1 Laboratoire d'Oncologie Nutritionnelle, Université Louis Pasteur EA 3430, Institut de Recherche contre les Cancers de l'Appareil Digestif (IRCAD), Strasbourg, France, 2 Unité de Recherches Cidricoles, Institut National de Recherche Agronomique (INRA), Biotransformation des Fruits et Légumes, Le Rheu, France and 3 Laboratoire de Pharmacognosie, Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France
* To whom correspondence should be addressed at: Department of Nutritional Oncology, IRCAD, 1, place de l'hôpital, BP 426, 67091 Strasbourg cedex, France. Tel: +33 388119023; Fax: +33 388119097; Email: francis.raul{at}ircad.u-strasbg.fr
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Abstract |
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Abbreviations: ACF, aberrant crypt foci; AdoMetDc, S-adenosylmethionine decarboxylase; AOM, azoxymethane; DMSO, dimethylsulfoxide; ERK1,2, extracellular signal-regulated kinases; JNK, c-jun N-terminal kinase; NP, nonprocyanidins; ODC, orinithine decarboxylase; P, procyanidins; PBS, phosphate-buffered saline; PKC, protein kinase C
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Introduction |
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Apples contain several classes of polyphenols: monomers, such as chlorogenic acid, (+)-catechin, ()-epicatechin, phloridzin, quercetin glycosides and other flavonoids, and condensed tannins, namely the procyanidins (4). The polyphenol content varies greatly from one variety to another, but procyanidins are predominant (5). They mainly consist of condensed ()-epicatechin units with a small proportion of (+)-catechin. Polyphenolic compounds are known to affect processes that are important for cancer development. Thus, they have antioxidant and scavenging effects on carcinogens, and may inhibit carcinogenesis by affecting molecular events in the initiation, promotion and progression stages (6).
In this work, we have evaluated the anti-proliferative mechanisms on human metastatic colon carcinoma-derived SW620 cells of two polyphenol fractions (monomers or oligomers) isolated from apples and studied the anti-carcinogenic properties of the most potent fraction in vivo. We present evidence to show that apple procyanidins trigger apoptosis, alter intracellular signaling pathways, and inhibit polyamine biosynthesis in human colon cancer cells, and that they antagonize promotion of colon carcinogenesis in rats.
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Materials and methods |
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The TPE extract was fractionated into fraction P (Procyanidins) and fraction NP (Non Procyanidins) on a fractogel column according to a method adapted from Souquet et al. (7). Polyphenols of fraction P were characterized and quantified by thiolysis, coupled with reverse-phase HPLC with diode array UV-visible detection. Fraction NP was analysed by reverse-phase HPLC without thiolysis (8).
On a weight basis, fraction NP contained 72.6% identified polyphenols: flavonoids (30.1%) and hydroxycinnamic acids (42.5%). Fraction P contained 78.4% procyanidins. Procyanidins present in fraction P mainly consisted of ()-epicatechin (95%) and (+)-catechin was found in a small proportion (5%). The mean degree of polymerization was close to 4. Fraction P was almost totally devoid of monomeric catechins and other phenols (<2%).
Cell culture
SW620 cells were obtained from the European Collection of Animal Cell Culture. Cells were seeded at 1 x 104 cells per well in 96-well plates or 1 x 106 cells in culture dishes (100 mm diameter). They were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 mM glucose, glutamax and supplemented with 3% heat-inactivated (56°C) horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% non essential amino acids, 5 µg/ml transferrin, 5 ng/ml selenium and 10 µg/ml insulin (Gibco, Invitrogen, Cergy-Pontoise, France).
Cell growth rate
Stock solutions of the fractions were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO in the culture medium was 0.1%. Cells were exposed to fractions NP or P 24 h after seeding and incubated for different times. Control cells were treated with 0.1% DMSO. Culture media were replaced every 48 h. Cell culture was stopped by the addition of trichloroacetic acid (50% v/v), and cell proteins were determined by staining with 200 µl Sulforhodamine B (SRB, 0.4%, w/v) (Sigma-Aldrich, Saint Quentin Fallavier, France). The relationship between cell number (protein content/well) and absorbance is linear from 0 to 2 x 105 cells per well.
