Fig 5 - uploaded by Robert J McKallip
Content may be subject to copyright.
Cannabidiol induces activation of the caspase cascade, loss of mitochondrial membrane potential, and release of cytochrome c. A, Jurkat tumor cells were exposed to various concentrations of CBD (2.5 or 5.0 M) or the vehicle or for 24 h, In addition, the role of CB2 in the CBD-induced changes in caspase activity was monitored by culturing Jurkat cells with CBD (2.5 or 5.0 M) as well as the CB2-selective antagonist, SR144528. Next, the cells were lysed, the cellular proteins were isolated, and Western analysis was performed. The levels of the procaspases as well as the presence of the cleaved form (CF) of various caspases were examined. B, Jurkat tumor cells were exposed to various concentrations of CBD (2.5 and 5.0 M) or the vehicle for 24 h. Fifteen minutes before the end of the incubations, DiOC 6 was added for a final concentration of 40 nM. The cells were harvested and analyzed by flow cytometry. The percentage of cells with loss of mitochondrial membrane potential is depicted. C, the effect of CBD exposure on the level of cytosolic cytochrome c in Jurkat cells was determined by culturing the cells with CBD (2.5 and 5.0 M) CB2-selective antagonist SR144528 (5.0 M) for 24 h. The cells were harvested and washed, and cytosolic proteins were analyzed for cytochrome c by Western blot analysis.
Source publication
In the current study, we examined the effects of the nonpsychoactive cannabinoid, cannabidiol, on the induction of apoptosis in leukemia cells. Exposure of leukemia cells to cannabidiol led to cannabinoid receptor 2 (CB2)-mediated reduction in cell viability and induction in apoptosis. Furthermore, cannabidiol treatment led to a significant decreas...
Contexts in source publication
Context 1
... the mechanism of cannabidiol- induced apoptosis, we examined the activation pattern of caspases after CBD exposure. To this end, Jurkat cells were exposed to various concentrations of cannabidiol (2.5 and 5 M) or the vehicle for 24 h. Next, the cells were harvested and the presence of the various caspases was determined by Western blot analysis (Fig. 5A). The results demonstrate that exposure to CBD at concentrations of 2.5 M or greater led to activation of the caspase cascade. More specifically, we ob- served cleavage of caspase-8, and reduction in procaspase-2, -9, and -10, which are thought to be involved in initiating the caspase cascade. In addition, the cleavage of the effector ...
Context 2
... investigated by examining the effect of CBD on Jur- kat mitochondrial membrane potential as well as the levels of cytosolic cytochrome c. Exposure of Jurkat cells to 2.5 M or greater CBD for 24 h led to a significant reduction in the mitochondrial membrane potential (Fig. 5B). Furthermore, 2.5 M or greater CBD led to a significant increase in the level of cytosolic cytochrome c (Fig. 5C). Together, these results suggest a direct role of the mitochondria in CBD- induced ...
Context 3
... of CBD on Jur- kat mitochondrial membrane potential as well as the levels of cytosolic cytochrome c. Exposure of Jurkat cells to 2.5 M or greater CBD for 24 h led to a significant reduction in the mitochondrial membrane potential (Fig. 5B). Furthermore, 2.5 M or greater CBD led to a significant increase in the level of cytosolic cytochrome c (Fig. 5C). Together, these results suggest a direct role of the mitochondria in CBD- induced ...
Similar publications
(-)-Cannabidiol (CBD) is a major, non psychotropic constituent of cannabis. It has been shown to cause numerous physiological effects of therapeutic importance. We have reported that CBD derivatives in both enantiomeric series are of pharmaceutical interest. Here we describe the syntheses of the major CBD metabolites, (-)-7-hydroxy-CBD and (-)-CBD-...
The endogenous cannabinoid system represents a promising therapeutic target to modify neurodegenerative pathways linked to Alzheimer's disease (AD). The aim of the present study was to evaluate the specific contribution of CB2 receptor to the progression of AD-like pathology and its role in the positive effect of a cannabis-based medicine (1:1 comb...
Citations
... The effect was still observed in the presence of CB receptor antagonists, suggesting a CB-independent cell death mechanism. Similarly, CBD, the non-psychoactive cannabinoid, demonstrated the potency to activate the caspase-8, -9, -3 and to increase the ROS production in Jurkat and Molt-4 leukemia cell lines, suggesting the possibility of extrinsic apoptotic pathways [37]. ...
... Neither the therapeutic nor the toxic dosage for CBN and CBG has been established in vitro or in vivo [40]; hence, the current work focused on a concentration range that is clinically achievable and well-tolerated based on previously conducted leukemia research with THC and CBD [30,34,37]. The presented in vitro data suggest that CBN and CBG can ...
