Cannabinoids and their derivatives have been the subject of scientific and clinical investigations and debates for decades, mostly due to their diverse effects on disease symptoms and complications as well as their excitant action on the nervous system [10,11,21,38]. As documented, accumulated evidence has demonstrated their antitumor activities in numerous cancer models [9,22,39]. However, their therapeutic potential for some malignancies, particularly leukemias, remains relatively unknown. The efficacy of conventional treatments for acute leukemias is largely unsatisfactory for most entities; therefore, this study was undertaken to investigate the antileukemic properties of plant-derived cannabinoids CBN and CBG in the leukemia THP-1 cell line. This study demonstrated the anticancer, antileukemic potential of plant-derived compounds that have not yet been thoroughly examined.

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). After the 24-h incubation (Figure 1A,C), CBG proved more effective in reducing the viability of THP-1 cells than CBN. However, after a 48-h incubation, the impact of the cannabinoids on cell viability was more significant, indicating that the activity of both cannabinoids is time-dependent (Figure 1B,D). The notable differences observed between the two incubation periods were supported by the microscopical analysis of cell morphology. After 24-h incubation, the cytotoxic effects of CBN and CBG were less observable (Figure 4B–D), with cells treated with 50 μM or 100 μM showing early apoptotic features but without complete cell death. After a 48-h incubation, later features of cell death were noted (Figure 5B–D). More cells were alive at 24 h as compared with 48 h (Figure 4), which can hint that cells have not necessarily completed the programmed cell death cycle.

The flow cytometric analysis demonstrated that CBN and CBG also had antiproliferative effects. After 24-h incubation with 5 μM CBN or 40 μM of CBG, the percentage of cells in the S and G2/M phases was significantly reduced (<1%) compared with the control, indicating that CBN is more efficient in restraining cell growth (Figure 2). It is important to note that the significant arrest of cells in G0/G1 at higher concentrations of CBN or CBG may be associated with apoptosis, as suggested by microscopic examinations and forward scatter analysis. After a 24-h incubation with the cannabinoids, changes in cell shape, cell shrinkage, and a decrease in living cells (Figure 4C,D) were observed, while the percentage of cells gradually declined with increasing concentrations of the cannabinoids (Figure 3). These findings by both cannabinoid effects suggest the involvement of multiple mechanisms and pathways.

The microscopic analysis of cannabinoids-treated THP-1 cells identified morphological alterations, which differed by CBN/CBG concentration and incubation duration. It was previously shown that some cannabinoid groups at a particular concentration range have a distinctive mode of action in hematological malignancies [41]. Investigating immunophenotypic markers (CD markers antibodies) or immune staining to detect blast maturation induced by cannabinoids can provide additional evidence of their activity. Such analyses will likely support the results obtained from the MTT assay and flow cytometry analysis.

One of the aims of this study was to investigate the effect of CBG and CBN on p53 expression in THP-1 cells. To our knowledge, currently, the effect of cannabinoids on p53 apoptotic signaling in leukemic cells hasn’t been completely elucidated, especially in the THP-1 cell line. The western blotting analysis demonstrated a pronounced increase in expression of p53 at concentrations of 50 and 100 μM compared with the control (Figure 6). At concentrations below 50 μM, no bands were detected of p53 in the western blot. The p53 status in the THP-1 cell line was reported to contain a partial deletion, 26 bases from the 1st letter of codon 174 [42], and the sequence length was observed to be shorter than the wildtype. It is well established that p53 activity and function in tumors are often suppressed in various cancer types [43]. However, in some types of cancers, like hematological malignancies, certain pathways become dysfunctional, triggering p53, and the truncated versions of the p53 protein can still perform aspects of their tumor-suppressor role. A similar finding to that of our study, where p53 expression increased in leukemic cells (Jurkat clone E6-1 cells carry a homozygous mutation in the p53 gene at codon 143), was observed in an investigation on the effect of the synthetic cannabinoid CP55940 [44]. The study showed that although Jurkat cells contain the mutated form, p53 was still regulated by the cell at different concentrations. Moreover, an attempt to inhibit the activity of the cannabinoid used resulted in no change in p53 expression, highlighting the cannabinoid’s mode of action on CB1 and CB2 receptors. Notably, beta-actin, the housekeeping gene employed in this assay, exhibited undetectable expression levels at higher concentrations of both cannabinoids. To further explore this observation, cells were treated with concentrations below 50 μM. At these lower concentrations, beta-actin expression was detectable; however, at 40 μM, the corresponding band was faintly visible. This outcome is further corroborated by post-treatment microscopic images (Figure 4 and Figure 5), which display pronounced cellular morphological alterations indicative of apoptotic processes. Beta-actin is a critical component of the cellular cytoskeleton, playing a key role in migratory behavior and cytoskeletal dynamics. Additionally, the actin family is essential in initiating apoptosis due to its involvement in maintaining cellular integrity and driving morphological changes [45]. However, comprehensive data on the direct effects of cannabinoids on the actin family remain limited. Nonetheless, several anticancer agents and compounds have been documented to target the actin cytoskeleton and corresponding pathways [46]. The Western blot results suggest a potential mechanism of action for CBN and CBG involving both the p53 pathway and the actin family.

