The Therapeutic Aspects of the Endocannabinoid System (ECS) for Cancer and their Development: From Nature to Laboratory

Abstract

The endocannabinoid system (ECS) is a group of neuromodulatory lipids and their receptors, which are widely distributed in mammalian tissues. ECS regulates various cardiovascular, nervous, and immune system functions inside cells. In recent years, there has been a growing body of evidence for the use of synthetic and natural cannabinoids as potential anticancer agents. For instance, the CB₁ and CB₂ receptors are assumed to play an important role inside the endocannabinoid system. These receptors are abundantly expressed in the brain and fatty tissue of the human body. Despite recent developments in molecular biology, there is still a lack of knowledge about the distribution of CB₁ and CB₂ receptors in the human kidney and their role in kidney cancer. To address this gap, we explore and demonstrate the role of the endocannabinoid system in renal cell carcinoma (RCC). In this brief overview, we elucidate the therapeutic aspects of the endocannabinoid system for various cancers and explain how this system can be used for treating kidney cancer. Overall, this review provides new insights into cannabinoids’ mechanisms of action in both in vivo and in vitro models, and focuses on recent discoveries in the field.

INTRODUCTION

Extracts from Cannabis sativa L. have been used for both medicinal and recreational purposes for many centuries. Cannabinoids are involved in a variety of physiological and pathological conditions, including inflammation, immunomodulation, analgesia, and cancer [1]. Nevertheless, the most active component of the plant, Δ⁹-tetrahydrocannabinol (THC), began to be explored in 1960 [2]. There are approximately 66 unique compounds, known as cannabinoids, derived from Cannabis sativa L. [3]. Cannabinoids are classified into three categories: phytocannabinoids, which occur uniquely in the Cannabis sativa L. plant (i.e., THC, CBD [cannabidiol], and CBN [cannabinol]); endogenous cannabinoids, which are produced in the human body and those of other animals (i.e., anandamide and 2-arachidonoyl-glycerol); and synthetic cannabinoids, which are produced in laboratory conditions (i.e., JWH-133, WIN 55,212-2, and SR141716).

Initially, it was believed that cannabinoids, which are highly hydrophobic, mediate their action by binding directly to biomembrane proteins. However, the first cloning of cannabinoid receptors from the mammalian brain in 1990 led to the discovery of the basic mechanism of cannabinoid action [4]. At this point, cannabinoid research expanded to the study of cannanioids’ therapeutic effects. Currently, two cannabinoid receptor subtypes have been identified: cannabinoid receptor type 1 (CB₁) and cannabinoid receptor type 2 (CB₂). Both subtypes belong to the G-protein coupled receptor superfamily, which is responsible for the transduction of intercellular signals. The CB₁ receptor is abundant in the brain and is also present in other tissues and organs of the body. Therefore, it is believed that the psychoactivity induced by cannabinoids is mostly regulated by CB₁ receptors. In contrast to CB₁, CB₂ receptors are expressed exclusively in immune system cells, including B and T lymphocytes and macrophages [5]. Activation of CB₁ receptors stimulates cell signaling through G_i and G_o activation, while on the other hand, CB₂ increases signaling only with G_i [6]. The activation of these G-protein-coupled and cannabinoid receptors inhibits the enzymatic activity of adenylate cyclase, which is responsible for the production of cyclic adenosine monophosphate (cAMP) inside cells [1]. However, the underlying mechanisms through which cannabinoids and cannabinoid receptors inhibit proliferation and migration, and induce apoptosis of cancer cells, remain obscure.

CANNABINOIDS IN CANCER TREATMENT: CHEMICAL APPROACHES TO THEIR SYNTHESIS

Cannabinoids are a family of compounds belonging to different chemical classes that can act on cannabinoid receptors (CB₁ and CB₂). They are grouped into three main classes: phytocannabinoids, which are naturally found in Cannabis sativa L. and other plants; endocannabinoids, which are naturally produced by mammalian species as endogenous CB₁ and CB₂ receptor agonists; and synthetic cannabinoids, which produced under laboratory conditions. Recent studies provide lines of evidence that cannabinoids, in addition to well-known pharmacological activities as an antiemetic and in the treatment of glaucoma, exhibit anticancer and antiangiogenic properties.

