Objective. Colorectal cancer represents a heavy burden for health systems worldwide, being the third most common cancer worldwide. Despite the breakthroughs in medicine, current chemotherapeutic options continue to have important side effects and may not be effective in preventing disease progression. Cannabinoids might be substances with possible therapeutic potential for cancer because they can attenuate the side effects of chemotherapy and have antiproliferative and antimetastatic effects. We aim to determine, through a systematic review of experimental studies performed on animal CRC models, if cannabinoids can reduce the formation of preneoplastic lesions (aberrant crypt foci), number, and volume of neoplastic lesions. Materials and Methods. A systematic, qualitative review of the literature was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Embase, and Scopus databases were searched. We use the following Medical Subject Headings (MESH) terms in PubMed: “colorectal neoplasms,” “colonic neoplasms,” “colorectal cancer,” “polyps,” “rimonabant,” “cannabidiol,” “cannabinoids,” “azoxymethane,” “xenograft,” and “mice.” Only studies that met the eligibility criteria were included. Results. Eight in vivo experimental studies were included in the analysis after the full-text evaluation. Seven studies were azoxymethane (AOM) colorectal cancer models, and four studies were xenograft models. Cannabidiol botanical substance (CBD BS) and rimonabant achieved high aberrant crypt foci (ACF) reduction (86% and 75.4%, respectively). Cannabigerol, O-1602, and URB-602 demonstrated a high capacity for tumor volume reduction. Induction of apoptosis, interaction with cell survival, growth pathways, and angiogenesis inhibition were the mechanisms extracted from the studies that explain cannabinoids’ actions on CRC. Conclusions. Cannabinoids have incredible potential as antineoplastic agents as experimental models demonstrate that they can reduce tumor volume and ACF formation. It is crucial to conduct more experimental studies to understand the pharmacology of cannabinoids in CRC better.
Colorectal cancer (CRC) is the third most common cancer worldwide, only behind prostate and lung in males, and behind breast and lung in females . It has high morbidity and mortality that represents a heavy burden for health systems worldwide. In the United States alone, with roughly 1.8 million new cases in 2018, healthcare costs exceed $14 billion annually . In addition, it is the fourth cause of cancer-related deaths [3, 4]. CRC represents a significant public health concern because temporal projections estimate that its global burden will increase by 60% to more than 2.2 million new cases and 1.1 million cancer deaths by 2030 .
CRC is a type of cancer with a complex and heterogeneous pathophysiology. It is the result of the transformation of healthy colonic epithelial cells into cancer . This process, called “adenoma-carcinoma sequence,” develops through an ordered series of events, in which the initial step is the transformation of normal colonic epithelium to aberrant crypt foci (ACF) . ACF progress to CRC, in 10–15 years . During this process, many risk factors play an essential role in pathogenesis, including unhealthy diet, smoking, alcohol use, physical inactivity, inflammatory bowel disease, and aging .
Breakthroughs in CRC therapy have decreased the mortality of patients with CRC. Current chemotherapeutic options continue to have important side effects due to cytotoxicity and may fail to prevent disease progression . Thus, there is a great interest in new therapeutic approaches for CRC, including phytochemical agents.
Cannabinoids might be substances with possible therapeutic potential for cancer because of their chemotherapeutic effect and their ability to attenuate anorexia, pain, and emesis; these are common side effects of chemotherapy [9, 10]. This has been proved in several experimental models of CRC, brain cancer, breast cancer, lung cancer, prostate cancer, leukemia, and melanoma . However, to the best of our knowledge, cannabinoids have not been tested in humans as medicines for CRC.
Animal models and cell lines of CRC have tested cannabinoids. This study aims to conduct a systematic review of the research about the effect of cannabinoids on in vivo azoxymethane (AOM) or xenograft CRC models. The outcomes used to assess the effects of cannabinoids, compared with no cannabinoid therapy, were a decrease in the number of preneoplastic lesions (aberrant crypt foci), number, and volume of neoplastic lesions.
2. Materials and Methods
The protocol for this study was registered in PROSPERO (International Prospective Register for Systematic Reviews) under CRD42019148356 . This systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Supplementary file) .
