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Bioscience Horizons Advance Access originally published online on April 16, 2009
Bioscience Horizons 2009 2(2):172-179; doi:10.1093/biohorizons/hzp020
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells

Caroline Saunders*

Department of Food Biosciences, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK

* Corresponding author: 47 Beech Lane, Earley, Reading, Berkshire RG6 5PT, UK. Tel: +44 1189 671022. Email: caroline.saunders50{at}ntlworld.com

Supervisor: Dr Jeremy P.E. Spencer, Molecular Nutrition Group, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 Author Biography 
 
Epidemiological evidence suggests that diets rich in fruit and vegetables protect against the development of colon cancer, primarily due to their high levels of polyphenols, in particular a group known as flavonoids. Tomatoes contain significant amounts of the glycosides of the flavonoids quercetin and naringenin. Both quercetin and naringenin have been shown to exert anti-proliferative effects on colon cancer cells, although the effects of whole tomato polyphenol extracts are unclear. The aim of this study was to determine the effect of three tomato varieties, with differing levels of flavonoids and total phenolics, on the proliferation of human colon adenocarcinoma cells. We show that all three varieties were able to significantly inhibit the growth of colon cancer cells, although this activity was found not to be linked to the levels of the flavonoids rutin and naringenin in the tomatoes, but rather to their total phenolic content. We show that the mechanism of growth inhibition was linked to the effects of tomato phenolics on the cell cycle, in that exposure of cells to the tomato extract induced a reduction in the percentage of cells in the S-phase, i.e. those actively synthesizing DNA. These data indicate that tomatoes may help to prevent colon cancer but that their flavonoid content does not appear to determine the magnitude of their biological effect.

Key words: colon cancer, tomato, flavonoids, naringenin, rutin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 Author Biography 
 
Colorectal cancer is one of the major cancers of the Western world with countries such as Australia and New Zealand having a 20-fold higher incidence than countries in Middle Africa.1 The two major risk factors in colon cancer aetiology have been proposed, genetic disposition and diet, although the variability between countries appears to result from the latter as migrant populations develop a similar colon cancer incidence to that of their new country within a generation.1 Indeed, epidemiological evidence recorded between 1966 and 1996 has suggested very strong links between diet and cancer of the breast, colorectum and prostate.2 One of the reasons that the colon is so susceptible is due to its direct exposure to dietary carcinogens such as N-nitroso compounds, such as those present in preserved meats, which cause DNA damage and lead to mutations and the initiation of carcinogenesis.3 In contrast, diets high in fruit and vegetables appear to provide protection against cancer development due to their levels of fibre and their high contents of phytochemicals, in particular flavonoids.4

Flavonoids are secondary plant metabolites, which are synthesized from phenylalanine via the Shikimate Pathway, and express many functions within the plant, including regulation of growth and UV protection.5 The basic structure of flavonoids consists of two benzene rings connected by an oxygen-containing pyrene ring (Fig. 1). On the basis of variations in the saturation of the basic flavan ring system, their alkylation and/or glycosylation and the hydroxylation pattern of the molecules, flavonoids may be divided into seven subclasses: flavonols, flavones, flavanones, flavanonols, flavanols, anthocyanidins and isoflavones (Fig. 1).6 Evidence indicates that flavonoids express a wide variety of biological activities including antioxidant nature,7 anti-inflammatory6 and anti-aggregatory8 effects and an ability to inhibit cancer cell growth.9–12 Tomatoes (Lycopersicon esculentum) are one of the world's common vegetables and contain significant amounts of the flavonol, rutin (quercetin 3-rutinoside) and the flavanone, naringenin and naringenin chalcone.13, 14 Rutin is present in tomato as a glycoside and as such undergoes very limited absorption in the small intestine, as the β-glycosidic bond of the rutinose moiety is resistant to hydrolysis by digestive enzymes.15 However, enzymes produced by the colonic gut microflora are capable of breaking this glycosidic linkage to yield quercetin, which has been shown to be bioavailable.9, 16 Flavonoids that are not well absorbed in the small intestine and the major proportion of ingested flavonoids pass to the large intestine where they may express biological function on host cells and bacteria.17 This is also true of naringenin which is produced from naringenin chalcone during tomato processing.6


