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Cbd oil for bowel cancer

Cannabidiol—from Plant to Human Body: A Promising Bioactive Molecule with Multi-Target Effects in Cancer

1 Department of Pharmacognosy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (B.K.); [email protected] (S.A.); [email protected] (I.Z.P.); [email protected] (C.D.)

2 Centre for Gene and Cellular Therapies in the Treatment of Cancer- OncoGen, Clinical County Hospital of Timişoara, Liviu Rebreanu Blvd. 156, 300736 Timişoara, Romania; [email protected]

Feng Chen Ifrim

3 Department of Marketing, medical technology, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania

Valentina Buda

4 Department of Pharmacology and Clinical Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timisoara, Romania

Stefana Avram

1 Department of Pharmacognosy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (B.K.); [email protected] (S.A.); [email protected] (I.Z.P.); [email protected] (C.D.)

Ioana Zinuca Pavel

1 Department of Pharmacognosy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (B.K.); [email protected] (S.A.); [email protected] (I.Z.P.); [email protected] (C.D.)

Diana Antal

5 Department of Pharmaceutical Botany, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (D.A.); [email protected] (F.A.)

Virgil Paunescu

2 Centre for Gene and Cellular Therapies in the Treatment of Cancer- OncoGen, Clinical County Hospital of Timişoara, Liviu Rebreanu Blvd. 156, 300736 Timişoara, Romania; [email protected]

6 Department of Functional Sciences, Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timisoara, Romania

Cristina Adriana Dehelean

7 Department of Toxicology, “Victor Babeş“University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected]

Florina Ardelean

5 Department of Pharmaceutical Botany, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (D.A.); [email protected] (F.A.)

Zorita Diaconeasa

8 Department of Food Science and Technology, Faculty of Food Science and Technology, University of Agricultural Science and Veterinary Medicine, Calea Manastur, 3-5, 400372 Cluj-Napoca, Romania; [email protected]

Codruta Soica

9 Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected]

Corina Danciu

1 Department of Pharmacognosy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (B.K.); [email protected] (S.A.); [email protected] (I.Z.P.); [email protected] (C.D.)

1 Department of Pharmacognosy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (B.K.); [email protected] (S.A.); [email protected] (I.Z.P.); [email protected] (C.D.)

2 Centre for Gene and Cellular Therapies in the Treatment of Cancer- OncoGen, Clinical County Hospital of Timişoara, Liviu Rebreanu Blvd. 156, 300736 Timişoara, Romania; [email protected]

3 Department of Marketing, medical technology, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania

4 Department of Pharmacology and Clinical Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timisoara, Romania

5 Department of Pharmaceutical Botany, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected] (D.A.); [email protected] (F.A.)

6 Department of Functional Sciences, Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timisoara, Romania

7 Department of Toxicology, “Victor Babeş“University of Medicine and Pharmacy, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [email protected]

8 Department of Food Science and Technology, Faculty of Food Science and Technology, University of Agricultural Science and Veterinary Medicine, Calea Manastur, 3-5, 400372 Cluj-Napoca, Romania; [email protected]

9 Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, Romania; [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

Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin with medicinal and psychoactive properties. It is the main source of phytocannabinoids, with over 100 compounds detected so far. In recent years, a lot of attention has been given to the main phytochemicals present in Cannabis sativa L., namely, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). Compared to THC, CBD has non-psychoactive effects, an advantage for clinical applications of anti-tumor benefits. The review is designed to provide an update regarding the multi-target effects of CBD in different types of cancer. The main focus is on the latest in vitro and in vivo studies that present data regarding the anti-proliferative, pro-apoptotic, cytotoxic, anti-invasive, anti-antiangiogenic, anti-inflammatory, and immunomodulatory properties of CBD together with their mechanisms of action. The latest clinical evidence of the anticancer effects of CBD is also outlined. Moreover, the main aspects of the pharmacological and toxicological profiles are given.

Keywords: cannabidiol, pharmacological and toxicological profile, breast cancer, prostate cancer, glioma, colon cancer, lung cancer, brain cancer, melanoma, immunomodulatory effects, clinical evidence

1. Introduction

Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin, with medicinal and psychoactive properties [1]. It is considered the oldest cultivated fiber plant, originating from Southeast and Central Asia [2]. Taxonomic controversies surrounding hemp have not yet resolved the issue of the Cannabis genus being monotypic, that is, including only one highly variable species, Cannabis sativa L., or polyspecific—enclosing four (Cannabis sativa, Cannabis indica, Cannabis ruderalis, and Cannabis afghanica) species with distinct geographical, chemotypic, and morphological features [3,4]. The current tendency converges towards the recognition of chemovars as the most appropriate denomination. This trend is supported by widespread crossbreeding and hybridization, as breeding barriers among Cannabis hybrids are absent. On the other hand, the common use of the appellation “strains” is considered improper, as this designation only applies to bacteria and viruses [5]. From a medicinal viewpoint, a demarcation of a fiber-type hemp (containing high levels of cannabidiol (CBD) but very low in psychotropic Δ9-tetrahydrocannabinol (THC) and of a drug-type Cannabis (containing up to 15% THC in the female inflorescences) can be made [6,7].

In Europe, great attention has been paid to the medical use of cannabis since 1840, and this was due to William O’Shaughnessy, an Irish physician who traveled to India and noticed the medicinal properties of Indian cannabis [8]. His experiments referred to cannabis use in epilepsy, tetanus, rheumatism, and cholera [9]. Later, various cannabis preparations (tinctures, extracts, cigarettes) were used in the treatment of migraines, asthma, insomnia, and even for opium-use withdrawal [8]. Despite its popularity, at the end of the 19 th century great variability in opinion on the therapeutic effects and preparations and also worries about drug abuse emerged [10]. The decline in use was due to the association of cannabis with addiction, mental deterioration, and crime [11], and to the replacement of cannabis preparations with synthetic drugs. This led to an international prohibition of cannabis use [11].

The identification of the major cannabinoids, THC and CBD, has been an important step for further research. Numerous studies have been conducted on THC after its isolation and characterization in the 1960s [12]. The cannabinoid receptors and the endocannabinoid system were discovered only in the 1990s, and this determined not only the evaluation of the pharmacological effects of phytocannabinoids, but also the synthesis of drugs that act on the endocannabinoid system [10].

Phytocannabinoids are a type of cannabimimetic compound which can interact with the endocannabinoid system [13]. Cannabis sativa L. is the main source of phytocannabinoids, with over 100 compounds detected so far [14]. The compounds accumulate in secretory hairs situated chiefly on the bracts of pistillate (female) flowers. Three different types of such trichomes have been described: bulbous glands, capitate-sessile glands, and capitate-stalked glands, resulting in a layered complex [15]. The capitate-stalked type glands contain the highest number of cannabinoids, and the biosynthesis of tetrahydrocannabinolic acid (THCA) by glandular cells has reliably been proven [16,17].

Cannabinoids are terpenophenolics comprising a diphenol and a monoterpene moiety. The synthesis of the former part occurs via the polyketide pathway by the stepwise condensation of three malonyl-Coenzyme A molecules with hexanoyl-Coenzyme A, in order to yield olivetolic acid [18]. The monoterpene unit, geranyl-diphosphate, results from the head-to-tail condensation of geranyl-diphosphate and dimethylallyldiphosphate through the non-mevalonate pathway. Subsequently, olivetolic acid undergoes prenylation by geranyl-diphosphate ( Figure 1 ). The product of this synthesis, cannabigerolic acid, is the key metabolic intermediary of cannabinoid biosynthesis [19]. It represents the substrate of three enzymes: tetrahydrocannabinolic acid synthase convertingcannabigerolic acid to Δ9-THCA [16], cannabidiolic acid synthase yielding cannabidiolicacid [20], and cannabinochromenic acid synthase producing cannabinochromenic acid [21]. Recent research could identify in planta both the acidic forms of the cannabinoids [22] as well as the decarboxylated forms (THC, CBD, cannabichromene, cannabigerol, cannabinol)—albeit in much lower amounts [23]. Non-enzymatic decarboxylation of these compounds is promoted by heating (during smoking or baking), sunlight, and storage. It is believed that the high number of diverse cannabinoids identified so far from Cannabis occur due to non-enzymatic modifications [24]. Studies performed on laser-microdissected capitate-stalked trichomes could suggest that cannabinoids are also present in the multicellular stipes of capitate-stalked hairs in addition to the secretory portion of the trichomes [23]. The exact contribution of the stalk cells to the biosynthesis of cannabinoids and the source of these compounds in the stems remains to be elucidated.

Key steps in the biosynthesis of the main phytocannabinoids from Cannabis.

2. Pharmacology, Toxicology, and Route of Administration

Cannabidiol is the second most abundant type of cannabinoid, showing higher concentrations than THC in many cannabis strains [25]. Compared to THC, CBD has non-psychoactive effects, an advantage for clinical applications of the anti-tumor benefits [26].

2.1. Pharmacokinetics

The pharmacokinetics of CBD depends on its pharmaceutical formulation and on its routes of administration [27]. It is a highly lipophilic compound, having a poor oral bioavailability (around 6%) [28,29]. The absorption following inhalation is similar to the intravenous one, having a peak plasma concentration obtained within 3–10 min and being higher than the one obtained after oral administration (the bioavailability of inhaled CBD being around 31%) [30,31]. The explanation for this variable absorption lies in the fact that after oral administration CBD suffers an intense first pass metabolism; thus, the inhalation route can avoid or reduce the extent of this first pass metabolism [32]. Also the transdermal formulation can surpass the first pass metabolism [33]. Plasmatic levels can be increased if CBD is administered orally with food or when a meal is consumed after its administration, as lipids can increase its absorption [34].

CBD is rapidly distributed in the lungs, heart, liver, and brain, and to the less vascularised tissues. It has a high distribution volume and can accumulate in adipose tissue in patients under chronic treatment, having the risk of a prolonged activity (several weeks after administration) [28,35]. Thus, CBD distribution is influenced by age, body size, composition, and the permeability of the blood—tissues barriers [27]. Due to its lipophylic structure, CBD is able to cross placenta and also to arrive in breast milk. Peak plasma concentrations and area under the curve were reported to be dose-dependent in human studies with a Tmax (time taken to reach the maximum concentration) of between 1 and 4h [34].

Further, CBD is metabolised mainly in the liver, by the main isoenzymes CYP2C19 and CYP3A4, and to a lesser extent by CYP1A1, CYP1A2, CYP2C9, and CYP2D6 [36]. After its hydroxylation to 7-hydroxy-cannabidiol (7-OH-CBD) and further metabolism, its metabolites (mostly with unknown pharmacological activity) will be predominantly excreted in faeces and to a lesser extent in the urine [28]. Cannabidiol has a long terminal elimination half-life, the average half-life being around 24h ± 6h after intravenous administration and around 31 ± 4h after inhaled administration. After repeated intake of oral formulations, CBD elimination half-life was reported to vary between two and five days [37] ( Figure 2 ). All these pharmacokinetic properties are important to be considered before CBD is administered as an anti-cancer agent.

Characteristics of cannabidiol’s (CBD) pharmacokinetic profile.

2.2. Pharmacodynamics

Cannabidiol is a non-psychoactive cannabinoid, compared with THC, the psychoactive compound which produces the main side effects of cannabis [38,39]. Compared with THC, which is a partial agonist of cannabinoid receptor 1 (CB1, located mainly in the central nervous system, but also present in organs, tissues, and peripheral nervous system) and CB2 (expressed in immune tissues, gastrointestinal tract, and in low concentrations in the central nervous system) receptors of the endogenous cannabinoid system, CBD has a weak affinity for the sites of the receptors (the orthostatic ones). Moreover, it was reported to possibly inhibit THC binding to CB1 through other mechanisms [40,41,42].

The main physiological function of the endogenous cannabinoid system consists of inhibiting the release of other neurotransmitters (acetylcholine, dopamine, histamine, serotonin, glutamate, GABA, etc.) in the nervous system, usually by stimulating CB1receptors [30]. Cannabidiol presents a complex mechanism of action consisting of: weak blocking of CB1 receptors; an inverse agonist of CB2 receptors;stimulation of vanilloid receptors type 1 (TRPV1—transient receptor potential vanilloid 1) and type 2 (TRPV2—transient receptor potential vanilloid 2); increasing the concentration of anandamide (by blocking its hydrolysis), a fatty acid neurotransmitter (its effects being mediated through CB1 receptors in CNS and CB2 receptors in the periphery); stimulation of endogenous adenosine signalling (by binding to equilibrative nucleoside transporter-1); inhibition ofG-protein coupled receptor 55 (GPR55); and the stimulation of the 5-HT1a (serotonin receptor 1A) receptor, PPARγ (nuclear peroxisome proliferation activated receptor γ) and glycine receptor subtypes [26,31] ( Table 1 ).

Table 1

Receptor Effect Ki; EC50; IC50 References
CB1 Antagonist Ki = 4350–4900 nM [43,44]
CB2 Inverse agonist Ki = 2860–4200 nM [43,45]
GPR55 Antagonist IC50 = 445 nM [45]
TRPM8 Antagonist IC50 = 80–140 nM [46,47]
TRPV1 Agonist EC50 = 1000 nM [46,48]
TRPV2 Agonist EC50 = 1250 nM [46,48]
TRPV3 Agonist EC50 = 3700 nM [46,49]
TRPA1 Agonist EC50 = 110 nM [46,48]
PPARϒ Agonist EC50 = 20,100 nM [50]

Legend: CB1—cannabinoid receptor 1; CB2—cannabinoid receptor 2; EC50—Half maximal effective concentration; GPR55—G-protein coupled receptor 55; IC50—half maximal inhibitory concentration; Ki—Inhibitory constant binding affinity; TRPA1—transient receptor potential ankyrin 1; TRPM8—transientreceptor potential melastanin 8; TRPV—transient receptor potential vanilloid.

