Mechanisms involved in the chemopreventive effect of
1. Introduction
Epidemiological studies on the relationship between dietary habits and disease risk have shown that food has a direct impact on health. Indeed, our diet plays a significant role in health and well-being, since unbalanced nutrition or an inadequate diet is known to be a key risk factor for chronic age-related diseases [1]. An example that illustrates this fact is the protective effect of the so-called Mediterranean diet. The lower occurrence of cancer and cardiovascular disease in the population located around the Mediterranean sea has been linked to the dietary habits of the region, in which the components of the diet contain a wide array of molecules with antioxidant and antiinflammatory actions [2].
Many diseases with a strong dietary influence include oxidative damage as an initial event or in an early stage of disease progression [3]. In fact, Western diets (typically dense in fat and energy and low in fiber) are associated with disease risk [4]. Therefore, dietary modification, with a major focus on chronic age-related disease prevention through antioxidant intervention, could be a good and cost-effective strategy [5]. The intake of whole foods and/or new brand developed functional foods rich in antioxidants would be suitable for this purpose. In this sense, dietary antioxidants such as polyphenols, carotenoids and peptides, as well as other bioactive chemopreventive components such as fiber and phytosterols have been regarded to have low potency as bioactive compounds when compared to pharmaceutical drugs, but since they are ingested regularly and in significant amounts as part of the diet, they may have noticeable long-term physiological effects [6].
For decades, the beneficial role of antioxidants was related to the reduction of unwanted and uncontrolled production of reactive oxygen species (ROS), leading to a situation referred to as oxidative stress [7]. Nowadays, the term “antioxidant” has become ambiguous, since it has different connotations for distinct audiences. For instance, for biochemists and nutritionists, the term is related to the scavenging of metabolically generated ROS, while for food scientists the term implies use in retarding food oxidation or for the categorization of foods or substances according to
In order to determine and verify the action of these bioactive compounds, it is clear that data from human intervention studies offer the reference standard and the highest scientific evidence considering the bioavailability and bioactivity of a food component, while
Taking this background together, and in order to obtain a more precise view of the
This review introduces the main features of the different
2. Simulated gastrointestinal digestion assays
Bioavailability is a key concept for nutritional effectiveness, irrespective of the type of food considered (functional or otherwise). Only certain amounts of all nutrients or bioactive compounds are available for use in physiological functions or for storage.
The term bioavailability has several working conditions. From the nutritional point of view, bioavailability is defined as the proportion of a nutrient or bioactive compound can be used for normal physiological functions [16]. This term in turn includes two additional terms: bioaccessibility and bioactivity. Bioaccessibility has been defined as the fraction of a compound that is released from its food matrix in the gastrointestinal tract and thus becomes available for intestinal absorption. Bioaccessibility includes the sequence of events that take place during food digestion for transformation into potentially bioaccessible material, absorption/assimilation through epithelial tissue and pre-systemic metabolism. Bioactivity in turn includes events linked to how the bioactive compound is transported and reaches the target tissue, how it interacts with biomolecules, the metabolism or biotransformation it may undergo, and the generation of biomarkers and the physiologic responses it causes [12]. Depending on the
The principal requirement for successfully conducting experimental studies of this kind is to achieve conditions which are similar to the
Bioactive compounds such as dietary fiber, carotenoids, polyphenols and phytosterols undergo very limited absorption, and may experience important modifications as a result of actions on the part of the intestinal microbiota. Small intestine
Continuous cultures allow us to control the rate and composition of nutrient feed, bacterial metabolism and the environmental conditions. These models simulate proximal (single-state models) or proximal, transverse and distal colonic regions (multistage models). Continuous cultures are used for performing long-term studies, and substrate replenishment and toxic product removal are facilitated - thereby mimicking the conditions found
Artificial continuous models including host functions/human digestive functions have been developed. Models of this kind control peristaltic movement, pH and gastrointestinal secretions. The SHIME model (Simulated Human Intestinal Microbial Ecosystem) comprises a 5-step multi-chamber reactor simulating the duodenum and jejunum, ileum, cecum and the ascending colon, transverse colon and descending colon [24]. In turn, TIM-1 is an intestinal model of the stomach and small intestine, while TIM-2 is a proximal colon simulator model developed by TNO (
