Marie Menard and Golden apples affect gene expression in the colon mucosa of rats fed high fat diet: potential
mechanisms for chemoprevention
Research Article
Elisabetta Bigagli1*; Vanessa Pitozzi2; Cinzia Castagnini1; Simona Toti3; Catherine M.G. C. Renard4; Lisa Giovannelli1; Cristina Luceri1
1Department of Neuroscience, Psychology, Drug Research and Child Health - NEUROFARBA – Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, Florence, Italy2Chiesi Pharmaceuticals S.p.A., Parma, Italy3ISTAT, Rome, Italy4UMR 408 SQPOV Sécurité et Qualité des Produits d’Origine Végétale, INRA, Université d’Avignon et des Pays du Vaucluse, F-84000 Avignon, France
Abstract Apples are among the most common fruits consumed on a regular basis by many populations. This fruit contains high amounts of polyphenols, depending on the variety. A high fat diet (23% corn oil, w/w) and low cellulose (2%, w/w) or the same diet supplemented with 7.6% lyophilised apples obtained from two cultivars (Golden Delicious and Marie Ménard, low and high in polyphenols, respectively) was admin-istered to Wistar rats. After 4 weeks of feeding we did not find any effect on basal DNA oxidative damage, both in the liver and in the colon mucosa. On the contrary, apple supplementation affected the expres-sion profiles of the colon mucosa, modulating a large number of genes. Functional analysis suggested that Golden apples may affect cell proliferation; in fact we observed the down-regulation of the DNA replica-tion pathway and modulation of genes involved in TGF β receptor signalling. Marie Ménard apples, rich in proantocyanidins and in hydroxycinnamic acids, down-regulated genes associated with the inflammatory response, and several cytokine and integrin-mediated cell adhesion pathways. Our results suggest that apples may possess chemopreventive and anti-inflammatory activities particularly in populations at high risk for colorectal cancer.
Introduction Colorectal cancer is the fourth leading cause of cancer mortality worldwide [1]. Its aetiology in-volves the interaction of genetic and environmental factors. Apart from genetic predisposition, several life style-related factors such as red meat consumption, smoking and obesity account for more than 80% of the overall incidence [2]. High-fat-diet (HFD) has emerged as one of the leading environmental risk factors for the development of colon cancer as supported by epidemiological studies [3-5] as well as by experimental models in rodents [6,7]. Consumption of a HFD is associated with a chronic state of low-grade inflamma-tion, which is known to promote tumour development and growth and for this reason is considered a risk factor for colon cancer [8-10] even in the sporadic form [11,12]. Diet may also affect carcinogenesis by modifying colon mucosa cell proliferation and apoptosis [13,14].
Chemoprevention is based on the use of suitable pharmacologic or dietary agents such as micro and macro nutrients and non-nutritive phytochemicals to prevent or slow down the progression of cancer [16,17].
Epidemiological studies have shown that regular consumption of fruits and vegetables is associ-ated with reduced risk of cancer and of chronic pathologies such as diabetes, obesity and cardiovascular diseases [18-20]. As a result, health authorities now recommend eating five to ten portions of fruits and vegetables each day.
Apples are one of the most common fruits consumed on a regular basis by many populations [21]. It has been estimated that 22% of dietary phenolics consumed in the USA are derived from apples [22]. The success of apples can be explained by various factors, including flavour, availability in the market through-out the year, varieties of commercial preparations (fresh fruit, juice, puree, cider) [23].
Apples contain high amounts of polyphenols; Sun et al. [24] measured the phenolic content of a wide range of commonly consumed fruits and found that apples had the second highest phenolic content after cranberries. This amount changes depending on the variety [25,26]; the total amount of polyphenols that can be extracted from 100 g of fresh apples ranges in fact between 110 and 357 mg [27,28]. Even higher amounts can be found in cider apples [29,30].
Polyphenols exhibit a large variety of biological activities: they are powerful antioxidants and free radical scavengers, modulate cellular signalling processes, have anti-inflammatory activity and may modify the composition of intestinal microflora [31-35].
The majority of the studies focused on apples were performed using single apple compounds, ex-tracts or juices; this approach may not permit the identification of effects due to the presence in the apples of a unique combination of different phytochemicals or to synergisms with other apple components such as fibre. Our previous studies suggest that apples may have anti-inflammatory activity [36] and that poly-phenol-rich Marie Ménard (MM) apples reduce inflammation in a genetic model of Inflammatory bowel disease (IBD) [33]. Jedrychowski et al. [37] also showed that the risk of colorectal cancer was inversely correlated with the number of apple servings per day (OR = 0.37, 95% CI: 0.15-0.91).
