Cbd oil dosage for breast cancer

Cannabidiol (CBD) as a Promising Anti-Cancer Drug

1 Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568, USA; [email protected] (E.S.S.); [email protected] (A.K.W.); [email protected] (D.M.J.)

Andrea K. Watters

1 Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568, USA; [email protected] (E.S.S.); [email protected] (A.K.W.); [email protected] (D.M.J.)

Danny MacKenzie, Jr.

1 Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568, USA; [email protected] (E.S.S.); [email protected] (A.K.W.); [email protected] (D.M.J.)

Lauren M. Granat

2 Department of Internal Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; [email protected]

Dong Zhang

1 Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568, USA; [email protected] (E.S.S.); [email protected] (A.K.W.); [email protected] (D.M.J.)

1 Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568, USA; [email protected] (E.S.S.); [email protected] (A.K.W.); [email protected] (D.M.J.)

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Associated Data

Abstract

Simple Summary

The use of cannabinoids containing plant extracts as herbal medicine can be traced back to as early as 500 BC. In recent years, the medical and health-related applications of one of the non-psychotic cannabinoids, cannabidiol or CBD, has garnered tremendous attention. In this review, we will discuss the most recent findings that strongly support the further development of CBD as a promising anti-cancer drug.

Abstract

Recently, cannabinoids, such as cannabidiol (CBD) and Δ 9 -tetrahydrocannabinol (THC), have been the subject of intensive research and heavy scrutiny. Cannabinoids encompass a wide array of organic molecules, including those that are physiologically produced in humans, synthesized in laboratories, and extracted primarily from the Cannabis sativa plant. These organic molecules share similarities in their chemical structures as well as in their protein binding profiles. However, pronounced differences do exist in their mechanisms of action and clinical applications, which will be briefly compared and contrasted in this review. The mechanism of action of CBD and its potential applications in cancer therapy will be the major focus of this review article.

1. Introduction

The use of Cannabis sativa plant extract as herbal medicine can be dated back as early as 500 BC in Asia. The human endocannabinoid system was uncovered after the discovery of the cannabinoid receptors [1]. It was initially thought that cannabinoids produce their physiological effects via non-specific interactions with the cellular membrane; however, research involving rat models in the late-1980s led to the discovery and characterization of specific cannabinoid receptors, CB1 and CB2 [2,3]. The CB1 receptor is expressed throughout the central nervous system (CNS), whereas the CB2 receptor is found primarily in the immune system and hematopoietic cells [4]. Soon after the discovery of CB1 and CB2, their endogenous ligands, or endocannabinoids, were also identified, including 2-arachidonolyglycerol (2-AG) and N-arachidonoylethanolamine (AEA, also called anandamide) ( Figure 1 A, i and ii) [5,6,7,8]. CB1 and CB2 belong to a large family of transmembrane proteins, called G protein-coupled receptors (GPCRs), and are now believed to be responsible for the majority of the physiological effects of the endocannabinoids ( Figure 1 B). Both receptors are coupled with Gαi/o, which can inhibit the adenylyl cyclase (AC) [4,9]. CB1 can also be coupled to Gαq/11 [10] and Gα12/13 [11]. CB2 has also been shown to act through Gαs [12]. For a more in-depth understanding of the downstream effects of the endocannabinoids and their receptors under physiological conditions, we refer you to other excellent reviews on the topic [13,14].

Endocannabinoid system. (A) Chemical structures of two endogenous cannabinoids, 2-arachidonylglycerol (i, 2-AG) and N-arachidonylethanolamine (ii, AEA), and two representative exogenous cannabinoids from Cannabis sativa, cannabidiol (iii, CBD) and Δ 9 -tetrahydrocannabinol (iv, Δ 9 -THC). (B) Schematic diagrams of the signaling transduction pathways of the endocannabinoid system. 2-AG and AEA are agonists of CB1 and CB2. Some of the downstream effects include: (1) upregulation of p42/p44 mitogen-activated protein kinases (MAPKs) by direct inhibition of adenylyl cyclase (AC) and direct activation of phospholipase C (PLC), leading to the induction of neuronal growth, interleukin production, and inflammation. PKA: protein kinase A. PKC: protein kinase C. (2) Activation of p38 MAPK, which induces inflammation and apoptosis. (3) Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways. Under certain conditions, these endocannabinoids can also induce transcription, cell survival, proliferation, and differentiation through similar pathways. Additionally, the cannabinoid receptors can also modulate ion channels including G protein-coupled inwardly-rectifying potassium channels (GIRKs) and voltage (V)-gated calcium channels.

The two primary endocannabinoids, 2-AG and AEA, can activate either CB1 or CB2 and are synthesized on-demand from phospholipid precursors in response to an elevation of intracellular calcium [15,16]. In addition to CB1 and CB2, 2-AG and AEA can also bind other transmembrane proteins, including orphan G protein-coupled receptor 55 (GPR55), peroxisome proliferator-activated receptors (PPARs), and transient receptor potential vanilloid (TRPV) channel type 1 (TRPV1) [17,18].

The TRPV channels are of particular interest concerning the anti-tumor functions of cannabidiol (CBD) ( Figure 1 A, iii), which will be discussed in more detail later. Six different TRPV channels have been identified in humans and can be subdivided into two groups: TRPV1, TRPV2, TRPV3, and TRPV4 belong to group I, while TRPV5 and TRPV6 fall into group II [19]. Though the exact functions of the TRPV channels are still under intense investigation, they are likely involved in regulating cellular calcium homeostasis. For example, TRPV1 and TRPV2 can be found in the cytoplasmic membrane as well as the endoplasmic reticulum (ER) membrane. They both play an important role in regulating the cytoplasmic calcium concentration from the extracellular sources as well as the calcium stored within the ER. Disruption of cellular calcium homeostasis can lead to increased production of reactive oxygen species (ROS), ER stress, and cell death.

A variety of cannabinoids exist in the Cannabis sativa plant (also known as the hemp or marijuana plant). There are more than 100 different cannabinoids and Δ 9 –tetrahydrocannabinol (Δ 9 -THC) ( Figure 1 A, iv) and CBD are the most well-known ones. The so called drug-type Cannabis sativa contains higher level of Δ 9 -THC and is used more widely for medical and recreational purposes, whereas the fiber-type cannabis contains less than 0.2% of Δ 9 -THC and is more often used in textiles and food [20,21]. Δ 9 -THC is thought to be the psychotic cannabinoid and many of its psychoactive effects are due to its interaction with the CB1 receptor, whereas its immune-modulatory properties are likely due to its interaction with the CB2 receptor. In contrast, CBD is non-psychoactive and has a relatively low affinity to both CB1 and CB2 [22].

The utility of cannabinoids in the treatment of cancer has long been of great interest. Recently, both CB1 and CB2 were found to be expressed in many cancer types. Intriguingly, both receptors were often undetectable at the site of the cancers’ origin before neoplastic transformation [23]. Additional evidence for the role of endocannabinoid system in neoplasia came when Wang and colleagues showed that CB1 has a tumor-suppressive function in a genetically modified mouse model of colon cancer [24]. On the other hand, CB1 is upregulated in hepatocellular carcinoma and Hodgkin lymphoma, and the extent to which CB1 was overexpressed correlated with disease severity in epithelial ovarian carcinoma [25,26,27]. Similarly, CB2 has also been found to be overexpressed in HER2+ breast cancers and gliomas [28,29]. Finally, it was shown that overexpression of both CB1 and CB2 was correlated with poor prognosis in stage IV colorectal carcinoma [30,31]. In 1976, Carchman and colleagues found that the administration of cannabinoids, such as Δ 8 -THC, Δ 9 -THC, and CBD, inhibited the DNA synthesis and growth of lung adenocarcinoma in cultured cells as well as mouse tumor models [32,33]. Similar effects were seen in both in vitro and in vivo models of various other cancers, including glioma, breast, pancreas, prostate, colorectal carcinoma, and lymphoma [34,35]. There are various proposed mechanisms of action behind these findings, including, but not limited to: cell cycle arrest, induction of apoptosis, as well as inhibition of neovascularization, migration, adhesion, invasion, and metastasis [36]. Despite the multitude of positive results with Δ 9 -THC-related cannabinoids in cancer research, the clinical use of these compounds is limited due to their psychoactive side effects.

In contrast to the Δ 9 -THC-related cannabinoids, CBD has no known psychoactive effects, and therefore, has recently been the focus of intense research in many therapeutic areas, including cancer. At present, the Food and Drug Administration (FDA) has only approved Epidiolex, purified CBD, for use in patients with seizures associated with the Lennox-Gastaut syndrome or Dravet syndrome [37]. Globally, more than 40 countries have approved medical marijuana/cannabis programs, whereas this is true of 34 states in the USA, plus the District of Columbia, Guam, Puerto Rico, and US Virgin Islands. While marijuana is considered a Schedule I controlled substance in the US, the Drug Enforcement Administration ruled that CBD is a Schedule V controlled substance [38]. When approved by the FDA, CBD must contain less than 0.1% of Δ 9 -THC.

It has been noted that CBD has a relatively low affinity to both CB1 and CB2 [22]. However, it was found that CBD can act as an antagonist to CB1 in the mouse vas deferens and brain tissues in vitro [39]. There is also evidence suggesting that CBD may act as an inverse agonist of human CB2 [22]. Other cellular receptors that CBD may interact with include TRPVs, 5-HT1A, GPR55, and PPARγ [40]. It has been hypothesized that CBD has robust anti-proliferative and pro-apoptotic effects. In addition, it may also inhibit cancer cell migration, invasion, and metastasis [1]. The utility of CBD in anti-tumor therapy and the potential mechanisms behind it will be discussed in more detail below. Since much of the anti-tumor activity of CBD seems to hinge on its regulation of ROS, ER stress, and immune modulation, we will first summarize the interplays between ROS, ER stress, and inflammation and their known effects on various aspects of tumorigenesis. Thereafter, we will further discuss the anti-tumor effects of CBD on a variety of cancers and the molecular mechanisms behind them.

2. The Interplays between Reactive Oxygen Species (ROS), ER Stress, Inflammation, and Cancers

2.1. ROS and Cancers

ROS refer to various oxygen-containing species that are energetically unstable and highly reactive with a variety of biomolecules, including amino acids, lipids and nucleic acids. Commonly seen ROS include superoxide (O2 − ), peroxide (O2 −2 ), hydrogen peroxide (H2O2), and hydroxyl free radical (OH − ) [41,42,43,44]. The most common sources of ROS are the electron transport chain in the mitochondria and the NADPH oxidase (NOX) family of transmembrane enzymes ( Figure 2 ). Certain enzymes and organelles, such as peroxisomes and ER, can also produce ROS. ROS can directly oxidize nucleic acids, proteins, and lipids thus altering or disrupting their functions [45].

Origins and effects of cellular reactive oxygen species (ROS). ROS are generated by complex I and III of the electron transport chain in the mitochondria and by NADPH oxidase (NOX) enzymes located at the cytoplasmic membrane (PM). ROS disrupt cellular processes by oxidizing the cysteine (Cys) residues of various proteins and damaging nucleic acids. Oxidation by ROS could cause the inactivation of phosphatases, activation of kinases and transcription factors (TF), and genomic alterations, leading to enhanced cellular proliferation and survival. ROS production is counteracted by the generation of antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), peroxiredoxin (PRX), thioredoxin (TRX), and catalase. In cancers, redox homeostasis is modified to favor ROS tolerance. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. NM: nuclear membrane.

To prevent constant damage to biomolecules, ROS are counter-balanced by various antioxidants inside the cells. Major anti-oxidant enzymes include superoxide dismutase (SOD), catalase, peroxiredoxin (PRX), thioredoxin (TRX), and glutathione peroxidase (GPX) [42].

In cancers, the redox balance is altered so that increased ROS production favors tumor progression and expansion while evading cell death. The pro-tumor effects of increased ROS generation include, but are not limited to, genomic instability and enhanced proliferation [42,43,44] ( Figure 2 ). ROS damage DNA by oxidizing guanine and forming 8-hydroxyguanine and 8-nitroguanine. This could lead to deletions/insertions, mutations in base pairing, and strand breaks followed by mutagenic repair [44,45]. Genome instability plays a key role in tumor progression through the accumulation of mutations that promote uncontrolled growth and evade cell death [43]. Proliferation is further enhanced through the oxidation and activation of the pro-growth intracellular signaling pathways, including mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a transcription factor vital for growth and migration, also becomes activated by ROS through inhibiting the phosphorylation of the inhibitor of NF-κB α (IκBα), or through promoting the S-glutathionylation of the inhibitor of NF-κB kinase subunit β (IKKβ). Finally, cancer cells can rewire their signaling transduction pathways to cope with elevated intracellular ROS. Most notably, this can be achieved through increased mitochondrial SOD activity or inactivation of the scavenging enzymes [42,46].

Nonetheless, toxic levels of ROS can induce cell death or autophagy in cancer cells. ROS modulate calcium channels, pumps, and exchangers activity by oxidizing their Cys residues [43]. The increase of intracellular mitochondrial calcium or the oxidation of lipids damages the mitochondrial membrane resulting in the release of cytochrome c, a potent activator of apoptosomes [42,45]. ROS can also directly affect caspase activity and cleavage of Bcl-2, and/or increase the expression of cell death receptors such as TRAIL and Fas [47]. Autophagy can be induced by the activation of the mTOR pathway.