2.3. Pharmacological Actions and Indications

In addition for its anti-tumor properties [51], CBD has been reported to induce the following effects: analgesic [52], neuroprotective [53], antiemetic [54], anticonvulsivant [55], anti-inflammatory [55], and antispasmodic [56].Thus, studies have shown its therapeutic potential not only in different types of malignant disorders [51], but also in the treatment of: epilepsy [57]; nausea and vomiting or other side effects caused by cytostatic therapy [54]; spasticity as well as other symptoms like tremor, bladder dysfunction, disease progression, inflammation, cognition in multiple sclerosis [58]; neuropatic and chronic pain [59]; spinal cord injury [60]; Parkinson’s and Alzheimer’s disease [61]; post-traumatic stress disorder and anxiety [62]; schizophrenia [63]; pulmonary disease [64]. Other therapeutic uses of CBD might be in the treatment of cannabis and tobacco addiction, although much more research is needed [65].

2.4. Toxicology

Cannabidiol has been reported to have low toxicity, is generally well tolerated, and has a good safety profile, although related studies are currently limited. It can interact with other co-administered drugs, causing different side effects depending on the interaction [65]. For the moment there are no reported risks of potential physical dependence (withdrawal and tolerance) or potential abuse [66,67,68].

Cannabidiol was reported to induce mainly fatigue and somnolence. On the other hand, THC was reported to induce dose-dependent performance (cognitive and psychomotor) impairment, as well as to increase anxiety, psychotic symptoms, heart rate, and blood pressure, and to alter perception. By mixing CBD with THC, the side effects of THC were reduced [67,68].

There are few data regarding its safety profile in children (limited pharmacokinetic information) [44]. Due to limited studies, and the ability of CBD to cross the placenta and to arrive in breast milk, it is not recommended for use in pregnancy and lactation [27,67,68].

Caution should be taken when CBD is administered in patients with hepatic impairment (risk of accumulation) or when taken with other drugs, because inducers/inhibitors of CYP3A4 or CYP2C19 can decrease/increase its plasmatic concentration [27,69]. Moreover, it acts as a CYP1A1 inducer and as an inhibitor of P-glycoprotein-mediated drug transport, which can affect the plasmatic concentration of co-administered drugs [27,69] ( Table 2 ).

Table 2

Inducers, and inhibitors for the main isoforms of cytochrome P450 that are implicated in the metabolism of CBD [27,69].

CYP 450 – Isoenzymes Substrates Inducers Inhibitors
CYP3A4 CBD
Alprazolam
Diazepam
Amlodipine
Simvastatine
Atorvastatine
Apixaban
Rivaroxaban
Carbamazepine
Fenitoine
Phenobarbital
Eritromicine
Claritromicine
Verapamil
Diltiazem
Fluconazol
Itraconazol
CYP2C19 CBD
Clopidogrel
Fenitoine
Diazepam
Carbamazepine
Rifampicine
Fluoxetine
Fluvoxamine
Ketoconazole
Omeprazole
CYP1A2 Theophylline
Clozapine
Naproxen
CBD
Tobacco smoke
Ciprofloxacine
Ofloxacine
Levofloxacine
Amiodarone
P-Glycoproteine (intestinal absorption) Loperamide
Morphine
Dabigatran
Atorvastatine
Simvastatine
Paclitaxel
Antraciclines
Carbamazepine
Rifampicine
Phenobarbital
CBD
Ketoconazol
Itraconazol
Eritromicine
Clarytromicine
Propafenone
Amiodarone

Both cannabis and tobacco smoking can induce CYP1A2 metabolism (with addictive effects when they are smoked together), causing significant interactions for substances that are substrates of this enzyme [27].

There were case reports of delirium and hypomania when CBD was associated with dysulfiram (unknown mechanism) and mania after association with fluoxetine (possibly CYP2D6 mediated) [67,68].

2.5. Route of Administration and Dosage

Cannabidiol is present for the moment in three pharmaceutical formulations, such as: Sativex ® , containing nabiximols (almost equal amounts of THC and CBD), Epidiolex ® (pure CBD), currently in Phase III trials, and Arvisol ® (oral tablet containing pure CBD), currently in Phase I trials for the treatment of schizophrenia and epilepsy [66].

Sativex ® is an oromucosal spray, 100 µL = 2.7 mg THC + 2.5 mg CBD, obtained from Cannabis sativa L. It is suggestedto be used as a symptomatic treatment (in combination with other current anti-spasticity medicine) in adults with moderate-to-severe spasticity caused by multiple sclerosis, in case of failure of previous treatments. It requires up to two weeks of titration period to achieve an optimal dose and its administration should be associated with food intake. Nabiximols can be used once (afternoon or evening) or twice a day (morning and evening), the average dose being 8 sprays/day (maximum 12 sprays/day), with a minimum 15 min gap between administrations. After 4 weeks of treatment, the specialist physician should evaluate the patient’s response to the treatment. It is not recommended to be used in patients less than 18 years old and with caution in the elderly. It is contraindicated in case of hypersensitivity to cannabinoids, known/suspected history/familial history of schizophrenia or other psychotic illnesses, or severe cardiovascular diseases [67].

Epidiolex ® is an oral solution containing 100 mg/mL of CBD, which is suggested for the treatment of seizures associated with Lennox–Gastaut syndrome or Dravet syndrome in patients older than 2 years. The starting oral dose is 2.5 mg/kg, administered twice daily and which can be increased after one week at 10 mg/kg/day, the maximum dose being 20 mg/kg/day (divided into 2 administrations). Side effects of somnolence, sedation, hepatotoxicity, suicidal behavior, ideation (being recommended to monitor the patients), decreased appetite, diarrhea, fatigue, asthenia, sleep disorder, insomnia, and an increased risk of infections were reported [68].

2.6. Cannabidiol and Hepatotoxicity: A Debate

The literature presents controversial studies regarding the hepatotoxic potential of CBD. On the one hand, Mallat et al. have shown that activation of the body’s endocannabinoid system, specifically the CB2 receptor, is therapeutically beneficial in the treatment of many liver diseases. Moreover, studies have shown that CBD can help fight cirrhosis by kill hepatic stellate cells [70]. Other studies in the field have also underlined that CBD can reduce the inflammatory signalling pathways, thus limiting damage caused by cirrhosis [71]. In line with these findings, Yang et al. have shown that CBD prevents alcohol-induced oxidative stress and autophagy [72]. According to Ashino et al., cannabinoids inhibit the enzymatic activity of CYP1A, thus they have the ability to reduce the risk of liver toxicity [73]. In preclinical studies-animal models, CBD has been shown to be effective in restoring liver function in liver injury [74].

From a different viewpoint, Ewing et al. have demonstrated that despite the many beneficial effects of CBD, it may possess a risk for liver toxicity [75]. This research was tested in the acute and sub-acute phase. In the acute phase 0; 246; 738 or 2460 mg/kg of CBD was delivered to male B6C3F1 mice and observed for 24 h. The mice on the highest dose indicated a significant increase in liver-to-body weight ratios and also in total bilirubin values. In the sub-acute phase, the eight-week-old mice delivered orally daily doses of 0; 61.5; 184.5; or 615 mg/kg for 10 days. 75% of the mice instantly died at the 61.5 mg/kg dose and the rest of mice, at the same dose, present overt toxicity, which is manifested as profound lethargy, loss of appetite, and body weight loss [75].

3. Anticancer Effects of CBD in In Vitro and In Vivo Studies

An increased number of studies have demonstrated that CBD and other structurally related cannabinoids present anticancer potential both in vitro on various cell lines ( Table 3 ) as well as in vivo in a multitude of animal models [76]. As previously mentioned, CBD belongs to the cannabinoid family and is a non-psychoactive compound that binds to specific G-protein-coupled receptors [77]. Particularly, CBD is able to interfere with different stages of the tumor process, it can inhibit cancer cell migrations and adhesions, and exerts anti-proliferative, pro-apoptotic, and anti-invasive effects [78,79,80]. Since the first study of Munson et al. in 1975 on the in vitro and in vivo anti-proliferative potential of CBD, clinical use has gradually grown year after year. Cannabidiol presents chemo-preventive effects in some types of cancer, such as breast, lung, colon, prostate, skin, and brain [81,82,83]. After activating the expression of various genes, proteins, enzymes, and signaling pathways, CBD in different forms and concentrations plays a key role in different complex mechanisms that have as a final result the blocking of cancer initiation, progression, and metastation in different types of cancer [84] ( Figure 3 ).

In vitro-in vivoanticancer mechanism of CBD in different types of cancerand the related effects on proliferation/apoptosis/gene expression/oxidative stress/tumor growth [76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119].

Table 3

Role of CBD among various cancer cell lines.

Type/Cancer Cell Line Cell Line In vitro In vivo Conc. Conclusion Ref.
Colorectal cancer HCT116 0–8 µM
100 mg·kg − 1
CBD induces apoptosis by regulating many pro- and anti-apoptotic proteins, and decreases tumor volume [105]
Colorectal cancer DLD-1 0–8 µM
100 mg·kg − 1
Colon cancer CaCo-2
HCT116

IC50 = 0.67 µM
5 mg/kg Reduced aberrant crypt foci (ACF) number of polyps and tumors [103]
Colon cancer CT26 5 mg/kg CBD induces apoptosis, showed anti-angiogenesis and anti-metastatic effect [106]
Colon cancer HCT116 5 mg·kg −1 CBD reduces colon cancer cells [104]
Prostate cancer PC3 1–5 µM CBD reduces exosome release. [108]
Prostate cancer LNCaP 1; 10; 100 mg/kg CBD decreased cell viability and tumor growth [109]
Prostate cancer DU-145 20–80 µg/mL CBD is a potent inhibitor of cancer cell growth and has lowest potency in non-cancer cells [110]
Prostate cancer LNCaP 20–80 µg/mL
Prostate cancer PC3
LNCaP
5–15 µM CBD induces apoptosis [111]
Lung cancer A549 5 mg/kg CBD decreased tumor growth [87]
Lung cancer H460 3µM CBD decreased tumor metastasis [88]
Lung cancer A549 3 µM ICAM-1 present an essential objective for CBD in executing its antitumorigenic function. [64]
Lung cancer A549
H460
3 µmol/L
5–10 mg/kg
CBD induces cancer cell apoptosis [90]
Brain tumor U87
U373
5–10 µM CBD induces apoptosis through activation of serotonin and vanilloid receptors [113]
Brain tumor GSC
IC50 = 3.5 µM
15 mg/kg CBD induces apoptosis through the production of ROS [114]
Brain tumor U251
SF126

IC50 = 1.1–1.3 µM
0.4 µM CBD induces apoptosis and reduces cell viability and invasion [115]
Brain tumor U87MG 10 µM CBD activates TRPV2 receptors to promote cancer cell death. [116]
Brain tumor U87MG 6.7 mg CBD enhances apoptosis and decreases cell proliferation. [118]
Brain tumor SH SY5Y
IMR-32
10 µM CBD induces apoptosis and reduces cancer cell migration and invasion [119]
Skin cancer Murine B16F10 melanoma tumors 5 mg/kg CBD reduces tumor size [92]
Breast cancer MDA-MB-231
IC50 = 6–10.6 µM
10 mg/kg Decreased tumor growth [51]
Breast cancer T47D
MDA-MB-231
10 mg/kg Decreased tumor metastasis [98]
Breast cancer MDA-MB-231 5 mg/kg CBD induces cancer cells apoptosis [97]
Breast cancer SUM-159 3–18 µM CBD induces both apoptosis and autophagy-induced death in cancer cells [99]
Endothelial cells HUVEC 1–19 µM CBD inhibited cell proliferations and exhibited potent antiangiogenic properties inhibiting cell invasion and migration [117]

Lung cancer is one of the most common causes of cancer deaths. According to the World Cancer Research Fund International, in 2018 there were two million new cases of lung cancer detected worldwide [85]. In Europe, Hungary had the highest rate, followed by Serbia and France. The most frequent risk factor for lung cancer is tobacco smoking, which is the cause of 90% of lung cancers [86]. Ramer et al. demonstrated that CBD caused inhibition of A549 cell invasion.The mechanism was assigned to a decreased secretion of plasminogen activator inhibitor-1 (PAI-1), which is responsible for the anti-invasive action [87]. In another study, the same group have also reported that PAI-1 plays an important role in the anti-metastatic potential of CBD. The anti-invasive effect was determined by a modified Boyden chamber assay using 1 µM CBD and 72 h incubation period, whereas the PAI-1 expression (a key factor for tumor invasion and metastasis; a high concentration of PAI-1 is considered a poor prognostic factor in many types of cancer, such as lung, colorectal, gastric, and breast) was determined by RT-PCRusing 1 µM CBD over a 48 h incubation period [88,89]. Inter-intracellular adhesion molecule (ICAM-1) plays an important role in the interaction between lymphokine-activated killer cells and cancer cells. In lung cancer, the application of CBD resulted in an upregulation in the expression of ICAM-1 event directly correlated with the prevention of metastasis of cancerous cells beyond the tumor site [64]. Ramer et al. demonstrated that 10 mg/kg/day of CBD reduces A549 and H460 lung cancer cell line viability in vivo in an animal model using athymic nude mice. The cellular mechanisms induced by CBD presume an up-regulation of cyclooxygenase (COX-2) and PPAR-γ, in vitro as well as in vivo [90].

The number of melanoma cases has permanently increased over the past few years compared to other types of cancer [91]. A recent study conducted by Simmerman et al. reported that CBD represents a potentially new therapeutic agent for malignant melanoma. In this study, murine B16F10 tumors were implanted in 8–12-week-old male mice and mice were treated with cisplatin (5 mg/kg/week) and CBD (5 mg/kg twice per week). Results have shown that the group treated with CBD exhibited similar behavior to the group treated with the consecrated anticancer drug cisplatin, namely, significantly reduced melanoma tumor growth and increased survival time and quality of life [92,93,94].