Figure 1
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  		Figure 1. Basic structure of flavonoids consisting of two benzene rings connected by an oxygen containing pyrene ring. Sub-classes of flavonoids: flavonols, flavanones, flavanols, anthocyanidins and with differing R groups that give each flavonoid its unique structure.

 
A number of case–control studies have found an inverse relationship between tomato intake and the risk of developing colorectal cancer.18, 19 Furthermore, in vitro and in vivo studies have demonstrated a wide range of biological activities related to tomato flavonoids, with quercetin and rutin able to inhibit abnormal colonic growths in rats20 and quercetin chalcone, capable of reducing the size of implanted colon tumours in mice.21 In humans, quercetin, in combination with the polyphenol curcumin, was shown to reduce the number and size of ileal and rectal adenomas in patients with familial adenomatous polyposis (FAP).22 Furthermore, quercetin, but not rutin, has been shown to express anti-proliferative effects on human colon cancer cell lines, in a dose-dependent and time-dependent manner.9, 10, 23, 24 Their ability to induce apoptosis9, 23 and/or evoke cell cycle arrest25–27 have been proposed as potential anti-cancer mechanisms. The latter has been proposed to occur via their ability to regulate cyclins, a family of proteins that are positive regulators of the cell cycle and which are commonly over-expressed in cancer.3 In response to growth factors and mitogens, cyclins associate with cyclin-dependent kinases (CDKs), which then phosphorylate cellular proteins resulting in the progression of the cell through the cell cycle.

A tomato digestate containing both rutin and naringenin chalcone has been shown to arrest cell cycle progression at the G0/G1 phase in colon adenocarcinoma cells through decreased expression of cyclin D1, a positive regulator of the cell cycle.27 Furthermore, many flavonoids present in tomatoes have been shown to be capable of inhibiting the cell cycle and modulating cyclin expression. For example, 2'-OH flavanone, a flavonoid with structural similarities to naringenin, induces cytotoxic effects on three colorectal carcinoma cells via a mechanism dependent on increased expression of p21, a potent CDK inhibitor capable of blocking cells in the G1/S phase of the cell cycle.28 In addition, quercetin has been shown to block the cell cycle at the G1/S phase in human colon cancer cells, by inhibiting the synthesis of a 17 kDa protein.26 In this study, we have determined the major flavonoid content and total polyphenol levels of three different tomato varieties and related this to their ability to inhibit the growth of human epithelial colorectal adenocarcinoma cells. In addition, we have attempted to determine how they act via assessment of their capability to induce cell cycle arrest.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 Author Biography 
 
Chemicals and Reagents
Human epithelial colorectal adenocarcinoma cells (Caco-2) were obtained from ECACC (Salisbury, Wiltshire, UK). Methanol [high-performance liquid chromatography (HPLC) grade] and N, N-dimethylformamide (analytical grade) were purchased from Fisher (Loughborough, UK); Anti-bromodeoxyuridine (BrdU) Pure was purchased from Becton Dickinson (Oxford, UK); DMEM was purchased from Cambrex Bio Science (Wokingham, UK). All other cell culture components were from Gibco Invitrogen (Paisley, UK). All other chemicals were from Sigma-Aldrich (Gillingham, UK).