Combined systems that include the fractions obtained from simulated human digestion (gastrointestinal and/or colonic fermentation) and the incorporation of cell culture-based models allow us to evaluate bioaccessibility (estimate the amount of bioactive compounds assimilated from the bioaccessible fraction by cell culture) and to conduct bioactivity studies. The Caco-2 cell model is the most widely used and validated intestinal epithelium or human colon carcinoma cell model. Although colonic in origin, Caco-2 cells undergo spontaneous differentiation in cell culture to form a monolayer of well-polarized cells at confluence, showing many of the functional and morphological properties of mature human enterocytes (with the formation of microvilli on the brush border membrane, tight intercellular junctions and the excretion of brush border-associated enzymes) [26]. However it must be mentioned that this cell line differs in some aspects from
The advantage of these systems versus those which only evaluate the influence of digestion is their greater similarity to the
3. Bioactivity of digested/fermented foods or related target bioactive compounds in cell lines
The chemopreventive properties of bioactive compounds have been investigated in cultured cells exposed to individual compounds. However, gut epithelial cells are more likely to be exposed to complex food matrixes containing mixtures of bioactive and antioxidant
A potential cell culture model for cancer or cardiovascular chemoprevention research involving dietary antioxidants (polyphenols, carotenoids and peptides) and other bioactive chemopreventive components such as phytosterols, should include some of the proposed mechanisms of action: inhibition of cell proliferation, induction of tumor suppressor gene expression, induction of cell cycle arrest, induction of apoptosis, antioxidant enzyme induction, and enhanced detoxification, antiinflammatory activities and the inhibition of cholesterol absorption [9, 15, 29, 30]. In addition, other mechanisms of chemoprevention could involve protection against genotoxic compounds or reactive oxygen species [31].
It recently has been stated that the measurement of cellular bioactivity of food samples coupled to
The chemopreventive effect of digested foods or bioactive constituents in cell lines is summarized in Table 1. From the 22 studies surveyed, and according to the digestion method used, it can be seen that most of them involve solubility (n = 17) versus dialysis (n = 5). Samples used are preferably of vegetal origin (n = 15), the target compounds responsible for the chemopreventive action being polyphenols, antioxidants (in general), antioxidant peptides, lycopene and phytosterols. Furthermore, these compounds are mainly studied in colon-derived cells (as a cancer model when not differentiated, or as an intestinal epithelial model when differentiated). Concentrations tested are physiologically achievable in colon cells, since the bioaccessible fractions obtained after digestion are considered to be fractions that can pass through the stomach and small intestine reaching the colon, where they can exert antioxidant activity
Bioactive compounds of digested foods present four different but in some cases complementary modes of action: (1) inhibition of cholesterol absorption (phytosterols), and (2) antiproliferative, (3) cytoprotective and (4) antiinflammatory activities (polyphenols and general antioxidants).
The inhibition of cholesterol absorption has been reported to be mainly due to competition between phytosterols and cholesterol for incorporation to the micelles as a previous step before absorption by the intestinal epithelial cells [35].
Antiproliferative activity has been linked to cell growth inhibition associated to polyphenols [28, 32, 36-38] and lycopene [39], which is mainly regulated by two mechanisms: cell-cycle arrest and apoptosis induction. The cell cycle can be halted at different phases: G0/G1 with down-regulation of cyclin D1 [39], S with down-regulation of cyclins D1 and B1 [28, 37] and G2/M [36]. Apoptosis induction in turn occurs as a result of caspase-3 induction and down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL [39].
The cytoprotective effect of polyphenols, peptides and antioxidants against induced oxidative stress is related to the preservation of cell viability [40-47], an increase in the activity of antioxidant enzymes (such as catalase, glutathione reductase or glutathione peroxidase) [41, 43, 47, 48], the prevention of reduced glutathione (GSH) depletion [46, 47, 49], a decrease in intracellular ROS content [46, 50, 51], the maintenance of correct cell cycle progression [41, 43, 47, 52], the prevention of apoptosis [43], and the prevention of DNA damage [42, 51, 52].
The antiinflammatory action of peptides and polyphenols is derived from the decrease in the release of proinflammatory cytokines such as IL-8 when cells are stimulated with stressors such as H202 or TNFα [53, 54].
Studies on the chemopreventive effect of foods or isolated bioactive constituents following colonic fermentation or gastrointestinal digestion plus colonic fermentation in cell lines are shown in Tables 2 and 3, respectively. The colonic fermentation procedure used in these assays has always been a batch model, except for one study combining batch and dynamic fermentation. In turn, when gastrointestinal digestion is involved, dialysis has been the method used. Foods of plant origin rich in fiber, and short chain fatty acids (mainly butyrate) and polyphenols as the target compounds have been used in such studies. The use of colon-derived cell lines is common in these assays, which have been performed using physiologically relevant concentrations and time periods of exposure of samples to cells ranging between 24 h and 72 h.