Phytocannabinoids

Phytocannabinoids are well-known natural products that are extracted and isolated from the Cannabis sativa L. plant (commonly known as marijuana). Marijuana has long been known for its medicinal and recreational properties. In 1964, Raphael Mechoulam and coworkers isolated one of the most active compounds from Cannabis sativa L., Δ⁹-tethrahydrocannabinol (Δ⁹-THC) [2]. They were the first to chemically synthesize this compound, which made possible further biological studies that confirmed Δ⁹-THC as its major active compound [7]. Since then, 60 unique phytocannabinoids, such as cannabidiol (CBD), cannabinol (CBN), and cannibigerol (CBG), together with endogenous cannabinoids, such as anan-damide (arachidonoylethanolamine or AEA) [8], and 2-Arachido-noylglycerol (2-AG) [9], have been identified by the Mechoulam research group. These seminal studies provided the first complete characterization of the endocannabinoid system (ECS).

Phytocannabinoids are physiologically produced in plant cell substructures called trichomes in the form of resin. From a biochemical point of view, all subclasses of phytocannabinoids originate from cannabigerol-type (CBG) molecules, according to the diverse cyclization products of this precursor Fig. (1). Two possible numberings of the C21 phytocannabinoids have been proposed, depending on either the dibenzopyran or monoterpenoid scaffold. In the present work, we refer to dibenzopyran numbering Fig. (2).

Fig. (1).

The chemical structures of different classes of phytocannabinoids.

Fig. (2).

Different numberings of the phytocannabinoid scaffold.

Dibenzopyran numbering Monoterpenoid numbering

Some progenitors of the main subclasses of pytocannabinoids derived from condensation of olivetolic acid and geranyl pyrophosphate are illustrated in Fig. (3).

Fig. (3).

Biosynthetic pathways for some cannabinoids.

Other classes of pythocannabinoids are derived from the condensation of geranyl pyrophosphate (GPP) with divarinolic acid instead of olivetolic acid. In the structure of the latter, propyl side chains replace the pentyl hydrocarbon residue. These chains include tethrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabivarin (CBV), cannabichromevarin (CBCV), and cannabigerovarin (CBGV) [10, 11].

Δ⁹-THC

The compound  (6aR,10aR)-delta-9-tetrahydrocannabinol is the main psychoactive compound of Cannabis sativa L. and is characterized by a relatively simple structure. Several of this isomer’s compounds are known, which vary in their double-bond positions (in nature, only Δ⁹ or Δ⁸ species are found), cis or trans ring junctions, and absolute stereochemical properties.

Four stereoisomers of THC are possible, but only the levorotatory ones occur in nature (Table 1). Accordingly, naturally occurring stereoisomers are energetically favored, due to the trans configuration being less strained than the cis configuration. Because of its double-bond position, Δ⁹-THC is thermodynamically less stable than Δ⁸-THC, as indicated by the easy Δ⁹ – Δ⁸ conversion under acid treatment. Several efforts have been made to synthesize Δ⁹-THC, the main isomer found in Cannabis sativa L. In 1967, Mechoulam et al. reported such synthesis from Verbenol and Olivetol using acid catalysis [12]; thereafter, many other synthethic procedures were published, including those that started with different natural products such as crysanthenol, p-menthadienol, p- menthenediol, and carene oxide, resulting in several methods for total synthesis [13-16]. Δ⁹-THC is currently commercialized with the INN of dronabinol (brand name Marinol) for medical uses, such as stimulating the appetite to prevent anorexia in patients with AIDS and as an antiemetic in patients resistant to conventional antiemetic treatment for cancer chemotherapy. Δ⁹-THC is an aromatic terpenoid with very low solubility in water but good solubility in polar organic solvents; as such, it is normally accumulated in fatty tissues after prolonged administration.

Cannabinol (CBN)

Cannabinol is a weak psychoactive cannabinoid isolated from Cannabis sativa L. and Cannabis indica [17]. It is structurally related to THC, from which it is obtained biosynthetically by oxidation. CBN differs from THC in that it possesses neither double-bonded isomers nor stereoisomers. It acts mainly as a weak CB₁ receptor agonist and possesses a higher affinity than THC for CB₂ receptors. It is also the main metabolite of Δ⁹-THC [18].