Breast cancer is the primary cause of death among women and is the second most common cancer overall [95]. There are many risk factors, such as age, history of breast cancer in the family, genetics (women who carry the breast cancer gene 1 (BRCA1), breast cancer gene 2 (BRCA2), and tumor protein p53 (TP53) genes), contraceptives, and lifetime duration of breastfeeding [96]. Using MDA-MB-231 and MCF-7 cells, Shrivastava et al. have explained that CBD induces cell death in selected cancer cells. Among the mechanisms, they have suggested that CBD induces endoplasmic reticulum stress and apoptosis by inhibiting the AKT/mammalian target of rapamycin (mTOR) signaling (5 µmol/L CBD for 24 h) and enhances reactive oxigen species (ROS) generation for selected breast cancer cells (5 µmol/L CBD for 12 h) [97]. Other studies have demonstrated that CBD inhibits the growth of different breast tumor cell lines (MCF-7, MDA-MB-231) with an IC50 value of about 6 µM and exhibits significantly lower potency in non-cancer cells [57]. Recent research showed that CBD induces apoptosis in two different human breast cancer cell lines: T-47D and MDA-MB-231. These effects were observed by MTT assay, DNA fragmentation, and ELISA apoptosis assays. The MTT screening showed that the IC50 values were 2 µM (MDA-MB-231 cells) and 5 µM (T-47D cells). At the same time, both cell lines showed improved nuclear localization of PPARγ following treatment with 1–7 µM CBD for 24 h. Moreover, treatment with CBD led to an interaction between PPARγ, mTOR, and cyclin D1 to the advantage of apoptosis induction [98]. In 2014, Elbaz et al. studied the anti-tumor mechanism of CBD: they showed that it inhibits epidermal growth factor-induced proliferation. The study concluded that CBD (3, 6, and 9 µM) can be used as a novel option to inhibit growth and metastasis of aggressive breast cancer cells [99]. McAllister et al. reported the efficacy of 1.5 µM CBD in the development of breast cancer metastasis in vivo as well as in vitro, using cell proliferation and invasion assays, flow cytometry and Western blotanalysis [100].

Colon cancer is the second most common cause of cancer patient mortality all over the world. Moreover, colorectal cancer is the third deadliest cancer in the United States [101,102]. Aviello et al. showed that CBD exerts a significant antiproliferative effect in two colorectal carcinoma cell lines (Caco-2 and HCT116). They used in vivo experiments, and the cells were treated with 0.01–10 µM CBD for 24 h in a male Institute of Cancer Research (ICR) mouse model of colon cancer. Treatment with just 1 mg/kg CBD significantly reduced aberrant crypt foci (ACF) polyps and tumors in male mice. Moreover, results have shown that by starting from this dosage, CBD presented an optimal chemo-preventive effect. The protective effect on colon cancer was associated with up-regulation of Caspase-3 [103]. Kargl et al. pointed out that GPR55 is implicated in the migratory behavior of HCT116 colon cancer cells and plays an important role in the prevention of metastasis. For this assertion, they used adhesion and migration assays. The GPR55 antagonist CID 16020046 (1, 2.5, 5 µM), CBD (1, 2.5 µM), a putative GPR55 antagonist and GPR55 small interfering RNA (siRNA) were used to block GPR55 activity of HCT116 colon cancer cells [104]. Another study has demonstrated that HCT116 and DLD-1 colorectal cancer cell cultures treated with different concentrations of CBD (0–8 µM) presents phenomena of apoptosis. The mechanism refers to the regulation of many proteins, of which Noxa showed significantly higher expression [105]. A recent study investigated the effect of CBD on the CT26 colon cancer line, in vivo, in an animal model using male BALB/c mice. Results have shown that 1–5 mg/kg CBD has an encouraging effect on reducing colon cancer growth and decreasing tumor size. These favorable effects may be due to growing activity of antioxidant enzymessuperoxide dismutase (SOD) and glutathione peroxidase (GPX) [106].

Prostate cancer is one of the most common types of cancer in men [107]. A recent study has demonstrated that CBD is a novel modulator of exosome and micro vesicle (EMV) release in several cancer cell lines (EMV plays an important role in limiting tumor growth). This study showed that 1–5 µM of CBD significantly reduced growth of PC3 prostate cancer cells [108]. Petrocellis et al. tested both in vitro and in vivo the effect of CBD against LNCaP prostate cancer cells. Results suggested that the anticancer mechanism involves the stimulation of intrinsic pathways of apoptosis. In vivo, CBD (1–100 mg·kg −1 ) significantly inhibited prostate cancer cell viability in an animal model using male MF-1 nude mice [109]. Sharma et al. worked on the evaluation of the antitumor activity of CBD in LNCaP and PC3 prostate cancer cell lines. The result indicated that 20–60 µg/mL CBD is a potent inhibitor of cancer cell growth. The effect was evaluated by ELISA assay and flowcytometry [110]. Another investigation into the effect of CBD on LNCaP prostate carcinoma cells demonstrated that 5–15 µM CBD inhibits cancer cell growth. The study concluded that, regarding this cell line, the pro-apoptotic activity of CBD was phosphatase-dependent. It is worth noting that the anti-tumoral effects of many cannabinoids include modulation of intracellular kinase [111].

Brain cancer is identified as one of the most terrible forms of cancer due to several impediments [112]. Massi et al. demonstrated that CBD led to a concentration-related inhibition of the U87 human glioma cell viability after 24 h of incubation with CBD 5–10 µM. Moreover, the phytocompounds inhibited the growth of U373 and U87 human glioma cell lines implanted in athymic female CD-1 nude mice [113]. Recently, Singer et al. have demonstrated that CBD inhibits the viability of 3832 and 387 glioma stem cell (GSC) lines and induces apoptosis by the production of ROS. Moreover, the in vivo treatment of intracranial GSC-derived tumors with CBD (15 mg/kg) inhibited tumor cell proliferation, activated pro-apoptotic caspase-3, and significantly prolonged the survival of mice. Even though GSCs adapted to CBD treatment, this fact has been suppressed by combining therapy of CBD with small molecule modulators of ROS (vitamin E). Taken together, these data suggest that a combination of CBD with vitamin E regulates ROS levels. This can represent a promising therapeutic model for glioblastoma management [114]. Furthermore Marcu et al. demonstrated that 0.4µM CBD inhibits the growth of different glioblastoma cell lines (U87-MG, U251, and SF126). They concluded that CBD is a more potent inhibitor of glioblastoma cell growth than THC [115]. The role of the transient receptor potential (TRP) channel is the regulation of cellular proliferation and differentiation. Nabissi et al. have shown that CBD represents a specific ligand for TRPV2; thus, CBD works as a TRPV2-selective activator by intensifying Ca 2+ influx in U87MG cells with an IC50 of 22.2 μM. Thereby, CBD could be used as a promising therapeutic agent against GBM cancer cell lines [116]. Solinas et al. proved that 1–10 μM of CBD induces apoptosis and inhibits human umblilical vein endothelial cell (HUVEC) migration using ELISA assay and angiogenesis array kit [117]. Another research group demonstrated that the cannabinoid loaded microparticules was shown to enhance apoptosis and decrease cell proliferation in glioma U87MG cells. The microparticules which contain only CBD (6.7 mg) was administeredin vivo on experimental athymic nude mice [118]. Alharris et al. demonstrated that 10 µM CBD induces apoptosis in neuroblastoma SH SY5Y and IMR-32 cell lines through activation of serotonin and vanilloid receptors, also significantly reducing cancer cell migration and invasion in vitro [119].

4. Immunomodulatory Effects of CBD

One of the struggles of cancer treatment consists in the possibility of activation of the immune system against the tumor. In recent years scientists’ efforts were focused on developing therapies that target tumor immunity [120,121]. Immunotherapy is considered a distinct category from classic cytotoxic therapies used for cancer treatment [121]. Based on the idea that it would be great to find a natural candidate for an anticancer agent that can on the one hand “kill” the cancerous cells via different mechanisms and pathways and on the other hand stimulate the immune system, this review also examines this aspect. Among various therapeutic effects, CBD also possesses immunomodulatory potential [122].

The stimulation of cannabinoid receptors (CBR) can lead immune cells to regulate the DNA binding of various nuclear factors, an effect mainly triggered by down-regulation of cyclic adenosine monophosphate (cAMP) formation [123]. Cyclic adenosine monophosphateanalogues can produce inhibition or stimulation in a dose-dependent manner of the immune responses and can affect cannabinoids’ effects on T-cell-dependent production of antibodies [123]. In contrast to THC, the non-psychoactive CBD was reported to have a low affinity for CBR [123].

Both in vitro and in vivo models have been used in order to evaluate the CBD effects on T-cells and macrophages. Results indicated that CBD has the ability to alter the reactivity of the immune system’s cells [124].

Cannabidiol has been reported to decrease the production of T-helper 2 cytokines such as IL-10, which is well known to play an important role in humoral immunity [125]. Furthermore, Malfait et al. showed that i.p. or s.c. administration of CBD to mice decreased tumor necrosis factor α (TNFα) and also reduced interferon gamma (IFN-γ) production [126]. Moreover, CBD was shown to reduce IL-1 and TNF in human peripheral blood mononuclear cells [127]. The TNFα and IL-1β expression in macrophages is regulated by the nuclear factor κB (NFκB), which augments the expression of anti-apoptotic molecules in cancer cells, leading to resistance of tumor cells to existing chemotherapies [128]. Reduction of TNFα and IL-1β expression in macrophages by CBD suggests its therapeutic anticancer potential.

Macrophages have an important role in innate and adaptive immunity and are one of the main producers of IL-12 [129]. Sacerdote et al. showed that both in vitro and in vivo administration of CBD elicited an increase in IL-12 production and a decrease in IL-10, respectively [130]. A potent antitumor cytokine, IL-12 is able to provoke tumor regression and reduce the formation of distant metastasis following systemic or peritumoral administration [130].

Another study performed on splenocytes derived from CB1 −/− /CB2 −/− mice showed that CBD administration caused suppression of IL-2 and IFN-γ expression and proliferation, suggesting an inhibition of T-cell function [131].

According to Carrier et al., CBD possesses immuno-suppressive effects through the enhancement of endogenous adenosine signaling [132]. The authors showed that CBD acts as a inhibitor of adenosine and thiamine uptake by inhibiting the equilibrative nucleoside transporter-1 (ENT-1) [132]. Due to these effects, CBD arises as an interesting compound in cancer patients’ therapy [131]. During tumor pathogenesis, the purine nucleoside, adenosine, is secreted by cancer and immune cells under metabolic stress and hypoxia [130]. Adenosine binding to A2A receptor stimulates IL-4 and IL-10 release that enhanced tumor cell growth by triggering the suppression of the antitumor immune response [130].

In a study performed on splenocytes, CBD treatment induced a reduction of IL-2, IL-4, and IFN-γ production. Moreover, when tested on mice prior to ovalbumin sensitization, CBD induced a significant inhibition of antigen-specific antibody production, indicating an effect on the suppression of humoral immunity [133].

In a recently published paper, Jensen et al. evaluated the immune gene expression following treatment with CBD using a zebrafish model that resemblances the human genome by around 70%. The authors concluded that CBD modulated the immune genes differently, up-regulating IL1B and IL17A/F2 and down-regulating transforming growth factor beta, alpha (TGFBA), S100A10B (S100 calcium-binding protein A10), immunoglobulin heavy constant Mu (IGHM) and CD4-1 [126]. The non-psychoactive component of Cannabis sativa L., CBD, proved to have a modulatory effect on tumor immunity, suggesting its therapeutic potential in cancer treatment. The compound is considered to be a component of the formulation known as “medical cannabis,” which is currently used in some countries [129]. Further studies are required in order to fully understand all the mechanisms involved in CBD antitumor activity.

5. CBD in Inflammation-associated Carcinogenesis

Although for all higher organisms inflammation is the most competent defensive response of the innate and adaptive immune system, when it becomes chronic it can eventually cause organ dysfunction and structural impairment. Various studies have shown that sphingolipids participate in the structural preservation of cell membranes and mediate cellular functions specifically: migration, proliferation, and apoptosis. Therefore, they are prone to regulate the fate of the cell and consequent onset of inflammation and cancer [134]. Sphingosine-1-phosphate (S1P) is an extracellular ligand for G protein-coupled receptor sphingosine-1-phosphate receptor 1 (S1PR1), and it can activate signal transducers and activators of transcription 3 (STAT-3), a pro-survival pathway implicated in the conversion of inflammation to oncogenesis. The cellular, extracellular, and tissue concentrations of S1P are regulated by its irreversible degradation by sphingosine-1-phosphate lyase (SGPL1). This key enzyme appears to be an auspicious drug target for the design of immunosuppressants [134].

Schwiebs et al. have shown that, based on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer and cancer-induced inflammation evolve through separate, observable molecular stages. The presence of two different mechanisms of carcinogenesis production was observed in a model of compartment-specific SGPL1 depletion in immune cell compartment and tissue cell compartment. In the tissue cell section, they noted fast tumor growth with particular modulation of the tumor microenvironment, and chronic, complex inflammation with succeeding, but relatively delayed carcinogenesis, in the immune cell section [134]. The theory that inflammation may lead to the commencement of cancer is rational considering the following common events: increased genomic injury and DNA synthesis, cellular multiplication, disruption of DNA restoration pathways, the promotion of angiogenesis, and inhibition of apoptosis [135]. Therefore, a potentially anti-inflammatory compound may have a chemopreventive effect.

Summarizing these aspects, chronic inflammation increases the probability of different types of cancer, suggesting that abolishing inflammation may represent a well-founded strategy for cancer prevention and therapy [135]. These findings suggest that CBD’s anti-inflammatory action highlights it as a potential anticancer agent worthy of clinical consideration for cancer therapy. The dual therapy consisting of an anti-inflammatory agent and conventional anticancer drugs may improve patient prognosis and metastasis [135].