The mechanism of action underlying the treatment of cells with colonic fermented foods or isolated bioactive constituents (see Table 2) mainly comprises antiproliferative activity (i) and/or cytoprotective action (ii). In the first case, antiproliferative activity (i) has been attributed to cell growth inhibition [55-59], mainly due to apoptosis induction [58-59] and/or the up-regulation of genes involved in cell cycle arrest (
The bioactivity observed with the incubation of cells lines with foods or isolated bioactive constituents following gastrointestinal digestion plus colonic fermentation (see Table 3) is derived from antiproliferative activity (i) regulated by cell growth inhibition [60-62], cell cycle arrest [60] and/or apoptosis induction [60, 62], or by a cytoprotective effect against induced oxidative stress (ii) as a result of preservation of cell viability [63], protection against DNA damage [31, 61, 63] and/or induction of antioxidant enzymes such as CAT, GST and sulfotransferase (SULT2B1) [31].
4. Conclusions and future perspectives
From the data here reviewed in disease cell models, it can be concluded that gastrointestinal digestion/colonic fermentation applied to whole foods or isolated bioactive constituents may have potential health benefits derived from cell growth inhibition through the induction of cell-cycle arrest and/or apoptosis, cytoprotection against induced oxidative stress, antiinflammatory activity and the reduction of cholesterol absorption.
Studies conducted with single bioactive compounds are unrealistic from a nutritional and physiological point of view, since they do not take into account physicochemical changes during digestion and possible synergistic activities. Thus, a combined model of human simulated digestion including or not including colonic fermentation (depending on the nature of the studied compounds) with cell lines should be carried out if
Although digested/fermented bioactive compounds appear as promising chemopreventive agents, our understanding of the molecular and biochemical pathways behind their mechanism of action is still limited, and further studies are warranted. In addition, the need for harmonization of the
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Chokeberry juice |
Caco-2 (human colon carcinoma) |
85 to 220 (µM total polyphenols) 2 h a day for a 4-day period |
Cell growth inhibition Viability decrease Cell cycle arrest at G2/M phase Up-regulation of tumor suppression gene |
Bermúdez-Soto et al. (2007) [36] |
Raspberries |
HT29, Caco-2 and HT115 (human colon carcinoma) |
3.125 to 50 (µg/mL) 24 h |
Prevention of H2O2 (75µM/5min)-induced DNA damage and decrease in G1 phase of cell cycle (HT29 cells) No effect on epithelial integrity (Caco-2 cells) Inhibition of colon cancer cell invasion (HT115 cells) |
Coates et al. (2007) [52] |
Green tea | Differentiated PC12 (model of neuronal cells) |
0.3-10 µg/mL (for H2O2) and 0.03-0.125 µg/mL (for Aβ(1-42)) Pretreatment 24 h and stressed 24 h |
Protection against H2O2 and Aβ(1-42) induced cytotoxicity (only at low concentrations) | Okello et al. (2011) [44] |
Blackberry ( |
SK-N-MC (neuroblastoma cells) |
1.5-6 µM total polyphenols 24 h |
Preservation of cell viability against H2O2 (300 µM- 24 h) –induced oxidative stress (not related to modulation of ROS nor GSH levels) | Tavares et al. (2012a) [45] |
The
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Wild blackberry species |
SK-N-MC (neuroblastoma cells) |
0-6 µM total polyphenols 24 h |
Preservation of cell viability and mitochondrial membrane potential against H2O2 (300 µM -24 h)-induced oxidative stress Decrease of intracellular ROS against H2O2 (200 µM -1 h)-induced oxidative stress (only Prevention of GSH depletion against H2O2 (300 µM -24 h)-induced oxidative stress Induction of caspase 3/7 activity against H2O2 (300 µM -24 h)-induced oxidative stress (preconditioning effect) |
Tavares et al. (2012b) [46] |
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Fruit beverages with/without milk and/or iron |
Caco-2 (human colon carcinoma) |
2%, 5% and 7.5% (v/v) in culture medium (3.4-22.7 mg/mL total polyphenols) 4 hours-4 days or 24 h |