Cannabidiol (CBD)

Cannabidiol is one of the most abundant phytocannabinoids isolated from Cannabis sativa L. (up to 40% of the extract) [19]. The biosynthesis of CBD starts from a common precursor of THC — CBG, which undergoes a C–C one ring cyclization catalyzed by cannabidiolic acid (CBDA) synthase — followed by decarboxylation. Multiple CBD double-bonded isomers and their corresponding stereoisomers are possible, but only one isomer is found in nature, the one with a double-bond in the same position as Δ9-THC. Cannabidiol is poorly soluble in water but has good solubility in polar organic solvents. In the presence of a strong base and oxygen, it is oxidized to quinone [20]. It can be converted into THC by acid catalyzed cyclization [21]. The synthesis of CBD has been accomplished by several research groups [13, 22, 23]. In contrast with THC, CBD does not exhibit psychomimetic activities and is capable of antagonizing these effects. For this reason, CBD has aroused increased research interest in its pharmacological properties. Several studies show CBD to have anti-inflammatory, anticonvulsant, antioxidant, antiemetic, anxiolytic, and antipsychotic properties; thus, it may serve as potential drug for the treatment of neuro-inflammation, epilepsy, oxidative injury, vomiting and nausea, and anxiety and schizophrenia, respectively [19]. CBD is commercialized in a 1:1 combination with THC under the trade name Sativex for the relief of neuropathic pain in multiple sclerosis. GW pharmaceuticals received an Orphan Drug Designation from the Food & Drug Administration (FDA) for a purified liquid extract of CBD (brand name Epidiolex) in the treatment of Dravet syndrome. The drug is currently in phases II and III of clinical trials for Dravet syndrome, common epilepsy, schizophrenia, and other rare syndromes. Recently, new evidence of other therapeutic activities of CBD has been found in the treatment of cancer and neurodegenerative disorders [24, 25].

Endocannabinoids

Endogenous cannabinoids, or endocannabinoids, are capable of binding to CB receptors; they are lipid-like structures corresponding to those depicted in Fig. (4). Endocannabinoids are obtained from arachidonic acid, a common precursor in the biosynthesis of prostaglandins, which are key regulators of inflammatory processes. These compounds are derived from the non-oxidative metabolism of arachidonic acid by formal condensation with ethanolamine or glycerol, starting from the precursors eicosanoic phospholipids and diacylglycerols, respectively. They are found primarily in the brain but are present in almost all human tissues [26]. Thus, endocannabinoids play a biological role in inflammation, insulin sensitivity, and fat and energy metabolism. In turn, endocannabinoid inhibitors may be precious tools for reducing the prevalence of metabolic syndrome. Furthermore, the modulation of the endocannabinoid system may display therapeutic potential for diverse chronic neurologic and immune conditions. Anandamide (N-arachidonoyl-ethanolamine) (AEA) and 2-arachidonoylglycerol (2-AG) are by far the most extensively investigated endocannabinoids.

Fig. (4).

The most common endogenous cannabinoids.

Synthetic Cannabinoids

Since the discovery of natural cannabinoids, novel synthetic analogues with similar structures have been prepared by different research groups, among which, Mechoulam’s and Huffman’s ones are notable examples. Some synthetic THC analogues, such as HU-210 show activity that is 100-fold higher than THC [27]. The pharmacological studies on these artificial cannabinoids have elucidated structure–activity relationships (SARs) and ECS metabolism [28]. Synthetic cannabinoids can be structurally related to THC or can have different scaffolds that do and do not contain heterocycle rings. Most synthetic cannabinoids are lipid-soluble and apolar, consisting of 22–26 carbon atoms. Optimal activity requires more than four and up to nine saturated carbon atoms. Big efforts were made by John W. Huffman et al., who synthesized more than 450 artificial cannabinoids. Some of these compounds, which are known be their acronyms WIN55212-2, JWH-018, JWH-073, JWH-133, and SR141716 (Rimonabant), were shown to be highly bioactive; this discovery paved the way for several pharmaceutical applications as well as illegal use in smoking mixtures called “spice” or “K2.” Synthetic cannabinoids can be classified into seven major structural groups:

• Naphthoylindoles (e.g., JWH-018, JWH-073 and JWH-398);

• Naphthylmethylindoles;

• Naphthoylpyrroles;

• Naphthylmethylindenes;

• Phenylacetylindoles (i.e., benzoylindoles, e.g., JWH-250);

• Cyclohexylphenols (e.g., CP 47,497 and homologues of CP 47,497);

• Classical cannabinoids (e.g., HU-210 and JWH-133).