2.2. Endoplasmic Reticulum (ER) Stress and Cancers

ER is an important organelle that plays a critical role in post-translational modification and folding of proteins, calcium homeostasis, and other biological processes [48,49]. Accumulation of unfolded and/or misfolded proteins triggers the unfolded protein response (UPR), which helps to re-balance the ER homeostasis. UPR temporarily halts protein synthesis and attempts to correct and re-fold proteins. In the case that the unfolded and/or misfolded proteins cannot be corrected in time, they will then be targeted for protein degradation.

UPR is a well-studied cellular process ( Figure 3 A). It is primarily regulated by the 78-kDa glucose-regulated protein (GRP78), which is also known as the binding immunoglobulin protein (BiP) [49]. Under non-stress conditions, GRP78 binds and inhibits three transmembrane proteins: inositol-requiring enzymes 1α (IRE1α), pancreatic endoplasmic reticulum kinase (PERK), as well as the activating transcription factor 6 (ATF6) [48,49]. Whereas under ER stress conditions, GRP78 binds the unfolded proteins, dissociates from PERK, IRE1α, and ATF6, and results in the activation of three distinct, but interconnecting, pathways. Downstream of the PERK and ATF6 cascades, CHOP activity is increased.

Endoplasmic reticulum (ER) homeostasis, stress, and the unfolded protein response (UPR). (A) ER homeostasis is mediated by 78-kDa glucose-regulated protein (GRP78). Under stress conditions, GRP78 dissociates from pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzymes 1α (IRE1α), as well as the activating transcription factor 6 (ATF6), leading to activation of their downstream signaling cascades in order to restore ER homeostasis. (B) When ER homeostasis fails to be restored, excessive UPR could lead to apoptosis, primarily via upregulation of C/EBP homologous protein (CHOP). PM: cytoplasmic membrane; eIF2α: eukaryotic initiation factor 2α; ATF4: activating transcription factor 4; GADD34: DNA damage inducible protein 34; XPB1: X-box-binding protein (XBP1s: spliced form); ERO1α: endoplasmic reticulum oxidoreductase 1α; PDI: protein disulfide isomerase; DR5: death receptor 5; TRAIL: TNF related apoptosis-inducing ligand; IP3R: inositol 1,4,5-triphosphate receptor; BAP31: B cell receptor-associated protein 31; Bid: BH3 Interacting Domain Death Agonist; TRAF2: tumor necrosis factor receptor-associated factor 2; RIDD: regulated IRE1-dependent decay; ASK1: apoptosis signal-regulating kinase 1; JNK: JUN N-terminal kinase; E2F7: E2F transcription factor 7; E2F1: E2F transcription factor 1.

CHOP induces apoptosis via multiple pathways ( Figure 3 B): (i) It increases the transcription of GADD34 [49]; (ii) It increases the transcription of ER oxidoreductase 1 alpha (ERO1α), which then re-oxidizes PDI and generates ROS; (iii) It increases the transcription of the inositol 1,4,5-triphosphate receptor (IP3R), which then increases the calcium level in the cytoplasm; (iv) It activates the extrinsic cell death pathway via death receptor 5 (DR5) and caspase-8 mediated activation of truncated Bid (tBid), which then translocates to the mitochondria and promotes the release of cytochrome c; (v) It activates the intrinsic cell death pathway by directly decreasing the expression of pro-survival factors, Bcl-2 and Bcl-xL, and increasing the expression of pro-apoptotic factors, such as Bax, Bak, Bim, Puma, and Noxa; (vi) It activates caspase-8 via TRAIL-DR5 on the cytoplasmic membrane, which cleaves B cell receptor-associated protein 31 (BAP31) and forms p20. p20 then releases calcium from the ER into the cytoplasm, which is taken up by mitochondria and results in the further release of cytochrome c.

During their development, tumors rely heavily on the UPR pathway for cell survival, possibly due to the hypoxic environment and metabolic stress accompanying the rapidly increasing tumor mass. For example, PERK and ATF4 activate vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1/2 (HIF1/2) for angiogenesis [48]. The silencing of the XBP1 gene prevented tumor growth and metastasis of triple-negative breast cancer (TNBC) in vivo [50]. Analysis using TNBC cell lines demonstrated that the upregulation of XBP1 enhanced HIF1α expression. Nonetheless, when the URP system becomes overwhelmed, pro-apoptotic factors become dominant, leading to cell death.

2.3. The Effects of Inflammation and Microenvironment on Tumor Survival, Migration, and Immune Evasion

Tissue microenvironment often plays an important role in supporting tumor establishment, expansion, and metastasis. The tumor microenvironment is primarily comprised of infiltrated leukocytes, including tumor-associated macrophages (TAMs), dendritic cells, and myeloid-derived suppressor cells (MDSC) [51]. The crosstalk between the infiltrated cells and tumor cells could suppress the immune response and create a pro-survival environment for tumor cells.

Evasion of the attack by the immune system is essential during the development of cancers. This is accomplished through dynamic interactions between different cytokines and their receptors in the tumor microenvironment. Tumors actively secrete different cytokines that attract a variety of infiltrating cells, such as TAMs, dendritic cells, MSDCs, and immunosuppressive regulatory T cells, which in turn help tumors to evade the attack by the immune system ( Figure 4 A). Cytokines released from myeloid cells can also induce genomic instability in tumor cells by directly damaging DNA or epigenetically altering the expression of genes ( Figure 4 B).

The interplays between tumor cells and inflammatory cells during tumorigenesis. (A) The effect of tumor cells on inflammatory cells. Tumor cells secrete many cytokines to alter the microenvironment to promote tumor growth and invasion and to blunt the anti-tumorigenic immune response. (B) Inflammatory cells affect the genomic stability of tumor cells. AID: activation-induced cytidine deaminase; DNMT1: DNA methyltransferase 1. (C) Inflammatory cells enhance tumor cell proliferation and survival through autocrine and paracrine signaling. (D) Inflammatory cells promote tumor cell migration, invasion, and metastasis through cytokine and chemokine production. COX-2: cyclooxygenase 2; MMP: matrix metalloproteinase; E-cad: E-cadherin; EMT: epithelial-mesenchymal transition; sLex: sialyl Lewis X; CXCR: CXC chemokine receptor; BV: blood vessel.

The key inflammatory mediators for tumor proliferation and survival include NF-κB and signal transducer and activator of transcription 3 (STAT3) ( Figure 4 C) [52]. IL-6, secreted by the myeloid cells, activates STAT3, which then upregulates cyclins D1, D2, and B as well as MYC to promote tumor cell proliferation. STAT3 expressed by the tumor cells further enhances IL-6 secretion by the myeloid cells via increased expression of NF-κB in these inflammatory cells, thus creating a positive feedback loop. IL-22, produced by the CD11c+ lymphoid cells, is also able to activate STAT3 in epithelial cells. In parallel, TNF-α and IL-1 secretion from leukocytes can upregulate the expression of NF-κB in tumor cells [52,53,54]. NF-κB, in turn, upregulates the expression of IL-1α, IL-1R, and MYD88, which can further enhance the activity of NF-κB, thus creating a positive autocrine loop [52]. The expression of NF-κB can be directly activated in immune cells by the inflammatory cytokines, TNF-α and IL-1, and by TLR-MYD88 from tissue damage [53,54]. Downstream of IL-6 signaling, NF-κB has also been shown to induce epithelial-mesenchymal transition (EMT), which then promotes tumor cell migration [54]. In a prostate cancer model, the interaction between receptor activator of NF-κB (RANK), on the surface of cancer cells, and RANK ligand, on the infiltrating leukocytes, promoted metastasis through the activation of NF-κB pathway. This NF-κB/IL-6/STAT3 positive feedback loop is present in all phases of tumorigenesis.

Furthermore, the expression of STAT3 in tumor-associated leukocytes also plays a key role in immune modulation. STAT3 expression in inflammatory cells allows for immune evasion of tumors, while STAT3 deletion in macrophages and neutrophils enhances Th1-mediated response with increased production of IFNγ, TNF-α, and IL-1 [55]. STAT3 expression in myeloid cells can inhibit the maturation of dendritic cells by downregulating their IL-12 expression and suppresses the immune response by upregulating the expression of IL-23 in TAMs [53].

Collectively, the activation of NF-κB and STAT3 signaling transduction pathways in cancer cells, as well as in the inflammatory cells in the tumor microenvironment, provide a great advantage for tumor cell proliferation, survival, migration, and immune evasion ( Figure 4 C,D).

3. The Anti-Cancer Effects of CBD

3.1. Glioma

Glioma is the most common primary brain malignancy. The grade IV glioma, also called glioblastoma multiforme (GBM) or glioblastoma, is one of the most aggressive types of cancer. The prognosis of GBM is very poor with only 4–5% survival within five years. Current treatment modalities include surgery, followed by radiotherapy and chemotherapy with Temozolomide (TMZ) or Carmustine (BCNU). Unfortunately, most GBM tumors are resistant to these treatments.

Cannabinoids have been studied to a great extent in gliomas due to the urgent unmet medical needs. The Table S1 summarizes the published studies focusing on CBD’s effects on gliomas either alone or together with BCNU, TMZ, tamoxifen, cisplatin, γ-irradiation, ATM inhibitors, and Δ 9 -THC [56,57,58,59,60,61,62,63,64]. In these studies, many GBM cell lines were used with a majority using U87MG [56,57,58,60,61,63,65,66,67,68,69,70,71,72,73,74]. The anti-proliferative effects of CBD on GBMs are quite clear, but the average IC50 values of CBD differed among different cell lines: C6 (8.5 µM) [67], U87MG (12.75 ± 9.7 µM), U373 (21.6 ± 3.5 µM) [65,75], U251 (4.91 ± 6.1 µM) [57,60], SF126 (1.2 µM) [57], T98 (8.03 ± 4.0 µM) [58,59,60,70,73], MZC (33.2 µM) [69], and GL261(10.67 ± 0.58 µM) [59]. Variation among different publications may be due to procedural differences, including assays used to measure the viability and/or time of CBD exposure.

CBD, alone or with other agents, has been shown to successfully induce cell death, inhibit cell migration and invasion in vitro, decrease tumor size, vascularization, growth, and weight, and increase survival and induce tumor regression in vivo [58,59,62,65,68,70,71,74]. Regarding CBD’s anti-proliferative action on GBM, data show that apoptosis occurs independent of CB1, CB2, and TRPV1, but is dependent on TRPV2 [58,65,66,67,69,72]. Specifically, Ivanov et al. found that CBD, γ-irradiation, and ATM inhibitor KU60019 upregulate TNF/ TNFR1 and TRAIL/ TRAIL-R2 signaling along with DR5 within the extrinsic apoptotic pathway [61,64]. CBD also activates the JNK-AP1 and NF-κB pathways to induce cell death. Less emphasis has been placed on the role of autophagy or cell cycle arrest in CBD-mediated effects on glial cells [57,58,64,72,74].

Many downstream effects of CBD have been investigated. Multiple papers reported an increased level of oxidative stress in CBD, but not Δ 9 -THC, treated GBM cell lines [58,65,73,76]. Massi et al. found that the level of ROS increases in a time-dependent manner, with significance after only five hours, when U87MG cells were treated with 25 µM CBD [76]. At the same time, glutathione, an antioxidant, was significantly decreased after six hours of CBD treatment. In contrast, there is no pronounced ROS increase in CBD treated normal glial cells. Co-treatment of CBD and antioxidants, including N-acetyl cysteine (NAC) and α-tocopherol (i.e., vitamin E), attenuated CBD’s killing effects [58]. Taken together, studies in GBM cell lines suggest that CBD induces cell death most likely by upregulating ROS. Scott et al. found that CBD also increased the expression of heat shock proteins (HSPs), which was found to be associated with the increased production of ROS because NAC hindered the role of HSPs [73]. Interestingly, the use of HSP inhibitors together with CBD were shown to increase the cytotoxic effect and reduce CBD’s IC50 value significantly, from 11 ± 2.7 µM to 4.8 ± 1.9 µM in T98G cells. This suggests that HSP inhibitors may be used as an adjunctive treatment with CBD. Recently, Aparicio-Blanco et al. administered CBD in lipid nanocapsules (LNCs) to GBM in vitro in an attempt to provide a prolonged-release formula of CBD [75]. LNCs loaded with CBD were more effective at decreasing the IC50 values when they were smaller in size and exposed for longer periods.

In GBMs, CBD inhibits the PI3K/AKT survival pathway by downregulating the phosphorylation of AKT1/2 (pAKT) and p42/44 MAPKs without effecting the total AKT and p42/44 MAPK protein levels [57,59,61,70,72,73]. This pathway may also be responsible for CBD-mediated autophagy in glioma stem-like cells, since in those cells, PTEN is upregulated while AKT is downregulated [72]. PI3K pathway plays an important role in the expression of TRPV2, which is a potential target of CBD. In U251, Δ 9 -THC and CBD together, but not separately, downregulated p42/44 MAPKs [57]. Whereas Scott et al. revealed that alone, CBD treated T98G and U87MG cells, albeit at a higher concentration (20 µM), decreased pAKT and p42/44 MAPKs levels, and more so when combined with γ-irradiation [59]. CBD can also activate the pro-apoptotic MAP kinase pathway. Ivanov et al. found that CBD treatment together with γ-irradiation led to the upregulation of active JNK1/2 and p38 MAPK, especially in U87MG cells [61]. However, using U251 cells, Marcu et al. showed that Δ 9 -THC and CBD did not increase the activity of JNK1/2 or p38 MAPK [57]. The discrepancy could be due to the genetic difference among different GBM cell lines.