... Neither the therapeutic nor the toxic dosage for CBN and CBG has been established in vitro or in vivo [40]; hence, the current work focused on a concentration range that is clinically achievable and well-tolerated based on previously conducted leukemia research with THC and CBD [30,34,37]. The presented in vitro data suggest that CBN and CBG can induce apoptosis in THP-1 cells and inhibit their growth in a dose-and time-dependent manner ( Figure 1). ...
Several cannabis plant-derived compounds, especially cannabinoids, exhibit therapeutic potential in numerous diseases and conditions. In particular, THC and CBD impart palliative, antiemetic, as well as anticancer effects. The antitumor effects include inhibition of cancerous cell growth and metastasis and induction of cell death, all mediated by cannabinoid interaction with the endocannabinoid system (ECS). However, the exact molecular mechanisms are still poorly understood. In addition, their effects on leukemia have scarcely been investigated. The current work aimed to assess the antileukemic effects of CBN and CBG on an acute monocytic leukemia cell line, the THP-1. THP-1 cell viability, morphology and cell cycle analyses were performed to determine potential cytotoxic, antiproliferative, and apoptotic effects of CBN and CBG. Western blotting was carried out to measure the expression of the proapoptotic p53. Both CBN and CBG inhibited cell growth and induced THP-1 cell apoptosis and cell cycle arrest in a dose- and time-dependent manner. CBN and CBG illustrated different dosage effects on THP-1 cells in the MTT assay (CBN > 40 μΜ, CBG > 1 μM) and flow cytometry (CBN > 5 μM, CBG > 40 μM), highlighting the cannabinoids’ antileukemic activity. Our study hints at a direct correlation between p53 expression and CBG or CBN doses exceeding 50 μM, suggesting potential activation of p53-associated signaling pathways underlying these effects. Taken together, CBG and CBN exhibited suppressive, cell death-inducing effects on leukemia cells. However, further in-depth research will be needed to explore the molecular mechanisms driving the anticancer effects of CBN and CBG in the leukemia setting.
... In healthy cells, treatment of HUVECs [13] and vascular smooth muscle cells [35] with CBD induced HO-1, but NAC hindered the induction of HO-1 by CBD in these studies, leading to the conclusion that the CBD-dependent HO-1 induction is dependent on ROS. Contradictory effects are found in lung cancer cells [36] and leukemia cells [37] in which a CBD treatment results in increased ROS levels. The reason why cancer cells respond to a CBD treatment with an increase in oxidative stress, while in normal cells, ROS levels decrease upon CBD treatment [38,39], is not fully understood. ...
Objective: Radiotherapy, which is commonly used for the local control of thoracic cancers, also induces chronic inflammatory responses in the microvasculature of surrounding normal tissues such as the lung and heart that contribute to fatal radiation-induced lung diseases (RILDs) such as pneumonitis and fibrosis. In this study, we investigated the potential of cannabidiol (CBD) to attenuate the irradiation damage to the vasculature. Methods: We investigated the ability of CBD to protect a murine endothelial cell (EC) line (H5V) and primary lung ECs isolated from C57BL/6 mice from irradiation-induced damage in vitro and lung ECs (luECs) in vivo, by measuring the induction of oxidative stress, DNA damage, apoptosis (in vitro), and induction of inflammatory and pro-angiogenic markers (in vivo). Results: We demonstrated that a non-lethal dose of CBD reduces the irradiation-induced oxidative stress and early apoptosis of lung ECs by upregulating the expression of the cytoprotective mediator heme-oxygenase-1 (HO-1). The radiation-induced increased expression of inflammatory (ICAM-2, MCAM) and pro-angiogenic (VE-cadherin, Endoglin) markers was significantly reduced by a continuous daily treatment of C57BL/6 mice with CBD (i.p. 20 mg/kg body weight), 2 weeks before and 2 weeks after a partial irradiation of the lung (less than 20% of the lung volume) with 16 Gy. Conclusions: CBD has the potential to improve the clinical outcome of radiotherapy by reducing toxic side effects on the microvasculature of the lung.
... However, the literature suggests the intriguing possibility of activating this mechanism using specific drugs. CBD, a cannabis sativa-derivative compound, which has been shown to trigger apoptosis in leukemic systems, has been researched in the past for its potential anticancer properties [199]. More recently, in ALL cellular models, it has been shown that CBD triggers certain processes typical of apoptosis, such as cytosolic migration of Cytc and caspase 9 activation, but causes cell death by MPT-driven necrosis by completely arresting oxidative phosphorylation and the mitochondrial production of ATP (Table 7) [195]. ...
