Research Progress on the Mechanism of the Antitumor Effects of Cannabidiol

2.1. CBD Regulates the Tumor Cell Cycle

Cell proliferation is regulated by the cell cycle, and blocking the division cycle of tumor cells is an important antitumor mechanism. Eukaryotic cells rapidly synthesize RNA and protein through the G1 phase, which provides material and energy for DNA synthesis in the S phase. The G2 phase assembles materials needed for cell division and prepares the cell for the M phase, or mitosis. Therefore, in addition to overriding the S and M phases to inhibit the proliferation of tumor cells, regulating the G1 and G2 phases is of great significance for cancer treatment. Several studies have proven that CBD treatment can render tumor cells susceptible to G0–G1 phase blockage.

In thyroid and breast cancer cells, CBD exerts its antiproliferative effect by causing cell cycle arrest during the G1/S phase transition, but it has no similar effect in non-neoplastic cells [10]. In pancreatic cancer, CBD inhibits cell cycle progression by binding to the GPR55 receptor and dose-dependently blocking its activity, resulting in the downregulation of the MAPK/ERK signaling pathway, the inhibition of the G1/S transition, a reduction in the DNA synthesis in the S phase, and the suppression of pancreatic cancer cell growth [11]. The cell cycle is regulated by cyclin and cyclin-dependent kinases (CDKs). Cyclin and CDKs can combine to form a heterodimer that promotes the procession of the cell cycle. The complex formed by cyclin E and CDK2 can promote the transition from the G1 phase to the S phase. Zhang et al. [12] studies showed that CBD inhibits the expression of cyclin E and CDK2 in gastric cancer by significantly downregulating the expression of the p53 protein and upregulating the expression of the p21 protein and the ataxia telangiectasia mutant gene, leading to cell cycle arrest in the G0–G1 phase and subsequently exerting an antiproliferation effect. Similar effects have also been reported for glioblastoma cells. CBD exerts its antiglioblastoma effects by specifically regulating the activities of ERK and multiple caspases (caspase-3, caspase-7, and caspase-9), resulting in the arrest of U251 and SF126 cells in the G0–G1 phase. Combined therapy consisting of CBD and THC can significantly enhance this inhibition [13].

2.2. Multiple CBD-Associated Pathways Can Induce Tumor Cell Apoptosis

Apoptosis refers to the autonomous and orderly death of cells. It is controlled by genes and is associated with processes that actively remove redundant or differentiated cells that are incompatible with the body, cells that have completed their functions but are no longer used/needed, and cells that are potentially dangerous. Therefore, apoptosis is considered an indispensable regulatory mechanism for the normal development of organisms; however, when out of control, apoptosis leads to various diseases, including tumorigenesis. A defect or obstruction in apoptosis can disrupt the normal proliferation, differentiation, and death programs of normal cells, and this disruption is one of the factors underlying tumorigenesis. At present, the clinical use of radiotherapy and chemotherapy drugs, which are mainly used to induce tumor cell apoptosis, shows efficacy. Recent research results have confirmed that CBD can exert its antitumor effects by inducing tumor cell apoptosis through several processes, including the disruption of mitochondrial homeostasis and the imposition of endoplasmic reticulum stress (ERS).

Mitochondria are the activity center of apoptosis regulation, and they play an important role in the apoptosis pathway [14]. Various antioxidant systems in cells and mitochondria can regulate the production of mitochondrial reactive oxygen species (ROS) to maintain intracellular ROS homeostasis under normal physiological conditions. The disruption of the mitochondrial ROS homeostasis is an important factor in the development of diseases in the body. In breast cancer MDA-MB-231 cells, CBD induces BH3 interacting-domain death agonist (BID) production and translocation, increases cytochrome C release, and ultimately promotes apoptosis by reducing the mitochondrial membrane potential. In addition, CBD treatment can increase the production of ROS. 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).