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Massi et al. explored how CBD modulates 5-lipoxygenase (5-LOX), COX-2, and the endocannabinoid system in GBMs [68,73,76]. They found that 5-LOX, but not COX-2, was decreased by CBD in vivo. CBD treatment also resulted in increased expression of fatty acid amide hydrolase (FAAH), which reduces the level of AEA, suggesting that CBD may inhibit the production of inflammatory mediators by indirectly attenuating the endocannabinoid system in GBMs.

In addition to γ-irradiation, CBD has also been tested with alkylating agents, especially TMZ, proving together to have synergistic anti-proliferative effects on glioma cells [60,62,63,74]. Kosgodage et al. found that CBD-treated cells, alone and with TMZ, increased extracellular vesicles (EV) containing anti-oncogenic miR-126 [63]. There were also reduced levels of pro-oncogenic miR-21 and prohibitin, which are responsible for chemo-resistant functions and mitochondria protective properties.

In pre-clinical GBM mouse models, oral administration of a Sativex-like combination of Δ 9 -THC and CBD, at a 1:1 ratio with TMZ, decreased tumor growth and increased survival [62,74]. These findings have led to two phase I/II clinical trials [77,78]. Preliminary results are only available for one study and are promising ( > NCT01812603) [79]. Patients with GBM were either treated with the Sativex, CBD:Δ 9 -THC (1:1), oro-mucosal spray with dose-intense TMZ, or placebo, and the first part of the study showed no Grade 3 or 4 toxicities. In the second part of this study, the same drug combination increased median survival compared to a placebo group with increased one-year survival of 83% and 56%, respectively. The most common adverse effects reported of the treatment were dizziness and nausea. Resistance to TMZ treatment may be reduced by using CBD: Δ 9 -THC combinations. When the full report is published, we are hopeful that the authors will discuss the safety and efficacy in more detail and help to determine which adverse effects can be attributed to Sativex versus TMZ.

There are also a few case studies that described the use of CBD in patients with high-grade gliomas [80,81]. Two patients were treated with procarbazine, lomustine, and vincristine along with CBD (one patient at 100–200 mg/day orally and the other at 300–450 mg/day orally) for about two years [80]. Both patients did not have any disease progression for two years after treatment. Adverse effects of the treatment included rash, moderate nausea, vomiting, and fatigue, without any lymphopenia, thrombocytopenia, hepatic toxicity, or neurotoxicity. In a case series describing nine patients with grade IV GBM, mean survival with the combination of surgery, radio- and chemo-therapy, and CBD (200–400 mg/day) was prolonged to 22.3 months, and two patients had no signs of disease progression for three or more years [81].

Taken together, the published results indicate that CBD alone, or in combination with Δ 9 -THC, TMZ, or γ-irradiation, show great promise in the treatment of glioma. Furthermore, the adverse effects of CBD alone, or together with Δ 9 -THC, appear to be relatively benign.

3.2. Breast Cancer

Breast cancer is the number one leading cause of new cancer cases and the second leading cause of cancer deaths of women in the United States [82]. CBD’s effects on breast cancer have been studied since 2006; research in the field has undergone recent expansion (Table S2). Various breast cancer cell lines have been used to demonstrate a dose-dependent response to CBD, including estrogen-receptor (ER)-positive cells (MCF-7, ZR-75-1, T47D), ER-negative cells (MDA-MB-231, MDA-MB-468, and SK-BR3), and triple-negative breast cancer (TNBC) cells (SUM159, 4T1up, MVT-1, and SCP2) [67,83,84,85,86,87,88]. As low as 1 to 5 µM of CBD induced significant cell death in MDA-MB-231 after 24 h [89]. CBD’s IC50 values for most cell lines are consistently low, indicating that breast cancer cell lines are generally sensitive to CBD’s anti-proliferative effects without a significant effect on non-transformed breast epithelial cells [87].

CBD exerts its anti-proliferative effects on breast cancer cells through a variety of mechanisms, including apoptosis, autophagy, and cell cycle arrest [67,83,87]. Ligresti et al. reported that CBD-treated MDA-MB-231 cells induced an apoptotic effect involving caspase-3, whereas CBD exerted its effects on MCF-7 through cell cycle arrest at the G1/S checkpoint [67]. That being said, cell cycle arrest at the G1/S checkpoint has been more recently demonstrated in MDA-MB-231 and 4T1 cells after CBD treatment [90]. While the activation of CB2 and TRPV1 receptors were seen in MDA-MB-231 cells, the effect was partial. More recent studies have found the anti-proliferative effects of CBD on breast cancer cells to be independent of the endocannabinoid receptors [87]. CBD has been consistently shown to generate ROS, which in turn inhibit proliferation and induce cell death [63,67,87,88,89]. CBD exerts its pro-apoptotic effects by downregulating mTOR, AKT, 4EBP1, and cyclin D while upregulating the expression of PPARγ and its nuclear localization [83,87]. Shrivastava et al. showed that inhibition of the AKT/mTOR signaling pathway and induction of ER stress also induced autophagy alongside apoptosis [87]. At increased CBD concentrations, or when autophagy was inhibited, the levels of apoptosis increased. They further showed that CBD may coordinate apoptosis and autophagy through the translocation and cleavage of Beclin-1.

CBD has also been shown to inhibit migration, invasion, and metastasis in aggressive breast cancer in vivo and in vitro [67,84,88,90]. McAllister et al. observed downregulated Id-1 protein by ERK and ROS in CBD-treated MDA-MB-231 and MDA-MB-436 tumors. This downregulation correlated with a decrease in tumor invasion and metastases [86,90]. Id-1 expression was also found to be downregulated in lung metastatic foci. Consistent with these observations, CBD failed to inhibit lung metastasis in Id-1 overexpressed breast cancer cells [88]. Interestingly, this same study showed that at a lower concentration (1.5 µM), which produced ROS and inhibited the expression of Id-1 in MDA-MB-231 cells, CBD did not induce autophagy or apoptosis [88]. More recently, CBD was shown to inhibit the proliferative, migratory, and invasive nature of TNBC cells by suppressing the activation of the EGF/ EGFR pathway and its downstream targets (AKT and NF-κB) [84]. MMP, phalloidin, and actin stress fibers are important in tumor invasion and were also suppressed by CBD. These results, as they pertain to EGF/EGFR pathway and the MMP, phalloidin, and actin stress fibers, were also confirmed in vivo. Primary tumor size has been shown to decrease along with the number of lung metastatic foci, volume, and vascularization in CBD-treated mice [84,90]. Intriguingly, when CBD was administered three times a week, rather than daily as was done by McAllister et al., the number of metastases were reduced and mice survived longer, but the primary tumor was not reduced [88,90]. The decreased angiogenesis and invasion were found to be due to a change in the tumor microenvironment, for example, a marked decrease in CCL3, GM-CSF, and MIP-2, which resulted in the inhibition of TAMs recruitment ( Figure 4 A) [84]. Finally, another study described a synthetic cannabinoid analog, O-1663, which was shown to be more potent than both CBD and Δ 9 -THC, and similarly induced cell death and autophagy [88]. O-1663 also inhibited breast cancer aggressiveness in vitro and in vivo. It significantly increased the survival in advanced breast cancer metastasis, inhibited the formation of metastatic foci ≥2 mm, and induced regression of established metastatic foci, all with no pronounced toxicity. Altogether, the evidence suggests that there are multiple mechanisms by which CBD impedes tumor migration.

Kosgodage et al. showed that breast cancer cells treated with CBD had increased sensitization to cisplatin. CBD significantly decreased the release of exosomes and microvesicles (EMV) (at 100–200 nm), which typically aid the spread of tumors and cause chemo-resistance [89]. However, in these same MDA-MB-231 cells, there was an increase in the release of the larger EMVs (201–500 nm). These cells displayed a concentration-dependent increase in ROS, proton leakage, mitochondrial respiration, and ATP levels. The authors attributed these effects to either a higher sensitivity or a sign of pseudo-apoptotic responses occurring, where the apoptotic factors such as ROS are still at a lower level resulting in the conversion of apoptosomes into EMVs. CBD inhibited paclitaxel-induced neurotoxicity through a 5-HT1A receptor system without conditioned reward or cognitive impairment [85]. It also decreased the viability of both 4T1 and MDA-MB-231 cells. Thus, CBD may be a viable adjunctive treatment for breast cancers as it can sensitize cells, allowing for potentially lower doses of such toxic chemicals to be prescribed.

Taken together, CBD has been consistently shown to be efficacious in many breast cancer cells and mouse models when it comes to its anti-proliferative and pro-apoptotic effects, while the mechanisms of these effects may vary. At this point, there is an urgent need for clinical trials looking at the anti-tumor effect of CBD for breast cancers, as this seems to be the next logical step in the progression of developing CBD as a treatment alternative for breast cancers.

3.3. Lung Cancer

Based on epidemiological studies by the American Cancer Society, lung cancer is the second most common cancer in both males and females [82]. Lung cancers are classified as small cell lung cancer (SCLC, 13%) and non-small cell lung cancers (NSCLC, 84%), which can be further subdivided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

Ramer and colleagues have published many studies on the effects of CBD on lung cancers (Table S3) [91,92,93,94]. They consistently used the WST-1 assay to assess the viability of lung cancers. CBD decreased the viability of two NSCLC cell lines, A549 (a lung adenocarcinoma cell line) and H460 (a large cell lung carcinoma cell line), with IC50 values of 3.47 µM and 2.80 µM, respectively [94]. There was a 29% and 63% reduction in A549 invasion after incubation with 0.001 µM or 0.1 µM CBD, respectively, for 72 h [92]. There was no significant cell death detected in A549 cells after treatment with 0.001 µM or 0.1 µM CBD. Various lung cancer cell lines (e.g., A549, H358, and H460) have been shown to express CB1, CB2, and TRPV1, which the anti-invasive function of CBD partly relies on [91,92,93]. CBD also significantly reduced tumor size and lung metastatic nodules (from an average of 6 nodules to only 1 nodule) in an A549 xenograft tumor model [92,93].

One mechanism of the pro-apoptotic effect of CBD is through the activation of COX-2, a pathway for endocannabinoid degradation, and PPAR-γ [94]. CBD treatment, at 3 µM in A549, H460, and primary lung tumor cells from a patient with brain metastasis, resulted in the upregulation of COX-2 and PPAR-γ both mRNA and protein. These observations were also confirmed in vivo. COX-2-derived products (PGE2, PGD2, and 15d-PGJ2) were also elevated in CBD-treated lung cancer cells. By suppressing COX-2 and PPAR-γ activity with antagonists or siRNA, CBD’s pro-apoptotic and cytotoxic effects were severely attenuated. Consistently, in a lung tumor mouse model, PPAR-γ inhibition by GW9662 reversed the tumor-suppressive effects of CBD.

While Ramer et al. discussed plasminogen activator inhibitor-1’s (PAI-1) pro- vs. anti-tumorigenic actions, they provided evidence supporting the former [92]. At 1 µM CBD, there was a decrease in PAI-1 mRNA and protein in A549, H358, and H460. This was confirmed in vivo using the A549 mouse model with 5 mg/kg CBD three times a week. In vitro, CBD’s anti-invasive property was reduced by siRNA knockdown of PAI-1 and was increased with the treatment of a recombinant PAI-1. The CBD-mediated decrease in PAI-1 is due, in part, to the activation of CB1, CB2, and TRPV1, as their antagonists reversed the effect. Therefore, CBD works as an agonist of CB1, CB2, and TRPV1 in lung cancers.

Tissue inhibitor of MMPs (TIMPs) were evaluated and are related to the anti-invasive effect of CBD and were found to be induced by CBD in a time- and concentration-dependent manner [93]. CBD-mediated upregulation of TIMP-1 was attributed to the activation of CB1, CB2, and TRPV1. CBD also activated p38 MAPK and p42/44 MAPK, two downstream targets of TRPV1. To connect CB1, CB2, and TRPV1 to the activation of MAPK and TIMP-1, Ramer et al. investigated the expression and function of intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein involved in tumor metastasis [91] ( Figure 5 A). Time- and concentration-dependent increase of ICAM-1 was observed in CBD-treated A549, H358, H460, and cells from a patient with brain metastatic NSCLC. An increase in the expression of TIMP-1 mRNA was also observed, but it occurred after an increase of ICAM-1 mRNA. The expression of ICAM-1 was dependent on the activation of p42/44 MAPK and p38 MAPK. In the in vivo A549 model displaying CBD’s anti-invasive properties, both ICAM-1 and TIMP-1 were also upregulated. Inactivation of ICAM-1 using a neutralizing antibody and siRNA led to a decrease in TIMP-1 activation as well as a reduction in CBD’s anti-invasive properties. These data suggest that the MAPKs activate ICAM-1, which then stimulates the function of TIMP-1 that, in turn, suppresses tumor invasion.

CBD’s effects on cancer cells and infiltrating immune cells. (A) Through its interactions with the CB1, CB2, and TRPV1 receptors, CBD induces cell cycle arrest and apoptosis in cancer cells. (B) CBD also binds the CB1 and CB2 receptors on the infiltrating inflammatory cells and disrupts the pro-tumorigenic cytokine production, thus leading to ineffective immunosuppression and promoting tumor cell death. ROS production by phagocytic cells disrupts the ER and mitochondrial homeostasis in tumor cells leading to apoptosis. UPR: unfolded protein response.