Cell cycle analysis
Cell cycle distribution was analysed by labelling cells with propidium iodide (P.I.). Assays were carried out as described by Nicoletti et al. (9). Briefly, 1 x 106 cells were seeded in 10 mm plates and harvested by trypsinization (0.5% trypsin/2.6 mM EDTA) at different time points after initial treatment with fraction P. Then cells were centrifuged and fixed in 1 ml methanol:phosphate buffered saline (PBS) (9:1, v/v), washed twice in PBS and re-suspended in 200 µl PBS containing 0.25 mg/ml RNAse A and 0.1 mg/ml P.I. (Sigma-Aldrich, Germany). After incubation in the dark at 37°C for 30 min, the fluorescence of 10 000 cells was analysed using a FACScan flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA).
Caspase-3 activity
Attached and floating cells were harvested, washed twice in PBS and stored at 20°C. Caspase-3 activity was detected by using Caspase-3 and Caspase 8 Assay Colorimetric Kits (Sigma-Aldrich, Germany). The assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in the release of a pNA moiety. The concentration of the pNA released was calculated from the absorbance values at 405 nm and the calibration curve of defined pNA solutions. Results were adjusted to the total protein content, and activity was expressed as nmol pNA/mg of total protein.
Signal transduction pathways
The determination of total (non phosphorylated) and activated (phosphorylated) extracellular signal-regulated kinases 1 and 2 (ERK1,2) and of c-jun N-terminal kinase (JNK) was performed using Fast Activated Cell-based ELISA (FACE) Kits (Active Motif, Rixensart, Belgium). SW620 cells were exposed to 0.1% DMSO (control), or to fraction P (50 µg/ml) for 24 h and then incubated with a primary antibody that recognizes either total or active ERK1,2 or JNK. Protein kinase C (PKC) activity was measured by using the MESACUP Protein Kinase Assay Kit (MBL International Corporation, Watertown, MA).
Measure of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities
Cells were homogenized in 100 mM TrisHCl buffer, pH 7.4. After centrifugation at 33 000 g for 25 min at 4°C, ODC and AdoMetDC assays were performed in the supernatants. ODC activity was measured by the rate of 14CO2 formation from [1-14C]L-ornithine (55 mCi/mmol, Amersham, Orsay, France) and AdoMetDC activity by measuring the rate of 14CO2 formed from [1-14C]S-adenosylmethionine (60 mCi/mmol, Amersham) (10).
Animals and treatments
The experiments were conducted according to the National Research Council Guide for use and care of laboratory animals with the authorization (No. 00573) of the French Ministry of Agriculture.
Male Wistar rats (n = 16) weighing 230245 g were housed under standardized conditions (22°C; 60% relative humidity; 12 h light/12 h dark cycle, 20 air changes/h) and fed a standard diet with free access to drinking water. All animals received intraperitoneal (i.p.) injections of azoxymethane (AOM)15 mg/kg body wt once each week for 2 weeks. One week after the last injection of AOM, rats were randomly separated into two groups. One group (n = 8) received daily 0.01% of the apple procyanidin mixture (fraction P) in water as drinking fluid. The AOM-treated control group (n = 8) received drinking water.
After 6 weeks, the colon of the animals were collected. The determination of aberrant hyperproliferative crypts was performed on a segment 5 cm in length corresponding to the distal part of the colon. The segment was washed with physiological saline, cut open, pinned out flat and fixed in 10% buffered formalin. The colon was stained with 0.2% methylene blue for 5 min, rinsed in KrebsRinger buffer, placed onto a glass slide and examined microscopically using a low power objective (5x) for the assessment of the number of hyperproliferative crypts and aberrant crypt foci (ACF). The criteria for the identification of hyperproliferative aberrant crypts were: (i) an increased size; (ii) a thicker epithelial cell lining; and (iii) an increased pericryptal zone relative to normal crypts.
Statistics
Data are reported as mean ± standard error of the mean (SE). Statistical differences between groups were evaluated by one-way ANOVA and specific differences were identified using the Student's t-test.
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Results |
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Activation of caspase-3
The activity of caspase-3 was measured in SW620 cells exposed to fraction P (50 µg/ml) for 48 and 72 h. As shown in Figure 1C, activation of caspase-3 was observed in cells exposed to procyanidins, which paralleled the increase observed in the population of hypodiploid cells whereas no caspase-3 activity was detected in untreated cells.
Perturbation of signal transduction pathways
We studied the effects of apple procyanidins on the amount of inactive and active phosphorylated forms of ERK1,2 and JNK. After 24 h of exposure to fraction P (50 µg/ml), a significant (P < 0.05) increase of the amount of total (non-phosphorylated) and active phosphorylated forms of ERK1,2 and of JNK were observed (Figure 2A). Apple procyanidins caused also a downregulation of PKC, leading to a 50% decrease of the enzyme activity (Figure 2B).