The mechanism of action of cannabinoids is generally described as involving their interaction with endocannabinoid receptors (CB1/CB2 receptor), which changes the receptor state and induces numerous signaling pathways inside the cell [47]. In several studies on hematological malignancies, the CB2 receptor has been the primary target due to its selective localization on immune cells. However, the underlying molecular anticancer mechanism of most cannabinoids remains poorly understood [48,49]. THC showed dose-dependent induction of apoptosis in leukemia cells (Jurkat and Sup-T1 cell lines) through the binding of CB2 receptors [35]. The anticancer efficiency of THC was also observed to increase in correlation with the incubation period, suggesting that cannabinoids are also time-dependent in their effects. Dronabinol was also shown to confer antileukemic activity against lymphatic and myeloid leukemia cell lines [34] and to induce apoptosis in Jurkat and MOLM13 cell lines in a dose-dependent manner, with a concentration of 75 μM completely killing cells while 15 μM (THC IC-50 concentration) only inhibited cell growth. A mix of cannabis extracts was observed to reduce the viability of the leukemia cell lines HL-60 and CEM at a concentration range of 1–50 μM after 48 h [30]. The study proposed that treating cancer cells with combined cannabinoids yields a more substantial anticancer effect with lower doses, as compared to a single compound at higher concentrations. The potency embodied by responses elicited by cannabinoids depends on the CB receptor expression level, and this can explain the different doses required for different cannabinoids to trigger specific effects [21,50]. CBN and CBG are both considered partial agonist ligands of the CB2 receptor but with different binding affinities [13,28]. Since the CB2 receptor is predominantly expressed in immune cells, it can be speculated that the associated anticancer effects induced by CBN and CBG on THP-1 cells are CB2 receptor-dependent. Cannabinoids that engage with the CB2 receptor are believed to trigger several signaling pathways that mostly involve MAPK, ion channels, adenylyl cyclase and beta-arrestin [51]. The signaling pathway by which cannabinoids induce p53 activation, as documented in the literature, is through the MAPK (Mitogen-Activated Protein Kinase) pathway, which includes p38, JNK, and ERK [27]. These kinases are capable of phosphorylating p53, thereby regulating its activity. Building on this understanding, the MAPK pathway plays a critical role in regulating p53 activity, particularly through phosphorylation events that enhance its stability and transcriptional activity, thereby promoting apoptosis in cancer cells [52]. This suggests that the engagement of CB2 receptors by CBN and CBG may activate MAPK signaling, which in turn could mediate the observed upregulation of p53 in THP-1 cells, further supporting their targeted anticancer effects. Only a small pool of leukemia studies has reported changes in leukemia cell machinery due to exposure to cannabinoids [49]. It has been demonstrated that THC upregulated the DUSP gene that encodes MKP3 in three different human acute leukemia cell lines (i.e., CEM, HL60, and MOLT-4) [36]. MKP3 is known to regulate members of the MAPK/ERK, stress-activated proteins that control cell survival. These signals were suggested to be triggered via the CB2 receptor. Exposure of Jurkat leukemia cells to THC led to apoptosis through the intrinsic and extrinsic apoptotic pathways [53]. Specifically, THC activated caspase-8 and caspase-10 as part of the extrinsic pathway and induced the cleavage of caspase-2 and caspase-9 as part of the intrinsic (mitochondrial) pathway. THC and other synthetic cannabinoid CB2 receptor agonists were shown to trigger apoptotic and antiproliferative mechanisms in various cancer models, including leukemic and nonleukemic malignancies. CBN and CBG are less potent compounds compared to THC and other synthetic agonists of the CB2 receptor [13], yet it is speculated that their antiproliferation and apoptotic effects are mediated via similar cellular mechanisms. It is important to note that the results of the viability assay (24-h incubation) and cell cycle analysis (Figure 1 and Figure 2) reflect the variability in potency of the CB receptor between CBN and CBG in THP-1 cells. Arguably, both techniques can be considered as measurements of proliferation and apoptosis, based on the fact that in some cancer cells, the induction of apoptosis is dependent on the cell cycle pathways [54]. However, CBN, unlike CBG, was previously suggested to have a unique mode of action in leukemia cell lines [41]. At low concentrations (30 μM), CBN induced differentiation, but not apoptosis, in a human myeloblastic leukemia cell line (ML-2 cell line); the CBN-treated cells expressed maturation markers and phenotypes in a dose- and time-dependent manner. This finding may explain the inhibition of THP-1 proliferation by lower doses of CBN as compared to CBG (Figure 2 and Figure 3).