Cell growth inhibition (no clear dose-response) Cell cycle arrest at S phase (7.5%) Down-regulation of cyclins D1 and B1 No apoptosis (cytostatic effect) |
Cilla et al. (2009) [28] |
Zinc-fortified fruit beverages with/without iron and/or milk |
Caco-2 and HT-29 (human colon carcinoma) |
7.5% (v/v) in culture medium (~50 µM total polyphenols) 24 h |
Cell growth inhibition (without citotoxicity) Cell cycle arrest at S phase No apoptosis and resumption of cell cycle after digest removal (cytostatic effect) |
Cilla et al. (2010) [37] |
Fruit juices enriched with pine bark extract |
Caco-2 (human colon carcinoma) |
4% (v/v) in culture medium 24-120 h |
Cell growth inhibition | Frontela-Saseta et al. (2011) [38] |
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Feijoada-traditional Brazilian meal |
HepG2 (human liver cancer cells) |
10-100 mg/mL 72 h (antiproliferation) and 1 h (antioxidant) |
Antiproliferative activity ("/ 80 mg/mL) Increase in cellular antioxidant activity (0.6 µM quercetin equivalents) |
Kremer-Faller et al. (2012) [32] |
Culinary herbs: rosemary, sage and thyme | PBL (peripheral blood lymphocytes) and Differentitated Caco-2 (model of intestinal epithelium) |
1:10 (v/v) in culture medium. Stressors (H2O2 2 mM and TNFα 100 µg/mL) Co-incubation 24 h or pre-incubation 3h then stress 24 h |
PBL: significant decrease in IL-8 release when co-incubation with H2O2 and pre-incubation prior H202 and TNFα Caco-2: significant decrease in IL-8 release only when co-incubation with TNFα |
Chohan et al. (2012) [54] |
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Fruit beverages with/without milk and/or iron/zinc |
Differentiated Caco-2 (model of intestinal epithelia) |
1:1 (v/v) in culture medium |
Preservation of cell viability No alteration of SOD |
Cilla et al. (2008) [40] |
Fruit beverages with/without milk or CPPs |
Differentiated Caco-2 (model of intestinal epithelia) |
1:1 (v/v) in culture medium or CPPs (1.4 mg/mL) | Preservation of cell viability (only fruit beverages) | Laparra et al. (2008) [41] |
Beef patties enriched with sage and oregano |
Caco-2 (human colon carcinoma) |
10-100% (v/v) 24 h |
Increase in cell viability at low concentrations (20-40%) but slight decrease at high concentrations (80-100%) Increase in GSH (only sage-enriched samples at 10%) Protection against H202 (200 µM/1h)-induced GSH depletion (at 10%) |
Ryan et al. (2009) [49] |
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Ellagic acid-, lutein- or sesamol-enriched meat patties |
Caco-2 (human colon carcinoma) |
0-20% (v/v) in culture medium 24 h |
Viability maintenance against H202 (500 µM/1h)-induced stress Prevention of H202 (50 µM/30 min)-induced DNA damage |
Daly et al. (2010) [42] |
Pacific hake fish protein hydrolysates | Caco-2 (human colon carcinoma) |
0.625-5 mg/mL 2 h |
Inhibition (at non cytotoxic doses) of intracellular oxidation induced by AAPH (50 µM/1-2 h) | Samaranayaka et al. (2010) [50] |
Human breast milk |
Co-culture of Caco-2 BBE and HT29-MTX (model of human intestinal mucosa) |
1:3 (v/v) in culture medium 30 min |
Decrease of H202 (1 mM/30 min)-induced ROS Prevention of H202 (500 µM/30 min)-induced DNA damage |
Yao et al. (2010) [51] |
Fruit beverages with/without milk and/or iron/zinc |
Differentiated Caco-2 (model of intestinal epithelia) |
1:1 (v/v) in culture medium Pre-incubation 24 h then stressed 2h with H202 5 mM |
Preservation of cell viability Increase in GSH-Rd activity (only Fe or Zn with/without milk samples) Prevention of G1 cell cycle phase decrease induced by H202 Prevention of apoptosis (caspase-3) induced by H202 |
Cilla et al. (2011) [43] |
Purified milk hydrolysate peptide fraction from digested human milk |
Caco-2 and FHs 74 int (human colon carcinoma and primary fetal enterocytes) |
0.31-1.25 g/L (peptide) and 150 µM (tryptophan) 2 h (peptide) and 1-12 h (tryptophan) |
Exacerbation of AAPH (50 µM/1-2 h)-induced oxidative stress (peptide) Up-regulation of Nrf-2 and subsequent up-regulation of GSH-Px2 gene as adaptive response to stress (tryptophan) |
Elisia et al. (2011) [48] |