Another apoptosis trigger is ERS, defined as the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum in response to hypoxia, acidosis, and other stress stimuli. High-intensity and sustained stress responses can induce apoptosis. In colon cancer cells, CBD induces ERS and increases the expression of death receptor DR5 by phosphorylating the double-stranded RNA-dependent protein kinase-like ER kinase-C/EBP homologous protein (PERK-CHOP) while also enhancing tumor cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [18]. In addition, CBD dose-dependently induces apoptosis in gastric cancer cell lines (AGS, MKN45, SNU638, and NCI-N87). The molecular mechanism of CBD action involves the induction of ERS and mitochondrial dysfunction by translocating the proapoptotic factor Smac to the cytoplasm to bind to the X-linked inhibitor of apoptosis protein (XIAP), thereby increasing the ubiquitination of XIAP in gastric cancer cells [19].

CBD can also induce tumor cell apoptosis in other ways. Sreevalsan et al. [20] demonstrated that CBD promotes phosphatase-dependent and/or CB receptor-dependent apoptosis in prostate cancer LNCaP and colon cancer SW480 cells. In lung cancer, CBD induced H460 and A450 cell apoptosis through the upregulation of the adhesion molecule ICAM-1 and tumor cell death medicated by lymphokine-activated killer cells (LAK) [21]. Micro-RNA (miRNA), which are short non-coding RNAs, may also be involved in tumor pathogenesis through the regulation of apoptosis [22]. Alharris et al. [23] published the first report showing that CBD can control tumor cell invasion, migration, and apoptosis through miRNAs. CBD can specifically downregulate miRNAhsa-let-7a and increase the expression of caspase-3 and GAS-7 genes. These changes, in turn, activate neuroblastoma (NBL) cell apoptosis.

2.3. CBD Induces Tumor Cell Autophagy

In addition to its proapoptotic effects, CBD also demonstrates the capacity to induce autophagy in neoplastic cells. Autophagy, recognized as type II programmed cell death, encompasses cell demise initiated by the breakdown of misfolded proteins or dysfunctional organelles. Autophagy plays a pivotal role in various stages of carcinogenesis, including tumorigenesis, progression, metabolism, and metastasis, thereby exhibiting a dualistic impact on numerous malignancies [24]. During the initial phases of tumorigenesis, autophagy potentially impedes carcinogenesis by eliminating faulty proteins and organelles [25]. Unfortunately, in the advanced stages of cancer progression, particularly when the tumor confronts stressors (e.g., restricted angiogenesis, nutrient deficiency, and hypoxia), autophagy assumes a prosurvival role and instead fosters tumor proliferation, metastasis, and invasion [26]. Cellular autophagy has therefore emerged as a focal point for antineoplastic therapy [27]. Consequently, the precise mechanism through which CBD elicits its antitumor effects through the instigation of autophagy in tumor cells has garnered substantial attention.

CBD induced the aggregation of the autophagy marker LC3-II protein in a human colonic epithelial cell line [28]. During autophagy, the LC3 protein is synthesized and hydrolyzed by ATG4 to produce cytoplasmic LC3-I, which then covalently binds to phosphatidylethanolamine under the action of Atg7 and Atg3. LC3-I is subsequently lipoylated to form LC3-II, which binds to the autophagosome membrane to form an autophagosome [29]. This process has been confirmed in several colorectal cancer (CRC) cell lines [30]. In addition, CBD-induced LC3-II overexpression has been found in glioma cells. However, in these cells, CBD enhanced the early autophagic flux rather than promoting the complete autophagic process. CBD-induced cell death could be blocked by early autophagy inhibitors (wortmannin and LY294002), whereas late autophagy inhibitors (CQ and Baf-A1) promoted cell death [31]. The autophagy process is closely related to apoptosis, and some studies have confirmed that CBD-induced autophagic responses sometimes coexist with apoptosis. One of these responses is associated with Beclin-1, a mammalian autophagy gene that is also a key target for regulating autophagy. Beclin-1 can bind to Bcl-2 family proteins to form a protein complex to regulate autophagy levels [32], and CBD can inhibit the connection between Beclin1 and Bcl-2, thereby increasing the level of autophagy. In breast cancer MDA-MB-231 cells, CBD treatment induced ERS, resulting in the accumulation of LC3-II and triggering autophagic cell death. In this case, a large amount of caspase-3 produced by apoptosis cleaves Beclin-1 and blocks autophagy. At the same time, the cleavage product is transferred to the mitochondria, where it induces apoptosis by promoting Cyt-c release. Interestingly, the dominance of apoptosis and autophagy seems to be determined by the concentration of CBD [15]. CBD also upregulates acute myeloid leukemia (AML)-1 transcription factors, binds to the transient receptor potential Vanilloid-2 (TRPV2) promoter in a PI3K/AKT-dependent manner, and activates TRPV2 to trigger the differentiation of glioma stem cells (GSCs), which activates the autophagy process and inhibits GSC growth [33]. In addition, some studies have demonstrated in ex vivo experiments that the combination of CBD and oxaliplatin treatment of CRC cells can reduce nitric oxide synthase 3 (NOS3) activity, leading to mitochondrial dysfunction-mediated ROS production and excessive ERS, and ultimately induce autophagy, and thus it has an antitumor role [34].