Tomato Samples and Extraction
Lyophilized tomato samples were supplied by Professor Peter Bramley, School of Biological Sciences, Royal Holloway, University of London. All samples were glasshouse grown under supplementary lighting. Three samples were used, including a sample selectively bred to be high in polyphenols, a low flavonoid mutant and a traditional Ailsa Craig variety. All samples were stored in the dark at room temperature. Polyphenols, hydroxycinnamates, flavonoids and their glycosides were extracted from lyophilized tomato by three sequential aqueous/methanol extractions. A methanol/water (2:1) solution was prepared and a total volume of 1 ml added to 100 mg lyophilized tomato. Samples were vortexed continuously for 30 min using a vibrating agitator. Samples were then microfuged at 10 000 rpm for 5 min at 4°C and the supernatants collected. The pellet was re-suspended in 1 ml of the aqueous methanol solution and the procedure repeated a further two times. Samples were purged with argon and protected from light throughout the extraction.

Quantification of Total Phenolics
The total phenolic content of the aqueous methanolic extract of each tomato sample was obtained using the Folin–Ciocalteau Micro method as previously described.29 Twenty microlitres of the tomato extracts (10 mg/ml) were mixed with 1.58 ml water and 100 µl of the Folin–Ciocalteu reagent, vortexed for 5 min. Following this, 300 µl of sodium carbonate solution (250 µg/ml) was added and the reactants were mixed and left at 4°C for 30 min. Absorbance was determined at 765 nm against the blank, and a gallic acid calibration curve (0–500 mg/l) was constructed and used to determine the total phenolic content of the samples, expressed as Gallic Acid Equivalents (GAE).

Assessment of Flavonoid Levels
Reverse phase HPLC (RP-HPLC) was performed to characterize and quantify the flavonoids rutin and naringenin. A Hewlett Packard (Agilent, Bracknell, UK) model 1100 series LC running HP ChemStation software with a Nova Pak C18 column (250 x 4.6; 4 µm) (Waters, Elstree, UK) was used to separate the phenolic constituents. The mobile phase consisted of (A) aqueous methanol (5%) containing HCl (0.1%) and (B) acetonitrile (50%) containing HCl (0.1%) and was pumped through the column at 1 ml/min with an injection volume of 50 µl. The following gradient system was used over a time course of 60 min (%A/%B): 95/5, 50/50, 0/100 and 95/5. HPLC profiles of extracts were measured by a diode-array detector set at four wavelengths: 254, 280, 320 and 365 nm and fluorescence detection at excitation 276 nm, emission 316 nm. Flavonoids were identified by matching their retention times and UV diode array spectra with those of authentic compounds. Calibration curves of rutin and naringenin were constructed using authentic standards (0–100 µM) and in each case were found to be linear with correlation coefficients of 0.998 (rutin) and 0.999 (naringenin).

Cell Culture, Treatment and Assessment of Proliferation
Caco-2 cells were grown as monolayers in T-75 culture flasks (Greiner-Bio-One, Frickenhausen, Germany). Cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 20% heat-inactivated foetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/ml penicillin and 100 µg/ml streptomycin and stored in a humidified 37°C incubator with 5% carbon dioxide. Medium was changed every 2 days and cells were passaged every 7 days using trypsin-versene (EDTA). Prior to the addition of tomato extracts, cells were seeded in 12-well plates (1.2 x 104cells/well). When cells were 30% confluent, they were exposed to the tomato extracts (0.33–3.3 mg). Following 24, 48, 72 and 96 h, cells were fixed and cell biomass was determined using the sulforhodamine B (SRB) assay, as described previously.30 Cells were fixed by the addition of 125 µl ice-cold TCA (10% final concentration; 4°C; 1 h). After fixing, media was removed, cells were washed and total biomass was determined using SRB (250 µl of 0.4% SRB; 0.5 h). Unincorporated dye was discarded by washing with 1% acetic acid, while cell incorporated dye was solubilized using Tris Base (10 mM, pH 10.5). Dye incorporation, reflecting cell biomass, was measured at 492 nm, using a GENios microplate reader (TECAN, Reading, UK).