Several case-control studies in Italy demonstrated a consistent inverse association between apple
consumption and the risk of various cancers, including colorectal cancer [38-40].
In the present analysis, we explored the effect of two varieties of apples with different proanthocya-nidin and hydroxycinnamic acid content, on rats fed a high fat diet, representative of an “at risk diet”, typical of Western countries and related to colorectal cancer development.
Materials & Methods
Diets and Animals: All the procedures adopted were carried out in agreement with the European Union Regulations on the Care and Use of Laboratory Animals (OJ of ECL 358/1, 12/18/1986) and the experi-ments were conducted according to Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 116/1992) after the ethical approval from the Italian Ministry for Scien-tific Research.
The experimental diets were prepared using components purchased from Piccioni (Gessate, Milan, Italy) and were based on the AIN76 diet, modified to contain high fat (23% corn oil, w/w) and low cellulose (2%, w/w) to mimic human Western diets. The apple-supplemented diet contained 7.6% of lyophilized apples and was modified to compensate for sucrose and fibres in the apple pulp. Golden Delicious apples, a common table variety, contained about 4 mg/g of proanthocyanidins and 0.5 mg/g of hydroxycinnamic acids, whereas Marie Ménard apples, a cultivar used for cider manufacturing, contained about 26 mg/g of proanthocyanidins and 6.3 mg/g of hydroxycinnamic acids (Table 1).
Male Wistar rats (Nossan, Correzzana, Milan, Italy) were divided into 3 groups of 10 animal each: controls (group C), rats fed a diet supplemented with Golden apples (group G) and rats fed a diet supple-mented with Marie Ménard apples (group MM).
After four weeks of treatment, rats were sacrificed and samples of liver and colon mucosa, (scraped manually from the connective layer with a glass slide) were harvested and stored in RNAlater (Qiagen, Mi-lan, Italy) at -800C.
Comet Assay: We used the comet assay as described [33] on samples of liver and colon tissue treated with a bacterial repair enzyme (formamidopyrimidine glycosylase, FPG, gift of A.R. Collins, University of Oslo, Norway), to determine oxidised DNA bases as FPG-sensitive sites. Data were analysed by one-way ANOVA, using GraphPad Prism software. Significance was assigned at p<0.05.
Microarray Analysis: Total RNA was extracted from the colon mucosa and from the liver using the RNeasy Midi kit (Qiagen, Milan, Italy). Equal amounts of RNA extracted from the colon mucosa and from the liver of control rats (n=6) were pooled and used as a common reference. We compared 12 RNAs, extracted from 6 rats for each experimental group (labelled with Cy5) to the pooled reference RNA (labelled with Cy3) (CyDye Mono-Reactive Dye Pack, Amersham, Cologno Monzese, Milan, Italy), for each type of tissue, using the indirect labelling method described by DeRisi (http://derisilab.ucsf.edu). All samples were hybridized using “in house” printed slides, constructed as previously reported (41) and containing about 6,000 spots.
The images were scanned using a Genepix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA); loading of the array list (to locate reporters on the microarray) and image analysis, were performed with the GenePixPro6.1 software. Microarray quality control, normalisation and differential analysis were performed as previously reported [41].
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To perform pathway statistics and visualize biological data on pathways affected by apple feeding, the visualization tool PathVisio, a freely available, open-source tool (http://www.pathvisio.org), was used.
Results
DNA Damage: Neither one of the apple varieties modified the level of oxidative damage in DNA isolated from colon mucosa and liver tissues, compared to control-fed rats (Figure 1).
Microarray Analysis: We first identified the number of genes differentially expressed in each group and in each tissue, by a t-moderated test. In the liver, we identified a very small number of genes modulated by the treatments, compared to controls: 2 genes were up- and 10 were down-regulated in rats fed Golden apples, and 2 genes were up- and 9 were down-regulated in rats fed Marie Ménard apples. None of these genes was modulated by both apple varieties.
In the colon mucosa, golden apples significantly (p<0.05) modulated 178 genes (135 up- and 43 down-regulated), whereas Marie Ménard apples modulated 979 genes, 748 up- and 231 down-regulated (Figure 2, panel A). Among these differentially expressed genes, 63 were modulated by both treatments, 5 genes being regulate in the opposite direction whereas the majority (58) in the same way (Figure 2, panel B).