Simple Summary
Resistance to the cell death of neoplastic cells represents one of the main limitations for cancer treatment. The following review describes, in different types of leukemia, the molecular mechanisms driving the new regulated cell death (RCD) processes and their de-regulation. Furthermore, renowned or newly characterized pharmacological strategies, able to modulate the specific mechanisms of RCD, will be addressed.
Abstract
Hematological malignancies are among the top five most frequent forms of cancer in developed countries worldwide. Although the new therapeutic approaches have improved the quality and the life expectancy of patients, the high rate of recurrence and drug resistance are the main issues for counteracting blood disorders. Chemotherapy-resistant leukemic clones activate molecular processes for biological survival, preventing the activation of regulated cell death pathways, leading to cancer progression. In the past decade, leukemia research has predominantly centered around modulating the well-established processes of apoptosis (type I cell death) and autophagy (type II cell death). However, the development of therapy resistance and the adaptive nature of leukemic clones have rendered targeting these cell death pathways ineffective. The identification of novel cell death mechanisms, as categorized by the Nomenclature Committee on Cell Death (NCCD), has provided researchers with new tools to overcome survival mechanisms and activate alternative molecular pathways. This review aims to synthesize information on these recently discovered RCD mechanisms in the major types of leukemia, providing researchers with a comprehensive overview of cell death and its modulation.
... The use of the antioxidant α-tocopherol (TOC) to limit the ROS level can decrease the expression of apoptotic protein induced by CBD, again implicating ROS in apoptosis [15]. Other studies have revealed that CBD activates a caspase cascade in leukemic Jurkat cells, leading to PARP cleavage, the upregulation of NADH oxidase expression, increased ROS production, and finally cell apoptosis [16]. Massi et al. [17] reported that CBD triggers apoptosis in glioma cells by activating caspase-8 and caspase-9 through the early generation of ROS and the depletion of glutathione (GSH). ...
Cannabidiol (CBD), a non-psychoactive ingredient extracted from the hemp plant, has shown therapeutic effects in a variety of diseases, including anxiety, nervous system disorders, inflammation, and tumors. CBD can exert its antitumor effect by regulating the cell cycle, inducing tumor cell apoptosis and autophagy, and inhibiting tumor cell invasion, migration, and angiogenesis. This article reviews the proposed antitumor mechanisms of CBD, aiming to provide references for the clinical treatment of tumor diseases and the rational use of CBD.
... Our mRNA-sequencing analyses confirmed the involvement of cell cycle/cell proliferation and apoptosis pathways in CBD-induced cytotoxicity in primary human Leydig cells and mouse TM3 cells. These findings align with previous studies' observations that CBD induced apoptosis in various cell types, including human leukemia, glioma, and gastric cancer cells (Jeong et al. 2019;Massi et al. 2006;Mckallip et al. 2006). Moreover, the pro-apoptotic property of CBD in normal lymphocytes has been reported to cause immunosuppressive effects (Kozela et al. 2010). ...
Cannabidiol (CBD), one of the major components extracted from the plant Cannabis sativa L., has been used as a prescription drug to treat seizures in many countries. CBD-induced male reproductive toxicity has been reported in animal models; however, the underlying mechanisms remain unclear. We previously reported that CBD induced apoptosis in primary human Leydig cells, which constitute the primary steroidogenic cell population in the testicular interstitium. In this study, we investigated the effects of CBD and its metabolites on TM3 mouse Leydig cells. CBD, at concentrations below 30 µM, reduced cell viability, induced G1 cell cycle arrest, and inhibited DNA synthesis. CBD induced apoptosis after exposure to high concentrations (≥ 50 µM) for 24 h or a low concentration (20 µM) for 6 days. 7-Hydroxy-CBD and 7-carboxy-CBD, the main CBD metabolites of CBD, exhibited the similar toxic effects as CBD. In addition, we conducted a time-course mRNA-sequencing analysis in both primary human Leydig cells and TM3 mouse Leydig cells to understand and compare the mechanisms underlying CBD-induced cytotoxicity. mRNA-sequencing analysis of CBD-treated human and mouse Leydig cells over a 5-day time-course indicated similar responses in both cell types. Mitochondria and lysosome dysfunction, oxidative stress, and autophagy were the major enriched pathways in both cell types. Taken together, these findings demonstrate comparable toxic effects and underlying mechanisms in CBD-treated mouse and primary human Leydig cells.