2.1. Eligibility Criteria
Population. The population should be animal species (no restrictions), used for in vivo models of CRC, either chemically induced (Azoxymethane or DSS) or by xenograft injection. Dose and time of exposure to azoxymethane were not exclusion criteria for this review. We excluded all studies that included only in vitro assessment and studies that evaluated species for noncolorectal cancer models.
Intervention. Studies had to evaluate the beneficial effects of the following cannabinoids: CBD, CBG, O-1602, LYR-8, WIN 55, 212–2, AEA, HU-210, rimonabant, anandamide reuptake inhibitors (VDM11), FAAH inhibitors, and MAGL inhibitors.
Comparators. Studies had to include at least one comparator group of the same animal species used for the intervention group, with similar characteristics (weight, age, sex, exposure to the same environment, and feeding), without exposition to cannabinoid therapy.
Studies. Studies should be experimental in vivo studies of CRC in mice, with at least one control group. We excluded conference abstracts, narrative reviews, and systematic reviews.
Primary Outcome. There should be a reduction in tumor volume (mm3), number of aberrant crypt foci (ACF), and number of tumors comparing intervention and control group.
Secondary Outcome. There should be an expression of apoptosis markers (Bax, caspase-3, caspase-9, annexin V, PI), expression of proinflammatory markers (STAT3, NFκβ, TNF-α), and levels of endocannabinoids.
2.2. Search Strategy
We performed a methodologic and systematic strategic search in the following electronic bibliographic databases: PubMed, Scopus, and Embase (from their inception to December 18, 2019). The last search was run on December 18, 2019. Only full available articles written in English were suitable for assessment. We used the following Medical Subject Headings (MESH) terms in PubMed: “colorectal neoplasms”, “colonic neoplasms”, “colorectal cancer”, “polyps”, “rimonabant”, “cannabidiol”, “cannabinoids”, “azoxymethane”, “xenograft”, and “mice”.
2.3. Study Selection and Data Collection
The authors EOG and LLT conducted the search independently. Duplicate articles were moved to a different folder and registered in the flowchart. Before the selection process, a test was conducted to evaluate the agreement between evaluators. All titles and summaries of the articles were assessed by EOG and LLT independently based on a selection criterion. The full text of previously selected studies was then reviewed and analyzed. Any disagreement was discussed, and if not resolved, a third author (AVV) was consulted. All selected articles were summarized in a flowchart according to the PRISMA protocol. We used a standardized form with a pilot test to collect the following data: title, author, publication year, type of animal model, sample size, type of cannabinoid, the dose of cannabinoid, the dose of AOM, type of outcome measure, length of the experiment, reduction in ACF formation, reduction in the number of tumors, tumor volume reduction, pathway or function modified by cannabinoids, an increase of endocannabinoid levels, expression of apoptosis markers, and expression of proinflammatory markers.
2.4. Quality Assessment
The risk of bias was independently evaluated by two authors (EOG and LLT) following the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool . The domains considered were random sequence generation, baseline characteristics, allocation concealment, random housing, blinding, random outcome assessment, outcome assessor blinding, incomplete outcome data, selective outcome report, and other sources of bias (contamination, the influence of funders, and analysis of errors) . We reviewed each article, and we sought if any of these biases were present. Any discrepancy was discussed between 2 authors (EOG and LLT), and if not resolved, a third investigator intervened (AVV).
2.5. Data Analysis
Proportions were used as descriptive statistics for primary outcomes. Secondary outcomes were described qualitatively. A meta-analysis or measures of consistency were not performed due to characteristics of the studies and heterogeneity of articles.
3. Cannabinoids: Pharmacology and Generalities
Endocannabinoids are lipid mediators, including amides, esters, and ethers of polyunsaturated fatty acids, which were isolated from the porcine brain [15–17]. Anandamide’s structure resembles Δ9-THC structure, and it is synthesized from membrane phospholipids by the enzymes N-acyl phosphatidylethanolamine phospholipase D (NAPE-D) and lysophospholipase D (lyso-PLD) . 2-Arachidonoyl-glycerol (2-AG) is an arachidonoyl ester, produced from diacylglycerols . Endocannabinoids diffuse into the extracellular space and bind to CB1 and CB2 receptors, TRPV1, TRPM8, and GPR55 . Anandamide and 2-AG are reuptake via an extraneuronal monoamine membrane transporter (EMT); then, they are degraded by the fatty acid amide hydrolase (FAAH) and the monoacylglycerol lipase (MAGL), respectively [16, 19]. Most known plant-derived cannabinoids include tetrahydrocannabinol (THC) and cannabidiol (CBD) . These are tricyclic terpenoid compounds bearing a benzopyran moiety soluble in lipids and nonpolar organic solvents [20, 21].