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Discussion |
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In studies using single layers of Caco-2 cells as a model of intestinal absorption, it was shown that only the dimers and to a lesser extent the trimers of catechins or epicatechins are able to cross the intestinal epithelium (12). It was reported that procyanidins are interacting with the cell membrane and cytoskeletal constituents of skin fibroblasts (13). These effects may explain in part the pharmacological action of the procyanidins. Thus, most of the procyanidins present in fraction P are presumed to interact with the cell membrane thereby perturbating signal transduction pathways. Indeed, our results show that the amount of inactive and active phosphorylated forms of ERK1,2 and JNK are significantly enhanced in cells exposed to the procyanidins.
Until recently the general view was that activation of the ERK pathway drives a survival signal that counteracts the pro-apoptotic effects of JNK (14). However, several recent reports indicate that activation of ERK may be important for the induction of apoptosis in cancer cells by various cytotoxic agents (15,16). Our data are in agreement with these findings.
There is also increasing evidence for a crosstalk between MAPK pathways (ERK, JNK, p38MAPK) and PKC. Several inhibitors of PKC cause apoptosis by a mechanism that involves a strong and sustained activation of ERK (17). PKC controls signal transduction pathways involved in the regulation of cell growth. It is a known target for a number of tumour promoters and is activated by the translocation of the enzyme from the cytoplasmic compartment to the cell membrane (18). In the present study we show that a 50% decrease of PKC activity is observed in SW620 cells exposed to fraction P. Procyanidinmembrane interactions may perturb PKC translocation from cytosol to membrane leading to a down-regulation of the enzyme.
Changes in PKC activity may also modulate polyamine biosynthesis. Indeed, it was reported that PKC inhibition by staurosporine inhibited both the induction of ODC and the promotion by phorbol ester of skin tumorigenesis (19). Inversely transgenic mice that overexpress PKC showed a 3- to 4-fold higher expression of phorbol ester-induced epidermal ODC when compared with wild-type littermates (20). In the present report we show that the downregulation of PKC triggered by apple procyanidins was associated with a 50% decrease of ODC and of AdoMetDC activities, the two key enzymes of polyamine biosynthesis. This may also contribute to the pro-apoptotic effects triggered by procyanidins since it has been shown that polyamine depletion may also induce apoptosis in tumour cells (21).
Procyanidins, due to their polymeric structure, are poorly absorbed along the gastro-intestinal tract and may exert only local activity. These compounds are among the most abundant dietary polyphenols and their concentration in the colon may reach several hundred micromoles per litre (22). Procyanidins and a few carotenoids constitute the only dietary antioxidants present in the colon because phenolic monomers and vitamins C and E are absorbed in the upper segments of the intestine (23). Based on the anti-proliferative and apoptotic effect of apple procyanidins at the cellular level, we hypothesized that oral administration of apple procyanidins may be appropriate to target the colonic mucosa, allowing these chemicals to act locally as chemopreventives. Therefore, we tested the effects of fraction P in a rat model of colon carcinogenesis. Colon carcinogenesis was induced in rats by i.p. administration of AOM. Administration of AOM causes morphological changes ranging from normal colon epithelium to carcinoma that are biologically quite similar to those seen in humans. One week after the last injection, one group of rats received apple procyanidins in their drinking water. The amount of procyanidins consumed daily by rats corresponded approximately to 6 mg per kg body wt. Considering the fact that the amount of procyanidins in dessert apples was shown to range between 0.4 and 0.8 g per kg in the flesh and between 1.3 and 2.8 g per kg in the skin (5), the amount (per kg body wt) of procyanidins ingested by the rats was close to the amount ingested by humans consuming daily two apples (4 to 10 mg per kg body wt).
Because of the potential progression of early lesions to malignancy, the premalignant hyperproliferative crypts and ACF formation initiated after AOM administration are important markers of the pathogenesis of colon cancer (24). We observed after 6 weeks, a 50% reduction in the total number of hyperproliferative crypts and of ACFs on the surface of the colon of rats receiving apple procyanidins in their drinking water. This indicates that apple procyanidins given in the drinking fluid inhibit the promotion/progression phases of colon carcinogenesis in addition to their potential protective effects as antioxidants in cancer initiation (3).
Taken together, our observations suggest the use of apple procyanidins in combination trials for colon cancer chemoprevention. In contrast with absorbable drugs, these natural, non toxic, dietary constituents can reach the colon, where they will be able to exert their antitumour effects.
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Supplementary material |
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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References |
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