All of these products normally act as agonists or inverse agonists of CB₁ and CB₂ receptors, and may show alternate selectivity towards one of the receptor types. Currently, some of these compounds (WIN55212-2, JWH-133, and HU-210) are under investigation as potential treatments for Alzheimer’s disease, because they show evidence of counteracting the inflammation caused by amyloid beta proteins [29].

JWH-133

JWH-133 is chemically related to THC and is a potent CB₂ selective agonist. This compound was synthesized by John W. Huffman’s research group [28]. Its properties against Alzheimer’s disease and cancer have also been recently investigated [29, 30].

WIN55,212-2

WIN55,212-2 is a naphthoylindole derivative (JWH analogue) possessing THC-like potent, non-selective, CB receptor agonist properties. It has potential therapeutic use as an analgesic and against Alzheimer’s disease [29, 31].

SR141716

Also known as Rimonabant (brand name Acomplia by Sanofi-Aventis), SR141716 is an inverse selective agonist of the CB₁ receptor [32]. It is a pyrazole derivative and is remarkably easy to synthesize [33]. SR141716 was approved as an anorectic in Europe for the treatment of obesity, given its efficacy in decreasing appetite. The FDA, however, refused its approval as anti-obesity drug because of the severe depression reported as a side effect.

CANNABINOIDS AND CANCER

The cannabinoid receptors CB₁ and CB₂ comprise the endocannabinoid system (ECS) inside a cell, and the activation of these receptors is important for a number of physiological processes that regulate nervous, digestive, reproductive, immune, and metabolic functions [34]. There is growing body of evidence that the ECS system and synthetic cannabinoids modulate the activity of enzymes and nuclear factors involved in cancer cell homeostasis, growth, migration, metastasis, and tumor angiogenesis [1, 35-38]. Remarkably, cannabinoids target cancer cells, while non-tumor cells and tissues are avoided. This apparent selectivity for tumor cells makes the ECS system an attractive potential target for cancer therapy. In 1975, the natural cannabinoid THC was recognized as a potential anticancer agent [39], inhibiting lung adenocarcinoma cell growth in vitro and in vivo. Since that time, the therapeutic potential of cannabinoid for various types of cancer have been widely investigated [35]. Today, capsules of THC and its synthetic analogues have been approved for treating nausea and emesis associated with cancer chemotherapy [5].

In 2006, the first human clinical study of THC was successfully performed in patients with recurrent glioblastoma multiforme [40]. The potential palliative effects of cannabinoids as appetite stimulators and pain inhibitors were also confirmed in phase III clinical trials in oncology [5, 41]. Now, several plant-derived, synthetic, and endogenous cannabinoids that exert an antiproliferation effect on various cancers are widely recognized [Table 2]. Cannabinoid administration has been proven to slow tumor growth, as well as to slow lung carcinoma, prostate cancer, glioma, and skin carcinomas in mice [39, 42-46]. Importantly, all of these studies found CB₁ receptors, CB₂ receptors, or both, which was later confirmed by molecular and pharmacological approaches that examined cannabinoid-receptor expression and used selective cannabinoid receptor agonists (JWH-133 and WIN 55,212-2) and antagonists (such as AM-630 and SR-141716A).

The binding of cannabinoids to their G-protein-coupled cannabinoid receptors affects various cellular pathways, for example, it inhibits adenylate cyclase and activates extracellular-signal-regulated kinase (ERK). This suggests that cannabinoids might also be involved in other pathways important for cell survival, such as MAPK [47]. The activation of MAPK by cannabinoids has been observed in human vascular endothelial cells, neural cell lines, and Chinese hamster ovary cells [5, 48, 49]. Both of these kinases (MAPK and ERK) are stimulated through G-protein-mediated mechanisms [50, 51]. Cannabinoid receptors are also coupled to activation of serine/threonine protein kinase B/Akt (PKB), resulting in increased phosphorylation of glycogen synthase kinase-3 (GSK3) on serine 21 residue [49]. The effect of cannabinoids on sphingomyelin breakdown, resulting in increased intracellular ceramide levels, has been also observed [52]. Finally, the activation of CB₁ or CB₂ receptors seems to induce apoptosis through de novo synthesis of ceramide in various types of cancer cells, including those associated with glioma, leukemia, colon, and pancreatic cancer [44, 53-55].