Cell Cycle Analysis
Caco-2 cells were seeded in 12-well plates (1.2 x 104cells/well) and grown until 30% confluent before exposure to tomato extracts (1.7 mg/ml; 48 h). After 48 h of exposure, BrdU (10 mM) was added and cells were incubated at 37°C for 30 min, prior to washing (PBS) and harvesting of cells (250 µl trypsin/well). Cells were re-suspended in ice-cold 70% ethanol (1 ml) and stored at 4°C for 24 h. Cells were centrifuged at 2000 rpm (5 min; 4°C), the ethanol removed and the cells re-suspended in HCl (0.1 M; 10 min) in order to permealize them. After washing, cells were incubated for 60 min with a BrdU antibody (5 µg/ml) in PBS containing 0.5% Tween 20, 1% foetal calf serum (FCS). Cells were then washed (PBS) and centrifuged (2000 rpm; 10 min; 4°C) prior to removal of the supernatant and addition of FITC-conjugated rabbit anti-mouse immunoglobulin (0.1 mM) in PBS, containing 0.5% Tween 20 and 1% FCS. This was mixed and incubated in the dark for 30 min. Following a final wash step (PBS) and centrifugation (2000 rpm; 10 min; 4°C), the supernatant was removed and propidium iodide (50 µg/ml) in PBS containing RNase (10 mg/ml) was added. The number of proliferating cells was determined by flow cytometry using a FACS Calibur benchtop flow cytometer (Becton-Dickinson, Oxford, UK) equipped with a 15 mW blue argon laser source (excitation wavelength: 488 nm) and were analysed using CellFIT Cell-Cycle Analysis Version 2.0.2 software.

Statistics
All data are expressed as means ± SD. The results of the Folin–Ciocalteu assay were subjected to a one-way ANOVA with Tukey's post hoc test. Statistical analysis of Caco-2 proliferation in response to tomato treatments was performed using a two-way ANOVA with Dunnet's post hoc test. Differences were considered significant when p < 0.05. All statistical analyses were performed using SPSS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 Author Biography 
 
Polyphenolic Content of the Tomato Extracts
Quantification of phenolics was determined by the Folin–Ciocalteu Assay and statistical analysis using a one-way ANOVA with Tukey's post hoc test. Results were expressed as GAE and indicated that the Ailsa Craig variety had a significantly higher phenolic content (p < 0.001) compared with the selectively bred high-polyphenol variety and the low-polyphenol mutant (Fig. 2). However, the high-polyphenol variety had significantly higher levels of total phenolics compared with the low-polyphenol mutant (p < 0.05) (Fig. 2).


Figure 2
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  		Figure 2. Total phenolic content (expressed as equivalents of gallic acid) of three different varieties of lyophilized tomatoes determined by the Folin–Ciocalteu assay and statistical analysis using a one-way ANOVA. Data are means of three technical replicates of each sample (mean ± SD; n = 3). A significant difference in total phenolic content between samples is represented by: *p < 0.05 and ***p < 0.001.

 
Flavonoid Identification and Quantification
HPLC analysis (365 nm) indicated that all three tomato varieties revealed that three major flavonoids were present, as defined by the appearance of peaks at detectable at 31.7, 34.1 and 50.4 min (Fig. 3). The Ailsa Craig variety (Fig. 3Ai) had significantly higher levels of rutin (quercetin-3-rutinoside) (RT: 34.1 min) and naringenin chalcone (RT: 50.4 min) than either the low mutant (Fig. 3Aii) or high-polyphenol tomatoes (Fig. 3Aiii). The tomatoes also contained another, more polar, flavonoid with similar spectral characteristics to rutin (RT: 31.7) (Fig. 3). Although we were unable to characterize this compound, it is likely to be either a more polar glycosidic derivative of quercetin or a myricetin glycoside (Fig. 1). LC-MS/MS is underway to identify this product. In quantitative terms, the Ailsa Craig variety had much higher levels of both rutin and naringenin chalcone compared with the low mutant or high-polyphenol varieties (Fig. 3B). Although our extraction and separation protocol is capable of revealing the hydroxycinnamates, all three tomatoes were found not to contain significant levels (Fig. 3).