Cannabinoids, unlike conventional cancer drugs, were shown in numerous cancer models to elicit apoptosis and inhibit cell division via different signaling pathways [14]. In addition, cannabinoids are being used in modern medicine to treat side effects associated with chemotherapy administration, such as chemotherapy-induced peripheral neuropathy and chemotherapy-induced nausea and vomiting [55]. Based on previous findings concerning the activity of cannabinoids in leukemia models, the working hypothesis in the current study was that the effects of CBN and CBG on THP-1 are mediated by their interaction with the CB2 receptor [35,36]. Yet, CBN and CBG can also induce CB2 receptor-independent anticancer effects. To determine the role of the cannabinoid receptors in the response to CBN or CBG, a CB2 receptor antagonist, such as SR144528 (a synthetic cannabinoid), can be added together with CBN or CBG [36]. Alternatively, CB2 receptor expression can be silenced using shRNAs or siRNA. This approach can explain the behavior of CBN and CBG in the presence of additional cannabinoids, knowing that the endocannabinoid system consists of endogenous cannabinoids such as N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2AG) [20]. The available in vivo and clinical data on cannabinoids as a treatment for cancer, especially leukemia, is still limited. Currently, the only published human trial testing any cannabinoid in an oncological setting was a small clinical pilot study in which THC was intracranially injected in patients with recurrent glioblastoma [56], and a phase 1b clinical trial of Nabiximols, a cannabinoid spray containing THC and CBD, was conducted in patients with recurrent GBM [57]. A study reported on a 14-year-old patient diagnosed with aggressive Philadelphia-positive ALL who took a cannabinoid extract (hemp oil) showed significant reductions in leukemic blast cell counts measured at different periods [58]. Specifically, after 15 days of cannabinoid treatment, the blast count dropped from 250,000 to 61,000, and after 39 days, the blast count dropped dramatically to 2000. Although observational, a dose-dependent response was achieved with minimal side effects.

Overall, the results suggest that the non-psychotropic cannabinoids CBN and CBG hold promise as agents for regulating cancer cell survival and growth in a leukemia context. The widespread recreational use of cannabis underscores its perceived safety profile, particularly regarding cellular toxicity and damage. Its long history of use, supported by growing scientific evidence, suggests that cannabinoids primarily exhibit antinociceptive effects through their interaction with the central nervous system and possess immunomodulatory properties with minimal side effects [59,60]. Our findings pave the way for novel investigations into the unique mechanisms of cannabinoids across diverse cancer models, as their effects may vary depending on specific cellular abnormalities. Although preliminary, this study supports the potential for further in vitro and in vivo research to elucidate the anticancer activity of these cannabinoids on leukemic cells, potentially contributing to the development of a new class of anticancer agents.