CANNABINOIDS AS ANTIANGIOGENESIS FACTORS: MOLECULAR ASPECTS OF CANNABINOIDS IN CANCER PREVENTION

The hallmark of cancer is the ability to proliferate despite the absence of mitogenic signals. Rapid proliferation of cancer cells requires large amounts of energy and oxygen; therefore, hypoxic cells secrete proangiogenic factors (e.g., HIF-1a, VEGF, PIGF, HGF, and Ang-2 [90-92] to induce the formation of new blood vessels to meet their need of increased oxygen. Thus, angiogenesis is one of the most important mechanisms through which solid tumors grow rapidly. Currently, more attention is being paid to the mechanism of angiogenesis, mostly due to its correlation with cancer metastasis, and targeting antiangiogenic molecules has become the most promising therapeutic approach to cancer treatment.

The range of known antiangiogenic factors is still growing; however, most treatment strategies are based on blocking signaling from vascular endothelial grow factors (VEGFs), and especially, using blocking monoclonal antibodies for VEGFR1 and VEGFR2, such as Sunitinib or Bewacizumab [93-95]. Other treatment strategies target various steps in angiogenesis or degrade newly formed blood vessels. However, most of these strategies fail due to the coexistence of strong proangiogenic factors such as VEGFs and EGFs.

Cannabinoids have been proven to inhibit angiogenesis in in vivo and in vitro studies [38]. Blazquez et al. observed that the local administration of JWH-133 altered the blood vessel morphology in C6 glioma and grade IV astrocytoma in rats [38]. All tumors treated with JWH-133 had a paler overall appearance and only very small and narrow capillaries forming differentiated and impermeable vascular networks compared to the controls. Moreover, the authors showed that cannabinoid administration decreased vascular endothelial cell migration, survival, and expression of major proangiogenic factors such as VEGF, Ang-2, and MMP2 proteins [38].

Analysis of microarray data showed that JWH-133 administration in C6 glioma-bearing mice induced altered expression of 10 genes directly or indirectly related to the VEGF pathway. The cannabinoid treatment decreased the expression of VEGF itself, HIF-1a, heme oxygenase-1, Id3, midkine, angiopoietin-2, and Tie-1 [96]. Cannabinoids WIN-55,212-2 and JWH-133 also decreased the expression of phosphorylated VEGFR-2 in the tumors of two glioma patients. The sphingolipid messenger ceramide might be responsible for cannabinoid-induced inhibition of VEGF and VEGFR2 protein expression. The same compounds were also efficient in the treatment of non-melanoma skin cancer by decreasing new blood vessel formation [46, 97].

Matrix metalloproteinases (MMPs) are other possible molecular targets for cannabinoid treatment [98]. MMPs belong to the family of enzymes that participate in the degradation of the vascular basement membrane and remodeling of the extracellular matrix (ECM) [99]. This process sends one of the signals required for endothelial cells to migrate and form new blood vessels. MMPs have been linked to tumor invasion and their increased expression is found in almost every type of cancer [100, 101]. Blazquez et al. investigated the role of cannabinoid agonists (THC, JWH-133, and anadamide) on MMP-2 expression inside glioma cell cultures and glioma-bearing mice [98], and found that THC administration decreased tumor growth and the expression of MMP-2 in C6.9 glioma cells. The MMP-2 level also decreased in biopsies obtained from patients with recurrent glioblastoma multiform after local administration of THC. However, the study also demonstrated the selective nature of THC because the level of other MMP protein family members (MMP-3, MMP-9, and MMP-14) remained unchanged Fig. (5) [98].

Fig. (5).