2.4. CBD Inhibits Tumor Cell Angiogenesis

Angiogenesis is a dynamic process of endothelial cell proliferation, adhesion, migration, and vascular ring formation that is regulated by both angiogenic mediators and inhibitors [35]. Uncontrolled angiogenesis can cause several disorders, including some immune diseases (rheumatoid arthritis), tumorigenesis, and tumor metastasis. Many studies have supported the possibility that CBD can exert its antitumor effects by inhibiting tumor angiogenesis.

Solinas et al. [36] found that CBD in glioma U87-MG cells significantly interfered with and decreased the protein expression of various regulatory factors involved in the angiogenic process, such as vascular endothelial growth factor (VEGF), matrix metalloproteinase-2/9 (MMP-2/9), matrix metalloproteinase inhibitor-1 (TIMP-1), urokinase-type fibrinogen activator (uPA), etc., but no specific signaling pathway was identified. Among them, the matrix metalloproteinase family (MMPs) can be involved in angiogenesis through the disassembly of the basement membrane and remodeling of the extracellular matrix [37]. In contrast, TIMP-1 is an endogenous metalloproteinase inhibitor that inhibits the activity of MMPs [38]. HIF-1α, as a major regulator of oxygen homeostasis, plays a key role in tumor angiogenesis and metastasis [39]. A recent study has shown that CBD can inhibit breast cancer growth by inhibiting SRC activity, thereby upregulating VHL expression, reducing HIF-1α synthesis in breast cancer cells, and inhibiting angiogenesis [40]. Tumor-associated macrophages secrete EGF and promote angiogenesis [41]. Elbaz et al. [42], in a study on the role of CBD in breast cancer, reported that CBD inhibits angiogenesis, thereby playing an antitumor role. It does so by regulating the tumor microenvironment through the modulation of cytokine production by tumor cells, which results in less recruitment of total macrophages and M2 macrophages to primary and secondary tumor sites.

Although the role of cannabinoids and their analogs in inhibiting angiogenesis has been extensively studied, the antiangiogenic effects of CBD have not been well investigated. In addition to the proven targets of CBD’s angiogenesis-inhibiting effects, a number of small molecules and proteins have emerged that potentially inhibit angiogenesis and are worth exploring. For example, endothelial TRPV4 has been demonstrated in recent years as a key regulator of vascular integrity and tumor angiogenesis, suggesting that targeting TRPV4 may represent a potential new strategy for vascular normalization and cancer therapy [43]. Given that CBD can activate TRPV4, CBD may exert its antiangiogenic effects by targeting TRPV4 [31]. In addition, GPR55, which was shown to affect endothelial cell proliferation by decreasing nerve growth factor (NGF) levels in the TME, can also act as a receptor for CBD action. Therefore, its relationship deserves to be investigated in depth to provide a new approach for inhibiting tumor angiogenesis [44].