... Herbal medicinal plants and their derivatives have been discovered and used as potential sources for the treatment of human cancers for decades. Of these, cannabinoids extracted from the hemp plant Cannabis sativa have been remarkably noted as a potential therapy for the treatment of several human tumors [5][6][7][8][9][10] . The anti-tumor effect of cannabinoids was reported in four main ways, including (1) inhibition of tumor cell proliferation, (2) induction of apoptosis, (3) inhibition of angiogenesis, invasion and metastasis, and (4) induction of anti-tumor immunity 11,12 . ...
Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.
... ROS is known to be important for CBD-induced cell death in glioma and leukaemia cells [42,43]. Reduced mitochondrial function leads to higher production of ROS through a process known as electron leakage in the electron transport chain of mitochondria [44]. ...
... Multiple studies have consistently demonstrated the involvement of endocannabinoids and cannabinoids in exerting antiproliferative effects through metabolic pathways, particularly those involving ROS [37,[41][42][43][44]. Park et al. observed a substantial elevation in ROS levels when treating head and neck squamous cell carcinoma (HNSCC) lines with the endocannabinoid anandamide (AEA) [45]. ...
Research suggests the potential of using cannabinoid-derived compounds to function as anticancer agents against melanoma cells. Our recent study highlighted the remarkable in vitro anticancer effects of PHEC-66, an extract from Cannabis sativa, on the MM418-C1, MM329, and MM96L melanoma cell lines. However, the complete molecular mechanism behind this action remains to be elucidated. This study aims to unravel how PHEC-66 brings about its antiproliferative impact on these cell lines, utilising diverse techniques such as real-time polymerase chain reaction (qPCR), assays to assess the inhibition of CB1 and CB2 receptors, measurement of reactive oxygen species (ROS), apoptosis assays, and fluorescence-activated cell sorting (FACS) for apoptosis and cell cycle analysis. The outcomes obtained from this study suggest that PHEC-66 triggers apoptosis in these melanoma cell lines by increasing the expression of pro-apoptotic markers (BAX mRNA) while concurrently reducing the expression of anti-apoptotic markers (Bcl-2 mRNA). Additionally, PHEC-66 induces DNA fragmentation, halting cell progression at the G1 cell cycle checkpoint and substantially elevating intracellular ROS levels. These findings imply that PHEC-66 might have potential as an adjuvant therapy in the treatment of malignant melanoma. However, it is essential to conduct further preclinical investigations to delve deeper into its potential and efficacy.
... Most studies have focused on the effect of CBD in cancer cells, and the majority of these have reported activation of mitochondria-led intrinsic apoptosis following CBD treatment in varying cancer cell lines [described in (Massi et al., 2006;McKallip et al., 2006;Shrivastava et al., 2011;Chan This article has not been copyedited and formatted. The final version may differ from this version. ...
... Downloaded from and Duncan, 2021)]. Implicated mechanisms of action for apoptosis included activation of pro-apoptotic caspase enzymes (Massi et al., 2006;McKallip et al., 2006;Shrivastava et al., 2011;Olivas-Aguirre et al., 2019), generation of ROS species (Massi et al., 2006;McKallip et al., 2006;Shrivastava et al., 2011;, and cytochrome C release (Massi et al., 2006;McKallip et al., 2006;Olivas-Aguirre et al., 2019), among others. ...
... Downloaded from and Duncan, 2021)]. Implicated mechanisms of action for apoptosis included activation of pro-apoptotic caspase enzymes (Massi et al., 2006;McKallip et al., 2006;Shrivastava et al., 2011;Olivas-Aguirre et al., 2019), generation of ROS species (Massi et al., 2006;McKallip et al., 2006;Shrivastava et al., 2011;, and cytochrome C release (Massi et al., 2006;McKallip et al., 2006;Olivas-Aguirre et al., 2019), among others. ...
Cannabidiol (CBD) is a pharmacologically active metabolite of cannabis that is FDA-approved to treat seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex in children aged one year and older. During clinical trials, CBD caused dose-dependent hepatocellular toxicity at therapeutic doses. The risk for toxicity was increased in patients taking valproate (VPA), another hepatotoxic antiepileptic drug, through an unknown mechanism. With the growing popularity of CBD in the consumer market, an improved understanding of the safety risks associated with CBD is needed to ensure public health. This review details current efforts to describe CBD pharmacokinetics and mechanisms of hepatotoxicity using both pharmacokinetic models and in vitro models of the liver. In addition, current evidence and knowledge gaps related to intracellular mechanisms of CBD-induced hepatotoxicity are described. The authors propose future directions that combine systems-based models with markers of CBD-induced hepatotoxicity to understand how CBD pharmacokinetics may influence the adverse effect profile and risk of liver injury for those taking CBD. Significance Statement This review describes current pharmacokinetic modeling approaches to capture the metabolic clearance and safety profile of cannabidiol (CBD). CBD is an increasingly popular natural product and FDA-approved antiepileptic drug known to cause clinically significant enzyme-mediated drug interactions and hepatotoxicity at therapeutic doses. CBD metabolism, pharmacokinetics, and putative mechanisms of CBD-induced liver injury are summarized from available preclinical data to inform future modeling efforts for understanding CBD toxicity.