The influence of CB₁ and CB₂ activity on angiogenesis in cancer: After binding to a specific agonist, both CB₁ and CB₂ receptors are activated. This leads to direct and indirect changes that have crucial effects on angiogenesis in a cancerous environment. Inactivation of the PI3K/Akt pathway leads to lower HIF-1 and VEGF levels — two of the most known proangiogenic molecules. CB₁ receptor activation leads to the direct inhibition of VEGF type 2 receptors, which are major angiogenesis inducers. When activated, CB₁ and CB₂ receptors strictly inhibit adenylyl cyclase (AC). PKAs, whose activity is strictly dependent on the cellular levels of cAMP, are inactivated and unable to phosphorylate CREB/ATF, decreasing nitric oxide synthase (NOS). Ras inactivation leads to inactivation of the whole pathway, including ERK and MAPK. Metalloproteinases production is stopped by the activation of both cannabinoid type 1 and type 2 receptors. Thus, all of these events have a direct impact on angiogenesis as well as the cell cycle and proliferation.

CANNABINOIDS AND THE TUMOR MICROENVIRONMENT

Interactions between the various cell types observed within a tumor mass influence its development, progression, and metastasis. The composition of the microenvironment and roles of particular cells vary, depending on the tumor type and organ of origin. These cells contribute to tumor development by secreting various pro-tumor factors or by inducing immunosuppression. One of the most important cell types to facilitate tumor development is tumor-associated macrophages (TAMs) [102-104]. TAMs are recruited into a tumor mass by various cytokines and chemokines secreted by cancer cells. Once they migrate to the tumor, they start to secrete various proangiogenic factors or enzymes that facilitate matrix remodeling. On the other hand, cancer cells influenced by macrophages start to express genes and antigens that are atypical (but specific to macrophages, such as chemokines CCL5 and CSF-1 and their receptors CXCR5 and CSF-1R) [105, 106]. Indeed, chemoattractants secreted by cancer cells that increase the migratory abilities of immune cells and their infiltration to a tumor mass can also enhance cancer cell migration in an autocrine manner. Due to the involvement of TAMs in cancer development, inhibition of their migration into a tumor mass has been proposed as one therapeutic approach [102].

There are no published papers about the influence of cannabinoids on the tumor microenvironment and TAMs. However, the expression level of CB₂ receptors is considerably upregulated in activated macrophages [107]. CB₂ receptors control several functions of macrophages, such as actin polymerization, migration, cell phenotype, and cytokine release from these cells [107]. Cannabinoid ligands share receptors with important chemotactic cytokines that direct the migration of immune cells. These receptors are coupled to heterotrimeric Gi proteins [108]. Ghosh et al. reported that the CB₁/CB₂ agonist CP55940, and CB₂-selective agonist JW-015, caused significant inhibition of chemokine CXCL12-induced chemotaxis of T lymphocytes [109]. Raborn et al. demonstrated that THC and cannabinoids that activate the CB₂ inhibit macrophage chemotaxis to CCL5 [108]. Because CCL5 is a very important ligand for G protein-coupled receptors CCR1 and CCR5, activation of the CB₂ leads to trans-deactivation of these receptors, which play specialized roles in leukocyte trafficking [110-112].

These results indicate that cannabinoids and chemokines may constitute integrative components of a network of intercommunicating G protein-coupled receptors that not only regulate the immune responses and chemokine/cytokine-regulated chemotaxis of leukocytes but also the migration of cancer cells. Additionally,cannabinoids, as highly lipophilic molecules, can perturb cellular membranes and alter ligand–receptor interaction, disrupt receptor-G protein complexes, and alter the cascade of signal transduction, thus decreasing the migratory abilities of cells [113-115].

The cannabinoid-based inhibition of immune cell migration to a tumor mass (in particular macrophages), or the alteration of chemokine/cytokine signaling in cancer cells, may constitute a beneficial approach in anticancer therapy. Therefore, the regulatory function of cannabinoids on the tumor microenvironment needs further investigation.

CANNABINOIDS IN ANTICANCER THERAPY FOR RENAL CELL CARCINOMA

Renal cell carcinoma (RCC) represents a serious problem in oncology — it is the cause of over 100,000 deaths each year. RCC is the most common type of renal cell tumor, consisting of a substantial number of malignant cancers and benign tumors. This type of cancer has a higher risk of appearance in males [116]. The treatment of choice for renal cancers are nephrectomy combined with immunotherapy [117], due to the high costs of other therapies or late detection. However, novel drugs give hope to cancer patients — for example, therapies concerning the tyrosine kinase receptor inhibitor or multipurpose drugs targeting not only cancer cells but also stimulating the immune response. However, there is no perfect therapy for eliminating tumor cells. Investigation of the anticancer properties of natural compounds, such as cannabinoids, have brought interesting results.