In a separate study, Haustein et al. investigated CBD-induced ICAM-1 expression on lymphokine-activated kill (LAK) cell-mediated cytotoxicity [95]. Treatment with 3 µM CBD induced ICAM-1 expression and LAK cell-mediated tumor cell lysis in A549 and H460, along with metastatic cells from a patient with NSCLC. The increased susceptibility to adhesion and lysis by LAK in CBD-treated cells was reversed using a neutralizing ICAM-1 antibody. This cell lysis effect was reversed with the usage of ICAM-1 siRNA, along with CB1, CB2, and TRPV1 antagonists. Lymphocyte function association antigen (LFA-1) reversed CBD-induced killing effects on LAK cells, suggesting that it works as a counter-receptor to ICAM-1 [95]. Finally, CBD did not induce LAK cell-mediated lysis and upregulation of ICAM-1 of non-tumor bronchial epithelial cells, suggesting this effect is specific to cancer cells.

Taken together, these studies suggest that through CB1, CB2, and TRPV1 receptors, CBD activates p38 MAPK and p42/44 MAPK, which first induce ICAM-1 and then TIMP-1. The upregulation of ICAM-1 and TIMP-1 then attenuates the invasion of lung cancers ( Figure 5 A).

At present, there are no published results on a clinical trial using CBD to treat lung cancer patients. However, in a recent case report, an 81-year-old male patient attempted to self-treat his lung adenocarcinoma using CBD oil [96]. When first diagnosed with a mass 2.5 × 2.5 cm in size and multiple mediastinal masses, the patient was denied chemotherapy and radiation therapy given his age and the toxicity profile of these treatments. However, a year later, computed tomography (CT) scan showed that the tumor and mediastinal lymph nodes began to regress. During that period, the primary factor that was changed was that he began taking 2% CBD oil. Adverse effects included slight nausea and sickening taste.

3.4. Colorectal Cancer

In the US, colorectal cancer (CRC) is the third leading cause of cancer deaths in both males and females [82]. Studies using two CRC cell lines, Caco-2 and DLD-1, as well as healthy and cancerous tissues from nine CRC patients, suggest that endocannabinoid production is significantly increased in precancerous adenomatous polyps and, to a lesser extent, cancerous colon tissue [97]. Normal human colorectal tissue does express both CB1 and CB2, along with AEA, 2-AG, and endocannabinoid-metabolizing enzymes such as FAAH. Transformed adenomatous polyps have increased levels of 2-AG compared to normal colorectal tissues. While DLD-1 cells express both CB1 and CB2, Caco-2 cells only express CB1. Depending on the stage of the cancer, endocannabinoids can either inhibit or promote the growth of CRC. Thus, based on the stage of the cancer, both activators and inhibitors of the endocannabinoid system may be useful in combating CRC.

CBD’s effects on CRC are summarized in Table S4. The dose-dependent killing of CRC cells by CBD has been demonstrated by many studies, however, the IC50 values of SW480 have been reported to be as low as 5.95 µM and as high as 16.5 µM over a 48 h period [98,99,100]. This dose-dependent killing response is specific to CRC cells and not normal human colorectal cells [101]. The IC50 value for CaCo-2 was reported as 7.5 ± 1.3 µM [67]. Under the physiologic oxygen conditions in the colon, estimated around 5%, Caco-2 were even more sensitive to CBD, showing a decline in proliferation at 0.5 µM compared to 1 µM under atmospheric oxygen (~20%) [102]. The same study found that under physiologic oxygen conditions, the anti-proliferative effects of CBD are likely due to its ability to induce mitochondrial ROS. Apoptosis has been described as the main pathway of cell death by CBD in CRC [98,101,103].

Sreevalsan et al. used SW480 cells with 15 µM of CBD to show that the apoptosis was phosphatase- and endocannabinoid-dependent [98]. After 24 h, CBD induced the expression of various dual-specificity phosphatases and protein tyrosine phosphatases, including DUSP1, DUSP10, serum ACPP, cellular ACPP, and PTPN6. Consistent with the hypothesis, apoptosis was reduced with the use of a phosphatase inhibitor, sodium orthovanadate (SOV). Knocking down CB1 and CB2 also inhibited apoptosis. Together, these studies indicate that the apoptotic effect of CBD in CRC is through the endocannabinoid system and the activation of its downstream targets, including various phosphatases.

CBD has been shown to induce Noxa-mediated apoptosis through the generation of ROS and excessive ER stress [101]. In HCT116 and DLD-1 cells, CBD treatment induced ROS overproduction, especially mitochondrial superoxide anion, and this was linked to Noxa activation. Jeong et al. also found that Noxa-activated apoptosis was dependent on excessive ER stress from ATF3 and ATF4 [101]. These proteins bind the Noxa promoter and stimulate its expression. Similarly, in vivo, CBD-treated CRC tumors also resulted in a significant decrease in tumor size and induction of apoptosis by Noxa.

Using HCT115 and Caco-2 cells, Aviello et al. found that 10 µM of CBD exerts anti-proliferative effects through multiple mechanisms [104]. CBD may act through indirect activation of the receptors by increasing endocannabinoids, specifically 2-AG, in CRC cell lines. In vivo, CBD at 1 mg/kg significantly reduced azoxymethane-induced aberrant crypt foci, polyps, tumors, and the percentage of mice bearing polyps. CBD’s antitumor mechanism was determined to be through the downregulation of the PI3K/AKT pathway and the upregulation of Caspase-3.

A few studies also investigated CBD as an adjunctive to chemotherapy for CRC [101,103]. CRC is often treated surgically in conjunction with the combination of 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX). Seeking to overcome the potential resistance to FOLFOX, Jeong et al. treated oxaliplatin resistant DLD-1 and colo205 cells with oxaliplatin and CBD (4 µM) and found that CBD was able to enhance oxaliplatin-mediated autophagy through decreased phosphorylation of NOS3, which is involved in the production of nitric oxide (NO) and plays a role in oxaliplatin resistance [100]. The combination of oxaliplatin and CBD caused mitochondrial dysfunction (decreased oxygen consumption rate, mitochondrial membrane potential, mitochondrial complex I activity, and the number of mitochondria) through reduced SOD2 expression. These results were confirmed in vivo as well.

An alternative targeted therapy for CRC cancer, TNF-related apoptosis-inducing ligand (TRAIL), has also displayed resistance that can be overcome with the addition of CBD (4 µM) in HCT116, HT29, and DLD-1 cells [103]. CBD and TRAIL increased apoptosis through the activation of ER stress-related genes, including PERK, CHOP, and DR5. In vivo, TRAIL with CBD showed a significant decrease in tumor growth and an increased number of apoptotic cells. Altogether, these FOLFOX and TRAIL therapy studies suggest that CBD may be considered as a therapeutic option for CRC or, perhaps, as an adjunctive treatment to work synergistically with conventional chemotherapies. Currently, there are no clinical trials related to CBD in CRC, however, these findings related to the synergistic effects of CBD with chemotherapies are very promising and make a good case for a clinical trial in the future.

3.5. Leukemia/Lymphoma

Our understanding of CBD’s effects on leukemia and lymphoma has expanded in recent years (Table S5). EL-4 and Jurkat cell lines are the commonly used models for lymphoma and leukemia, respectively. CBD induced a dose- and time-dependent killing effect on these leukemia and lymphoma cell lines, whereas peripheral blood monomolecular cells were more resistant to CBD [105,106,107,108,109].

McKallip et al. [106] found that in both EL-4 and Jurkat cells, CBD’s anti-proliferative effects were mediated through CB2, but independent of CB1 and TRPV1 [106]. However in a separate study Olivas-Aguirre et al. showed CBD’s effects to be independent of the endocannabinoid receptors and plasma membrane Ca 2+ channels in Jurkat cells [110]. These conflicting results need to be resolved by future studies. Despite this, the majority of research on leukemia/lymphomas confirmed apoptosis as the mechanism by which CBD-mediated cell death occurs, either alone or in combination with other treatment modalities, including γ-irradiation, Δ 9 -THC, vincristine, and cytarbine [105,106,107,110]. One study also demonstrated that CBD decreased tumor burden and induced apoptosis in vivo [106]. Kalenderoglou et al. found that CBD can induce cell cycle arrest in Jurkat cells, with increased cells in G1 phase [108]. CBD treatment also resulted in changes to cell morphology, including decreased size of cells, extensive vacuolation, swollen mitochondria, disassembled ER and Golgi, and plasma membrane blebbing [108,110].

Similar to the results of other cancers as discussed above, CBD also induced ROS in leukemia and lymphoma [106,110,111]. Treating Jurkat and MOLT-4, another leukemia cell line, with ≥2.5 µM CBD for 24 h induced increased ROS levels [106]. Treating cells together with ROS scavengers, α-tocopherol and NAC, reduced CBD’s killing effects. CBD exposure also increased NOX4 and p22 phox while inhibiting NOX4 and p22 phox decreased ROS levels and inhibited CBD-induced cell toxicity. Consistent with these observations, ROS levels were significantly increased after only two hours of CBD treatment in EL-4 cells, with a concomitant decrease in cellular thiols [111].

Kalenderoglou et al. explored CBD’s effects on the mTOR pathway in Jurkat cells [108]. They found that CBD reduced the phosphorylation of AKT and ribosomal protein S6. They also tested CBD’s effects with different nutrient and oxygen conditions and found that CBD’s anti-proliferative effects alone or together with doxorubicin were greater with 1% serum than 5% serum. Olivas-Aguirre et al. found that when Jurkat cells were treated with lower concentrations of CBD, proliferation still occurred (at 1 µM CBD) and autophagy was increased at 10 µM CBD [110]. However, at higher concentrations (30 µM), the intrinsic apoptotic pathway was activated, resulting in cytochrome c release and Ca 2+ overload within the mitochondria. In Burkitt lymphoma cell lines, Jiyoye and Mutu I, AF1q stimulated cell proliferation and reduced ICAM-1 expression, through which cells became resistant to chemotherapies [104]. After exposure to CBD for 24 h, the chemo-resistant effect was dramatically attenuated.

3.6. Prostate Cancer

Prostate cancer is the most common cancer and the second most common cause of cancer-related deaths in men [82]. The detailed summary of studies describing CBD’s effects on prostate cancer can be found in Table S6. The prostate cancer cell lines used in those studies can be divided into androgen receptor (AR)-positive (LNCaP and 22RV1) and AR-negative (DU-145 and PC-3). CBD can inhibit the expression of the androgen receptor in AR-positive cell lines [112]. Regarding the endocannabinoid receptors, depending on the specific cancer cell type, either CB1, or CB2, or both, are upregulated in prostate cancer cells relative to normal prostate cells [112,113]. Specifically, 22RV1 only expresses CB1 while DU-145 only expresses CB2. Though CB1 and CB2 can be found in both LNCaP and PC-3, their levels are much more prominent in PC-3. TRPV1 is expressed in all four prostate cancer cell lines, with the highest expression found in DU-145 cells.

CBD induced anti-proliferative effects and apoptosis-mediated cell death (via the intrinsic pathway) in prostate cancer cells, which may be dependent on CB2, but not CB1, and the transient receptor potential cation channel subfamily M member 8 (TRPM8) receptor in LNCaP cells [112,113]. Additionally, treatment with CBD was shown to downregulate the expression prostate-specific antigen (PSA), vascular endothelial growth factor (VEGF), and pro-inflammatory cytokines [113]. CBD treatment resulted in cell cycle arrest at G0/G1 transition in LNCaP and PC3 cells and G1/S transition in DU-145 cells.

Similar to the CRCs, Sreevalsan et al. found that dual-specificity phosphatases and protein tyrosine phosphatases were also induced by CBD in LNCaP cells [98]. Inhibition of the phosphatases with the phosphatase inhibitor, SOV, decreased PARP cleavage. Additionally, CBD enhanced the phosphorylation of p38 MAPK. Most recently, Kosgodage et al. found that in PC3, CBD treatment (1 µM and 5 µM) reduced the release of EMV [89,114]. CBD was also shown to reduce mitochondrial-associated proteins, prohibitin, and STAT3, which may account for the decrease of EMV.

At this point, only one study testing CBD’s effectiveness on prostate cancer has been conducted in vivo. More quality studies using mouse models are required before moving to the clinical trial phase.

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3.7. Other Cancer Types:

The effects of CBD on a variety of other cancers have also been reported, however to a lesser degree (Table S7). Cervical cancer cell lines treated with CBD had time- and concentration-dependent killing effects that were shown to be mediated by apoptosis and independent of cell cycle arrest [93,115]. Treatment with CBD resulted in the upregulation of p53 and Bax, a pro-apoptotic protein, and downregulation of RBBP6 and Bcl-2, two anti-apoptotic proteins, in SiHa, HeLa, and ME-180 cells [115]. CBD also decreased the invasion of HeLa and C33A, which was dependent on CB1, CB2, and TRPV1. Ramer et al. also found this anti-invasive property of CBD to be associated with the upregulation of p38 MAPK and p42/44 MAPK, along with their downstream target, TIMP-1, which is similar to lung cancers as discussed above ( Figure 5 A).