Among the genes significantly up-regulated by MM compared to HF we found the repair enzyme 8-oxoGua DNA glycosylase 1 OGG1 with a Fold Change (FC) of 1.93 and the apurinic/apyrimidinic endonu-clease 1 (APEX1) (FC 1.85 vs HF). The adenomatous polyposis coli APC genes was also slightly up-regulated (FC 1.21 vs HF). The oncogenic gene RAS was slightly down-regulated (FC 0.8 vs HF).
Among the genes significantly modulated by both treatments we found the nuclear factor (eryth-roid-derived 2)-like 2 (Nr2f2) and heme oxygenase 2 (HMOX-2) with a FC of 1.53 and 1.36 respectively.
Functional analysis was performed only for the microarray data on colon, considering the lack of effect observed in liver gene expression profiles. In the colon, pathway analysis identified 10 maps signifi-cantly modulated by MM feeding: among them, we found several cytokine signalling pathways, the inflam-matory response, the integrin-mediated cell adhesion and the Delta-Notch pathways. Analysing the colon mucosa of G-treated rats we found the down-regulation of the DNA replication and the up-regulation of the TGF-β-receptor pathways (Table 1).
Discussion
The identification of effective preventive measures can significantly contribute to reduce the inci-dence and mortality of colon cancer. A consistent inverse association between apples and risk of various cancers has been reported [26,39].
Polyphenols are abundant in several foods that form the basis of the Mediterranean diet, have been associated with lower cancer incidence, and possess a number of health-promoting activities [31-33; 35] and apples are a rich source of these compounds [26].
The aim of the present study was to test the effect of the whole apple fruit, administered to rats as lyophilized powder, mixed with the diet. We used two variety of apples: Golden apples, easily available in
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the market, and Marie Ménard apples, used in the cider manufacturing, containing 6.5 times more proan-thocyanidins and about 12 times more hydroxycinnamic acids than the Golden variety.
The chemopreventive property of phytochemicals have been generally ascribed to their antioxidant activity via inactivation of reactive oxygen species, which plays a vital role in the initiation and progression of colon cancer [42].
In our model, neither one of the two cultivars was able to reduce basal oxidative DNA damage. Gra-ziani et al. demonstrated that polyphenols extracted from Annurca apples, rich in catechin, epicatechin and chlorogenic acid, prevented cell damage induced by indomethacin in rat gastric mucosa [43]. It is possible that the antioxidant effect of apples can be observed only when oxidative stress or cellular damage are ex-perimentally induced. However, we have demonstrated that 4-coumaric acid, a phenolic acid with antioxi-dant properties in vitro [44], added to a standard lab chow, was able to reduce basal oxidative DNA damage in the colon mucosa of healthy rats [45].
Apple dietary supplementation affected gene expression profiles in cells of the colon mucosa, while it did not modulate gene expression in liver cells. Considering that the most relevant effects on gene expres-sion were observed in Marie Ménard-fed rats, it is possible to speculate that they are due to the proantho-cyanidin content. Although proantocyanidin oligomers from apples were shown to be absorbed in the di-gestive tract and to be present in rat plasma [46], polymeric proanthocyanidins are poorly absorbed along the gastro-intestinal tract; consequently, they may have only local effects on the gut mucosa.
Functional analysis suggested that Golden apples may affect cell proliferation and extracellular ma-trix production. In fact, we observed down-regulation of the DNA replication pathway and modulation of genes involved in TGF beta receptor signalling. Among the genes belonging to this latter pathway, we found over-expression of regulatory genes, such as transforming growth factor beta (TGF-β), the cell cycle regula-tor p53 and the Fas death domain–associated protein DAXX, implicated in the modulation of apoptosis [47]. Down-regulation was observed for Jun activation domain-binding protein 1 (Jab1) and for the fifth subunit of the COP9 signalosome exhibiting oncogenic activity and recently found to be over-expressed in 80.3% of breast tumor tissues [48]. These data are in agreement with several in vitro studies, demonstrating that individual phenolic components of apples or apple extracts modulate cell proliferation. Eberhardt et al. [49] reported that apple extracts inhibited proliferation of human intestinal CaCo-2 cells at concentrations above 20 mg ml-1; on HepG2 liver tumour cells, tested at 50 mg ml-1, the inhibitory effect was greater in extracts containing apple skins. Veeriah et al. [50] found that a flavonoid mixture from apples inhibited proliferation of human intestinal HT29 cells and modulated the expression of genes associated with trans-formation of xenobiotics. Individual flavonoids (several of which have been detected in apples) have been shown to inhibit proliferation of CaCo-2 and HT29 cells and to increase apoptosis [51].
