1. Introduction
Cancer is a multistage process conventionally defined by three stages: initiation, promotion and progression [
1]. Development of colon cancer typically is initiated from normal epithelial cells via aberrant crypts and progressive adenoma stages to carcinomas
in situ and then metastasis. Along this process, oxidative stress has the potential to affect numerous signaling pathways related to the proliferation of initiated cells and enhanced malignant transformation [
2]. In the initiation stage, the generation of reactive oxygen species (ROS) has been involved in DNA damage and in the development of aberrant crypt foci (ACF) [
3,
4], the earliest identifiable precancerous lesions in colon cancer [
5]. Similarly, in the post-initiation/promotion stages, ROS also contribute to abnormal gene expression and modification of second-messenger systems in epithelial cells within the ACF. In this manner, several signaling pathways involved in cell survival are deregulated, resulting in an increase in cell proliferation and/or a decrease in the apoptosis of the initiated cell population [
5]. Therefore, inhibition of oxidative stress together with the modulation of signaling routes related to cell survival/proliferation exerted by natural antioxidant compounds seems to be an effective approach in preventing and slowing down the initiation and progression of colon cancer. In fact, the identification of natural bioactive compounds that downregulate cell proliferation or upregulate apoptotic processes could be a complementary and useful strategy to control the development and progression of colon cancer, and it has become an essential subject for study in current research [
6,
7]. In this regard, cocoa and its flavonoids have shown other anticarcinogenic properties independently of their conventional antioxidant activity [
7,
8,
9,
10]. Consequently, cocoa polyphenols could be considered as promising candidates for colon cancer chemoprevention.
Cocoa, the dried and either unfermented or fermented seeds derived from
Theobroma cacao, has the highest flavanol content of all foodstuffs on a weight basis and is a significant contributor to the total dietary intake of flavonoids [
11,
12,
13]. In fact, for many individuals, cocoa products constitute a larger proportion of the diet than foodstuffs containing bioactive compounds with similar properties, such as green tea, wine or soy beans [
14].
However, health effects derived from cocoa flavonoids depend on their bioavailability, which is also affected by their chemical structure [
15]. In this regard, fermented cocoa contains high quantities of flavanols (−)-epicatechin (EC), (−)-catechin and their dimers procyanidins B2 (PB2) and B1 (
Figure 1), although other polyphenols, such as naringenin, luteolin, apigenin, quercetin, isoquercitrin (quercetin 3-
O-glucoside), quercetin 3-
O-arabinose and hyperoside (quercetin 3-
O-galactoside), have also been found in minor amounts [
16]. Interestingly, in comparison to other flavonoid-containing foodstuffs, cocoa and its derivative products exhibit a high concentration of larger procyanidins that are poorly absorbed through the gut barrier, and consequently, their beneficial effects would be restricted to the gastrointestinal tract, where they may have an important antioxidant and anticarcinogenic local function [
6,
7,
8,
9,
10]. Thus, those oligomers and polymers of flavanols that are not absorbed in the intestine could be metabolized by the microbiota into low molecular weight phenolic acids, which are more bioavailable, and might be well absorbed through the colon [
17,
18]. Additionally, recent findings have demonstrated that some of the microbial metabolites derived from cocoa consumption also retain biological properties [
19].
Figure 1.
Main flavonoids present in fermented cocoa. Chemical structures of (−)-epicatechin and (−)-catechin and their respective dimmers, procyanidins B2 and B1.
Figure 1.
Main flavonoids present in fermented cocoa. Chemical structures of (−)-epicatechin and (−)-catechin and their respective dimmers, procyanidins B2 and B1.
Evidence for chemoprevention by bioactive substances is achieved from a combination of epidemiological, animal and basic mechanistic studies. Consequently, the interest for understanding the mode of action of cocoa and its flavanols has been recently rising, especially in cultured colonic cells, although it has not been fully elucidated. In addition, it also remains to be demonstrated whether these mechanisms are involved in cancer prevention in humans. In the present work, different in vitro studies that have identified the potential targets and mechanisms whereby cocoa and their polyphenolic compounds could interfere with colonic cancer cells are reviewed. Then, the potential beneficial effects of cocoa in animal models of colon cancer are summarized, and finally, some evidence from human studies is also provided.
3. Chemopreventive Effects in Animal Models
Colon cancer prevention by cocoa and its major components can also be studied in animal models of genetic and chemically-induced colon cancer. Although colonic cell culture models have clearly demonstrated the antioxidant and chemopreventive abilities of cocoa and its flavonoids, only experimental models for colorectal cancer could offer the opportunity to assess the contribution of this natural dietary compound to the potential prevention of colon cancer. In this regard, carcinogen-induced rodent models have been shown to mimic many features of human non-familial colorectal cancer (non-genetic based) [
35], which is the most common type of colon cancer and which occurs sporadically. Thus, induction of colon tumors could be achieved by the administration of carcinogens, such as nitrosamines, heterocyclic amines, aromatic amines, 1,2-dimethylhydrazine and azoxymethane (AOM). Among these, the AOM model has been extensively used to examine the chemopreventive effect of numerous compounds on colon cancer [
36]. Administration of AOM to rodents induces the development of the ACF colonic preneoplastic lesions that may progress into cancer with time [
1]. Indeed, ACF represent the earliest identifiable intermediate precancerous lesions during colon carcinogenesis in both laboratory animals and humans [
37].