CBD (1 µM and 5 µM) also decreased the cell viability of a hepatocellular carcinoma cell line, Hep G2, in a dose-dependent manner after 24 h [89]. Similar to the breast and prostate cell lines, MDA-MB-231 and PC3, respectively, CBD-treated Hep G2 cells reduced the release of EMV and the expression of CD63, prohibitin, and STAT3. Additionally, treating Hep G2 cells with CBD sensitized them to cisplatin. Neumann-Raizel et al. used the mouse hepatocellular carcinoma cell line, BNL1 ME, which expresses functional TRPV2 channels, to demonstrate the effects of CBD in conjunction with doxorubicin [116]. CBD (10 µM) was shown to activate TRPV2 and inhibit the P-glycoprotein ATPase transporter, allowing for increased entry and accumulation of doxorubicin into the cell since it is transported across the cytoplasmic membrane through TRPV2 and pumped out of the cell using the P-glycoprotein ATPase transporter. These effects were likely responsible for CBD’s ability to decrease the dose of doxorubicin required to reduce cell viability and proliferation.

Regarding thyroid cancers, CBD induced an anti-proliferative effect in KiMol through the activation of apoptosis and cell cycle arrest [67]. KiMol was shown to contain increased levels of CB1, CB2, and TRPV1, but inhibitors of CB1, CB2, and TRPV1 only slightly decreased the anti-proliferative effects of CBD. CBD (5 mg/kg twice per week) produced anti-tumor effects in a mouse thyroid tumor model as well.

Taha et al. studied patients with stage IV non-small cell lung cancer, clear cell renal cell carcinoma, and advanced melanoma treated with nivolumab immunotherapy (anti-PD-1 agents) and patients who had additionally used cannabis, including CBD and Δ 9 -THC [117]. They showed a decreased response rate to treatment in groups using cannabis with nivolumab, whereas patients not using cannabis were 3.17 times more likely to respond to treatment with nivolumab. However, cannabis use resulted in no significant difference in overall survival and progression-free survival. This group suggested that there may be a possible negative interaction between cannabis and immunotherapy.

CBD decreased cell proliferation and colony formation in a concentration-dependent manner in gastric cancer cells without affecting normal gastric cells [67,118,119]. The gastric adenocarcinoma cell line, AGS, has abundant expression of TRPV1 without the detection of CB1 or CB2 [67]. Zhang et al. found that CBD induced cell cycle arrest by inhibiting the expression of CDK2 and cyclin E in SGC-7901, another gastric cancer cell line [119]. In addition, CBD increased the expression of ATM and p21, while decreasing that of p53. CBD’s anti-proliferative effects in SGC-7901 were also attributed to mitochondrial-dependent apoptosis, as it increased the activity of Caspase-3 and Caspase-9, the release of cytochrome c, and the expression of Apaf-1, Bad, and Bax proteins and decreased the expression of Bcl-2. CBD-induced cell cycle arrest and apoptosis were associated with increased ROS levels. In multiple gastric cancer cell lines, Jeong et al. showed that CBD caused apoptosis by inducing ER stress, which then upregulated the second mitochondria-derived activator of caspase (Smac) [118]. Smac upregulation resulted in downregulation of X-linked inhibitor of apoptosis (XIAP) through ubiquitination/proteasome activation. CBD was also shown to induce mitochondrial dysfunction ( Figure 5 A), as shown by CBD-driven decreases in oxygen consumption rate, ATP production, mitochondrial membrane potential, and NADH dehydrogenase ubiquinone 1α sub-complex subunit 9. In vivo, mice injected with MKN45, another gastric cancer cell line, showed slower tumor growth and smaller tumor size with CBD treatment (20 mg/kg) three times a week. Like the in vitro studies, CBD promoted apoptosis and decreased the expression of XIAP in the tumors.

Melanoma cancer cell lines (B16 and A375) express the endocannabinoid receptors, CB1, and CB2 [120]. Previous studies have also shown that activation of these receptors with Δ 9 -THC decreased melanoma growth, proliferation, angiogenesis, and metastasis in vivo [120]. While Δ 9 -THC looks promising as a treatment modality of melanoma, there has been little research on the effects of CBD on melanoma. A recent study by Simmerman et al. tested CBD in a murine melanoma model (B16F10) [121]. They set up three groups of mice: control (ethanol- and PBS-treated), cisplatin-treated (5 mg/kg intraperitoneal once per week), and CBD-treated (5 mg/kg intraperitoneal twice a week). Survival time was significantly increased, and tumor size was significantly decreased in CBD-treated mice compared to control mice, but to a lesser effect when compared to that of cisplatin-treated mice. Quality of life was subjectively described, and CBD-treated mice were found to have a better quality of life, improved movement, and less hostile interaction/fighting compared to both controls and cisplatin-treated mice. This study did not include a group of CBD and cisplatin combination treatment. More research is required to understand the effects of CBD on human melanoma cells.

Pancreatic cancers, especially pancreatic ductal adenocarcinoma (PDAC), have seen few improvements in treatment and survival. Ferro et al. used PDAC cancer cell lines, including ASPC1, HPAFII, BXPC3, and PANC1, as well as the KRAS Wt/G12D /TP53 WT/R172H /Pdx1-Cre +/+ (KPC) mice as models of PDAC to demonstrate GPR55 accumulating in PDAC tissue, and that its disruption resulted in improved survival and reduced proliferation both in vivo and in vitro [122]. This mainly occurred via cell cycle arrest at the G1/S transition by reducing the expression of cyclins, without increasing apoptosis. Additionally, they found downstream MAPK/ERK signaling to be inhibited in cells depleted of GPR55. In vivo, treatment of KPC mice with CBD (100 mg/kg) increased survival similar to gemcitabine (GEM) (100 mg/kg), and when CBD and GEM were used together survival was increased about three-fold compared to the control. With this combination, cell proliferation was also reduced. CBD was also able to counteract the increased ERK activation by GEM, a proposed mechanism of acquired GEM resistance.

4. Summary and Conclusions

As evidenced by the large volume of literature reviewed above, CBD has demonstrated robust anti-proliferative and pro-apoptotic effects on a wide variety of cancer types both in cultured cancer cell lines and in mouse tumor models. In comparison, CBD generally has milder effects on normal cells from the same tissue/organ. The anti-tumor mechanisms vary based on tumor types, ranging from cell cycle arrest to autophagy, to cell death, or in combination. In addition, CBD can also inhibit tumor migration, invasion, and neo-vascularization ( Figure 5 A), suggesting that CBD not only acts on tumor cells but can also affect the tumor microenvironment, for example by modulating infiltrating mesenchymal cells and immune cells. The dependency of CBD on the endocannabinoid receptors, CB1 and CB2, or the TRPV family of calcium channels, also varies, suggesting that CBD may have multiple cellular targets and/or different cellular targets in different tumors ( Table 1 ). Mechanistically, CBD seems to disrupt the cellular redox homeostasis and induce a drastic increase of ROS and ER stress, which could then exert the cell cycle arrest, autophagy, and cell death effects ( Figure 5 A). For future studies, it is crucial to elucidate the interplays among different signaling transduction pathways, such as ROS, ER stress, and inflammation, in order to better understand how CBD treatment disrupts cellular homeostasis in both tumor cells as well as infiltrating cells, leading to cancer cell death and inhibition of tumor migration, invasion, metastasis, and angiogenesis. The final step of developing CBD as an oncology drug is through extensive and well-designed clinical trials, which are urgently needed.

Future Aspects for Cannabinoids in Breast Cancer Therapy

2 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; [email protected]

Mária Suváková

3 Institute of Chemistry, Faculty of Sciences, University of Pavol Jozef Šafárik in Košice, Šrobárova 2, 04154 Košice, Slovakia; [email protected]

Walter Jäger

4 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria; [email protected]

Theresia Thalhammer

2 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; [email protected]

1 Institute of Biology and Ecology, Faculty of Sciences, University of Pavol Jozef Šafárik in Košice, Šrobárova 2, 04154 Košice, Slovakia

2 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; [email protected]

3 Institute of Chemistry, Faculty of Sciences, University of Pavol Jozef Šafárik in Košice, Šrobárova 2, 04154 Košice, Slovakia; [email protected]

4 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria; [email protected]

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Abstract

Cannabinoids (CBs) from Cannabis sativa provide relief for tumor-associated symptoms (including nausea, anorexia, and neuropathic pain) in the palliative treatment of cancer patients. Additionally, they may decelerate tumor progression in breast cancer patients. Indeed, the psychoactive delta-9-tetrahydrocannabinol (THC), non-psychoactive cannabidiol (CBD) and other CBs inhibited disease progression in breast cancer models. The effects of CBs on signaling pathways in cancer cells are conferred via G-protein coupled CB-receptors (CB-Rs), CB1-R and CB2-R, but also via other receptors, and in a receptor-independent way. THC is a partial agonist for CB1-R and CB2-R; CBD is an inverse agonist for both. In breast cancer, CB1-R expression is moderate, but CB2-R expression is high, which is related to tumor aggressiveness. CBs block cell cycle progression and cell growth and induce cancer cell apoptosis by inhibiting constitutive active pro-oncogenic signaling pathways, such as the extracellular-signal-regulated kinase pathway. They reduce angiogenesis and tumor metastasis in animal breast cancer models. CBs are not only active against estrogen receptor-positive, but also against estrogen-resistant breast cancer cells. In human epidermal growth factor receptor 2-positive and triple-negative breast cancer cells, blocking protein kinase B- and cyclooxygenase-2 signaling via CB2-R prevents tumor progression and metastasis. Furthermore, selective estrogen receptor modulators (SERMs), including tamoxifen, bind to CB-Rs; this process may contribute to the growth inhibitory effect of SERMs in cancer cells lacking the estrogen receptor. In summary, CBs are already administered to breast cancer patients at advanced stages of the disease, but they might also be effective at earlier stages to decelerate tumor progression.

Keywords: breast cancer, Cannabis sativa, cannabinoid receptor, cannabidiol, CBD, delta-9-tetrahydrocannabinol, THC

1. Introduction: Cannabis sativa and Cannabinoids

Cannabis sativa (C. sativa) was known among ancient Asian, African, and European agricultural societies. Due to its hallucinogenic effects, Cannabis sativa was applied in religious ceremonies, but it was also widely used in fiber manufacturing, nutrition and medicine. However, in the early part of the last century, C. sativa lost its importance in industry and medicine [1,2]. At present, application of C. sativa in industry and medicine is experiencing a revival. Since 1990, C. sativa became important as a source of compounds to treat cancer and life-threating diseases. The C. sativa plant contains >500 chemical and biologically active compounds [3]. So far, 60 structures have been identified as belonging to the family of cannabinoids (CBs). CBs share a lipid structure featuring alkylresorcinol and monoterpene moieties (terpenophenols) [2,4].

Two CBs have been intensively investigated for their pharmacological properties: delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD); THC, but not CBD, exerts potent psychotropic effects ( Figure 1 ). A high THC/CBD ratio is responsible for the euphoric, relaxing, and anxiolytic effects of medical cannabis (marijuana), whereas, a high CBD/THC ratio has a rather sedating effect [5].

Chemical structures of cannabinoids. Phytocannabinoids—THC: Delta-9-tetrahydrocannabinol; THCA: Delta-9-tetrahydrocannabinolic acid; CBD: Cannabidiol; CBDA: Cannabidiolic acid; CBN: Cannabinol; CBG: Cannabigerol; CBC: Cannabichromene THCV: Tetrahydrocannabivarin. Endocannabinoids—AEA: Anandamide; 2-AG: 2-Arachidonoylglycerol; Met-F-AEA: 2-methyl-2’-F-anandamide; ACEA: Arachidonyl-2’-chloroethylamide. Synthetic cannabinoids—AM251: N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbox amide; JW133: (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-d5; WIN55,212-2: (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; HU-331,CBDHQ: 3-Hydroxy-2-[(1R)-6-isopropenyl-3-methyl-cyclohex-2-en-1-yl]-5-pentyl-1,4-benzoquinone; O-1663: 5-(1,1-Dimethylheptyl)-2-(4-phenylcyclohexyl)-1,3-benzenediol.

Cultivation from different varieties of C. sativa produces two main varieties with distinct concentrations of CBs, and the discrimination from the THC/CBD ratio divides commercial cannabis strains into three principal chemotypes. Chemotype I flowers have the highest THC content (18–23%). Industrial C. sativa flowers (chemotype II and III flowers) contain less than 0.3% THC and CBD levels are 10–12% when calculated for dry weight [6]. Since there are still systematic differences in reports on the CB content and the relative stability of CB levels from different laboratories, a better standardization of CB analysis is urgently required.

Based on the ability of CBs to inhibit inflammation and block cancer cell proliferation, plant-derived and synthetic CBs have been investigated for their applications as antitumor drugs. Indeed, a growing number of reports on the role of receptors for CBs in tumor cells suggest that CBs with different properties that can block or activate CB-receptors (CB-Rs) may be useful in cancer treatment [7,8].

2. Mechanism of Cannabinoid Action

The term ‘endocannabinoid’ was invented in the mid-1990s after the discovery of membrane receptors for THC and their endogenous ligands. It now comprises a whole signaling system consisting of the ‘classical’ CB-Rs, their endogenous ligands, which are lipid signaling molecules called endocannabinoids, and the associated biochemical machinery, including precursor molecules, enzymes for synthesis and degradation, and transporter proteins, such as fatty acid binding protein and heat shock protein 70 [9]. There is now a growing number of endocannabinoid molecules known, which share a similar structure and are natural ligands of the two CB-Rs, CB1-R and CB2-R. They seem to be involved in an increasing number of pathological conditions. Plant-derived CBs (phytocannabinoids, phyto-CBs) as well as synthetic CBs interfere with the endocannabinoid system, and a number of pharmacological effects of phyto-CBs can be explained by this interference [10].