6. Anti-angiogenic Effects of CBD

As already described above, the multi-target effect of CBD includes anti-proliferative and pro-apoptotic activities, and as more recently described, anti-angiogenic properties [117]. Angiogenesis, the formation of new blood vessels from preexisting ones, is an essential multistep generating growth, invasion of cancer cells, and metastasis. This process can be modulated by targeting several key factors, by inhibiting growth factors, such as vascular endothelial growth factor (VEGF), integrins, angiopoietins, or by activating inhibitory effectors as thrombospondin andinterferons [117].

Cannabinoids were found to be responsible for down-regulating VEGF receptors in different cancer types. Apoptosis of endothelial cells was observed by activating CB receptors [136], and a reduction of pro-angiogenic factors was also observed [137].

So far, few studies have investigated the effects of CBD as an angiogenesis modulator. Solinas et al. discovered strong anti-angiogenic effects of CBD, both in vitro, reducing growth, migration, and invasion of HUVEC cells, and in vivo, using the Matrigel sponge assay in mice. Multiple mechanisms in modulating angiogenic factors relatingto impaired angiogenesis were observed [138].

An interesting difference compared to the results obtained for cannabinoids in general was that CBD did not induce either apoptosis or necrosis on HUVEC cells at high concentrations (12 µM), but it did induce endothelial cell migration. The anti-migration activity was supported by the molecular modulation of several factors. Cannabidiol inhibits matrix metalloproteinase-2, 9 (MMP2, MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1), thus impairing cell motility and invasion, suppresses the activity of urokinase-type plasminogen activator (uPA), and serpinE1/plasminogen activator inhibitor-1 (PAI1), which are involved in extracellular matrix degradation. By inhibiting chemokineligand 16 (CXCL16) and IL-8, cell motility and network formation are impaired, whereas down-regulating endothelin-1 (ET-1), platelet-derived growth factor subunit A (PDGF-AA), and VEGF expression, micro vessel density is reduced [117]. Vascular endothelial growth factor was found to be down-regulated by CBD in glioma and prostate cancer. Another study indicated that CBD also inhibits HIF-1a (the regulatory subunit of the hypoxia-inducible transcription factor) in U87-MG glioma cells, therefore suggesting its involvement in cell survival, motility, and angiogenesis in a hypoxic environment [138].

A more recent publication explored how CBD could inhibit angiogenesis in colon cancer, and it was shown that it significantly decreased the level of proinflammatory cytokines IL-6 and IL-8 and increased the activity of malonaldehyde (MDA), an antioxidant enzyme. Others found that CBD reduces the levels of an mTOR substrate and STAT5, inducing vasorelaxation, thus contributing to the underlying mechanism of the anti-angiogenic effect in human endothelial cells [138,139].

Another recent study evaluated the effect of CBD in breast cancer and found that it can modulate the tumor microenvironment by reducing the recruitment of macrophages, which leads to angiogenic inhibition [140].

Next to its low toxicity and the non-psychoactive activity, the anti-angiogenic properties and the multi-target anti-tumor effects indicate that CBD is an interesting candidate for anticancer clinical applications [138].

7. Clinical Evidence of the Anticancer Effects of CBD

Today, CBD has become extraordinarily popular around the world. It is now accessible in a growing number of products with different administration modes [141]. There are many types of dietary supplements like capsules, gummies, tinctures, and oils. For topical administration, creams, lotions, and ointments are frequently employed, but the most common is CBD oil. Oil has become a preferred mode of administration for many CBD users for multiple reasons. The foremost motive is that the oil is very easy to administer; also, it allows consumption of a high dose of CBD in a lightly ingestible form [142].

Currently, there are hundreds of active manufacturers and sellers of CBD, and their number is rapidly growing, because CBD is capable, as shown by important studies in the field, of inhibiting the development of an increased number of cancer cells both in vitro and in vivo [143].

Information collected to date in relation to the anticancer effects of CBD are nearly completely limited to preclinical studies conducted on cell lines and also on animal models [84]. Although there is a lot of literature on preclinical in vitro and in vivo studies that describe the anticancer mechanism of CBD on various types of cancer, the number of clinical trials which have as a research theme the study of the effect of CBD on different types of cancer is limited [144]. To discover the full scope of its positive effects on cancer, more human studies are needed to investigate the toxicological parameters. The risks about the long administration’s effects are unknown, especially for children. Drug interaction studies are necessary from both a therapeutic and a safety viewpoint. To determine the risks and also the benefits by the help of clinical trials remains desirable, but this will take a longtime and require a big budget [145].

In terms of clinical trials, CBD was most studied for glioblastoma. A clinical trial that analyzed the effect of CBD as a single agent for solid tumor was conducted in 2014 (clinical trial: > NCT02255292). Another placebo-controlled phase II clinical trial analyzed the effect of the combination of THC and CBD with adjuvant chemotherapy with temozolomide for patients with glioblastoma, and reported positive results using this approach (clinical trial: > NCT01812603) [146].

An outstanding example is the use of CBD products for the self-medication of cancer with the aimof thoroughly curing it. In 2018, Sulé-Suso et al. reported an important case: an 81-year-old man, who was diagnosed with lung adenocarcinoma in 2016, refused chemotherapy and radiotherapy and began self-administration of CBD oil. He started first with two drops twice daily for a week and then nine drops twice daily for a month. One month later, a significant reduction on the number and size of mediastinal lymph nodes was observed on CT scan [147].

In 2018, a clinical trial with 119 cancer patients was conducted over a four-year framed period. Patients were given CBD oil three days on and three days off, with an average dose of 10 mg twice a day (max 30 mg for increased tumor mass). In this study, patients with different types of cancer (e.g., breast, prostate, and esophageal) were included. From 119 cancer patients, the most stunning case was a 5-year-old male patient with anaplastic ependymoma, a very rare brain tumor. The patient had all the standard treatments (surgery, chemotherapy, radiotherapy). He started the treatment with CBD oil in February 2016, and in December 2016 the relevant scans showed that tumor volume had decreased by approximately 60%. The other patients from this study have reported that the side effects completely disappeared, and although the duration of treatment was six months, many continued the treatment [148]. Stella et al. reported a case in which two patients were diagnosed with high-grade gliomas. In addition to chemotherapy and a drug regimen, CBD oil (ranging from 300 to 450 mg/day) was also involved. Both patients had admissible clinical and imaging responses as well as positive responses to treatment at periodic assessments [149]. Another case study presents an 81-year-old woman who was diagnosed with ovarian cancer in March 2017. The patient declined all interventions due to the treatment toxicity. She started with alternative therapy CBD oil (1 drop sublingually/day) and Laetrile tablets, which contain purified amygdalin (500 mg 4 times/day). After two months, CT imaging showed a dramatic decrease in the size of the tumor [150]. In a recent comprehensive review, Dumitru et al. also observed that the phase II clinical trials results indicate an increased life span for patients treated with CBD. Currently CBD is in a 180-day clinical trial-randomized double-blind, placebo-controlled parallel multi-center study including 160 patients for the evaluation of the effect of the cannabinoid combined with standard treatment for patients that suffer from multiple myeloma, glioblastoma multiform, and GI malignancies (clinical trial: > NCT03607643) [151].

All these clinical cases prove that CBD demonstrates promising results, as it has undeniably helped different types of cancer patients. Although the literature describes the results of some clinical trials that include the pharmacokinetics and pharmacodynamics of CBD, there is an urgent need for further clinical trials to gain a complete picture of the effect of this molecule within the human body.

8. Conclusions

This review provides an update on the phytochemistry of CBD. Moreover, it highlights important aspects of the pharmacokinetics, pharmacodynamics, toxicology, route of administration, and dosage of this bioactive molecule. An in-depth screening of the literature led to the conclusion that preclinical studies (in vitro and in vivo) have shown that CBD possesses a very complex mechanism of action through which it can inhibit tumor formation and propagation in different types of cancer. However, despite the large number of preclinical studies, further research is needed because at this point there is not yet enough clinical evidence to prove that CBD can safely and effectively treat any or one particular type of cancer in humans. To date, most clinical positive outcomes following treatment with CBD have been reported for glioblastoma. However, all the data described above suggest that CBD is a promising candidate for an anticancer agent.

Abbreviations

5-HT1A Serotonin receptor type 1A
ACF Aberrant crypt foci
AD Alzheimer’s disease
Aᵦ42 Amiloid beta 42
BCE Before Common Era
BRCA-1 Breast cancer gene 1
BRCA-2 Breast cancer gene 2
cAMP Cyclic adenosine monophosphate
CB1, CB2 Cannabinoid receptor 1,2
CBD Cannabidiol
CBR Cannabinoid receptor
CD-1; 4 Cluster of differentiation 1; 4
CGS Glioma stem cells
CID 16020046 Inverse agonist at the former orphan receptor GPR55
COX Cyclooxygenase
CT Computed tomography scan
CXCL16 Chemokine (C-X-C motif) ligand 16
DNA Deoxyribonucleic acid
EAE Experimental autoimmune encephalomyelitis
EC50 Half maximal effective concentration
EGF/EGFR Epidermal growth factor/epidermal growth factor receptor
ELISA Enzyme-linked immunosorbent assay
EMA European Medicine Agency
EMV Exosome and microvesicle
ENT-1 Equilibrative nucleoside transporter-1
ET-1 Endothelin-1
FDA Food and Drug administration
GABA Gamma-aminobutyric acid
GI Gastro-intestinal
GPR55 G-coupled protein receptor-55
GSC Glioma stem cell
GW9662 Potent antagonist of Peroxisome proliferator-activated receptor gamma
GPX Glutathione peroxidase
HUVEC Human umbilical vein endothelial cells
HIF-1a Regulatory subunit of the hypoxia- inducible transcription factor
i.p. Intraperitoneal
IC50 Half maximal inhibitory concentration
ICAM Inter-intracellular adhesion molecule
ICR Institute of Cancer Research
IFN-γ Interferon Gamma
IGHM Immunoglobulin heavy constant Mu
IL-2,6,8,9,12,IL-1A; 17A/F2 Interleukin 2, 6,8,9,12, 1A, 17A/F2,
iNOS Inducible nitric oxide synthase
Ki Inhibitory constant binding affinity
KPC KRAS, p53, Cre mice
MDA Malonaldehyde
MIP-1α,1ᵦ,2 Macrophage inflammatory protein 1α,1ᵦ, 2
MMP2,9 Matrix metalloproteinase-2, 9
mRNA Messenger RNA
Mtor Mammalian target of rapamycin
NFκB Nuclear factor κB
n.d. Not determined
PAI-1 Plasminogen activator inhibitor-1
PDGF-AA Platelet-derived growth factor subunit A
PERK Extracellular signal-regulated kinase phosphorylation
Phospho-Akt Phosphorylated AKT
PPARγ Nuclear peroxisome proliferation activated receptor
PSA Prostate-specific antigen
PTEN Phosphatase and tensin homolog
ROS Reactive oxygen species
RT-PRC Real-time polymerase chain reaction
s.c. Sub cutaneous
S100A10B S100 calcium-binding protein A10
S1P Sphingosine-1-phosphate
S1PR1 Sphingosine-1-phosphate receptor 1
Serpin E1 Serpin Family E Member 1
SGPL1 Sphingosine-1 phosphate lyase
SiRNA Small interfering RNA
SOD Superoxide dismutase
STAT-3,5 Signal transducers and activators of transcription 3,5
TGFBA Transforming growth factor beta, alpha
THC Tetrahydrocannabinol
THCA Tetrahydrocannabinolic acid
TIMP1 Tissue inhibitor of matrix metalloproteinases1
TNF-α Tumor necrosis factor α
Tp53 Tumor protein p53
TRP Transient receptor potential
TRPA Transient receptor potentialankyrin
TRPV1 Transient receptor potential vanilloid type1
TRPV2 Transient receptor potential vanilloid type 2
TRPM8 Transient receptor potentialmelastatin 8
uPA Urokinase-type plasminogen activator
VEGF Vascular endothelial growth factor
VR1, VR2 Vanilloid receptor 1, 2

Author Contributions

Conceptualization, C.D., F.C.I., V.P., and C.A.D.; writing—original draft preparation: chapter 1, D.A. and F.A.; chapter 2, C.S. and I.Z.P.; chapter 3, V.B.; chapter 4, V.B. and Z.D.; chapter 5, B.K.; chapter 6, S.A., I.Z.P.; chapter 7, B.K., C.D.; chapter 8, B.K., Z.D.; writing—review and editing, F.C.I., C.D., C.A.D., C.S., V.P.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Cbd oil for bowel cancer

Cannabidiol (CBD) is in the news and on the internet, but what is it, and how can it help you?

Fight Colorectal Cancer (Fight CRC) spoke with two experts about CBD and the Food and Drug Administration (FDA) approved uses.

Victor Melgar, Chief Operations Officer of NALA Health, covered CBD for general human use, while Ashley Glode, PharmD, BCOP, Associate Professor at the University of Colorado Skaggs School of Pharmacy and Pharmaceutical Science, provides Fight CRC with a medical perspective for CBD and medical cannabis.

CBD for General Use

Victor Melgar, Chief Operations Officer of NALA Health tells us that NALA was started with the simple idea that CBD could pave the way for all natural healing and remedies through the power of cannabis. However, CBD is only part of the equation. In fact there are over 100 active and inactive cannabinoids found in the cannabis plant.

What is CBD?

CBD is one of over 100 different Cannabinoids that are extracted from agricultural hemp and cannabis. This cannabinoid does not contain the same psychoactive compounds that give the “high” effect that is in THC, which is why CBD is known as the “medicinal compound” of the plant.

Studies have shown that CBD treats pains, aches, anxiety, and stress and was originally praised by renowned herbalist Nicholas Culpeper for treating gout, skin inflammations, and muscular/joint pain in 1625. According to the US National Library of Medicine, cannabidiol has been shown to treat seizures and syndromes.