Figure 3
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  		Figure 3. (A) RP-HPLC chromatogram recorded at 365 nm of tomato extracts (concentration 10 mg/ml), (i) Ailsa Craig; (ii) low polyphenol and (iii) high polyphenol. (B) Content of flavonoids, rutin and naringenin derivatives, in tomatoes extracted with aqueous methanol (2:1) and expressed as µg/g lyophilized tomato. Quantified using ‘area-under-curve’ analysis relative to an internal standard and by comparison with calibration curves of rutin and naringenin.

 
Effect on Colon Adenocarcinoma Cell Proliferation
The cytotoxic effects of the three tomato extracts were indicated by a reduction in cell biomass compared with vehicle-treated cells which was determined by the SRB assay. A statistically significant (p < 0.05) inhibition of cell growth was apparent following exposure to Ailsa Craig (Fig. 4A) and high polyphenol (Fig. 4B) at both concentrations and at both time points and the low polyphenol (Fig. 4C) after 72 h exposure only. At the 72 h exposure time point, there was a statistically significant difference in the ability of the Ailsa Craig extract to inhibit cell proliferation compared with the low-polyphenol sample (p < 0.05) (Fig. 5). Cells exposed to the high-polyphenol tomato extract (1.7 mg/ml) and vehicle-treated cells were analysed by flow cytometry for the number of cells actively synthesizing DNA (i.e. in the S-phase of the cell cycle) (Fig. 6). Emission spectra of FITC indicated a shift in cell cycle distribution in cells exposed to the high-polyphenol extract, as indicated by a decreased cellular incorporation of BrdU (Fig. 6).


Figure 4
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  		Figure 4. Growth inhibition of Caco-2 cells induced by each methanolic tomato extract treatment. Black bars: 0.5 mg/ml and grey bars: 2.5 mg/ml, after 48 and 72 h initial exposure. (A) Ailsa Craig (B) high polyphenol and (C) low polyphenol. All values are given as means ± SD (n = 4). A significant difference of growth inhibition at an individual time point and concentration, compared with vehicle-treated cells, is represented by: *p < 0.05; **p < 0.01 and ***p < 0.001.

 


Figure 5
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  		Figure 5. Comparison of growth inhibitory effect of the three different tomato varieties, on proliferation of Caco-2 cells, relative to vehicle-treated cells, after 72 h of exposure to sample. Values are given as means ± SD (n = 4). A significant difference of growth inhibition between samples is represented by: *p < 0.05.

 


Figure 6
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  		Figure 6. Effect of high-polyphenol methanolic tomato extract on active DNA synthesis determined by the incorporation of BrdU into cell DNA during S-phase of cell cycle. Analysis was by flow cytometry. Black line: vehicle-treated cells; red line: high-polyphenol extract showing a decrease in cells in DNA synthesis phase (S-phase) of the cell cycle (n = 1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 Author Biography 
 
A number of case–control studies have shown an inverse relationship between tomato intake and the risk of developing colorectal cancer.16, 17 Furthermore, in vitro and animal studies have shown that tomatoes, and phenolic compounds present in tomatoes, are able to inhibit colon cell proliferation.25 In this study, we demonstrate that three different tomato extracts are capable of significantly inhibiting colon adenocarcinoma cell growth. However, this ability to effect growth did not appear to be dependent on the flavonoid content of the tomatoes. The most abundant flavonoids in the tomatoes we studied were the flavonol glycoside, quercetin 3-rutinoside (also known as rutin) and the flavanone, naringenin chalcone.13, 14 Interestingly, although the high-polyphenol variety had been selectively bred to have an increased content of polyphenols, the traditional Ailsa Craig variety had higher levels of total polyphenols and >10 times the rutin and naringenin chalcone levels than this tomato. The low-polyphenol mutant as expected had lower levels of total phenolics compared with both other varieties and also had very low levels of rutin and undetectable levels of naringenin chalcone. Although all the tomatoes induced anti-proliferative effects, the degree of growth inhibition (Fig. 5) appeared to be dependent on the total phenolic content of the tomatoes (Fig. 2) rather than their levels of rutin and naringenin (Fig. 3B). For example, the large difference in levels of rutin and naringenin in the Alisa Craig variety compared with the other two tomatoes was not reflected in their ability to influence cancer cell growth inhibition.