Medical cannabis still remains a prescribed medication in some of therapeutic protocols and Cannabis sativa L. has started to become one of the most commonly used additional medications to treat nausea (caused, for example, by chemotherapy), stimulate the appetite, and deal with chronic pain [5, 118].

Cannabinoids also have an important influence on cancer cells themselves, what was mentioned before in this manuscript. There are many ways they inhibit tumor proliferation and survival. Fig. (6) illustrates a couple of ways CB₁ and CB₂ receptors are integrated into intracellular pathways. However, all of mechanisms are not identical in every cell and do not occur simultaneously.

Fig. (6).

ECS system-related pathways: The activation of CB₁/CB₂ receptors has an immense effect on the fate of cells, leading them to apoptosis. Adenylyl cyclase is inhibited by the activation of both cannabinoid type 1 and type 2 receptors. Inhibiting various pathways — including the PI3K/Akt pathway and cAMP-dependent PKA and Ras phosphorylation — results in stimulating cell death through numerous secreted molecules, such as Bad, Bcl2, and Fos. Secreting apoptotic proteins, such as p27 and p21 via ceramide synthesis, speeds up the process of apoptosis.

In the case of RCC, research is still lacking on cannabinoids as anticancer compounds. However, the expression of cannabinoid receptors (CB₁, CB₂, TRPV1, and GPR55) in kidney cells and RCC has been confirmed [119]. The expression of cannabinoid receptors CB₁ and CB₂ was first confirmed in the human proximal tubule (HK2) cell line, and also lysate from rat kidney [145]. Larrinaga et al. investigated the expression of cannabinoid receptors in human fetal and adult kidneys [120], finding that CB₁ (CNR1a) mRNA was present in all samples; however, CB₂ (CNR2) mRNA was undetectable in all samples. Further studies are needed to help clarify these differences and the role of ECS in renal function.

Renal cell carcinoma (RCC) is the most common epithelial malignancy of the kidney in adults, accounting for 3% of all neoplasms [121]. Considering the specific cannabinoid receptors in kidneys, as well as the pro-apoptotic, antiproliferative, anti-metas-tatic and antiangiogenic effects of cannabinoids, this field should be explored. So far, there are only a few published papers about the ECS in RCC.

Some results also suggest that patterns of cannabinoid receptor expression could be used in differential diagnosis of kidney cancers. Chromophobe RCC (ChRCC) and renal oncocytoma (RO) are two histologically distinct neoplastic entities of renal tumors. These tumors originate from distinct sites in the kidney and show different molecular abnormalities. In ChRCC samples, the mRNA expression for CB₁ receptors was 12-fold lower than in the surrounding normal tissue [86]. However, RO samples showed no difference in the level of CB₁ mRNA expression between the tumor and surrounding tissue. The mRNA expression for CB₂ receptor was undetectable in both ChRCC and RO. Interestingly, protein expression analysis of these RCC neoplasms failed to provide any evidence for the expression of CB₁ [36, 86]. Thus, the CB₁ protein may undergo modifications in these tumors at the post-transcriptional level. Therefore, further studies are required to understand the role of cannabinoids and their receptors in RCC.

CONCLUSION

More research is needed to answer all questions about the potential use of cannabinoids to treat cancer. The first question is which type of cannabinoids should be used to treat cancer patients — natural or synthetic? Different cannabinoids seem to have different effects on various cancer types, which may be caused by the different expression of cannabinoid receptors in various cancer types. Similarly, no studies have shown the effects of cannabinoids on the efficacy of chemotherapy. The psychoactive effect of cannabinoids should also be considered before its routine use.

Table 1.

Stereoisomers of Δ⁹ THC.

Table 2.

The cannabinoid modulation of different cancers and pathways.