How is this all possible? The human body contains an endocannabinoid system (ECS) and already produces cannabinoids daily, which are regulated by two receptors, CB1 and CB2. These receptors are found in our immune and nervous systems, in which they assist in moderating inflammation, our immune response to pathogens, motor control, appetite stimulation, and perception of pain.

There are also non-cannabinoid receptors that are activated by CBD such as serotonin, orphan, vanilloid, and nuclear receptors. These activations are the cause of anti-anxiety and anti-inflammatory relief!

What are the benefits of taking CBD?

Many studies have shown the benefits of taking CBD to reduce seizures in adults and children with various conditions. More studies conducted have shown CBD helps alleviate inflammation-based symptoms, anxiety, stress, insomnia, pain, and seizures. CBD helps alleviate cancer-related symptoms at high doses.

Will CBD make me high?

CBD has no psychoactive effect and therefore, you won’t feel “high.” However, depending on how many milligrams you ingest, you can feel sleepy, very relaxed, and possibly a bodily “floating” sensation.

Will I fail a drug test?

CBD by itself will not show up on an employer’s drug test. The FDA states that CBD products must be at least 99.7% pure CBD and allows only 0.3% THC to make it legally consumer ready. CBD may stay in your system anywhere from 24 hours to a few weeks. That timeframe can change depending on a variety of factors, including metabolism, consumption method, frequency of use, and dosage. Therefore, if a large amount of THC is found in the CBD you are using, it may show on a drug test. Make sure you read the label of the product you are using and that it meets these regulations and guidelines.

Purchasing CBD from a reputable company is a must, and you can read more about CBD regulations below.

Can CBD help me?

Each cannabinoid helps in ways big or small, depending on the combination, ratio, and dosage you are taking.

First, determine what symptom or problem you are experiencing. For example, CBD + CBN would be more effective for sleep-related problems. CBD + CBN + CBG might be more effective for inflammation-based problems. Even though you can take CBD and CBG by themselves, it is more effective to take cannabinoids in pairs or groups because when you combine cannabinoids, you create something known as the “entourage effect,” which allows each cannabinoid to be boosted by the other or a counterpart. By working backward, you can find the right combination for you.

How do I choose a CBD?

Key terms you should look out for are broad spectrum, full spectrum, and CBD isolate. Broad spectrum CBD products contain many cannabinoids in varying amounts such as CBD, CBN, CBC, CBG, to name a few, with no trace amounts of THC.

Full-spectrum products may contain all cannabinoids, including THC. Some full-spectrum products may have CBD isolate, pure CBD with no trace amounts of the other cannabinoids. While CBD works great on its own, due to the entourage effect, it is best to combine CBD with other cannabinoids.

How do I take CBD?

CBD can be taken sublingually (under the tongue), via inhalation or ingestion, or applied topically.

Products exist for each route of consumption. For example, sublingual products include CBD oils of all types taken underneath the tongue. Inhalation products include CBD flower and CBD concentrates, such as shatter and wax. Ingestion products are CBD edibles, such as CBD honey or CBD-infused food. Topical products are absorbed through the skin, so products can include CBD lotion, massage oils, salves, creams, and transdermal patches.

Typically, inhalation is the fastest route to absorb CBD and ingestion is the slowest. However, all four ways are effective depending on your symptom and dosage.

Where does CBD come from?

CBD is manufactured in a variety of ways. It typically goes through an extraction process.

There are three main ways to extract CBD from the cannabis plant:

  1. Carbon dioxide (CO2) extraction: This method uses CO2 to separate CBD oil from the cannabis plant. It is a popular extraction method for CBD products and is capable of successfully producing a high concentration of CBD.
  2. Steam distillation: With this method, steam helps separate the oil from the plant material. It is a popular method for extracting essential plant oils, but it is not as effective as the CO2 method.
  3. Solvent extraction: Although this method is effective if solvents are left behind, the process does pose a potential health risk. Solvent extraction can also affect the flavor of the extract.
  4. Lipid extraction: This process is gaining popularity, as some companies are now trying to avoid CO2 and solvents. After extraction, the resulting CBD oil is considered full-spectrum. Hemp-sourced CBD has a THC concentration of 0.3% or less. The extract must go through a cooling and purification process to obtain a CBD isolate product. Further processing leaves behind a crystalline isolate, or CBD crystals.

Is CBD legal?

Regulation for CBD depends on the state; however, many states follow the federal guidelines. Due to the Farm Bill that was passed in 2018, CBD oil that follows the 2018 Farm Bill’s established regulations can be bought, sold, and used within all 50 states.

However, it’s important to note this is only true when CBD oil contains no more than 0.3% THC content. Anything above this number is deemed illegal and will not be recognized by the FDA as a legitimate CBD product. To ensure quality and precision a company must have the following verification processes in place:

  1. Source of CBD product
  2. Adherence to FDA guidelines
  3. General quality control protocols
  4. Third-party tested certificates of analysis

The source of the CBD products is very important. Is the source marijuana- or hemp-based? Does it adhere to FDA guidelines for CBD product development, which include having a facility, consistent measuring, and testing metrics? Is there quality control to ensure sanitizing equipment and to make sure no contaminants are found in the batches created? A third-party tested certificate of analysis is a very important verification if a third-party is testing each product to make sure that each product meets regulations and to ensure no contaminants are found.

CBD for Medical Use

Ashley Glode, PharmD, BCOP, Associate Professor at the University of Colorado Skaggs School of Pharmacy and Pharmaceutical Science provided Fight CRC with a medical perspective for CBD and medical cannabis.

Dr. Glode’s clinical practice site is the Phase I/GI/Head and Neck/Sarcoma Multidisciplinary Clinic at the University of Colorado Cancer Center, Colorado’s only National Cancer Institute designated cancer center.

Her clinical, research, and scholarship interests include supportive care interventions to maximize treatment dose intensity while minimizing toxicities and improve patient quality of life, and evaluate the use of integrative therapies, dietary supplements, and cannabis in cancer patients.

Is CBD or Medical Cannabis approved by the FDA as a treatment for cancer patients?

Not at this time. CBD, or Epidiolex, was FDA approved July 31, 2020 for the treatment of certain types of seizure disorders. The THC-based product of dronabinol (Marinol) is FDA approved for anorexia in patients with AIDS and chemotherapy-induced nausea and vomiting.

How can someone keep track of important considerations, such as dosage and potency, when there are so many cannabis products available now?

It is very important for patients to understand the label on their specific product and know the dosage of the product they are ingesting. It is important for patients to review this information with the staff at the store where the product is being purchased.

The components we know most about are THC and CBD. Products may be more predominantly THC or CBD, or be more of a 1:1 blend. THC and CBD exert different effects in the body; therefore, a patient may want a predominantly CBD- or THC-based product based on the indication. I recommend patients keep a journal of what they took and when, what their goal was (such as a decrease in pain from a 7 to a 4, resolution of nausea so they may eat a meal, etc.), if they achieved that goal, and any side effects they may have experienced. This helps guide any adjustments that need to be made to products or dosage that is used.

How can a patient effectively prepare to discuss therapeutic cannabis use with their care team?

A patient should be open and honest about what they have tried, if anything at all; what research they have done; and what their goal(s) of use are. It is important for their providers to understand where they are in their cannabis journey to make the best recommendation.

We try to weigh the risks and benefits with the limited information we have on potential drug interactions and safety concerns to do what is best for each patient. Each cannabinoid product is different in what it contains and patients may experience different effects.

Ultimately, we as the patient’s care team are learning too since there are limited studies with the products patients are using. Patients should bring us with them on their journeys and share their experiences with us.

What Patients are Saying

“I use CBD or delta 8 (another cannabis compound) daily since North Carolina has legalized medical marijuana.”

JJ Singleton, stage IV CRC survivor

“I use a dermal patch after chemo to help with nausea and appetite. I have some cream that I use on my temples to help with sleep. Both are CBD and THC 1:1”

Tim McDonald, stage IV CRC survivor

“After my 6 1/2 hour surgery I couldn’t sleep for days. I finally got some vape CBD and it really was helpful! FYI, vaping in the hospital is frowned upon!”

Jay Overy, stage II rectal cancer survivor

“I use CBD for anxiety.”

Mandi Griffin, stage III CRC survivor

“I did use it for anxiety but my body got too used to it, so now I’m on anxiety meds. So my experience wasn’t as good.”

Courtney Maurer, stage III CRC survivor

Additional Resources

Check out the additional resources and community stories we have around the use of cannabis and other alternative medicines. Remember to talk to your doctor about the complementary or alternative therapies you are considering before trying them so you can discuss how they may interact with your current treatment course. When you have all of the information, you can make an informed decision about what’s right for you!

Cannabinoids, Medical Cannabis, and Colorectal Cancer Immunotherapy

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

Colorectal cancer is a major public health problem. Unfortunately, currently, no effective curative option exists for this type of malignancy. The most promising cancer treatment nowadays is immunotherapy which is also called biological or targeted therapy. This type of therapy boosts the patient’s immune system ability to fight the malignant tumor. However, cancer cells may become resistant to immunotherapy and escape immune surveillance by obtaining genetic alterations. Therefore, new treatment strategies are required. In the recent decade, several reports suggest the effectiveness of cannabinoids and Cannabis sativa extracts for inhibiting cancer proliferation in vitro and in vivo, including intestinal malignancies. Cannabinoids were shown to modulate the pathways involved in cell proliferation, angiogenesis, programmed cell death and metastasis. Because of that, they are proposed as adjunct therapy for many malignancies. By far less information exists on the potential of the use of cannabis in combination with immunotherapy. Here, we explore the possibility of the use of cannabinoids for modulation of immunotherapy of colon cancer and discuss possible advantages and limitations.

Introduction

Nowadays, colorectal cancer (CRC) is considered to be the third most deadly and the fourth most commonly detected cancer in the world (1). Despite the presence of highly advanced screening techniques, the incidence rate has been steadily increasing globally (2). It is estimated that the global burden of colorectal cancer is expected to rise by 60% to more than 2.2 million newly diagnosed cases and 1.1 million deaths by 2030 (3). Factors, like sedentary lifestyle, increased consumption of alcohol, tobacco, red meat, genetic predisposition, chronic inflammatory processes of the gastrointestinal tract, are triggering factors of this type of malignancy (4). Adenomatous polyps are known to be the main precursors of CRC. The transformation rate of these polyps into carcinoma is ~0.25% per year. When these lesions have a high grade of dysplasia and villous architecture, the risk of being transformed into malignancy rises to 50% (5).

Understanding the pathogenesis of colorectal cancer is very important for choosing the right therapy. Etiology of CRC is complex and includes the accumulation of acquired epigenetic and genetic modifications that transform normal epithelial cells into malignant ones. The classical tumor progression model is called the development of the polyp- carcinoma sequence which involves three main steps. The first step is the formation of benign neoplasms like adenomas and sessile serrated polyps. The second step is characterized by the progression of benign tumors into more histologically advanced neoplasms, and the last step -their transformation into carcinoma. This process might take many years without showing any signs and symptoms. When CRC has developed, it still might take several years before it is diagnosed. CRC is caused by mutations in oncogenes, tumor suppressor genes, and genes involved in DNA repair mechanisms. One of the first mutations typically occurs in adenomatous polyposis coli (APC), a tumor suppressor gene, followed by mutations in KRAS, TGF-β, BAX, BRAF, and other genes (6).

Most cases of CRC are sporadic (70–80%), while the inherited and familial CRC cases account for roughly 5 and 25%, respectively. Sporadic cancers arise due to point mutations, and the molecular pathogenesis of these cancers is very heterogeneous in nature. The inherited group of this particular malignancy is due to the inherited mutations and can be subdivided into two groups: polyposis and non-polyposis. The polyposis type includes mostly familial adenomatous polyposis which is characterized by the presence of numerous possibly malignant polyps in the colon. The non-polyposis variant is represented by Lynch syndrome (7). The familial CRC is also due to the inherited mutations, and it runs in the families without the presence of particular inherited syndromes (8).

Recently, two molecular pathological classifications have been proposed based on the broad-range genomic and transcriptomic analysis of CRC. The first one is called The Cancer Genome Atlas (TCGA) that has three groups: hypermutated (13%), ultramutated (3%), and chromosomal instability (84%). The hypermutated category is characterized by a high mutation rate, defective mismatch repair (dMMR) with a good prognosis, but poor prognosis after relapse. The ultramutated type has an extremely high mutation rate with DNA polymerase epsilon proofreading mutation and generally good prognosis. The majority of CRC are distinguished by chromosomal instability (CIN) with features of a low mutation rate but a high frequency of DNA somatic copy number alterations. The second gene expression-based classification is called Consensus Molecular Subtypes (CMS) that has four groups. CMS1 (14%) is characterized by microsatellite instability (MSI), BRAF oncogene mutation and a vigorous immune activation. The poor survival rate after recurrence has been noticed in patients with this subtype. CMS2 (37%), also called canonical, exhibits a high chromosomal instability and activation of WNT and MYC signaling. CMS3 (13%), known as metabolic, has numerous KRAS mutations and deregulated pathways of metabolism. CMS4 (23%), called mesenchymal, is described by the presence of stromal infiltration, the highly expressed mesenchymal genes, the activation of transforming growth factor-beta, a worse overall and relapse-free survival compared to patients from other groups (7, 9). These classifications have provided information about a proper treatment selection and patients’ prognosis, thus being very important for ongoing and future clinical trials.

The main therapeutic options available nowadays for patients with CRC are surgery, chemotherapy, immunotherapy, radiotherapy. The 5-year survival rate of patients with early stages of CRC is almost 90%. Due to subtle symptoms, more than half of patients are diagnosed when they have already developed advanced malignancies. The 5-year survival rate is only 10% or less when patients have metastases (10).