This is in agreement with previous studies which suggest that the flavonol glycoside, rutin, is not able to induce anti-proliferative effects until the glycosidic bond of the rutinose moiety is cleaved to yield quercetin, which has previously been shown to exert strong anti-proliferative effects.10 Such cleavage may occur in the large intestine via the action of the colonic gut microflora, indicating that although rutin is incapable of preventing the growth of cancer cells in vitro, it may still be able to induce positive reductions in the growth of cancer cells in vivo. The correlation between total phenolic content and the degree of inhibition of colon adenocarcinoma cell growth suggests that other polyphenolic compounds, or the synergistic effect of a number of different polyphenols, are likely to be responsible for the observed growth inhibition. As our extraction and analysis protocol was capable of detecting and quantifying hydroxycinnamates, our data also suggest that this group of polyphenols is also unlikely to be responsible for the anti-proliferative effects as all the tomatoes contained very low levels. Further experiments are necessary in order to identify and characterize the polyphenols in the tomatoes which are responsible for their biological actions on cancer cells.

Our data suggest that the mechanism by which the high-polyphenol tomato extract exerts its anti-proliferative effects is linked to its ability to induce a shift in the distribution of cells in the S-phase of the cell cycle. Cancer cells treated with the high-polyphenol tomato extract underwent a reduction in proliferation which was accompanied by a reduction in the number of cells actively synthesizing DNA, relative to vehicle-treated cells. This is in agreement with a previous study which showed the ability of a tomato digestate to inhibit growth of HCT-116 colon adenocarcinoma cells was mediated by its ability to block the cell cycle in G0/G1. This block was accompanied by down-regulation of cyclin D1, a positive regulator of the cell cycle.27 Furthermore, polyphenols in olive oil have been shown to inhibit proliferation of Caco-2 cells by inducing a cell cycle block in G2/M.29 This block was induced via the upstream inhibition of the MAP kinase, p38 and the transcription factor, CREB. Further work is required in order to determine the precise mechanism by which tomato polyphenols induce the anti-proliferative effects in cells, although our initial observations suggest that they are also able to prevent cell cycle progression.

In summary, the polyphenol extracts of all three tomato varieties were able to inhibit the growth of colon adenocarcinoma cells and the growth inhibition did not appear to be due to their flavonoid or hydroxycinnamate content. The growth inhibitory effect was accompanied by a shift in cell cycle distribution, suggesting that fewer cells were present in the S-phase of the cell cycle following treatment. Future studies are needed in order to identify the specific component(s) present in tomatoes which are responsible for the growth inhibitory effects and to determine the mechanism by which they induce their effect on the cell cycle.


   Acknowledgements
 
I would first like to thank Dr Paul Fraser and his team, from Royal Holloway University, for supplying us with the tomato samples used in this study. I would also like to thank all members of the Nutrition Research Group at the University of Reading for all their help with this project, with special thanks to Dr Jeremy P.E. Spencer and Mrs Vanessa Collins.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
Author Biography 
 

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   Author Biography 
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 Author Biography 
 
    Caroline Saunders completed a BSc in Nutrition and Food Science at the University of Reading and is now about to undertake a PhD at Reading on the subject of flavonoids, genotype and cognitive function. Her undergraduate project allowed her to gain valuable experience and an insight into the field of research which helped make the decision to continue to do a PhD.
Submitted on 30 November 2008; accepted on 18 December 2008


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