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Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain and * Department of Nutrition and Food Science, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain
2To whom correspondence should be addressed. E-mail: vnoe{at}ub.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • cDNA arrays • cocoa • epicatechin • gene expression • antioxidant
Flavonoids are naturally occurring compounds found in fruits, vegetables, and nuts with potential health benefits in reducing the incidence of various chronic illnesses, including cancer, stroke, and coronary heart disease (1–3). By virtue of their chemical nature, flavonoids can act as antioxidants and may help to maintain the body’s natural defenses against a variety of diseases associated with oxidative stress, such as inflammation, and proliferative and cardiovascular diseases (4,5). Waterhouse et al. (6) suggested for the first time that the intake of cocoa and chocolate could contribute to a large proportion of the dietary antioxidants. Since then, several reports have been published demonstrating that cocoa and its products can be sources of flavonoids (7–9).
The primary flavonoids in cocoa and chocolate are the flavan-3-ols, epicatechin and catechin (monomeric units), and polymers of these, the proanthocyanidins, also termed procyanidins (9). Depending on the method used in its production, cocoa powder can contain as much as 10% flavonoids on a dry-weight basis (5). The apparent bioavailabilty of flavonoids in humans ranges from 1 to 26%, depending on the chemical structure (e.g., quercetin, epicatechin, and soy isoflavones), and shows a large interindividual variability. Epicatechin from chocolate is rapidly absorbed (10), and the increase in its plasma concentration is dose dependent. Moreover, in addition to the monomeric flavonoids catechin and epicatechin, dimeric procyanidins have been detected in human plasma following consumption of flavonoid-rich cocoa (11).
The only data available about the contribution of cocoa and chocolate to the total flavonoid intake were reported by Arts et al. (12) in a Dutch nutritional survey indicating that cocoa products contribute in 20% of the dietary catechins intake in this population. According to the food consumption data of the Spanish Department of Agriculture, Fishery and Food, in children and teenagers, cocoa products can play an important role as a source of dietary flavonoids, because cocoa powder consumption in our country is 6–8 kg of cocoa powder per child per year (13). However, the benefit that this cocoa consumption level may represent due to its antioxidant properties has not yet been determined.
Flavonoids have multiple actions, both in vitro and in vivo. However, the exact mechanisms of action of the monomeric flavan-3-ols, oligomeric procyanidins, and their metabolites in vivo remain to be elucidated (5). In addition to the free-radical scavenging activity displayed by flavonoids, it is likely that these compounds could interact with intracellular signaling mechanisms to elicit a variety of biologic effects in the vascular and the immune systems.
Food components play a role in influencing, directly or indirectly, the expression of genes encoding proteins involved in energy metabolism, cell growth, and cell differentiation. Arrays represent a new powerful technology for high throughput screening of differential expression and create an exciting new field for nutrition and health care. According to Muller & Kersten (14), nutrigenomics attempts to study the genome-wide influences of nutrition and patterns of gene expression, protein expression, and metabolite production in response to particular compounds can be viewed as "dietary signatures." Several reports of the effects of food components that result in marked increases and suppression in the expression of multiple genes can be found in the literature [reviewed in (15)] In addition, functional genomics enable the comparison of the action of food components at the molecular level and provide tools to generate new hypotheses on their mechanism of action.
The objective of the present work was to perform a functional genomic analysis to study the effects of epicatechin, the main cocoa flavonoid, and cocoa powder extract on gene expression in human cells. We report several genes whose differential expression provides more information of the antioxidant properties of these compounds and suggests new mechanisms of action of flavonoids at the molecular level.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Epicatechin. (–)-Epicatechin was purchased from Sigma. Standard solutions were prepared in DMSO at a concentration of 10 mmol/L in dimmed light and were stored at –20°C.
Cell culture. The human colon adenocarcinoma cell line Caco-2 was used throughout the study as an experimental model, because it serves as a common in vitro model for estimating the fraction absorbed of compounds via the intestinal tract (18). Cells were grown in F-12 medium (Gibco) supplemented with 7% (v:v) fetal bovine serum (Gibco), 100,000 U/L sodium penicillin G, and 0.1 g/L streptomycin, and were maintained at 37°C in a humidified atmosphere containing 5% CO2.