Cancer Type	Signaling Pathways Involved	Effects	Experimental System	Receptors Involved	Cannabinoids Tested	References
Glioma	ERK, MAPK, cAMP, LOX, PI3K/Akt/mTOR	Decrease tumor volume and apoptosis	In vitro In vivo (rat, mouse)	CB1, CB2	THC, WIN55,212-2, JWH-133, AM-1241, CBD, SR144528, SR141716	[44, 56-62]
Breast	NF-κB, PA-1, cAMP, PKA, MAPK, S1P/ceramide, CXCR4/CXCL12	Cell cycle arrest, Apoptosis and inhibit tumor growth	In vitro In vivo (mouse)	CB1, CB2	AEA, THC, CBD, WIN55,212-2, JWH-015, SR 141716A	[63-68]
Lung	MAPKs, PI3K/Akt/mTOR, JNK, ERK	Decrease tumor growth, invade cancer cells, and induce apoptosis	In vitro In vivo (mouse)	CB1, CB2	THC, CBN, CBD, AM-251, AM-630	[39, 69-72]
Leukemia	cAMP, ECS	Decrease tumor growth, apoptosis	In vitro In vivo (mouse)	CB2	THC, JWH-015, SR144528	[73-75]
Melanoma	COX-2, LOX, Akt	Apoptosis, inhibit cancer cell growth	In vitro In vivo (mouse)	CB1, CB2	JWH-133, WIN55,212-2, AM251, AEA, AM251, SR141716, SR144528	[46, 76, 77]
Prostate	NF-κB/cyclin D/cyclin E, JNK, Akt	Arrest cell cycle, apoptosis, and decrease tumor volume	In vitro In vivo (mouse)	CB1, CB2	AEA, JWH-015, WIN55,212-2, SR 144528, SR141716A	[37, 78-81]
Thyroid	cAMP, ERK, MAPK	Decrease tumor volume, apoptosis	In vitro In vivo (mouse)	CB1, CB2	AEA, JWH133, SR141716A	[82-84]
Pancreatic	(Ca2+) Calcium signaling, p8-ATF-4-TRB3	Inhibit cancer cells, reduce tumor growth	In vitro (mouse)	CB1, CB2	ACPA, GW, WIN 55,212-2, SR141716, SR144528	[55, 85]
Kidney	?	?	In vitro	CB1? CB2?	?	[36, 86]
Ovary	?	?	In vivo (mouse)	CB1, CB2	?	[87]
Bladder	(Ca2+) Calcium signaling	Decrease cancer cell proliferation by delaying the cell cycle progression	In vitro	CB1, CB2	2-AG, AEA, AM-281, CP55,940	[88, 89]

List of abbreviations

2-AG

2-arachidonoylglycerol,

ACPA

arachidonylcyclopropylamide

AM-630

6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone

Ang2

angiopoietin-2

cAMP

cyclic adenosine monophosphate

CB₁

Cannabinoid receptor type 1

CB₂

Cannabinoid receptor type 2

CBD

Cannabidiol

CBDA

Cannabidiolic-acid

CBN

Cannabinol

CCL5

CC chemokine ligand-5

CCR1

CC chemokine receptor 1

CCR5

CC chemokine receptor 5

ChRCC

Chromophobe RCC

CP55,940

(-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol

CSF-1

colony stimulating factor-1

CXCR5

C-X-C chemokine receptor type-5

ECM

extracellular matrix

ECS

endocannabinoid system

ERK

extracellular-signal-regulated kinase

GSK3

glycogen synthase kinase-3

GW

1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-(1-morpholine)ethyl)-5-methoxyindole

HGF

hepatocyte growth factor

HIF-α

hypoxia-inducible factor-1 alpha

JNK

c-Jun-NH2-kinase

JWH-133

(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran

MAPKs

mitogen-activated protein kinases

MMPs

matrix metalloproteinases

NOS

nitric oxide synthase

PIGF

placenta growth factor

PKB

serine/threonine protein kinase B/Akt

RCC

renal cell carcinoma

RO

renal oncocytoma

SAR

Structure-activity relationships

SR141716

5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide

TAM

tumor-associated macrophage

THC

Δ⁹tetrahydrocannabinol

TNF-α

tumor necrosis factor alpha

VEGF

vascular endothelial growth factor

VEGFR1

vascular endothelial growth factor receptor 1

VEGFR2

vascular endothelial growth factor receptor 2

WIN-55,212-2

(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate

CONFLICT OF INTEREST

The authors indicate no potential conflicts of interest.