Marie Ménard apples also exhibit interesting effects on other cancer-relevant gene pathways. In the colon mucosa of MM-fed rats we observed a down-regulation of a number of genes encoding for phos-phatidylinositol 3’-kinases (PI3K), intracellular transducers involved in a wide range of cancer-associated signalling pathways [52]. The most pronounced effect was on pathways associated with the inflammatory response, where we found the down-regulation of several cytokines and pro-inflammatory genes. It has also been reported that proanthocyanidins modulate the expression of inflammatory genes such as COX2 and iNOS in murine macrophages stimulated with endotoxins [53,54], and apple extracts (200–2000 nM) produced increasing inhibition of the TNF-alpha signal via NF-kB in human endothelial HUVEC cells [55].
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We described the down-regulation of genes associated with the inflammatory response in the colon mu-cosa of rats fed A. thaliana seeds containing proanthocyanidins [41]. Our group [34] also demonstrated that high-polyphenol Marie Ménard apples reduce inflammation in a genetic model of Inflammatory bowel disease (IBD) by controlling the expression of Cox-2 and iNOS.
Among the genes significantly up-regulated by MM treatment compared to HF diet we found the repair enzyme 8-oxoGua DNA glycosylase 1 OGG1. OGG1 removes the oxidized purine from DNA as the first step in base excision repair and it is the second line of defence against oxidative DNA damage [56]. Few studies on the DNA repair genes OGG1 reported no changes in gene expression levels in vivo after a fruit and vegetable or antioxidant-rich diet [56]. Among BER gene polymorphisms, the S326C variant of OGG1, associated to decrease repair ability, was associated to a moderately increased risk of colorectal cancer [57].
The apurinic/apyrimidinic endonuclease 1 (APEX) is a key enzyme in base excision repair (BER) pathway, responsible for the repair of damaged DNA caused by oxidative reagents and endogenous and exogenous carcinogens [58].
We also found up-regulation of the intracellular redox-sensitive transcription factor Nrf2 and the constitutive heme oxygenase (HMOX2) in both G and MM fed rats. Nrf2 is essential for the protection against oxidative stresses but has also been known to attenuate inflammation [32]. HMOX2 is involved in DNA repair and its expression was induced by gallic acid in K562 cell line [59].
The slight, but significant up regulation of APC gene found in MM fed rats, is also another interest-ing finding of our study, since loss of function mutations in Apc are responsible for up to 90% of all cases of colon cancer [60]. The down-regulation of RAS gene is in line with the results reported by Psahoulia et al. [61] in quercetin-treated cells.
Most of the studies conducted so far on the health promoting activities of apples, were performed in experimentally induced colon cancer or in experimental model of inflammation and very few data are avail-able on the effects of apples on wild type animals [62]. Our data are among the first to demonstrate that one month apple supplementation affected the expression profiles of the normal colon mucosa, modulating a large number of genes involved in biological pathways relevant for colon cancer development.
We therefore suggest that apples may possess chemo-preventive and anti-inflammatory activities which may encourage the implementation of chemoprevention trials using food as a safer alternative to dietary supplements in high risk population such as those suffering from inflammatory bowel diseases or those consuming high fat diets typical of Western countries.
Acknowledgments
Supported by the European Network of Excellence in Nutrigenomics, NuGO (FOOD-CT-2004-506360), the EU programme FLAVO (FOOD-CT-2004-513960) (VP), Ministero dell’Università e della Ric-erca Scientifica e Tecnologica, Italy, University of Florence, Italy. The authors thank Dr. Carlotta De Filippo for the microarray printing.
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TablesJournal of Advances in Oncology Bigagli E et al.,
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Apple composition (mg/g) Golden Marie Ménard
Fibres 203.5 193.3
Hydroxycinnamic acids
caffeoylquinic 0.49 6.06
p-coumarylquinic 0.04 0.25
Flavan-3-ols
monomers 0.43 2.78
proanthocyanidins 4.03 26.07
degree of polymerization 1.98 3.6
Dihydrochalcones 0.38 0.62
Flavonols 0.05 0.3
Malic acid 20 11
Table 1: Polyphenolic composition of lyophilized Marie Ménard and Golden apples
Table 2: Results of the PathVisio analysis of pathways affected by apple feeding. All pathways with a p for pathway enrichment < 0.05 and with positive z-scores are shown
Figures
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