The most studied compounds of the endocannabinoid system are anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) ( Figure 1 ). Each can activate both CB-Rs and both are synthesized on demand in response to elevations of intracellular calcium [11]. The biosynthesis of AEA, which was the first endocannabinoid identified, starts from the activation of N-acyltransferase (NAT), which transfers an acyl group to the membrane phospholipid phosphatidylethanolamine. In this way, N-acyl-phosphatidylethanolamine (NAPE) is generated. The NAPE-specific phospholipase D forms AEA from NAPE. The major biosynthetic pathway for 2-AG involves the sequential hydrolyses of inositol phospholipids via diacylglycerol (DAG) by phospholipase C and DAG lipase.

AEA and 2-AG are produced on demand by cells and work to maintain homeostasis [9]. They have a short half-life and are quickly degraded through transport protein reuptake and hydroxylation by either fatty acid amide hydrolase (FAAH) for AEA or monoacylglycerol lipase (MAGL) for 2-AG. Finally, arachidonic acid (AA) and ethanolamine, from AEA, and AA and glycerol, from 2-AG, are formed. Endocannabinoids are responsible for retrograde synaptic signaling in the central nervous system. They move across the synaptic cleft in order to bind and activate the presynaptic CB1-R, causing an inhibition of neurotransmitter release.

These compounds serve as a new class of endogenous signaling molecules involved in a plethora of physiological functions related to behavior, memory, temper, addiction, and reward systems, as well as cellular metabolism and energy regulation. Their synthesis occurs ‘on demand’ (no storage) with a very short half-life. Drugs influencing the endocannabinoid system (e.g., inhibitors of FAAH and MAGL) were developed to treat neurological diseases and neuropathic pain in cancer patients [12,13]. However, a tragic incidence at a phase I clinical trial with an FAAH inhibitor put its application into question [14].

Endocannabinoids work via specific G-protein coupled receptors (GPRs) CB-Rs (CB1-R and CB2-R). While AEA acts as a partial CB1-R agonist and is a weak CB2-R agonist, 2-AG is a strong CB1-R agonist. CB1-R and CB2-R belong to the seven-transmembrane-spanning receptor superfamily. The distinct tissue distribution of CB1-R and CB2-R allows a selective and cell-specific effect of receptor activation. CB1-R is highly expressed in brain areas related to cognitive functions, memory, anxiety, pain, sensory and visceral perception, motor coordination, and endocrine functions. Low expression levels are observed in the peripheral nervous system, testicles, heart, small intestine, prostate, uterus, bone marrow and vascular endothelium. CB1-R activations inhibit forskolin-stimulated adenylyl cyclase through activation of a pertussis toxin-sensitive G-protein, to inhibit N-, P-, and Q-type calcium channels, and activate inwardly rectifying potassium channels.

CB2-R is present at high levels in cells of the immune system. In glial cells, the spleen and tonsils, CB1-R levels are low. CB2-Rs are also present at a lower level in the heart, endothelium, bones, liver, and pancreas. Furthermore, a functionally relevant expression of CB2-Rs was also found in the brain [15]. Intracellular CB2-R dependent signaling pathways include Gi/o-dependent inhibition of adenylyl cyclase, stimulation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and cyclooxygenase-2 (COX-2) signaling, and activation of de novo ceramide synthesis. Both CB-R types are highly expressed in a variety of cancerous tissues, and it is well established that CB2-R plays a crucial role in carcinogenesis and cancer progression. Therefore, CB2-R is now emerging as target for cancer treatment, although the exact role of CB2-R in cancer progression is still not completely elucidated [16].

At molecular levels, the activation of CB-Rs confers signals of endo, phyto, and synthetic CBs ( Figure 1 ) via inhibition or activation of a variety of signaling pathways [17] ( Figure 2 ). An important signal transduction pathway regulated by CB-R is linked to the synthesis of ceramide with palmitoyl-transferase as the rate-limiting enzyme in ceramide synthesis [18]. Long-term treatment with ceramide, which activates the proto-oncogene serine/threonine-protein kinase (RAF1), leads to sustained activation of p42/p44 MAPK and induction of apoptosis, as demonstrated in a glioma cell line. This activation could be blocked by CB-R agonists, including THC, by the synthetic CB WIN55,212-2, and the endocannabinoids AEA and 2-AG. However, the duration of the activation of p42/p44 MAPK seems to be critical to the apoptotic response because a protective role of CBs against ceramide-induced apoptosis was also reported [19].

Mechanism of CB-R-mediated antitumor activity in breast cancer cells. By binding to CB1-R and CB2-R, CBs inhibit breast cancer cell proliferation through various mechanisms. They block cell cycle progression at the G1/S phase via CB1-R and at the G2/M phase via CB2-R activation. They induce breast cancer cell death via apoptosis, mediated by the activation of the transcription factor jun-D. In HER2-overexpressing breast cancer cells, they block cancer cell proliferation in culture and tumors by inhibiting Akt and ERK signaling. They also inhibit cell migration and angiogenesis via CB2-R. CB1-R activation inhibits the FAK/SRC/RhoA pathway leading to inhibition of cell migration. Cell migration blockade is also achieved by CB2-R activation through the inhibition of COX-2 and ERK signaling, which is important for triple-negative breast cancer. AC: adenylate cyclase; Akt: protein kinase B; CB-R: cannabinoid receptor; COX-2: cyclooxygenase-2; EMT: epithelial-mesenchymal transition; ERK: extracellular-signal-regulated kinase; FAK: focal adhesion kinase; GPR: G-protein coupled receptor; HER2: human epidermal growth factor receptor 2: MAPK: mitogen-activated protein kinase; mTOR: mammalian target of rapamycin; PI3K: Phosphoinositol-3-kinase; Raf: serine/threonine-protein kinase; RhoA: transforming protein RhoA; SRC: proto-oncogene tyrosine-protein kinase Src.

Importantly, CBs also bind and activate several other receptors, including the GPRs, GPR18, GPR55, and GPR119. Of particular interest is GPR55, which is activated by lysophospholipid and also by the endocannabinoids AEA and 2-AG. Downstream targets of GPR55 include phospholipase C (PLC), transforming protein RhoA (RhoA), Rho-associated protein kinase (Rock), extracellular-signal-regulated kinase (ERK), and p38 MAPK [20]. CB-Rs form heterodimers with other GPRs, e.g., GPR55, which consequently affects the functions of both receptors. Other GPRs, which are activated by CBs, are acetylcholine receptors and 2-alpha adrenoreceptors as well as opioid-, adenosine-, 5-hydroxytryptophan-, angiotensin-, prostanoid-, dopamine-, melatonin-, and tachykinin receptors. Furthermore, the peroxisome proliferator-activated receptors (PPARs) α and γ are also considered to be receptors for endocannabinoids [21].

3. Cannabinoids from Cannabis sativa

3.1. Cannabidiol (CBD)

CBD and its precursor cannabidiolic acid (CBDA) are the main phyto-CBs in industrial used C. sativa [3,22]. CBD works as an allosteric negative modulator of CB1-R and CB2-R activity [23,24]. Some of its pharmacological effects are caused by it binding to other GPRs and other receptors (see previous chapter). For example, the anticonvulsant, antispasmodic, anxiolytic, antiemetic, and neuroprotective effects of CBD are thought to be conferred by several GPRs in neuronal cells. CBD acts as a partial agonist for GPR18 and GPR55 and antagonizes the effects of THC [24].

The pharmacokinetic properties of all CBs are highly dependent on the route of administration. A high intra and intersubjective variability is common in humans. Extensive studies in animals, including rodents and dogs, indicated that a high amount of administered CBD is excreted unchanged or in its glucuronidated form. The most abundant metabolites are the hydroxylated 7-carbonyl CBD derivatives, which are excreted into urine either in their unconjugated form or as glucuronides. Lipid soluble CBs and their metabolites, found in blood and urine, can be stored in fat cells for up to several weeks. Typically, CB and its metabolites appears in the urine within 60 min with high concentrations for ≤4 h [25].

The 7-carbonyl metabolites confer anti-inflammatory properties in mice. In vitro studies revealed that they reduce nitric oxide (NO) formation and prevent the formation of reactive oxygen species (ROS). They also block the production of tumor necrosis factor (TNF)-α and other pro-inflammatory cytokines and transcription factors [e.g., interleukin (IL)-1β, IL-2, IL-6, IL-8 and nuclear factor (NF)-κB], and their effects are comparable to that of CBD. CBD is known to inhibit the metabolism of AA to leukotriene B4 via 5-lipoxygenase as part of its anti-inflammatory effect [26]. Although CBD was found to reduce the formation of ROS and NO in various cell lines and animal models of inflammation, there are also reports showing that CBD can induce ROS formation in cancer cells, leading to cytotoxicity [27].

3.2. Delta-9-tetrahydrocannabinol (THC)

THC is the main psychotropic constituent of C. sativa and is a CB1-R and CB2-R partial agonist. Thereby, the CB-R expression level and signaling efficiency of CB-Rs together with the release of endogenous CBs will influence its effects. Euphoria is among the most often observed psychotropic effects, but dysphoric reactions, including anxiety and panic reactions, as well as paranoia, are known. The absorption kinetics of THC (similar to those of other CBs) depend on the exposure route. Inhaled THC is rapidly distributed in the bloodstream, with peak levels observed at 2–10 min. Concentrations decline rapidly within 30 min and the formation of the psychoactive 11-hydroxy metabolite stops. After oral consumption, THC reaches peak levels after 2–4 h and the half-life of THC is 20–30 h. The oral bioavailability of the highly lipophilic THC and of other CBs is low and variable (6–20%). The hepatic cytochrome p450 system primarily metabolizes THC to many hydroxylated metabolites, which are mostly inactive [13]. However, the main active metabolite of THC is 11-hydroxy-delta-9-tetrahydFrocannabinol (11-OH-THC) with potent psychoactive activity. This metabolite is further degraded to mostly inactive metabolites, including 11-nor-delta-9-tetrahydrocannabinol-carboxylic acid, which is detectable in urine. The excretion of the metabolites through feces and urine lasts from hours to days, with a more prolonged elimination after chronicity of use. The presence of the metabolite in the urine indicates exposure to THC within the last 3 days.

The acute toxicity of CBs is low in adults, but toxic effects occur mostly through THC. Inhaled doses of 2–3 mg THC and ingested doses of 5–20 mg THC can lead to impaired attention and memory, as well as in executive functioning, and conjunctivitis is a common symptom. Higher doses in adults and oral 5–300 mg in pediatric patients can cause more severe symptoms such as hypotension, panic, anxiety, delirium, respiratory depression and ataxia. Furthermore, chronic application of THC may lead to attention and memory deficits, as well as loss of the ability to process complex information. In children, neurological abnormalities, including lethargy and hyperkinesis, can be signs of severe toxicity. As THC crosses the placenta and accumulates to significant concentrations in breast milk, THC consumption by pregnant and breast-feeding women may harm unborn and newborn babies [28].

Physiological effects of THC primarily take place in the central nervous system. Activation of CB1-R by THC leads to a disturbance in the gamma aminobutyric acid/glutamatergic neurotransmission system and the release of dopamine [24]. Thereby, the expression level and signaling efficiency of CB1-R determines the psychotropic effects of THC.

As described for CBD, antiproliferative actions of THC in tumor cells are caused by the activation of CB-Rs, which influence various signaling mechanisms ( Figure 2 ). Activation of CB2-R impairs cell cycle progression by downregulating cell division control 2 (Cdc2) and inducing cell cycle arrest at the G2/M phase. Furthermore, CB2-R causes an activation of a member of the activating protein 1 transcription family, transcription factor jun-D, preventing cell proliferation and inducing apoptosis [29,30].

CB2-R activation also induces PPARγ-regulated pathways in carcinoma cells. In this way, CBs promote the expression of intercellular adhesion molecule 1. This process results in an enhancement of cancer cell adhesion to lymphokine-activated killer cells and causes cancer cell lysis.

THC was shown to antagonize the tumor-promoting GPR55, both at the single receptor level and within the CB2-R-GPR55 heterodimers. These heterodimers of CB2-R and GPR55 influence tumor growth by modulating cyclic adenosine monophosphate (cAMP) signaling and the ERK-1/2 pathways [31,32].

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3.3. Minor Phytocannabinoids

Other CBs from C. sativa were also found to have anti-inflammatory and analgetic effects. Some of these CBs were found to improve the effects of inflammatory diseases in the gut and stimulate bone formation. These effects are conferred by the activation of CB-R and other receptors. Their concentration varies between different C. sativa strains but is generally as low as 2%. However, the concentrations of these CBs may reach significant levels in special cultivated strains. Additive or synergistic interactions between CBD, THC with minor phyto-CBs, or non-CBs, such as terpenes, in the extracts may increase the therapeutic efficiency of the extract for the treatment of inflammation and cancer [12,33].

Other CBs from C. sativa

Cannabinol (CBN) is a non-psychoactive CB with a higher concentration in aged plants, or in degraded or oxidized CB preparations. Pharmacologically relevant quantities are formed as a metabolite of THC. CBN is a partial CB1-R agonist, but it has a higher affinity to CB2-R than to CB1-R.

Cannabigerol (CBG) was found to improve digestive functions and has powerful antiemetic and anti-inflammatory effects. CBG is a partial agonist for CB1-R and CB2-R [34]. It may be used for the treatment of neurological disorders.