... The anti-proliferative and pro-apoptosis effect of CBD has been reported in breast cancer, colorectal cancer, glioma (most extensively studied), lung cancer (pro-apoptosis reported only), lymphoma, pancreatic cancer and prostate cancer (Ligresti et al., 2006;Massi et al., 2006;Massi et al., 2004;McKallip et al., 2006;Ramer et al., 2013;Sharma et al., 2014;Shrivastava et al., 2011;Sreevalsan et al., 2011). Several other studies reveal CBD's effect on tumour progression, reduction, or inhibition of metastasis, both in vitro and in vivo, on breast cancer and lung cancer (Elbaz et al., 2015;Haustein et al., 2014). ...
Introduction: Melanoma, the most lethal and aggressive variant of skin cancer, poses a concerning health issue due to existing therapeutic deficiencies, necessitating the development of novel treatments. Tumour microenvironment (TME) is a crucial player in cancer progression and treatment responses. TME, a heterogeneous and dynamic ecosystem, is actively deployed by cancer to orchestrate disease-permissive influences via a complex network of spatial interaction and communication. This thesis proposes two innovative therapeutic approaches concerted around re-sculpting the pro-tumorigenic microenvironment.
Aims: To assess the therapeutic efficacy of the proposed drug combination of vemurafenib and the in-house CSF1R inhibitor SN38114 or cannabidiol (CBD) in both in vitro and in vivo settings.
Methods: Therapeutic efficacy was evaluated via transplantation of patient-derived melanoma tumour xenografts into homozygous NIH-III mice. Immunohistochemistry analyses of xenograft tumours were conducted to virtualise the effect of the combinations. In vitro investigations of the cytotoxicity of single agents and combination treatments were performed on a panel of colorectal cancer cell lines, melanoma cell lines, and tumour microenvironment cell lines.
Results: A synergetic tumour growth inhibitory effect on BRAF V600E mutated NZM 20 human melanoma xenograft tumours was observed with the combination of vemurafenib and CBD, while in vitro cell viability assays revealed ineffectiveness. Significant attenuation of Ki-67 and CD31 expression was observed in vivo tumours treated with vemurafenib and CBD. Furthermore, we observed decreased expression of type one collagen and alpha-smooth muscle actin in the BRAF wild type NZM 40 human melanoma xenograft model treated with CBD.
Conclusion: We demonstrate the potential synergetic anti-tumour effect of a combination of mitogen-activated protein kinases inhibitor vemurafenib and cannabidiol on a patient-derived melanoma tumour xenograft model. We propose the immune-modulating role of cannabidiol in human melanoma and reveal enhanced anti-proliferative and anti-angiogenesis effects of cannabidiol with vemurafenib in vivo. We further suggest CBD's potential dual anti-invasiveness impact in the BRAF wild-type human melanoma xenograft model. This study advocated the medical use of CBD as a viable adjunctive option in combination with other therapeutic agents.
... 14,15 In adaptive immune cells, CBD inhibited IFN-γ production and induced apoptosis in T cells. 16,17 As a result, CBD is often used as an anti-inflammatory drug to treat a variety of inflammatory conditions. 18,19 Even though CBD has exhibited a range of advantageous effects, research in the clinical trial phase of CBD is still limited and controversial. ...
... 11 The majority of published articles suggest that CBD has immunosuppressive effects. [14][15][16][17] Nevertheless, some of these studies did not investigate the direct cytotoxicity of CBD on immune cells. 18,26 The data of the toxicity effect of CBD on immune cells is limited. ...
... Our results of the toxicity of CBD are in line with the previous studies, which reported that CBD could induce immune suppression, both in innate and adaptive immune responses. [14][15][16][17] The effects of CBD on the suppression of immune cell function may be due to the toxicity of CBD that affects both innate immune cells, monocytes and NK cells, and adaptive immune cells, T and B lymphocytes if the high concentration of CBD were used in the study. The researchers who perform experiments on the function of CBD need to monitor the toxicity of CBD on the tested cells concurrently in their research. ...
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.