To investigate the effects of epicatechin and cocoa extract on gene expression, the concentrations of these compounds used in this study were pharmacological in magnitude, based on dietary intakes reported in (14,15). Caco-2 cells (5 x 106) were incubated for 24 h with either 100 µmol/L (29.03 g/L) epicatechin or 0.2155 g/L of catechin equivalents of cocoa polyphenolic extract, both in DMSO [final concentration of DMSO in the medium <1% (v:v)]. The concentration of epicatechin used in the incubations has been shown not to be toxic for Caco-2 cells or to affect the cell cycle distribution (19).
cDNA arrays. Caco-2 cells were harvested, and total RNA was prepared by following the procedure recommended by Clontech. The integrity of the RNA (2 µg) was assessed after agarose gel electrophoresis in the presence of formaldehyde. Gene expression was analyzed by hybridization to specific cDNA arrays (Human Hematology/Immunology arrays from Clontech). This type of array was used because it contained genes related with stress response. The hybridization procedure has been described elsewhere (20). Array data analyses were performed by using the Atlas Image 2.01 software (Clontech) and the GeneSpring 6.1 program (Silicon Genetics) (20). The expression of each gene was reported as the ratio of the value obtained after each treatment relative to control after the normalization of the data (upregulated: ratio > 1.5; downregulated: ratio < 0.6). A cutoff of 1.5, representing a 50% overexpression or underexpression compared with the control, was chosen, because small changes in gene expression may represent important changes downstream of the differentially expressed genes. Lists of differentially expressed genes with a P-value <0.05 were generated by using data from 3 independent experiments for each condition. The genes in these lists were further classified according to their function.
Quantitative RT-PCR.
Levels of specific messenger RNAs (mRNA) were assessed by quantitative RT-PCR with [
-32P]-dATP to produce a radioactive product that could be detected with great sensitivity during the exponential phase of the reaction. The same cDNA preparations were amplified with primers for the different selected genes’ mRNAs. Adenosyl phosphorribosyl transferase (APRT) mRNA was used as a control for both the reverse transcription and the PCR reactions.
Total RNA was extracted from Caco-2 cells by using the Ultraspec RNA reagent (Biotecx) in accordance with the manufacturer’s instructions. RT-PCR reactions were typically carried out as described in Noé et al. (21). The primers used were as follows:
Preliminary experiments were carried out by using different numbers of cycles to determine the linear conditions of PCR amplification for all the genes studied: 18 cycles for FTH1 (generating a fragment of 338 bp), 20 cycles for TOP1 (333 bp); 22 cycles for C/EBPG (387 bp), BAT2 (325 bp), STAT1 (342 bp), and MLF2 (362 bp); 23 cycles for APRT (270 bp), 24 cycles for MAPKK1 (333 bp) and MRP1 (316 bp); and 25 cycles for XRCC1 (517 bp). The quantification of the intensity of the radioactive bands was carried out by phosphorimaging with the ImageQuant software (Molecular Dynamics). The signals corresponding to the APRT mRNA were used to normalize the changes in the levels of mRNA for each particular case.
Western blot analyses. Whole extracts were obtained from control or treated Caco-2 cells, according to Kraus et al. (22). A total of 5 µL of the extract was used to determine protein concentration by the Bradford assay [(23), Bio-Rad]. The extracts were frozen in liquid N2 and stored at –80°C.
Total extracts, 100 µg, were resolved on SDS-polyacrylamide gels (24) and were transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore) by using a semidry electroblotter. The membranes were probed either with anti-MEK-1 antibody C-18 at 1:50 dilution, anti-STAT1 p84/p91 antibody E-23 at 1:500 dilution, anti-TOP1 antibody H-300 at 1:500 dilution, or anti-MRP1 antibody H-70 at 1:100 dilution (all from Santa Cruz Biotechnology). Signals were detected by secondary horseradish peroxidase-conjugated antibody (1:5000 dilution) and enhanced chemiluminiscence, as recommended by the manufacturer (Amersham). Blots were reprobed with antibodies against ß-actin (Sigma) at a 1:2000 dilution or Oct-1 (C-21, Santa Cruz Biotechnology) at a 1:100 dilution to normalize the results.
Statistical methods. Array data analyses were performed through a parametric comparison by using all available error estimates as a filter based on the variances estimated by the Cross-Gene Error Model in GeneSpring 6.1 (Silicon Genetics). The Cross-Gene Error Model performs a variance components analysis for the accurate comparison of mean expression levels between experimental conditions (25). In this model, separate estimates of 2 different kinds of random variation are used to estimate the variability in gene expression measurements: 1) measurement variation, which comprises the lowest level of variation, corresponding to the variation of the measurement of a gene on a single chip around the true value that would be achieved by a perfect measurement of the expression level of the gene for that sample; and 2) sample-to-sample variation, which is variation between replicates that represents biological or sampling variability.