Δ9-Tetrahydrocannabinol (THC) and anandamide have the highest affinity for the CB1 receptor, while CBD exhibits low affinity for these receptors [20, 21]. However, CBD has been proved to enhance endocannabinoid levels and indirectly activate CB receptors .
CB1 receptors constitute one of the most abundant receptors in the central nervous system. In the case of CB2 receptors, these are expressed in cells of the immune and hematopoietic system, spleen, and tonsils, modulating cytokine release and cellular immune migration . Both receptors are metabotropic and belong to the G protein-coupled receptor (GPCR) family, and their activation produces inhibition of the adenylyl cyclase via G proteins (Gi/o) . This decreases cAMP in the cell and activity of protein kinase A .
Cannabinoids may have alternative molecular targets other than classical CB1 and CB2 receptors . Recently, orphan GPCRs like the GPR 55, GPR18, and GPR110 have been identified as new targets . There is also increasing evidence that they can interact with ionotropic receptors such as the transient receptor potential cation channel subfamily V member 1 (TRPV1), and the transient receptor potential cation channel subfamily M member 8 (TRPM8) .
The transient receptor potential vanilloid receptor 1 (TRPV1) and the transient receptor potential cation channel subfamily M member 8 (TRPM8) are ionotropic channels that allow Na+ and Ca++ entry to the cell . Cannabidiol (CBD) and cannabigerol (CBG) close the TRPM8 channel, whereas CBD opens TRPV1 .
GPR55 is another GPCR, which is coupled to a Gα12/13 protein . Several cancer lines like OVACAR3 (ovarian cancer cell line), PC-3, and DU145 (prostate cancer cell lines) exhibit expression of this orphan receptor . Furthermore, Piñeiro et al. showed an autocrine activation of this receptor through his main endogenous agonist lysophosphatidylinositol (LPI) . The receptor acts via activation of Gα12 and Gq family proteins, which activate Ras homolog gene family, member A (RhoA) kinase . Overexpression of GPR55 produces increased levels of pERK in HEK-293, breast carcinoma, and glioma cells, while pAKT levels are increased in ovarian and prostate cancer cells [25, 27].
The high expression of GPR55 is also linked to high proliferation indices in human breast tumors and Glioblastoma . The best-studied cannabinoid with actions on the GPR55 in colonic tissue is O-1602 . It is highly speculative that the compound exerts its antineoplastic effects on CRC tissue through the GPR55 receptor, as the cannabinoid has shown agonist activity on this receptor . More research is needed before we can conclude the actions of cannabinoids on this receptor.
Ceramide’s synthesis begins with the enzyme serine palmitoyltransferase (SPT) . Gustaffson et al. have demonstrated that the cannabinoids Win55,212-2 and R (+)-methanandamide induce ceramide accumulation mainly through CB1 and CB2 activation, which acts on SPT [30, 31]. Both studies were performed in mantle cell lymphoma cells (L718, L1547, L1676, and Rec-1) [30, 31]. Furthermore, in neural tissues (rat glioma C6 line and H4 neuroglioma), R (+)-methanandamide and JWH-133 (CB2 agonist) also induce ceramide accumulation [32, 33]. Ceramide provokes a loss of mitochondrial membrane potential and caspase activation, subsequently [30, 31].
3.1. Reported Effects of Cannabinoids on CRC
Most cultured colonic cancer cells used for in vitro assessment express CB1, CB2, TRPM8, and GPR55 (G protein-coupled receptor) [34–39]. Additionally, adenomatous polyps and colorectal cancer tissue have increased the amounts of the endogenous cannabinoids AEA and 2-AG (3-fold versus 2-fold, respectively) . This has been suggested to be a mechanism of self-protection against further tumor progression [40, 41]. Cannabinoids and phytocannabinoids have, therefore, effects on colonic cancer tissues since CRC tissues produce those (endogenous cannabinoids) and express some of their receptors (Figure 1).