Among new potential therapeutic approaches, treatment with cannabinoids and Cannabis sativa extracts have been shown to be efficient in inhibiting cancer growth in vitro and in vivo (11). C. sativa plant contains phytocannabinoids, terpenoids, flavonoids, fatty acids and other molecules. Cannabinoids act through the endocannabinoid system which is composed of receptors like cannabinoid 1 (CB1), cannabinoid 2 (CB2), transient receptor potential channels of the vanilloid subtype 1 and 2 (TRPV1, TRPV2), G protein-coupled receptors 18, 55, 119 (GPR18, GPR55, GPR119), endocannabinoids such as 2-arachidonoylglycerol and anandamide (2-AG, AEA), and enzymes responsible for their metabolism. The main biosynthetic emnzymes are NAPE-phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL); the main degradation enzymes are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The main function of the endocannabinoid system is to maintain homeostasis (12). The CB1 receptor is mainly expressed in CNS, and the CB2 receptor, being the most prevalent in the immune system, is mostly present in peripheral organs. Both receptors are G-protein-coupled cell surface receptors that are coupled to the adenylyl cyclase and cAMP-protein kinase A pathways and the MAPK and PI3K pathways (13).

The Importance of the Immune System in CRC

In the past, tumors were defined as just a collection of homogeneous cancer cells. The aggressiveness of neoplasia has been described by its clinicopathological features. Recent progress in immunology and molecular biology has allowed us to become more familiar with the fundamental mechanisms of metastatic potential of tumors. Many studies in this field have broaden the knowledge and emphasized the importance of the immune system in the regulation of cancer growth. The main players of this process are innate immune cells like neutrophils, macrophages, mast cells, eosinophils, myeloid-derived suppressor cells (MDSCs), and adaptive immune cells such as T and B lymphocytes (14, 15).

Over the past decade, the knowledge of tumor microenvironment (TME) has become a key for understanding complex multistep tumorigenesis and developing novel treatment regimens and drugs (16). The cancer microenvironment includes resident and non-resident cells that are interconnected by different mediators, and each of them have a specific function. The communication between these cells and tumor cells within their surroundings essentially regulates the destiny of tumor progression. Immune cells can either inhibit or favor tumor growth ( Table 1 ). New preclinical research has shown that non-antigen-presenting atypical cells are first targeted by the innate immune system; then, the inflammatory response promotes the formation of new blood vessels and the proliferation of tumor cells. Unfortunately, tumors can turn on the immunosuppressive mechanisms and escape the host immunosurveillance. The adaptive immune response needs the identification of non-self-antigens by the communication between proteins and the major histocompatibility complex of antigen-presenting cells and the receptors of CD8+ and CD4+ T cells through antigen presentation. Tumors might lose their antigenicity due to acquired faults in the antigen presentation, or they might be identified as self (25–27).

Table 1

Pro-tumorigenic and anti-tumorigenic effects of immune cells.

Immune cells Roles in cancer (anti-tumorigenic and pro-tumorigenic) References
Dendritic cells (DC) Release cytotoxic cytokines
Antigen presentation to T cells
(17)
Suppress T cell functions via expression of CTLA-4
Promote tumor growth and progression
T cells (CD8+, CD4+) Direct lysis of cancer cells
Release cytotoxic cytokines
(18)
Release cancer promoting cytokines
Treg cells Inhibit chronic inflammation (19)
Suppress anticancer immune responses
Enhancement of pro-inflammatory cytokine production
Macrophages Release cytotoxic cytokines
Antigen presentation to T cells
(20)
Promote angiogenesis, tumor proliferation, chemotaxis, invasiveness, and metastasis
Myeloid derived suppressor cells (MDSC) Limited (21)
Release immunosuppressive molecular mediators
Suppress T cell functions
Recruit immunosuppressive immune cells
NK cells Release cytotoxic cytokines
Directly kill cancer cells
(22, 23)
Granzyme A expressed on NK cells promotes cancer development by enhancing inflammation
Mast cells Inhibit cancer cell growth, increase in inflammatory anti-tumor reaction (24)
Promote cancer growth by stimulation of neoangiogenesis, tissue remodeling and by modulation of the host immune response

There are three phases of tumor immunoediting: elimination, equilibrium, and escape ( Figure 1 ). During the first stage, immune cells eliminate the neoplastic cells that express surface proteins. Through the equilibrium phase, some cells persist as a result of their potential to camouflage surface molecules or by suppressing macrophages and T cells via the expression of substances like PD-1/2 on the antigen-presenting cells. In the last phase, some cells can escape from being killed, and this subsequently leads to evasion and proliferation of resistant clones. In addition, the degradation of the extracellular matrix by matrix metalloproteinases and new blood vessels formed as a result of abnormal angiogenesis promotes the formation of metastases (15).

Phases of immunoediting in colorectal cancer. Elimination includes the removal of neoplastic cells, equilibrium describes the survival of a fraction of transformed cells, and escape describes the evasion and proliferation of these cells.

As to the expression of cannabinoid receptors in the cells of the immune system, it has been demonstrated that the receptors are expressed in both adaptive and innate immunity. For example, CB1, CB2, and GPR55 receptors are expressed on the NK cells, CB1, CB2 – on the mast cells, T lymphocytes – on the B cells. Therefore, it can be hypothesized that phytocannabinoids can influence the function of the immune system, regulate inflammation and possess antitumor effects, etc (28).

The Role of Inflammation in Colorectal Carcinogenesis

Inflammation plays a crucial role in colorectal carcinogenesis, and it is considered nowadays as one of the emerging hallmarks of cancer (29). A better understanding of CRC and inflammation can lead to the development of new tumor biomarkers and more personalized and effective therapies. It is well-known that patients who suffer from chronic conditions such as inflammatory bowel disease have a much higher risk of developing CRC (30). Inflammation is considered an important driving force of colitis-associated CRC cancers, while its role in sporadic and hereditary cancers is less clear. The evidence demonstrates that non-steroidal anti-inflammatory drugs may prevent or postpone the CRC development (31). A meta-analysis of randomized trials showed that during follow-up after 20 years of using aspirin for 5 years, the mortality and incidence rate of CRC would be reduced by 30–40% (32).

Based on the CMS classification of CRC, CMS1, and CMS4 are considered inflammatory, with the former having a poor prognosis after relapse, and the latter—having the worst survival rate. In general, inflammation plays a dual role in the neoplasia. Targeting malignant cells by cytotoxic T lymphocytes or diminishing the non-specific inflammation by T-regs can lead to an anti-tumorigenic response. This type of response is called protective and is associated with Th1 polarization and a lower recurrence of CRC. The Th1 subtype produces IFN-γ and enhances the cell-mediated toxicity, while the Th2 subtype releases IL-4 and enhances a humoral B cell response. The most common pro-inflammatory cytokines are TNF-α, IL-6, IL-12, IL-2, and the most common anti-inflammatory ones are IL-10, IL-4, IL-5, TGF-β, and IFN-α ( Table 2 ). The cells of the innate and adaptive immunity and other cells such as fibroblasts, mesenchymal cells and pericytes are important in the cancer-associated inflammation (57). The communication between these cells happens via a web of cytokines produced and secreted by immune cells after being stimulated. The role of theIL-10 signaling pathway remains controversial in CRC. A higher level of IL-10 is linked to a worse patients’ survival, while studies on animals show that it has a protective role by suppressing inflammation (46, 47). IL-6 is an activator of the STAT-3 signaling pathway and is often found in CRC patients; and it is also linked to a worse survival and increased risk of relapse (37, 57, 58). The stromal fibroblasts, obtained from colon cancer, produced prominent amounts of IL-6. The last one induced tumor angiogenesis by enhancing VEGF production (38). IL-6 facilitates the metastatic colonization of colorectal cancer cells. In IL6-/- mice the metastasis of CT26 cells into the liver were reduced and the function of CD8+ T cells was improved in vivo. Moreover, IL-6 deficient mice responded to anti-PD-L1 injection effectively by suppression of metastatic colonization, while this effect was not observed in IL6+/+ mice (39). IFN-γ is produced by CD4+, CD8+, and NK cells, and it induces apoptosis of cells. A loss of one copy of this interferon in Apcmin/+ mice showed a much faster progression to colon adenocarcinoma. CRC cells can minimize the anti-tumorigenic effects of interferon signaling by the type I interferon receptor chain that leads to a poor response to anti-PD1 checkpoint inhibitors (44). The expression of TNF-α is much higher in colorectal cancer than in adjacent normal colorectal tissue. The increased expression of this cytokine strongly correlates with the more advanced tumors (59). After TNF-α stimulation, was noticed increase in Metastasis-Associated in Colon Cancer 1 (MACC1) oncogene at both mRNA and protein levels. MACC1 induces cancer cell proliferation, survival and metastasis. The expession levels of this oncogene was reduced by knocking down the p65 NF-kB. In addition, the induction of MACC1, was hindered by monoclonal anti-TNF- α antibody, adalimumab (34). TNF- α increased levels of pro-inflammatory cytokines, such as IL-6 and IL-8 in vitro on HT-29 colorectal cancer cells (35). Another study showed that the effect of peptide vaccine, AH1, on CT26 colon tumor-bearing mice caused a modest inhibition of tumor growth, but the combination with F8-TNF increased the anticancer activity drastically. F8-TNF is an antibody fusion protein which delivers TNF to the tumor extracellular matrix. The synergism between the peptide vaccine and TNF fusion protein was explained by F8-TNF causing rapid tumor hemorrhagic necrosis and as a result leaving small amount of residual cancer cells. In addition, was noticed a significant increase in AH1-specific CD8+ T cells in tumors and draining lymph nodes (60). IL-12 inhibited human colon cancer cells (HRT18, HT29, and HT115) motility and invasion, suggesting its important role in metastasis (41). IL-4 is actively released by colon cancer stem-like cells, and gives tumors a death-resistant phenotype. Neutralizing IL-4 with its antibody significantly sensitizes cancer cells to chemotherapy (49). Early transgenesis of IL-5 in colitis-assosiated CRC mouse model increased the severity of colitis, induced the rate of polyps formation and as a result higher tumor load (51). In patient was reported a case of extreme eosinophilia caused by IL-5 producing disseminated colon cancer (61). TGF-β promotes the survival, invasion and metastasis of CRC cells (53). TGB-β in the tumor microenvironment enhances T-cell exclusion and inhibits the excavation of Th-1 phenotype. Mice with metastatic colon cancer and blocked TGF-β signaling pathway have tumors sensitive to anti-PD-1 anti-PD-L1 therapy. In contrast, mice with unblocked TGF-β signaling, showed a limited response to immune checkpoint inhibitors (54). Systemic administration of IFN-α to mice with colon cancer significantly inhibited the growth of the tumor and its vascularization; induced apoptosis of tumor cells and in metastasis-associated hepatic endothelial cells (56).

Table 2

The main effects of pro- and anti-inflammatory cytokines.

Cytokines Production site Effects Relevance to colorectal carcinogenesis References
Pro-inflammatory cytokines
TNF-α Macrophages, T lymphocytes, NK cells, mast cells, eosinophils Inflammation stimulation, resistance to infection and cancers TNF-α regulates the induction of MACC1 via the NF-κB subunit p65.
Increases levels of IL-6 and IL-8.
(33)
(34)
(35)
IL-6 T cells, macrophages Stimulation of cellular differentiation, inflammation and the development of effector T cells;
induces synthesis of acute phase proteins.
Linked to a worse survival, increased risk of relapse.
Induction of tumor angiogenesis by enhancing VEGF production.
IL-6 facilitates the metastatic colonization of colorectal cancer cells.
(36)
(37)
(38)
(39)
IL-12 Dendritic cells, macrophages Encourages the advancement of the Th-1 response, enhances the cytotoxic activity of NK cells and CD8+ T cells, has anti-angiogenic effects. Inhibition of colon cancer cells motility and invasion. (40)
(41)
IL-2 T cells, dendritic cells A signal transducer and activator of transcription (STAT5), influences the differentiation of T helper cells, activates cytotoxic lymphocytes Limited data (42)
IFN-γ T helper cells (Th1), NK cells Regulates the Th1/Th2 balance, promotes macrophage activation, enhances antigen presentation and leukocyte migration, activates STAT1. Anti-tumorigenic effect by slowing cancer progression. (43)
(44)
Anti-inflammatory cytokines
IL-10 Monocytes, lymphocytes, mast cells, macrophages, T helper cells (Th2), regulatory T cells Limiting a host immune response to pathogens, tissue homeostasis maintenance, the prevention of autoimmune conditions development; decreases antigen presentation and phagocytosis, enhances T reg cells Dual role: suppressing inflammation, linked to a worse patients’ survival. (45)
(46, 47)
IL-4 Mast cells, eosinophils, basophils, T cells Regulates the Th1/Th2 balance, induces an alternative macrophage activation and immunoglobulin class switch to IgE and IgG Gives tumors a death-resistant phenotype.
Causing chemoresistance.
(48)
(49)
IL-5 T helper cells (Th2), mast cells Stimulates the proliferation of B cells and their differentiation to Ig-secreting cells. Exacerbate the disease severity. (50)
(51)
TGF-β White blood cells Controls cell proliferation, differentiation, wound healing; inhibition of B cells and activates macrophages; promotes T cells differentiation. Promotes the survival, invasion and metastasis of CRC cells. Reduces response to immune checkpoint inhibitors. (52)
(53)
(54)
IFN-α Plasmacytoid dendritic cells, macrophages Chemokinesis and migration induction of T cells, anti-viral activity. Promotes apoptosis of cancer cells, inhibits angiogenesis and tumor growth. (55)
(56)

Current Treatments of CRC

Finding the best choice of treatment can be done by combining and analyzing information about the tumor-associated factors (tumor localization, the presence of metastasis, the presence of biomarkers, etc.) and the patient-related factors (prognosis, concomitant diseases, etc.).