Cannabichromene (CBC) has mild psychotropic effects and may stimulate bone growth [35]. It also has anticonvulsive effects. It is may be used in the treatment of hypomotility, catalepsy and hypothermia.

Tetrahydrocannabivarin (THCV) works as a potent CB-R partial agonist in vitro. THCV interacts with CB1-R when administered in vivo, behaving as a CB1-R antagonist at low doses and as an agonist at higher doses [24]. THCV has antibacterial and antiviral properties and is also thought to prevent obesity. It may additionally have some anti-convulsive properties [36].

3.4. Drugs Based on CBs from C. sativa

Nabilone (Cesamet ® ) and Dronabinol (Marinol ® ) are synthetic molecules that mimic the pharmacological activity of THC. Their chemical structures are presented in Figure 1 .

Nabiximol (Sativex ® ) was first approved as a botanical drug in the UK in 2010. The aerosol mouth spray contains an extract from the C. sativa plant and flowers derived from two cannabis plant varieties. It contains nearly equal amounts of THC and CBD, but also minor quantities of CBs, flavonoids and terpenes from the plant.

3.5. Synthetic Cannabinoid Analogues

To target CB-R mediated pathways, compounds with different chemical structures were screened for CB-R receptor ligand activity. A number of these compounds were investigated in cell culture and animal tumor models to determine their antineoplastic effects. For relevant reviews, see references [37,38,39,40,41]. Their chemical structures are depicted in Figure 1 and their effects are discussed in the following chapters.

4. Cannabinoids in Breast Cancer

4.1. Molecular Effects of CBs in Breast Cancer

Breast cancer is the most frequently diagnosed cancer in women worldwide. There is also an increasing tendency for aggressive subtypes of breast cancer, particularly in women of younger ages [6,42]. Although the main intrinsic molecular subtypes are breast cancer hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative luminal A and B tumors, HER2-enriched tumors and triple-negative tumors, which are usually the most aggressive type, have been identified. As these molecular subtypes differ in the course of the disease and the clinical outcome, individualized therapies will achieve a better outcome for individual patients [43]. Interestingly, data from preclinical in vitro and in vivo studies identified various antitumor activities of plant-derived and synthetic CBs, although there are some studies in which CBs might also promote tumor progression. The relevant data are summarized in the following chapters and the kinetic data for individual CBs are summarized in Table 1 .

Table 1

Antitumoral activity of CBs in hormone-dependent and –independent breast cancer cell lines.

CB Cell Line IC50 Antitumoral Activity Receptor Mechanism Citation
THC MDA-MB-231
MDA-MB-468
SKBR-3
MCF-7
EVSA-T
T-47D
5.0 ± 1.2 µM
4.4 ± 0.3 µM
4.5 ± 0.4 µM
10.2 ± 0.7 µM
4.0 ± 0.1 µM
6.7 ± 0.2 µM
Induction of apoptosis
Cell cycle arrest,
Inhibition of G2-M transition via
downregulation of Cdc2
CB2-R [44]
MDA-MB-231
MCF-7
4T1
n.d. Increased production of IL-4 and IL-10
Suppression of the cell-mediated Th1 response
and enhancement of the Th2-response
CB1-R
CB2-R
[45]
MDA-MB-231
MDA-MB-468
1.2 µmol/L
2.5 µmol/L
Antiproliferative activity
Reduction of invasiveness via ID-1
n.d. [46]
MCF-7
MDA-MB-231
14.2 ± 2.1 µM
24.3 ± 4.2 µM
Inhibition of cell growth CB2-R [47]
THCA MCF-7
MDA-MB-231
9.8 ± 0.4 µM
18.2 ± 5.3 µM
Inhibition of cell growth CB2-R [47]
CBD MDA-MB-231
MCF-7
SK-BR-3
ZR-75-1
MCF-10A (n.m.)
n.d. Inhibition of cell viability
Induction of apoptosis/autophagy
No influence on cell viability
CB1-R
CB2-R
TRPV
[27]
MDA-MB-231
MCF-7
8.2 ± 0.3 µM
10.6 ± 1.8 µM
Inhibition of cell viability
Cell cycle arrest at the G1/S transition
Induction of apoptosis via pro-caspase-3 cleavage to caspase-3, induction of endoplasmic reticulum stress, inhibition of mTOR and Akt
CB2-R [47]
MDA-MB-231
T-47D
2.2 µM
5.0 µM
Induction of apoptosis, inhibition of mTOR, upregulation of PPARγ n.d. [48]
MDA-MB-231
MDA-MB-436
1.3 µmol/L
1.6 µmol/L
Antiproliferative activity
Invasiveness reduction via ID-1
n.d. [46]
CBDA MDA-MB-231 >100 μM Inhibition of cell migration by modulating the activity and expression of COX-2 CB1-R
CB2-R
[49,50,51,52]
MDA-MB-231 25 μM Inhibition of cAMP-dependent protein kinase A via activation of the small GTPase, RhoA CB1-R
CB2-R
[51]
MDA-MB-231 >25 µM Invasiveness reduction via ID-1 and SHARP1 n.d. [52]
MCF-7
MDA-MB-231
21.7 ± 3.2 µM
>25 µM
Inhibition of cell growth CB2-R [47]
CBN MDA-MB-231
MDA-MB-468
1.2 µmol/L
2.6 µmol/L
Antiproliferative activity
Invasiveness reduction via ID-1
n.d. [46]
CBG MDA-MB-231
MDA-MB-468
2.3 µmol/L
2.1 µmol/L
Antiproliferative activity
Invasiveness reduction via ID-1
n.d. [46]
MCF-7
MDA-MB-231
9.8 ± 3.4 µM
20.4 ± 2.6 µM
Inhibition of cell growth CB2-R [47]
CBC MCF-7
MDA-MB-231
14.2 ± 1.4 µM
>25 µM
Inhibition of cell growth CB2-R [47]
AEA MDA-MB-231 n.d. No growth inhibition

CB1-R [53]
MCF-7
EFM-19
BT-474
T-47D
0.5 µM
1.5 ± 0.3 µM
1.9 µM
1.9 µM
Cell cycle arrest, inhibition of G1/S transition CB1-R [54]
MCF-7
EFM-19
1.4 ± 0.9 µM
1.5 ± 0.3 µM
Inhibition of adenylyl cyclase and activation of MAPK, thereby exerting a downregulation of PRLr and trk n.d. [55]
MCF-7
T-47D
1.4 ± 0.9 µM
1.9 ± 0.2 µM
Inhibition of proliferation, inhibition of forskolin-induced cAMP formation, stimulation of RAF1 translocation and MAPK activity CB1-R
CB2-R
TRPV
[56]
MDA-MB-231 n.d. Regulation of lipid rafts CB1-R [57]
2-AG EFM-19 n.d. Cell cycle arrest, inhibition of G1/S transition CB1-R [54]
MCF-7
T-47D
1.4 ± 0.3 µM
5.0 ± 1.1 µM
Inhibition of proliferation, inhibition of forskolin-induced cAMP formation, stimulation of RAF1 translocation and MAPK activity CB1-R
CB2-R
TRPV
[56]
Met-F-AEA MDA-MB-231
T-47D
n.d. Inhibition of adhesion and migration on type IV collagen without modifying integrin expression CB1-R [53]
MDA-MB-231 n.d. Inhibition of proliferation by degradation of b-catenin and decrease in cyclin D1, c-Myc and MMP-2
Cell cycle arrest, inhibition of G1/S transition
Upregulation of E-cadherin accompanied by the reduction of vimentin, fibronectin and N-cadherin
CB1-R [58]
MDA-MB-231 n.d. Inhibition of angiogenesis by the reduction of pro-angiogenic factors VEGF
Reduction of metalloproteinases, TIMP1 and TIMP2
n.d. [59]
(R)-Met-AEA EFM-19 0.8 µM Cell cycle arrest, inhibition of G1/S transition CB1-R [54]

2-AG: 2-Arachidonoylglycerol; AEA: anandamide; Akt: protein kinase B; AMP: adenosine monophosphate; CBC: cannabichromene; CBD: cannabidiol; CBDA: cannabidiolic acid; CBN: cannabinol; CBG: cannabigerol; CB-R: cannabinoid receptor; Cdc: cell division control; COX: cyclooxygenase; GTP: guanosine triphosphate; IC: inhibitory concentration; ID-1: inhibitor of DNA binding 1; IFN: interferon; IL: interleukin; MAPK: mitogen-activated protein kinase; Met-F-AEA: 2-methyl-2’-F-anandamide; MMP: matrix metalloproteinase; mTOR: mammalian target of rapamycin; Myc: avian virus myelocytomatosis; n.d.: not determined; n.m.: non-malignant; PPAR: peroxisome proliferator-activated receptor; PRLr: prolactin receptor; RAF: proto-oncogene serine/threonine-protein kinase; Rho: transforming protein RhoA; SHARP: SMART/HDAC1 associated repressor protein; TGF: tumor growth factor; Th: T helper; THC: tetrahydrocannabinol; THCA: tetrahydrocannabinolic acid; TIMP: tissue inhibitor of metalloproteinases; trk: tyrosin kinase; TRPV: transient receptor potential cation channels; VEGF: vascular endothelial growth factor.

4.2. Cannabinoid Receptor Signaling

Breast cancer cell lines express CB2-R at high levels but levels of CB1-R are rather low [44]. On a microarray performed on human breast cancer samples with different histological features, CB1-R immunoreactivity was found in 28% of carcinomas and CB2-R was identified in 72% of carcinomas. No significant CB1-R and CB2-R immunoreactivity was detected in non-transformed mammary tissue [45]. CB2-R expression in breast cancer correlates with the aggressiveness of the tumors. Estrogen- and/or progesterone receptor-negative tumors, which are more aggressive than tumors expressing steroid-hormone receptors, express higher levels of CB2-R, which usually have a better prognosis [46]. In particularly difficult to treat triple-negative tumors (lacking the expression of receptors for steroid hormones and HER2/neu (human epidermal growth factor receptor 2/erb-B2, and tumors expressing HER2/erb-B2 but no steroid hormone receptors, increased CB2-R levels are nearly always observed. These tumors are usually poorly differentiated, contain highly proliferative and invasive growing cells, and have a higher probability for early local tumor recurrence and formation of distant metastases. Therefore, they usually have a poorer prognosis than steroid hormone receptor positive tumors [47,48]. To treat these tumor entities, targeting CB-associated pathways could be a promising treatment option and might also work in patients suffering from a relapse with an anti-HER2 targeted therapy.

In addition to CB2-R and CB1-R, alternative CB-Rs are also of interest for breast cancer therapy. High expression levels of GPR55 were found in human breast tumors and were related to worse prognoses. GPR55 was also highly expressed in MDA-MB-231 cells, a human breast cancer cell line with considerable metastatic potential (compared with less-metastatic MCF-7 cells) [49]. The proliferative effects mediated by GPR55 are thought to be a result of ERK activation and downstream expression of proto-oncogene c-FOS [20].

4.3. The Effect of Cannabinoids in Breast Cancer Cell Lines

4.3.1. Phytocannabinoids and Synthetic Analogues

In 2006, Ligresti et al. demonstrated that CBD caused a potent and selective inhibition of breast cancer cell growth [50]. A number of breast cancer cell lines, such as estrogen receptor (ER)-positive MCF-7, ZR-75-1, and T47D cells, and ER-negative cell lines MDA-MB-231, MDA-MB-468 and SK-BR3, are sensitive to the antiproliferative effects of CBD [27,50,51,52,53]. CBD interferes with cell cycle progression and causes an increase in the number of breast cancer cells in the resting G0 stage and in the G1 compartment. At higher concentrations, CBD causes cell death [46]. Shrivastava et al. showed that in CBD-treated breast cancer cells, a complex interplay between apoptosis and autophagy exists. In the MDA-MB-231 breast cancer cell line, CBD leads to an increase in the generation of ROS, which finally results in an induction of apoptosis and autophagy [27]. Using the MDA-MB-231 breast cancer cell line, it was further shown that beclin 1, a protein that interacts with either B cell lymphoma-2 or PI3K, plays a central role in the induction of autophagy and cell death. CBD causes apoptosis through the production of ROS by changing the mitochondrial permeability transition pore opening, as first demonstrated in human monocytes [54]. CBD inhibits protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling and induces autophagy and cell death under oxidative stress conditions. An interplay among decreased mTOR and cyclin D1 together with an upregulated PPARγ expression promotes the induction of apoptosis, a process that is independent of the expression of ERs [55].

In both ER-positive and ER-negative breast cancer cells, CBD activates the intrinsic apoptotic pathway by changing the mitochondrial membrane potential, activating the translocation of the BH3 interacting-domain death agonist to the mitochondria, and releasing cytochrome C from mitochondria [27].

CBD also inhibits the invasiveness of aggressive MDA-MB-231 and MDA-MB-436 breast cancer cell lines by downregulating inhibitor of DNA binding 1 (ID-1), a transcriptional regulator, which stimulates the metastasis of breast cancer [51]. In a mouse model of advanced breast cancer with lung metastases, CBD reduced the degree of metastasis by downregulating ID-1. However, CBD caused only a moderate increase in survival in this model. The resorcinol derivative O-1663 was proposed as a selective for CB2-R, which prolonged the survival more efficiently than the parent compound in mice with advanced breast cancer. O-1663 inhibited ID-1, stimulated ROS production, and increased autophagy and apoptosis [56].

