochromeconjugated zombie viability dye and fluorochrome-conjugated monoclonal antibodies specific to each cell sub-population. Then, the percentage of cell death in each sub-population was assessed using flow cytometry. Results: CBD at concentrations of 40 and 80 µM showed toxicity effects on PBMCs. At these concentrations, CBD induced both cell morphological changes and cell death. While 20 µM CBD induced different effects, ranging from none to mild and high toxicity. The toxicity of CBD at 20 µM concentration depends on the individual. In contrast, CBD at ten µM and below showed no toxicity to PBMCs. The observed toxic effects of CBD occurred in all sub-populations of PBMCs, including CD4 T cells, CD8 T cells, B cells, NK cells, and monocytes. Conclusion: CBD has toxicity effects on immune cells. These effects depend on CBD concentrations, PBMC concentrations, and the duration of CBD exposure. Our findings emphasize the importance of awareness for CBD users when consuming CBD.

Cannabidiol (CBD) appears to possess some neuroprotective properties, but experimental data are still inconsistent. Therefore, this in vitro study aimed to compare the effects of CBD in a wide range of concentrations on oxidative stress and excitotoxic-related cell damage. Results showed that low concentrations of CBD ameliorated the H2O2-evoked cell damage of primary cortical neuronal cell culture. However, higher concentrations of CBD alone (5-25 μM) decreased the viability of cortical neurons in a concentration-dependent manner and aggravated the toxic effects of hydrogen peroxide (H2O2). Neuroprotection mediated by CBD in primary neurons against H2O2 was not associated with a direct influence on ROS production nor inhibition of caspase-3, but we found protective effects of CBD at the level of mitochondrial membrane potential and DNA fragmentation. However, CBD had no protective effect on the glutamate-induced cell damage of cortical neurons, and in higher concentrations, it enhanced the toxic effects of this cell-damaging factor. Likewise, CBD, depending on its concentration, at least did not affect or even enhance cortical cellular damage exposed to oxygen-glucose deprivation (OGD). Finally, we showed that CBD in submicromolar or low micromolar concentrations significantly protected human neuronal-like SH-SY5Y cells against H2O2- and 6-hydroxydopamine (6-OHDA)-induced cell damage. Our data indicate that CBD has a dual effect on oxidative stress-induced neuronal death-in low concentrations, it is neuroprotective, but in higher ones, it may display neurotoxic activity. On the other hand, in excitotoxic-related models, CBD was ineffective or enhanced cell damage. Our data support the notion that the neuroprotective effects of CBD strongly depend on its concentration and experimental model of neuronal death.

Background: Breast cancer (BC) is a very heterogeneous disease. Currently, decision-making regarding treatment of individual patients is based on several factors, most important of them are expression of estrogen receptors (ER), progesterone receptors and receptor for epidermal growth factor 2 (HER2). Common endocrine therapies include selective estrogen receptor modulator tamoxifen (TAM), aromatase inhibitors (AIs) and selective estrogen receptor degrader fulvestrant. Inhibitors of cyclin dependent kinases 4/6 (iCDK 4/6) in combination with AIs or fulvestrant, are recently being used in treatment of metastatic BC. Trastuzumab, an anti-HER2 monoclonal antibody, is a cornerstone treatment in HER2 positive disease. Next to other chemotherapeutic drugs, cisplatin is commonly used in triple-negative BC. Cannabinoids, a therapeutic option in the palliative treatment of cancer patients, have recently been demonstrated to induce anti-tumor response in certain cancer types, including BC. Reportedly, cannabinoids have been shown to exert antiproliferative and antimetastatic activities against BC cells. The effects of cannabinoids on signaling pathways in cancer cells are conferred via G-protein coupled cannabinoid receptors (CBRs) type 1 (CB1) and type 2 (CB2), other receptors (e.g. GPR55, TRPV2) or in a receptor-independent way. Cannabidiol (CBD) is a ligand of CB1 and CB2 receptors and is not psychoactive. Recent studies have shown that TAM acts as inverse agonist on CBRs but the significance of this interaction is unknown. Objectives: The aim of the study was to evaluate the effect of CBD on cell viability of selected BC cell lines of different subtypes (ER+, HER2+, triple-negative) alone and in combination with different BC therapies (TAM, fulvestrant, fulvestrant + iCDK 4/6, trastuzumab and cisplatin). Methods: The expression of CBRs in BC cell lines (MCF7, MDA-MB-231, MDA-MB-361, T-47D, SK-BR-3) was confirmed using western blot (WB). We used polyclonal antibodies against CB1 and CB2 receptors (ab23703 and ab45942, Abcam), and were among the first to test the monoclonal antibodies (sc-518035 and sc-293188, Santa Cruz Biotechnology) that exhibit lower levels of the requested specificity. All tested cell lines (T-47D, MDA-MB-231, SK-BR-3) were exposed to different concentrations of CBD. In addition, T47D cell line (ER+) was exposed to combination of CBD and endocrine therapy (TAM, fulvestrant), as well as combination of fulvestrant and palbociclib. Triple-negative MB-231 cell line was exposed to combination of CBD and cisplatin and SK-BR-3 cell line (HER2+) to CBD and trastuzumab. Results and Conclusions: CB1 and CB2 receptors are expressed in all tested BC cell lines. CBD decreased cell viability of all tested cell lines, however in MB-MDA-231 and SK-BR-3 cell lines only in above 25 µM concentrations. We are first to demonstrate that CBD increases the potency of TAM in inhibiting T-47D cell line viability. So far we could not confirm similar effect of CBD in combination with cisplatin, trastuzumab, fulvestrant alone or the combination of fulvestrant and iCDK 4/6. Citation Format: Luka Dobovišek, Metka Novak, Fran Krstanović, Polonca Ferk, Simona Borštnar, Tamara Lah Turnšek, Nataša Debeljak. Cannabidiol increases potency of tamoxifen in inhibiting breast cancer cell viability [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-11.