CRC patients with a metastatic disease receive a combination of chemotherapy and immunotherapy. The 1st line chemotherapy includes fluoropyrimidines such as capecitabine and 5-fluorouracil (5- FU) alone or with leucovorin (LV), oxaliplatin (5-FU/LV/oxaliplatin – FOLFOX), irinotecan (5-FU/LV/irinotecan – FOLFIRI), capecitabine/LV/oxaliplatin – CAPOX. The 2nd line chemotherapy – FOLFOX or CAPOX for patients who are resistant to irinotecan. Patients who are refractory for oxaliplatin combinations will be prescribed FOLFIRI or irinotecan as monotherapy. Usually, the treatment lasts up to 6 months, but the duration significantly depends on individual cases (62).

The most common side effects of chemotherapy for CRC are leukopenia, polyneuropathy, diarrhea, thrombocytopenia, hyperemesis, hepato-renal dysfunctions, and the deterioration of the general condition. The severity of side effects is usually more profound in elderly patients and in patients with preexisting comorbidities (63). Due to toxicity concerns, chemotherapy might not be suitable for many patients. Oncologists might not recommend this type of treatment due to some advanced stages of chronic diseases (liver, kidney, and heart failures) and a poor physical performance (64).

Immunotherapies

Immunotherapy is one of the most promising therapeutic modalities for patients with CRC (65). Targeted therapy has revolutionized cancer treatment. Immunotherapy is a type of curative approach that helps the immune system to eradicate tumors. It can be classified into two main groups: active (vaccines) and passive (monoclonal antibodies, adoptive cell therapy) ( Table 3 ). Also, some biological therapies can particularly target certain designated tumor antigens, while others work non-specifically by enhancing the natural immune responses (75).

Table 3

Advantages and disadvantages of immunotherapeutic agents.

Immunotherapy Advantages and disadvantages Status of approval in CRC References
Whole tumor vaccines Composed of all known and unknown tumor antigens, easy production Not approved (66)
Low immunogenicity and efficacy
Peptide vaccines The known specificity for the tumor-associated antigen Not approved (67)
(66)
Low efficacy
Viral vector vaccines Specific for the tumor-associated antigen, naturally immunogenic Not approved (68)
(66)
Cytokine storm induction
Dendritic cell vaccines Tumor-associated antigen specificity, the generation of the own immune response Not approved (69)
High cost and time-consuming preparation
Adoptive cell therapy High tumor specificity, the elimination of the need to produce an immune response Not approved (70)
(71)
High cost, long preparation time, target-dependent toxicities
Antibody-based immunotherapy Target immunosuppressive pathways, the enhancement of the anti-tumor immune response Bevacizumab
Cetuximab
Panitumumab
Ipilimumab
Nivolumab
Pembrolizumab
(72)
(73)
(74)
Toxicity

There are some types of cancer vaccines that have been studied in CRC treatment, such as a whole tumor -, peptide -, viral vector -, and dendritic cell (DC) vaccines. The aim of these agents like any other immunization strategy is to induce the antitumor immune response that will eradicate cancer and supply the organism with continuing surveillance to protect from its return.

Whole Tumor Vaccines

Some advantages of working with whole tumor vaccines are: they are easy to produce and are composed of all known and unknown tumor antigens. In contrast, the most significant disadvantage of these vaccines is a very low immunogenicity that can target normal cells and as a result, a low efficacy. Several approaches were made to augment the immunogenicity of whole tumor vaccines. A trial with Newcastle disease virus infection was performed that showed a 98% 2-year survival rate in resected CRC patients in comparison with 67% in patients who received a whole tumor vaccine combined with a Bacillus Calmette–Guérin (BCG) vaccine. The results suggest that the immunogenicity of these compounds has been improved (27).

Peptide Vaccines

Peptide vaccines are more specific for atumor-associated antigen, but the efficacy is still considerably low due to a small amount of T cell responses. A phase I/II trial performed in CRC patients showed that a combination of p53 vaccine with interferon-alpha elevated the amount of interferon-gamma (76). The next type of vaccine is called viral vector vaccines. They are specific for a tumor-associated antigen and naturally immunogenic. The drawback of using them is their ability to cause a cytokine storm. The most used viruses in CRC are adenoviruses, poxviruses, and alphaviruses. Most of these vaccines target a carcinoembryonic antigen (CEA), a protein expressed by CRCs. Preclinical data show that the recombinant Vaccinia virus expressing CEA (rV-CEA) can enhance the adaptive and innate immune responses in mice. Also, it suppressed the proliferation of colon adenocarcinoma in animal models. However, clinical trials done in patients with advanced stages of colorectal cancer demonstrated a lack of responses (77). The dendritic cell vaccines are characterized by the tumor-associated antigen specificity and generation of an organism’s own immune response. The negative aspects are high costs and a very time-consuming preparation process. After the complete excision of CRC liver metastasis, the phase II vaccine clinical trial showed fewer and delayed relapses in the vaccine arm in comparison with the observation arm (78). Results of DCs vaccines are very encouraging, and soon their efficacy can be significantly improved.

Adoptive Cell Transfer Therapy

Adoptive cell transfer therapy is another type of immunotherapy. The main advantages of this cure type are the elimination of the need to produce the immune response and high tumor specificity. In contrast, some disadvantages are high costs, long preparation time, and target-dependent toxicities. In this therapy, the autologous T cells are withdrawn from the tumor, lymph nodes or peripheral blood of a patient and modified ex vivo by making them expand and adding some co-stimulatory molecules and cytokines. Then a passive transfer of these T cells into the host is done for the direct tumor destruction. The most recent discovery of this type of passive immunotherapy is the development of engineered T cells that express the chimeric antigen receptors specifically for carcinoembryonic antigen (79). A phase I trial performed in patients with CRC resistant to the standard treatment protocol regimen by using the autologous T cells modified to express a murine CEA T cell receptor showed a significant decrease in serum CEA in all three patients; in one of them a clinical response by the presence of metastasis regression in the liver and lungs was demonstrated. At the same time, these patients experienced transient inflammatory colitis (80). The recently reported results of a case study in patients with advanced colorectal cancer showed a notable clinical response to the combination of capecitabine and adoptive cell transfer (αβT cells and NK cells) prescribed after laparoscopic resection of colon cancer and some liver metastasis. Two weeks after laparoscopy, a drastic increase in CEA levels was observed. Adoptive cell transfer allowed to decrease the serum level of CEA, eventually bringing it to normal. A noticeable size reduction of the unresectable liver metastasis was observed. During the follow-up examination in 19 months, no progression or relapse was noted, and the levels of CEA remained within normal limits (81).

Antibody-Based Immunotherapy

Highly specific monoclonal antibodies have been very effective in cancer treatment for decades. Proteins against epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in combination with chemotherapy have shown better outcomes of malignant CRC (mCRC). Anti-EGFR agents like Cetuximab or Panitumumab as a monotherapy or combined with cytotoxic drugs are prescribed only when there is an absence of KRAS mutations (62). Bevacizumab, a humanized monoclonal antibody against VEGF, suppress the tumor growth and angiogenesis as well as modulates the immune system of a host by increasing the population of B and T cells (82).

The dramatic efficacy of antibody-based immunotherapy was proven with the use of another type of monoclonal antibodies (mAbs) known as checkpoint inhibitors (ICIs). The currently used ICIs have been shown to provide significant clinical responses to patients with mCRC, specifically with the mismatch repair-deficient/microsatellite instability-high (dMMR-MSI-H) type. They target the inhibitory immune receptors: programmed cell death 1 (PD-1) and its ligand PD-L1, cytotoxic T-lymphocyte associated antigen 4 (CTLA-4). The latter one is expressed in the naive T-cells, effector T-cells, and regulatory T-cells (T-regs). It stimulates the deactivation of T-regs via binding to the antigen-presenting cells. The PD-1 receptor is present in the CD4, CD8 lymphocytes, NK cells, MDSCs, T-regs, and B cells. Together with its ligand, this receptor causes the exhaustion of T-cells by minimizing the tumor-infiltrating lymphocytes and T-cell proliferation. Consequently, tumors acquire immunoresistance. Anti-CTLA-4 and anti-PD-1 agents activate T cells and cause a stronger anticancer response (83). dMMR-MSI-H CRC tumors have a 20 times higher mutational load than mismatch repair proficient microsatellite instability low tumors (pMMR-MSI-L). Besides, they are more infiltrated by TILs, macrophages and have the elevated levels of immune-stimulatory cytokines compared with pMMR-MSI-L. The latter one has a less effective response to ICIs and a worse prognosis (84).

As of August 2020, there were three FDA-approved ICIs that were used for patients with dMMR-MSI-H mCRC. The first one was Nivolumab, an anti-PD-1 agent approved by the FDA in July 2017 after successful results of a phase II CheckMate 142 trial for the second-line treatment of patients with dMMR-MSI-H mCRC that progressed on treatment with oxaliplatin, fluoropyrimidine, and irinotecan. In this trial, it was reported that at 12 months of follow-up, the objective response rate was present in 31% of patients and 69% of control individuals. The 12-months progression-free survival (PFS) was 50%, and the overall survival (OS) was 73%. The most common side effects related to the therapy were pruritus, rash, diarrhea and fatigue. BRAF, KRAS mutations and PD-L1 expression did not affect the response to the prescribed targeted therapy (85).

The second ICI that FDA approved in May 2017 was Pembrolizumab, an anti-PD-1 substance, the efficacy of which was proven in phase one of the Keynote 016 trial. Initially, it was demonstrated that patients with mCRC dMMR-MSI-H experienced a 40% response rate (RR), while patients with pMMR-MSI-L – 0% RR. Later on, it was documented that a 2-year PFS was 53% in the first group. Severe side effects were present only in 14% of patients, including thrombocytopenia, leukopenia and pancreatitis. This curative monotherapeutic option was prescribed for dMMR-MSI-H mCRC patients who deteriorated on or after oxaliplatin, fluoropyrimidine, and irinotecan therapies.

The next immunotherapeutic approach approved by FDA for refractory CRC that progressed on oxaliplatin, fluoropyrimidine, irinotecan therapies was a combination of Nivolumab with Ipilimumab (anti-CTLA-4 agent). The approval was granted in July 2018, after the report of the results of the phase II CheckMate trial 142. During the follow-up at 13.4 months, the objective response rate was 54.7%, with a partial response−51.3%, a complete response−3.4%, and the disease control rate for 3 months or more−80%. PFS at 12 months was 71%, OS−85%. Thirteen percent of patients were obliged to stop treatment because of the drug-related side effects. This combination demonstrated the superior efficacy than anti-PD-1 monotherapy. But the adverse effects of grade 3–4 were more prominent in combination therapy compared with one agent treatment, 32–20%, respectively (86).

Concerning patients with pMMR-MSI-L, more research on immunotherapeutic regimens should be done. There is a need to find drugs that will target an immune response and will also promote the T-cell infiltration. Due to a low mutational and neoantigen load, it is difficult to reach these aims. Current regimens include radiotherapy, chemotherapy, and anti-angiogenic substances for enhancing the immune activation, the killing of tumor cells, and the elevation of tumor antigens. Later on, the treatment may be combined with ICIs and other biologics. There are currently some ongoing clinical trials that evaluate the effects of chemotherapy with either anti-PD-1, anti-PD-L1 ans external beam radiation therapies or radiofrequency ablation (87). The ongoing trial > NCT01633970 phase Ib assessed the efficacy of Atezolizumab (anti-PD-L1) and Bevacizumab plus FOLFOX; it showed OS of 7% and stable disease in 64% of patients. Another approach that has been well-studied is a combination of the mitogen-activated protein kinase inhibitors (MEK) like Cobimetinib and Atezolizumab. MEK inhibitors can further sensitize MSS mCRC for targeted therapy. A phase Ib clinical trial ( > NCT01988896) assessed this combination in patients with refractory KRAS-mutant CRC and pMMR-MSI-L CRC and demonstrated RR of 17%, where five patients out of 23 had stable disease, and four patients developed PR. No advanced therapy-related adverse effects were noted. Later on, 84 patients were included, and results were updated. The RR was 8%, the disease control rate-−31%. The 6-month PFS and 12-month OS were 27 and 51%, respectively. This approach is very promising as it shows that MEK inhibitors can increase the response to immunotherapy in MSS mCRC patients. Some promising results were presented during the ongoing > NCT03406871 trial that combined Nivolumab and Regofarenib (multi-kinase inhibitor); 18 out of 19 patients had objective tumor response (seven of which were MSS CRC, 11—MSS gastric cancer and 1—MSI-H CRC). More personalized approaches to treatment of pMMR-MSI-L are still required (84).

The Relevance of ECS To CRC

ECS actively regulates gut homeostasis. All components of ECS are highly expressed in the intestinal tissue, meaning that this system directly affect the proper functioning of gastro-intestinal system. CB1 and CB2 receptors are expressed in healthy colon epithelium, submucosal myenteric plexus, and smooth muscles, plasma cells in the lamina propria; CB2 receptor is also present on the intestinal macrophages (88, 89). TRPV1 receptor is expressed on colonic nerve fibers (90). The GPR55 receptor is present in the mucosa and the muscle layer of the colon (91). The endocannabinoids, 2-AG an AEA are also present in healthy colonic tissue (92). The main degradation enzymes of endocannabinoids, FAAH and MAGL enzymes are distributed on colonic epithelium glands, lamina propria, and myenteric plexus. The NAPE-PLD and DAGL biosynthetic enzymes are expressed on colonic smooth muscles, lamina propria, and epithelium glands; DAGL is also present on myenteric plexus (89).

To understand the role of ECS in the gut, it is important to distinguish the effects of increased and decreased cannabinoid tone in the gastro-intestinal system. In general, CB1 receptor antagonists reduce the cannabinoid tone in the gut and lead to vomiting, diarrhea, increased gastric emptying, and gastro-intestinal transit. In contrast, CB1 and CB2 receptor agonists, as well as MAGL inhibitors and FAAH blockers lead to an increase in intestinal cannabinoid tone by reducing vomiting, gastric acid secretion, and gastric emptying, as well as reducing hypermotility, diarrhea, and visceral pain (93). CB1 receptor silencing by selective CB1 receptor antagonist AM251 in ApcMin/+ mice led to an increase in the number of intestinal polyps, while CB1 receptor activation caused tumor cell death. In contrast, silencing of the CB2 receptor did not show any effect on polyp growth (94).