An important finding was that CBD improved the response to treatment with cytarabine and vincristine in cancer cells [57]. In vitro studies showed greater antitumor activity when combining CBs and radiotherapy. Survival of patients treated with CBs could be significantly increased by the incorporation of CBs in smart biomaterials for sustained delivery [58].

As CBD is derived from CBDA by decaboxylation, it was investigated whether CBDA is also biologically active [59]. Indeed, CBDA prevents migration of triple-negative MDA-MB-231 human breast cancer cells via CB2-R activation by modulating the expression and activity of COX-2 [59,60,61,62]. Furthermore, CBDA inhibits the growth and migration of breast cancer cells via the inhibition of cAMP-dependent protein kinase A via activation of the small GTPase, RhoA [61]. It also causes a downregulation of the enhancer of breast cancer metastasis ID-1 [62].

Resembling the effects of CBD, THC has pro-apoptotic effects in a number of breast cancer cell lines (EVSA-T, MDA-MB-231, MDA-MB-468, SKBR-3, MCF-7 and T-47D) [46]. It reduces cell cycle progression and induces apoptosis in hormone-sensitive and hormone-resistant human breast cancer cell lines. In this way, THC induces cell cycle arrest at the G2/M transition, causing downregulation of Cdc2 and inducing ROS formation to induce cancer cell death. This mechanism is also seen in a number of other cancer cell types e.g., glioma cells [46].

CBs are favorable for antitumor therapies, as they are potent inhibitors of the inflammatory process, primarily via CB2-R activation. As demonstrated in MCF-7 and MDA-MB-231 cells, THC suppressed the cell-mediated T helper (Th)1 response and enhanced Th2-associated cytokine secretion [63]. Furthermore, THC prevented activation of inflammatory signaling pathways, such as the NF-κB, MAPK, and JAK-signal transducer and activator of transcription (STAT) pathways in immune cells. Therefore, CBs may be a potent treatment option against breast cancer subtypes accompanied by strong inflammation, as well as against non-malignant inflammatory disorders [64].

Triple-negative breast tumor cells express basal markers, such as epidermal growth factor receptor (EGFR) and cytokeratin 5/6 at high levels. In these tumors, higher expression levels of basal markers, such as EGFR, are associated with a poorer outcome. Although EGFR inhibitors are effective in treating cancer, the early onset of drug resistance limits their therapeutic success [65]. In SUM159 and SCP2 human tumor cells, as model cells for triple-negative breast cancer, CBD effectively inhibited epidermal growth factor (EGF)-induced tumorigenic properties of these cancer cells by obstructing signaling pathways for EGFR, Akt, ERK, and NF-κB. Furthermore, CBD is able to block the secretion of matrix metalloproteinases (MMPs) and the effects of EGF on the cytoskeleton [66].

Studies in breast cancer cell lines and animal models showed that an extract from C. sativa was more potent than CBD and THC. Minor CBs in the extract may also contribute to the observed anticancer activity by modulating various targets in the pro-oncogenic pathways leading to an “entourage effect” against cancer cells [67].

4.3.2. Endocannabinoids

Of the endocannabinoids, AEA was characterized for its antitumoral properties in vitro and in vivo. AEA modulates the cAMP/protein kinase A and MAPK kinase pathway to exert antiproliferative effects in breast cancer cells [68]. AEA inhibits the proliferation of breast cancer cells through nerve growth factor (NGF) and prolactin, by downregulating the NGF receptor and prolactin receptor, respectively [69]. The inhibitory effect of AEA on prolactin receptors may regulate the cancer-directed immune system, as prolactin is a potent endogenous proliferative agent of B and T cells [70]. Furthermore, AEA inhibits cell cycle progression by preventing G1/S transition, as demonstrated in the human prolactin sensitive breast epithelioid EFM-19 cell line [70]. Similar to AEA, the endocannabinoid 2-AG also exerts antiproliferative activity in MCF-7 and T-47D cells [69], as well as in EMG-19 breast cancer cells [70]. The synthetic AEA analogue Met-F-AEA has an increased binding affinity and selectivity for CB1-R compared with AEA, which leads to dose-dependent inhibition of cell proliferation in MDA-MB-231 cells [71]. This analog also inhibits the epithelial-mesenchymal transition of cancer cells, thereby preventing invasive growth and metastases of cancer cells [72], indicating that in addition to CB2-R, CB1-R is also a main target for the observed anticancer effect.

Other synthetic cannabinoid derivatives, e.g., ACEA, a selective CB1-R agonist, and AM251, a selective CB1-R antagonist, were investigated for their effects on breast cancer stem cells. While ACEA decreased the invasive potential, AM251 increased the invasive power of breast cancer stem cells, indicating that CB1-R contributes to the stem cell properties in breast cancer [73]. Furthermore, a number of other synthetic CBD analogues, such as O-1663 and HU-331, showed antiproliferative activity on breast cancer cells; these analogues were previously reviewed [38].

4.4. Preclinical Evidence of the Effects of CBs in Animal Models

It was demonstrated that CBD had favorable effects in a mouse model of cisplatin-induced nephropathy. Cisplatin induced the expression of superoxide-generating enzymes, enhanced the formation of ROS and inducible NO synthase, and promoted apoptosis. It reduced inflammation by inhibiting TNF-α and IL-1β in the kidneys of the mice, leading to an improved renal function [74]. At a dose of 5 mg/kg body weight, CBD inhibits breast tumor growth and reduces tumor volume in xenografts in athymic nude mice, leading to prolonged survival of the animals [50,53].

Important for HER2-positive tumors, an association between the levels of CB-Rs and HER2 in human breast cancer was identified in the study by Caffarell et al. (2010) [45]. In total, 91% of the CB2-R-positive tumors were also positive for HER2 [75]. The levels of CB2-R in breast cancer are strongly related to the aggressive growth of the tumor, as CB2-R activation triggers signaling pathways that drive the proliferation and survival of cancer cells, tumor angiogenesis and epithelial-mesenchymal transition, promoting tumor cell migration, and invasion. Among these pathways are the PI3K/Akt and the ERK/MAPK cascades [75].

For the consideration of CBs in the therapy of breast cancer patients with difficult to treat HER2 expression tumors, a combination of CBs with HER2-targeted therapies, such as the tyrosine kinase inhibitor lapatinib, may be promising, as previous studies showed that CBs enhance the antitumor effects of the drugs. Moreover, this effect was also shown for the application of CBs with standard chemotherapeutic drugs such as cisplatin [67].

In a clinically relevant mice model of HER2-posititive cancer (the polyoma middle T oncoprotein transgenic mice, MMTV-neu mouse) selective overexpression of HER2 in the mammary epithelium resulted in the formation of focal tumors in the breast and lung metastases. These tumors also expressed CB2-R. Long-term treatment of these mice with either THC or the synthetic JWH-133 delayed the onset and progression of the tumors [47]. JWH-133 is as effective as THC in reducing tumor progression, without the psychoactive effects of THC. This tumor preventive effect was attributed to the blocking of the PI3K/Akt/mTOR signaling pathway via downregulation of the Akt kinase. Furthermore, the formation of metastatic lesions in the lung was reduced by downregulation of the metalloproteinase MMP2, which degrades the extracellular matrix, and the upregulation of the metallopeptidase inhibitor tissue inhibitor of metalloproteinases 1. JWH-133 also reduced vascular endothelial growth factor (VEGF) secretion, preventing tumor angiogenesis [45]. An antineoplastic effect was also shown for Met-F-AEA as it blocked the activity of the p21 ras oncogene and reduced tumor angiogenesis and VEGF expression [76,77].

A recent study explained how THC can exert an antitumor effect in HER2 positive breast cancer cells; HER2 forms heterodimers with CB2-R and the expression of these heterodimers correlates with a poor patient prognosis. By binding to CB2-R, THC is able to disrupt these HER2-CB2-R complexes, which leads to the inactivation of HER2 and its degradation [78].

CBD inhibited the growth of triple negative breast tumors in a 4T1.2 mouse model, where the tumor volume and tumor weight was greatly reduced. Furthermore, reduced tumor vascularization, reduced expression of EGFR, as well as reduced phosphorylation of Akt and ERK, will prevent tumor progression [66]. The synthetic CB derivative WIN-55,212-2 caused breast cancer suppression through a coordinated regulation of the COX-2/prostaglandin E2 signaling cascade [44]. In this model, WIN55,212-2 administered in combination with doxorubicin, enhanced the anticancer effect of the standard chemotherapeutic drug. WIN55,212-2 induced cell cycle arrest and apoptosis and inhibited the proliferation, migration, and invasion of other cancer types, including prostate cancer [44]. Similarly, the synthetic CB analogue HU-331 was shown to inhibit tumor growth in nude mice xenografts without significant signs of toxicity in healthy organs [79].

Furthermore, CBD was also found to modulate the tumor environment [66]. CBD reduces the secretion of cytokines such as granulocyte-macrophage colony-stimulating factor from cancer cells. Consequently, a reduced recruitment of macrophages from the tumor microenvironment by the cancer cells will suppress the angiogenesis in the tumor [28]. This will limit the supply of nutrients and oxygen required for tumor growth [31].

4.5. Effect of Cannabinoids Related to Estrogen

Estrogens, particularly the most potent estrogen 17β-estradiol (E2), bind to ERα and ERβ to mediate the transcription of target genes, which regulate cell metabolism, cell growth, differentiation, and survival. Transcription starts after binding of the estrogen/ER complexes to estrogen response elements in DNA. Furthermore, a variety of non-genomic effects of estrogens are mediated by their influence on cellular signaling pathways for the regulation of growth and differentiation.

In addition to the classical ERs, a G-protein coupled receptor for estrogen (GPER, also known as GPR30) has been identified in the plasma membrane of a great variety of cells. It is a major mediator of estrogen’s rapid cellular effects [80]. So far, the role of GPER in breast cancer progression is still not fully elucidated. Nevertheless, it has been shown that in patients with ER-positive breast cancer treated with tamoxifen, the expression of GPER is negatively correlated with relapse-free survival. In these patients, GPER is considered to be an independent prognostic parameter for a poor outcome. In triple-negative breast cancer, GPER expression seems to be associated with a younger age and a more aggressive disease (reviewed in [81]). The progression of estrogen-related cancer is promoted by GPER activation through the MAPK, PI3K, and PLC signaling pathways [81]. These pathways are also affected by CB-R signaling and potential interaction is expected. A concise review on overlapping pathways between CB-Rs and estrogens was previously conducted [82].

Pharmacological targeting of ERα has been proved to be effective for the prevention and treatment of breast cancer [83]. The majority of newly diagnosed breast cancer (>70%) express ERα and ERβ and are sensitive to estrogen-mediated growth stimulation. Estrogen induces the expression of genes associated with cellular proliferation and survival and contributes to breast cancer development and progression. Importantly, estrogen-sensitive tumors are successfully treated by an antihormonal therapy, and the expression of ERα is a positive prognostic marker for the patient’s risk of a future outcome [43,84]. Also, high levels of ERβ are associated with a better prognosis for the survival [85]. Although CBs do not bind to ERs [86], THC was found to exert antiestrogenic activities in breast cancer cell lines. Both estrogen and CBs influence pathways associated with cell growth, cell death, and tumor progression, and their antagonistic effects on pathways involving adenylate cyclase, MAPK, ERK, PI3K, and J-Jun may maintain homeostasis between cell survival and cell death [82]. Thereby, the CB-R-induced activation of ERβ-mediated transcriptional activation will disrupt ERα signaling, leading to a reduced expression of estrogen-regulated genes that promote cell growth [87].

In women at reproductive ages, E2 is secreted by the ovaries and taken up by breast cancer cells [88]. However, in post-menopausal women with the highest rate of breast cancer, E2 is locally formed from inactive estrogen precursors, such as estrone sulfate via estrogen sulfatase and from androgens via aromatase. Estrone sulfate, which is the most prominent estrogen in post-menopausal women and may be formed from androgenic precursors, are taken up from circulation [88].

For the treatment and recurrence prevention of ER-positive breast cancer, a long-term treatment with the selective estrogen receptor modulator (SERM) tamoxifen is now standard. Tamoxifen effectively blocks estrogen-related growth of cancer cells and increases the disease-free and overall survival in patients with ER-positive breast cancer. It is still the therapy of choice for the treatment of ER-positive breast cancer in premenopausal women [65,78]. Studies involving cancer cell lines showed that tamoxifen, a hydroxylated, biologically active metabolite, and several other newer SERMs, act as inverse agonists for CB1-R and CB2-R with considerable affinity (between nM and low µM concentrations). In ER-lacking cancer cells, tamoxifen modulates adenylate cyclase activity and causes an increase in the intracellular cAMP by modulating CB-R activity [89]. In ER-negative breast cancer cell lines, tamoxifen also increases intracellular calcium levels via CB2-R activation [64].

Additionally, several newer SERMs were identified as CB-R agonists. Chemical structures of selected SERMs are presented in Figure 3 . Tamoxifen analog ridaifen-B from the group of ridaifen compounds was found to inhibit the growth of various cancer cell lines. However, they lack the affinity for ER binding. Ridaifen analogs are inverse agonists of CB2-R and have a potent anti-inflammatory effect. In lipopolisaccharide-activated macrophages, ridaifen analogs cause a reduction in the levels of NO and block pro-inflammatory cytokine secretion. The compounds also exhibit a pronounced anti-osteoporotic effect as they inhibit bone resorption by osteoclasts, preventing differentiation of bone marrow macrophages to osteoclasts, an effect which is partly due to CB2-R activation [90].