2.5. CBD Suppresses Tumor Cell Invasion and Migration

The remarkable characteristics of malignant tumors include their capacity for distant invasion and migration of cells. These metastatic cells are also important determinants of mortality among patients with clinical cancers. Research findings substantiate CBD’s capability to inhibit the invasion and migration of various types of tumor cells.

The epithelial–mesenchymal transition (EMT) has garnered extensive attention due to its potential role in transforming benign tumors into aggressive and metastatic malignancies. The EMT has been established as a pivotal process associated with tumor invasion and metastasis. During EMT progression, numerous genes undergo altered expression, including the downregulation of epithelial phenotype-associated proteins (e.g., E-cadherin, keratins, and tight junction protein-1) and the upregulation of mesenchymal phenotype-associated proteins (e.g., N-cadherin, vimentin, and fibronectin) [45]. Targeting EMT-related signals therefore holds promise as a strategy for halting tumor progression. CRC cells treated with CBD show the inhibition of the EMT process due to the disruption of the Wnt/β-catenin signaling pathway. This intervention increases E-cadherin expression and decreases N-cadherin expression, thereby effectively obstructing the Wnt pathway and preventing cancer cell migration [46]. Similarly, in breast cancer cells, CBD demonstrates the capability to restore the epithelial tissue lost due to cell dispersion by facilitating the repositioning of E-cadherin and β-catenin at cell adhesion junctions. CBD also thwarts the nuclear translocation of β-catenin by blocking the IL-1β-induced IL-1β/IL-1RI/β-catenin signaling pathway that regulates migration and progression, one of the factors crucial in driving the EMT [47]. Consequently, CBD might counteract tumor invasion and migration by reversing the EMT. Another target of CBD with the potential to inhibit invasion is the fibrinogen activation system. In that system, plasminogen activator inhibitor type 1 (PAI-1), a member of the serine protease inhibitors, can inhibit fibrinogen activator and interfere with malignant tumor cell invasion [48]. Some studies have confirmed that CBD inhibits PAI-1 expression in lung cancer A549 cells through a cannabinoid receptor 1 (cannabisreceptor1, CB1)/cannabinoid receptor 2 (cannabisreceptor2, CB2)/TRPV1-dependent pathway, which inhibits cell invasion.

MMPs synthesized and secreted by tumor cells can target a variety of proteins in the extracellular matrix (ECM) and degrade the ECM, resulting in tissue structural relaxation and promoting tumor cell invasion and metastasis [49]. CBD inhibits the activation of the EGF/EGFR signaling pathway and the activity of its downstream targets, AKT, ERK, and NF-κB to inhibit tumor cell proliferation and, at the same time, inhibits breast cancer cell invasion and migration through the downregulation of MMP- 2 and MMP-9 secretion, inhibiting breast cancer cell invasion and migration [32]. The transcriptional regulator Id-1 has been reported to be dysregulated in the expression of several types of cancers and is a key determinant of tumorigenesis and metastasis [50]. In breast cancer cells, McAllister et al. [51] showed by analyzing three different classes of cannabinoid compounds that the compound with the most pronounced inhibitory effect on tumor cell proliferation, invasion, and migration was CBD, and further study of the related mechanism of action indicated that CBD could inhibit the expression of the transcriptional regulator Id-1 in breast cancer cells by increasing both p-ERK and ROS, thereby reducing cell proliferation and invasion. Additionally, in glioma cells, CBD plays a similar role, significantly downregulating Id-1 expression and reducing glioblastoma cell invasion after CBD action [52]. In addition, the CBD activation of P38 and MAPK after treatment of lung cancer cell lines A549, H358, and H460 cells induced the expression of TIMP-1, which reduced tumor cell invasion, while in vivo experiments confirmed that CBD had a significant inhibitory effect on lung metastasis in the A549 mouse model [53]. An earlier study using a mouse breast cancer metastasis model also confirmed that CBD reduced the total breast cancer metastasis to the lungs to 75%, inhibited the growth of lung metastases, and showed greater potency in terms of its antitumor invasive ability than in its ability to inhibit the growth of primary tumors [54].