For the RT-PCR and Western blot analyses, values are expressed as the mean ± SE. Data were evaluated by unpaired Student’s t test, and analysis was performed by using InStat v.3.0 software (GraphPad Software). Differences with P-values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For epicatechin, 3 genes were analyzed from the list of upregulated genes: BAT2, C/EBPG, and FTH1; and 4 genes were analyzed from the list of downregulated genes: MAPKK1, MRP1, STAT1, and TOP1. For the polyphenolic cocoa extract, 2 genes were analyzed from the list of upregulated genes: BAT2 and MLF2; and 7 genes were analyzed from the list of downregulated genes, C/EBPG, FTH1, MAPKK1, MRP1, STAT1, TOP1, and XRCC1.
In epicatechin-treated cells, mRNA levels for FTH1 were increased by 54.6% compared with control cells. We also validated, by quantitative RT-PCR, the underexpression of MAPKK1 (68.5%), STAT1 (41.4%), and TOP1 (26.4%) upon incubation with epicatechin (Fig. 1). In cells treated with cocoa extract, MLF2 mRNA was increased by 337% compared with the control values. It was also demonstrated that treatment with a cocoa extract caused a decrease in the mRNA levels for C/EBPG of 82.1%, MAPKK1 of 36.8%, MRP1 of 47.3%, STAT1 of 68.3%, TOP1 of 35.7%, and XRCC1 of 13.8% (Fig. 2). All these changes were significant (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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MRP1 is a membrane protein that acts as a glutathione (GSH) S-conjugate efflux pump by transporting drugs that are conjugated with glutathione. Yamane et al. (26) described the induction of MRP1 at the mRNA level with pro-oxidants and its downregulation upon overexpression of antioxidant GSH. The underexpression of MRP1 mRNA and protein upon incubation with cocoa extract reported here could explain, at the transcriptional level, the inhibition of MRP1-mediated transport upon incubation with flavonoids observed by other authors (27,28).
MAPKK1 corresponds to the dual specificity kinase that phosphorylates extracellular regulatory kinase, ubiquitously expressed and critically involved in the regulation of signaling pathways involved in cell proliferation and differentiation. It was reported that an increase in reactive oxygen species production results in activation of MAPK pathways [reviewed in (29)]. Modulation of the MAPKs pathway by flavonoids has been reported by different authors (30–32). In addition, stimulation of MAPKs may be the potential signaling pathway used by green tea polyphenols (GTP) to activate antioxidant-response element-dependent genes (33,34) and could represent a mechanism for the protective effect attributed to GTP. In our conditions, the observed decrease in MAPKK1 mRNA and protein levels upon epicatechin incubation could be part of the mechanism to explain the antioxidant properties of this flavonoid.
Ferritin is a 24-subunit protein composed of 2 types of subunits, H and L, and plays a central role in the maintenance of the intracellular iron balance. Inflammatory cytokines, such as tumor necrosis factor and interleukin-2, upregulate ferritin synthesis, preferentially the H chains, thus determining an increase of the catalytic sites and a reduction of cell iron availability (35). Overexpression of the H subunit leads to a reduced production of reactive oxygen species after exposure to hydrogen peroxide, suggesting a role for ferritin in the protection against oxidative damage (36). In this direction, the overexpression of FTH1 mRNA observed upon epicatechin incubation could lead to the reduction of the reactive reduced iron pool in the cell as a protective mechanism against an oxidant injury.
STAT1 belongs to a family of transcription factors that transduce a signal from a cytokine receptor upon activation by phosphorylation of a specific tyrosine residue. STAT1 and STAT3 are activated by the oxidative stress induced by oxidized LDL, which can be prevented by the addition of vitamin E or N-acetylcysteine, suggesting that activation of STAT proteins can be regulated by the cellular redox status (37). The underexpression of STAT1 observed upon cell incubation with either epicatechin or cocoa polyphenolic extract could be due to the antioxidant properties of these compounds and may play a protective role in inflammation.