The components of ECS are significantly dysregulated in CRC. The endocannabinoids (2-AG and AEA) were 3-fold higher in adenomas and 2-fold higher in CRC in comparison to normal colon mucosa (92). The expression of CB1 receptor is decreased in CRC (95). The CB2 receptor expression is increased in CRC and is considered as a poor prognostic factor for this type of cancer (96). Levels of FAAH and MAGL were also increased in patients with CRC (97). ECS is a very important factor of CRC pathogenesis, suggesting a potential impact of cannabinoids in this disease.

The medicinal plant that has recently gained a lot of attention in the cancer field is Cannabis sativa. Many in vitro and in vivo experiments have shown that cannabinoids and cannabis extracts inhibit proliferation, stimulate apoptosis and autophagy, suppress angiogenesis and metastasis (98–100). The main active cannabinoids responsible for these effects are cannabigerol (CBG), cannabidiol (CBD), and tetrahydrocannabinol (THC). It was demonstrated that CBG activated apoptosis, prompted ROS production, increased CHOP mRNA, and suppressed cell growth in CRC cells (Caco-2, HCT-116) (101). It was found that the inhibitory effect of CBG on colorectal cancer cells viability was time dependent. In TRPM8 silenced cells, the inhibitory effect of CBG on cell growth was prominently suppressed in comparison with non-silenced cells. The induction of apoptosis was shown by an increase in the activity of caspases 3 and 7, the presence of DNA fragments, an increase in the expression of CHOP. In the same paper, it was shown that CBG (3 or 10 mg/kg) inhibited the growth of xenograft tumors (HCT-116) in a mouse model by 45.3% and chemically induced colon carcinogenesis in models by azoxymethane (AOM) in which CBG at a concentration of 5 mg/kg completely suppressed the formation of aberrant crypt foci (ACF), reduced the number of tumors by one half, and did not affect polyp formation (101).

CBD was also demonstrated to have the antiproliferative effects in colorectal cancer models. In some in vitro studies, CBD protected DNA from oxidative stress, elevated the levels of endocannabinoids, and suppressed colorectal cancer cell proliferation via CB1, TRPV1, PPAR-γ receptors (102). Selective antagonists rimonabant and AM251 (CB1R antagonist), capsazepine (TRPV1R antagonist), GW 9662 (PPAR-γ receptor antagonist) suppressed the antiproliferative effects of CBD. The chemoprevention of CBD was confirmed using in vivo models of AOM-induced colon cancer. CBD (1 mg/kg) reduced ACF by 67%, the number of tumors by 66% and polyps by 57%. When the concentration was elevated to 5 mg/kg, it only prevented the formation of polyps. This effect was due to the activation of caspase-3 and a decrease in the phosphorylated form of Akt-protein (102). In another study, the pro-apoptotic effect of CBD in CRC cells (HCT-116, DLD-1) was shown and was suggested to be the result of Noxa activation, the elevation of ROS production and the induction of endoplasmic reticulum stress. When the levels of Noxa were suppressed by siRNA, the expression of apoptosis markers became significantly reduced. Similarly, after the blockage of ROS production, the level of Noxa were reduced. CBD induced apoptosis in a Noxa-ROS-dependent manner (103). Moreover, while using CT26 cell line-induced colon cancer in mice, CBD at concentrations of 1 and 5 mg/kg was reported to have the anti-angiogenic and antimetastatic effects via the inhibition of VEGF, with the latter dose being more effective. In animals receiving CBD, a significant increase in the activity of antioxidant enzymes, including SOD, GPX, GR, TAC, and a decrease in MDA were noted (104).

The effects of full botanical extracts, such as high CBD botanical drug substance (BDS), on colon cancer were also studied. Such extracts are typically prepared from cannabis flowers that are rich in CBD, or CBD isolate is added (spiked) to a certain concentration. It was hypothesized that other components of cannabis plant extracts may act synergistically with CBD and can be useful from a therapeutic point of view. It was shown that CBD BDS had the significant antiproliferative properties on cancer cells (HCT-116, DLD-1), while healthy colonic epithelial cells were not affected. No difference was noted in the potency and efficacy between CBD BDS and CBD when the same doses were used (0.3–5 μM). CBD BDS effects were counteracted by selective antagonists to CB1 and CB2 receptors. CBD BDS had a more pronounced affinity to both CB1 and CB2 receptors than pure CBD. In vivo studies showed that using chemically induced carcinogenesis by AOM, C. sativa extract with a high content of cannabidiol inhibited ACF by 86%, polyps by 79% and tumor formation by 40%. In xenograft models, CBD BDS significantly reduced the tumor volume, but no difference in the growth of tumors was observed after 1 week of treatment (105).

THC was shown to induce apoptosis in colorectal cancer cells via the activation of CB1 receptors and the inhibition of PI3K-AKT, the RAS-MAPK cascade and BAD activation. Colorectal cancer cells (SW480, HCT-15, HT29, Caco-2, HCT-116, and SW620) that were exposed to THC (2.5–12.5 μM) resulted in a dose-dependent reduction in cell survival. In contrast, smaller concentrations from 100 nM to 1 μM had no noticeable effect on colorectal cancer cell proliferation and survival. THC increased the levels of caspase-3 and PARP (caspase-3 substrate). THC caused the dephosphorylation and activation of BAD (106). The anti-cancer potential of cannabinoids in CRC is summarized in Table 4 .

Table 4

Cannabinoids anti-cancer potential in CRC.

Phytocannabinoids Model type Antitumor effect/mechanism of action References
CBG in vitro (Caco-2, HCT116)
in vivo (athymic nude female mice, xenograft-HCT116; azoxymethane induced colon cancer model)
Pro-apoptotic, antiproliferative; prompted ROS production, increased CHOP mRNA, increased levels of caspase 3, 7 activity;
in xenograft tumors – reduced tumor growth, in AOM tumors – completely suppressed ACF, reduced number of tumors
(101)
CBD (Caco-2, HCT116)
in vivo (male ICR mice, AOM induced CRC)
Antiproliferative; activation of PPAR-γ, TRPV1, CB1R, DNA; protection from oxidative stress, elevated levels of endocannabinoids;
the chemopreventive effect in AOM model –reduced number of tumors, ACF, polyps; activated caspase-3, suppressed phospho-Akt protein
(102)
in vitro (HCT116 and DLD-1) Pro-apoptotic; Noxa activation, ROS elevation, induction of ER stress (103)
in vivo (male BALB/c mice, xenograft-CT26) Anti-angiogenic, antimetastatic; VEGF inhibition (104)
THC in vitro (SW480, HCT-15, HT29, Caco-2,HCT116, SW620) Pro-apoptotic; CB1 activation and inhibition of PI3K-AKT, RAS-MAPK cascade, BAD and caspase-3 activation (106)

Slow development and approval of new anti-neoplastic drugs for CRC is due to the lack of proper preclinical models. 2D in vitro models allow to perform high throughput screenings and are simple to work with, but allow only to study cell-to-cell or cell-to-matrix interactions, not a whole TME; that is why they are not physiologically relevant and not clinically predictive. On the other hand, in vivo animal models allow to study the whole organism interactions with proper TME and intra-tumor heterogeneity, but these models are not suitable for large scale screenings, are very time-consuming, and are not “human.” Thus, both, in vitro and in vivo models serve as a valuable tool to study colorectal carcinogenesis (107). However, due to mentioned differences, correlation between these models is not very strong (108). Clinical trials, on the other hand, are golden standard for testing and approval of any potential drug.

It is important to mention one clinical study that has investigated the largest number of cancer patients receiving medical cannabis between 2015 and 2017 in Israel. Two thousand nine hundred seventy patients suffering from the breast (20.7%), lung (13.6%), pancreatic (8.1%), and colorectal cancer (7.9%) were receiving medical cannabis as a palliative treatment to alleviate symptoms such as pain, poor appetite, malaise, sleep disorders, and nausea. Four types of cannabis were used in this study: sativa strains high in THC, without CBD; indica strains high in THC without CBD; strain with an equal amount of CBD and THC, and CBD-rich strains. Interestingly, most patients received more than one strain. Nine hundred two (24.9%) patients died and 682 (18.8%) patients terminated the treatment after 6 months of follow up. Out of the remaining patients, 60.6% of them responded to the treatment; 95.9% had a significant improvement in their condition, 3.7%—no change noticed, 0.3% -deteriorated. Before initiating the treatment, only 18.7% of patients said to have a good quality of life, while at 6 months post-treatment −69.5%. Among the all cancer-associated symptoms, nausea, vomiting, depression, migraine, and sleep disorders, were the most improved. The most common side effects of cannabis treatment at 6 months of follow up were dizziness, xerostomia, and increased appetite. The psychoactive adverse effects were noticed by 2.8% of patients only. Notably, out of 344 patients taking opioids, 36% of them discontinued taking them. It was concluded, that medical cannabis is a well-tolerated and safe palliative therapeutic option for cancer patients (109).

The Role of Cannabis on the Innate and Adaptive Immune Responses

Being immunomodulatory agents, cannabis extracts and single cannabinoids can affect both the innate and adaptive immune responses. Generally, cannabinoids are considered as immunosuppressive compounds. They influence the innate immune responses by suppressing the activity of NK cells, dendritic cells, the migration of neutrophils and macrophages with their antigen presentation and phagocytosis processes (110), and by triggering the induction of MDSCs (111, 112). Inflammation is the main mechanism of the innate immune responses. In general, cannabinoids, such as THC and CBD, cause the downregulation of pro-inflammatory cytokines and the upregulation of anti-inflammatory cytokines. By doing this, they actively suppress the inflammation process (57). However, some studies demonstrate that these compounds have different effects on inflammation by either enhancing or suppressing it. For example, CBD can activate the immune response by elevating mRNA expression of TNF-α, IL-6, as it was shown in mice in response to the LPS-induced pulmonary inflammation (113). In contrast, CBD inhibited IL-6 and IL-8 in an in vivo mouse colon cancer model based on the cell line CT26 (104). These contradicting results might be tissue- and dose-specific.

Cannabinoids may affect the adaptive immune responses by influencing the humoral and cellular immunity. The T cell immunity can be influenced by cannabinoids in different ways: they can affect the proliferation and the number of T cells by polarizing the cytokine response to either Th1 or Th2 (114). Cannabinoids have been shown to suppress the proliferation of T cells, to cause their apoptosis and support the Th2 polarization (115, 116). Some of the initial in vitro and in vivo studies of THC showed an immunosuppressive effect on the T cells and B cells when high concentrations were used, while the immunostimulatory effects was observed at low concentrations (110). The experimental research conducted in vivo with SIV-infected macaques that were receiving THC for the period of 17 months demonstrated an increase in T cells, the reduction in viral load and an increase in the expression of Th2 cytokines (117). Another study performed with HIV patients showed a higher concentration of CD4+ and CD8+ T cells in THC- positive patients vs. THC-negative counterparts (118). Concerning the role of CBD, it was also shown that it could act as an immunosuppressant of Th2 in vitro and in vivo by polarizing the cytokine response to Th2 and working as an immunostimulant to Th1 (119). Concerning the humoral immunity, some reports from human studies showed the reduced number of B lymphocytes and the decreased amount of IgM and IgG after cannabinoid ingestion in the form of bhang (120).

Future Perspectives of Enhancing Immunotherapy by Cannabinoids and Cannabis Sativa Extracts

The immunomodulatory effects of cannabis are well-documented. Nowadays, there are many well-known cannabis cultivars, and each one has a unique composition of different compounds. Many studies have demonstrated the effects of single cannabinoids, such as THC and CBD, on inflammation and cancer cell growth (98). Other components of the plant (such as minor cannabinoids, terpenes, terpenoids, flavonoids, and others) may act synergistically with cannabinoids and can be useful from a therapeutic point of view. The modulating effect of these compounds is known as “an entourage effect;” such modulation is typically positive which means that the medicinal effect of the whole plant extract is more significant than the effect of isolated compounds (121). Like with any other drug, the effects significantly depend on the concentration. In the future, with more research being done, we might gain more insight into the potential immunostimulatory effect of individual cannabinoids or cannabis extracts. This knowledge can help medical professionals to integrate cannabis extracts into cancer targeted therapy, potentially as adjunct therapy. The special extracts with strong anti-neoplastic activities should be identified that are not cytotoxic to normal cells and can sensitize cancer cells to further treatment without reducing the immune responses. Then, these extracts can be combined with immunotherapy, and such combination may have a synergistic action. The results of the retrospective analysis performed with patients with melanoma, renal carcinoma and non-small cell lung cancer when cannabis was used in combination with an immunotherapeutic agent Nivolumab showed a decrease in RR but no changes in PFS and OS. More studies are needed to investigate the possible interactions between cannabinoids and immunotherapy drugs (122).

A thorough exploration of cannabis research and associated drugs should be performed. Currently, we have limited data about cannabis interactions with other drugs, especially with targeted therapy. Since the immune checkpoint inhibitors are a type of the most successful and effective immunotherapy for CRC patients. Therefore, research on the possibility of enhancing the immunotherapy by cannabis extracts should be conducted ( Figures 2 , ​ ,3 3 ).

The potential of cannabinoids for cancer immunotherapy. The upper panel shows how cannabinoids can increase tumor immunogenicity. The release of tumor antigens might be increased due to the direct cytotoxicity of cannabinoids in cancer cells. Next, the presentation of enhanced tumor antigens occurs followed by an increase in T cells-mediated immune response and T cells lysis of tumor cells. The lower panel shows how cannabinoids can reverse tumor immunosuppression. Macrophages can be reprogrammed into an antitumor phenotype with the help of cannabinoids. M1 immunostimulatory macrophages secrete the anti-tumorigenic cytokines and effectively phagocytize cancer cells.