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CPPs from digested cow’s skimmed milk |
Differentiated Caco-2 (model of intestinal epithelia) |
1, 2 and 3 mg/mL Pre-incubation 24 h then stressed 2h with H202 5 mM |
Preservation of cell viability Increase in GSH content and induction of CAT activity Decrease in lipid peroxidation Maintenance of correct cell cycle progression |
García-Nebot et al. (2011) [47] |
Purified hen egg yolk-derived phosvitin phosphopeptides | Differentiated Caco-2 (model of intestinal epithelia) |
0.05-0.5 mg/mL 2 h |
Reduced IL-8 secretion in H202 (1 mM/6 h)-induced oxidative stress | Young et al. (2011) [53] |
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Tomatoes |
HT29 and HCT-116 (human colon carcinoma) |
20-100 mL/L 24 h |
Cell growth inhibition Cell cycle arrest at G0-G1 phase and apoptosis induction (caspase-3) Down-regulation of cyclin D1 and anti-apoptotic proteins Bcl-2 and Bcl-xL |
Palozza et al. (2011) [39] |
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Orange juice enriched with fat-free phytosterols |
Differentiated Caco-2 (model of intestinal epithelia) |
2 mL test medium/well 4 h |
Reduced micellarization of cholesterol Decrease in cholesterol accumulation by Caco-2 cells |
Bohn et al. (2007) [35] |
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Fibre sources: linseed, watercress, kale, tomato.soya flour, chicory inulin and wheat | HT29 (human colon carcinoma) |
2.5-25% (v/v) in culture medium 72 h |
Cell growth inhibition (all samples except watercress) Prevention of HNE (150µM/30 min)-induced DNA damage (only soya flour) |
Beyer-Sehlmeyer et al. (2003) [55] |
Wheat bran-derived arabinoxylans | HT29 (human colon carcinoma) |
0.01-50% (v/v) in culture medium 24-72 h |
Cell growth inhibition Prevention of HNE (200µM/30 min)-induced DNA damage (at 25-50%) Induction of GST activity (at 10%) |
Glei et al. (2006) [56] |
Inulin-type fructans |
LT97 and HT29 (human colon adenoma and carcinoma) |
1.25-20% (v/v) in culture medium 24-72 h |
Cell growth inhibition (at 5-10%) Apoptosis induction (cleavage of PARP) only in LT97 cells (at 5-10%) |
Munjal et al. (2009) [58] |
Wheat aleurone |
LT97 and HT29 (human colon adenoma and carcinoma) |
5-10% (v/v) in culture medium 24-72 h |
Cell growth inhibition Apoptosis induction (caspase-3) Up-regulation of genes |
Borowicki et al. (2010a) [59] |
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Apples | LT97 and HT29 (human colon adenoma and carcinoma) |
100-900 µg/mL 24-48 h |
Cell growth inhibition (LT97 more sensitive than HT29 cells) | Veeriah et al. (2007) [57] |
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Resistant starches | Differentiated Caco-2 (model of intestinal epithelia) |
10% (v/v) in culture medium 24 h |
Preservation of cell viability Prevention of H202 (75 µM/5 min)-induced DNA damage Maintenance of barrier function integrity (TEER) |
Fässler et al. (2007) [63] |
Wheat aleurone | HT29 (human colon carcinoma) |
10% (v/v) in culture medium 24-72 h |
Cell growth inhibition Cell cycle arrest in G0-G1 phase Apoptosis induction (caspase-3) |
Borowicki et al. (2010b) [60] |
Wheat aleurone | HT29 (human colon carcinoma) |
5-10% (v/v) in culture medium 24-72 h |
Induction of antioxidant enzymes (CAT and GST) Up-regulation of genes Prevention of H202 (75 µM/5 min)-induced DNA damage |
Stein et al. (2010) [31] |
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Bread |
HT29 (human colon carcinoma) |
2.5-5% (v/v) in culture medium 24-72 h |
Cell growth inhibition Prevention of H202 (75 µM/5 min)-induced DNA damage |
Lux et al. (2011) [61] |
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Bread | LT97 (human colon adenoma) |
5-20% (v/v) in culture medium 24-72 h |
Up-regulation of genes from DNA repair, biotransformation, differentiation and apoptosis Increase in GST activity, GSH content and AP activity (differentiation) Cell growth inhibition Apoptosis induction (caspase-3) |
Schölrmann et al. (2011) [62] |
Acknowledgments
This work was partially supported by Consolider Fun-C-Food CSD2007-00063 and the Generalitat Valenciana (ACOMP 2011/195).
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