CAAT/enhancer-binding proteins (C/EBP) are a family of leucine zipper transcription factors. The binding activity and/or mRNA and protein levels of various C/EBPs are differentially modulated in response to inflammatory stimuli and recombinant cytokines. C/EBP-binding motifs have been identified in the functional regulatory regions of genes encoding the inflammatory cytokines, interleukines (IL) such as IL-6, IL-1b, and tumor necrosis factor , and other cytokines such as IL-8 and IL-12 (38,39). The ability of cacao polyphenols to modify the expression of different cytokines involved in immune responses both at the level of transcription and translation has been reported in several in vitro studies (40–43). In addition, cocoa flavonoids may have antiinflammatory properties in vivo as suggested by Osakabe et al. (44). The reduced expression of C/EBPG observed in cells treated with cocoa polyphenols could lead to the underexpression of a variety of genes containing C/EBP-binding motifs in their promoter sequences that would reduce the inflammatory response.
Human DNA topoisomerase I (TOP1) is a nuclear protein that plays a key role in DNA replication, transcription, and recombination. Because many neoplastic cells are characterized by high levels of TOP1 activity (45), this enzyme has become one of the cellular targets for anticancer therapy, and inhibitors for TOP1, e.g., camptothecin, have been used with clinical applications (46). The underexpression observed in TOP1 mRNA and protein levels upon incubation with either epicatechin or cocoa extract could decrease the final activity of TOP1 at the transcriptional level.
XRCC1 appears to operate as a molecular scaffold protein that interacts with and stimulates enzymatic components of single-strand break repair (47). XRCC1, through its BRCT I domain, interacts with poly (ADPribose) polymerase (PARP-1) (48). PARP-1 is required for assembly or stability of XRCC1 nuclear foci after oxidative DNA damage, in agreement with a model in which poly (ADPribose) synthesis serves to recruit XRCC1 to sites of DNA strand breakage (48). Both XRCC1 and PARP-1 are downregulated in Caco-2 cells upon incubation with cocoa extract, suggesting that the presence of these antioxidant flavonoids could prevent the oxidative DNA damage that would trigger PARP-1 and XRCC1 overexpression.
Little is known about the promyelodysplasia/myeloid leukemia factor 2 (MLF2). The MLF2 gene is highly related to the previously identified MLF1 (49), an oncogene involved in acute myeloid leukemia and myelodysplastic syndrome (50). In myeloid leukemia cells, the translocation t(3;5) t(3;5)(q25.1;q34) produces chimeric gene transcripts containing 5' sequences encoding nucleophosmin (NPM), fused in-frame to those of MLF1. MLF1 is normally located in the cytoplasm, whereas NPM-MLF1 is targeted to the nucleus, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in leukemia cells (51). The final effect of the overexpression of MLF2 observed upon cocoa extract incubation remains unknown, although it is worth mentioning that nucleophosmin mRNA is underexpressed upon incubation with either epicatechin or cocoa extract in the arrays.
In summary, by using cDNA arrays we determined the changes in gene expression upon incubation with epicatechin or cocoa extract. The differential expression of MRP1, MAPKK1, STAT1, and FTH1 could explain the antioxidant effect of flavonoids at the molecular level, because these genes are involved in different oxidative pathways. In addition, the changes observed in CEPBG, TOP1, MLF2, and XRCC1 expression could suggest novel mechanisms of action for these compounds. In the future, array analyses could be used as a screening for the study of the mechanism of action of food components at the molecular level and could serve as a basis for diet responsiveness.
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FOOTNOTES |
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3 Recipient of a predoctoral fellowship from the Ministerio de Ciencia y Tecnología, Spain.
4 Abbreviations used: APRT, adenosyl phosphorribosyl transferase; BAT2, HLA-B-associated transcript 2; C/EBP, CAAT/enhancer-binding protein; C/EBPG, CCAAT/enhancer binding protein gamma; DMSO, dimethyl sulfoxide; FTH1, ferritin heavy polypeptide 1; GSH, glutathione; GTP, green tea polyphenol; IL, interleukin; MAPK, mitogen-activated protein kinase; MAPKK1, MAPK kinase 1; MLF, myeloid leukemia factor; mRNA, messenger RNA; MRP1, ATP-binding cassette, subfamily c member 1; NPM, nucleophosmin; PARP-1, poly (ADPribose) polymerase; STAT1, signal transducer and activator of transcription 1; TOP1, DNA topoisomerase 1; XRCC1, x-ray repair complementing defective repair 1.
Manuscript received 16 April 2004. Initial review completed 24 May 2004. Revision accepted 11 July 2004.
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