The potentially important role of cocoa and their phenolic compounds for colon cancer prevention was first proven by Weyant and coworkers [
38]. In this work, the authors demonstrated that the cocoa flavanol catechin added to a diet (0.1 and 1%, which has been estimated by the authors to correspond to a daily dose of 8 mg/kg (+)-catechin in humans, that is 480 mg for a 60-kg human that might be obtained from approximately 25 g cocoa) reduced the formation of intestinal tumors by 75% and 71%, respectively, in a genetic mice model of multiple intestinal neoplasia that spontaneously develops multiple intestinal tumors. Further mechanistic studies related this effect to (+)-catechin-induced changes in integrin-mediated intestinal cell-survival signaling, involving structural alteration of the actin cytoskeleton and decreased focal adhesion kinase (FAK) tyrosine phosphorylation. All of these suggested that (+)-catechin could prevent the progression of initiated enterocytes to the adenoma stage, as FAK has been implicated in the regulation of cell migration, one of the earliest changes in adenoma development [
39].
In another study, male Wistar rats were fed with a cocoa-enriched diet (12%, which according to the body surface area normalization method [
40] would correspond to a daily dose of 78 g of cocoa, containing 1560 mg of polyphenols, for a 60-kg human ) for eight weeks starting two weeks before the AOM administration (20 mg/kg bw). As estimated, all rats injected with AOM developed aberrant crypts (100% incidence) [
41]. Nevertheless, the cocoa-rich diet diminished the AOM-induced ACF formation and especially those ACF with larger number of crypts (≥4 crypts), which exhibit a higher tendency to progress into malignancy. Therefore, a cocoa-supplemented diet was able to abolish the early phase of chemically-induced colon carcinogenesis. This inhibitory effect of cocoa on AOM-induced preneoplastic lesions was related to its anti-oxidative and anti-inflammatory properties. Thus, cocoa circumvented the oxidative stress induced by AOM, as it recovered to control levels the reduced content of GSH and the activities of GPx, GR and GST induced by the toxic. Consequently, the cocoa-enriched diet avoided the subsequent enhancement in the phosphorylated levels of AKT and ERK provoked by AOM, as well as the decrease in the values of cyclin D1 and cell proliferation, measured as proliferating cell nuclear antigen levels (PCNA). Cocoa flavanols also repressed intestinal inflammation induced by AOM by inhibiting the NF-κB signaling and downregulating pro-inflammatory COX-2 and iNOS expression [
41]. Furthermore, cocoa supplementation upregulated Bax and downregulated Bcl-xL levels in the colon tissue of AOM-treated rats, suggesting that cocoa was also able to induce apoptosis as another complementary mechanism of chemoprevention during the progression of carcinogenesis [
41]. In the same rat model of AOM-induced colon cancer, a diet including dark chocolate has been reported to reduce cell proliferation and some gene expression involving inflammation (COX-2 and p-65-NF-ĸB), resulting in a lower number of early preneoplastic lesions [
42].
To conclude with the animal experimentation, it has been very recently observed that dietary cocoa (5% and 10%, which according to the body surface area normalization method would approximately correspond to a daily dose of 32.5–65 g of cocoa, containing 650–1300 mg of polyphenols, for a 60-kg human) inhibits colitis-associated cancer in a mouse model of AOM/dextran sulfate sodium (DSS)-induced chronic inflammation by preserving the redox status of the animals and limiting cell proliferation [
43]. Thus, cocoa ingestion for 62 days highly reduced lipid oxidation and prevented the decrease in the activities/levels of antioxidant defenses (GSH, SOD, CAT, GPx and GR) induced by AOM/DSS administration. Moreover, cocoa-rich diets effectively decreased the expression of iNOS and COX-2 and activated the Nrf2/Keap1 pathway, as well as its downstream targets, such as NQO1 and UDP-GT [
43]. In the same experimental conditions, a cocoa-enriched diet was also able to suppress the IL-6/STAT3 pathway and to induce apoptosis, as increased levels of Bax and caspase-3 together with diminished Bcl-xl values were observed in cocoa-fed mice [
44].
5. Conclusions
This review reports the potential chemopreventive actions of cocoa and its main flavanols against colon cancer (
Figure 2). In this line, their potential cellular molecular mechanisms of action include the regulation of different antioxidant defenses and numerous key proteins of signal transduction pathways related to inflammation, cell proliferation, differentiation and apoptosis. These actions could contribute to preserve a balanced redox status and to prevent the initiation and progression of an uncontrolled cell growth, as well as to avoid a pro-inflammatory environment. In addition, animal studies have proven that cocoa and its main flavanols would prevent and/or slow down the initiation promotion of colon cancer. However, amounts administrated are probably higher than what a person should normally consume and, despite these doses could be achievable through supplementation, more moderate quantities of cocoa would be desirable. Importantly, human intervention studies have reported some favorable changes in biomarkers for antioxidant status. Therefore, it could be suggested that daily consumption of small amounts of flavanols and procyanidins from cocoa or chocolate, together with an ordinary dietary intake of flavonoids, would constitute a natural approach to potentially prevent colon cancer with minimal toxicity. Nevertheless, cocoa and its derivative products merit further investigations, since the molecular mechanisms of action are not completely elucidated. Additionally, extensive well-controlled and well-designed human epidemiological and intervention studies are needed to fully assess the potential of cocoa in terms of optimal dose, route of administration, cancer targets and preventive activities.
Figure 2.
Mechanisms involved in the potential chemopreventive effects of cocoa and its flavonoids against colorectal cancer. The arrows indicate an increase (↑) or decrease (↓) in the levels or activity of the different analysed parameters. ACF, aberrant crypt foci.
Figure 2.
Mechanisms involved in the potential chemopreventive effects of cocoa and its flavonoids against colorectal cancer. The arrows indicate an increase (↑) or decrease (↓) in the levels or activity of the different analysed parameters. ACF, aberrant crypt foci.