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© 2008 American Society for Nutrition J. Nutr. 138:1840-1845, October 2008

Biochemical, Molecular, and Genetic Mechanisms

Polyamine Metabolism and Transforming Growth Factor-β Signaling Are Affected in Caco-2 Cells by Differentially Cooked Broccoli Extracts1–5,

Caroline S. M. Furniss6,*, Richard N. Bennett6,7, James R. Bacon6, Gwen LeGall6 and Richard F. Mithen6

6 Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK and 7 Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas-Departamento de Fitotecnia e Engenharia Rural, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal

* To whom correspondence should be addressed. E-mail: caroline.furniss{at}bbsrc.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Methods and Materials
 Results
 Discussion
 LITERATURE CITED
 
The health benefits of consuming cruciferous vegetables are widely considered to be due to the biological activity of glucosinolate degradation products. However, it is conceivable that other phytochemicals within crucifers may also have biological activity that may contribute to health benefits. In this study, we analyzed global gene expression in Caco-2 cells exposed to extracts derived from broccoli that had been heat treated to different extents to result in contrasting profiles of glucosinolates and their degradation products. Extracts microwaved for 0, 1, and 4 min contained 9.5, 25.5, and 0 µmol/L sulforaphane and induced changes in expression of 381, 1017, and 101 genes, respectively (>2 fold; P < 0.01). Seventy-two genes showed similar changes in expression after treatment with all 3 extracts. These included genes involved in polyamine catabolism and transforming growth factor (TGF)-β signaling. Consistent with these changes in gene expression, subsequent studies demonstrated that exposing cells to these extracts, including the 4-min extract that contained no glucosinolate degradation products, increased putrescine and N-acetyl-spermine concentration, and suppressed the TGFβ1-mediated induction of phosphorylated Smad 2. This is the first report, to our knowledge, of phytochemicals from a cruciferous vegetable affecting both a signaling pathway and a catabolic process.


   Introduction
TOP
 ABSTRACT
 Introduction
Methods and Materials
 Results
 Discussion
 LITERATURE CITED
 
Epidemiological studies suggest a diet rich in broccoli can reduce the risk of cancer at several sites (1–6). It has been widely assumed that the major bioactive components are isothiocyanates (ITC)8 and indoles derived from the enzymatic hydrolysis of glucosinolates. Cruciferous vegetables, however, contain many other potentially bioactive phytochemicals, including flavonols such as quercetin and kampferol glycosides (7), hydroxycinnamic acid esters (8), carotenoids, folates, and vitamin C (9). Attempting to distil the biological activity of any one of these phytochemicals is complex. Upon ingestion and absorption, these compounds are metabolized to a range of products, each one of which may have contrasting activity to its parental compound. Moreover, it is likely that there may be interactions among these compounds so that the biological activity of consuming broccoli may not be deduced through the analyses of the biological activity of each of its metabolites individually. Furthermore, cooking is likely to have a considerable effect on biological activity, as some components will be more heat labile than others. In broccoli (Brassica oleracea L. ssp. italica), the main glucosinolate is 4-methylsulphinylbutyl glucosinolate (glucoraphanin). Like all glucosinolates, the glucose moiety is removed by hydrolytic activity of myrosinase (EC 3.2.3.1), producing an unstable intermediate. This is converted to the ITC sulforaphane (SF) or to the corresponding nitrile through the action of the epithiospecifier protein (ESP) in combination with Fe2+ ions. Myrosinase also hydrolyses the indole glucosinolates (glucobrassicin and its derivatives) to reactive ITC and carbonium ions. These immediately react with water to give carbinols, such as indol-3-carbinol, which in turn can react with ascorbic acid to give ascorbigens (10). Homogenizing raw broccoli produces a mixture of ITC and nitriles, while cooking for a short length of time results in only ITC, due to the ESP being denatured (11). Cooking for longer destroys myrosinase activity, resulting in no glucosinolate breakdown (12). Thus, by processing broccoli for different lengths of time, the ratio of glucosinolates, ITC, and nitriles can be manipulated. Cooking also affects other phytochemicals in broccoli. Steaming or boiling broccoli leads to an increase in carotenoids and a decrease in phenolic compounds, although steaming actually has little effect on the levels of kampferol and quercetin (13).

Many previous studies in cell models have shown that SF can induce phase II enzymes such as quinone reductase, glutathione transferase, thioredoxin reductase, and UDP-glucoronsyl transferase, mediated via the electrophile-responsive element (14–18). SF has also been shown to induce cell cycle arrest and apoptosis in a number of different cell types, including prostate (19,20), colon (21), T-cell leukemia (22), liver (23), bladder (24), and lung (25). Similar responses have been observed when bladder and breast cells are treated with broccoli sprout and cauliflower extracts, respectively (26) (27), where the major component was SF. The breakdown products from the indole glucosinolates found in broccoli, such as indole-3carbinol, ascorbigen, and 3,3'-diindolylmethane, have also been shown to stimulate apoptosis and confer cell protection against DNA damage in colon cell lines (28,29). In vitro studies have shown the major polyphenolic compounds in broccoli to have antioxidant properties (30); dietary quercetin-glycosides induce quinone reductase activity in Hepa1c1c7 cells. In human colon cells, quercetin has been shown to reduce the expression of the Ras protein and inhibit growth in pancreatic and colon cancer cells through inhibition of epidermal growth factor receptor expression and tyrosine kinase activity [(31) and references therein].

In the current study, we have addressed the question of to what extent do compounds other than those derived from glucosinolate degradation contribute to the biological activity of broccoli. The tissues and cells of the gastrointestinal tract are exposed both to the complex mixture of plant compounds from topological exposure and to the metabolites of a subset of these products following absorption and metabolism, including microbial metabolism that may occur in the colon and subsequent metabolism in the enterocyte and liver. In this study, we exposed the Caco-2 cell model to crude extracts of broccoli that were cooked for different lengths of time to alter the manner of glucosinolate degradation.


   Methods and Materials
TOP
 ABSTRACT
 Introduction
 Methods and Materials
Results
 Discussion
 LITERATURE CITED
 
    Cell culture and materials. All chemicals were purchased from Sigma Aldrich unless otherwise stated. The human Caucasian colon adenocarcinoma cell-line Caco-2 was obtained from the European Collection of Animal Cell Cultures. All cell culture media and reagents were from Invitrogen. The cells were cultured in DMEM supplemented with 1% nonessential amino acids, 2 mmol/L glutamine, 1 U/L penicillin, 1 g/L streptomycin, and 10% fetal calf serum and were maintained in 5% CO2 at 37°C. Cells were seeded at 2 x 104 cells/cm2 in 6-well plates (real-time RT-PCR and polyamine analysis) and 10-cm (55 cm2) dishes (microarrays). Cells were allowed to reach 80% confluence before being treated with broccoli extract (or equivalent volume of water). Cell passages 39–46 were used. Cell viability (percent) was measured using WST assay (Roche). Broccoli (var. Marathon) was purchased from a local supermarket and had been grown in Fife, Scotland.

    Broccoli extract preparation and analysis. We cooked 100-g portions of broccoli with 150 mL water in an 800-W domestic microwave oven for 0, 1, 1.5, 3, and 4 min. Immediately after cooking, the broccoli and water were homogenized to a soup using a food blender and centrifuged at 13,000 x g; 4°C, 50 min. The supernatant was removed and divided into aliquots for analysis and for use in cell treatments. Liquid chromatography-MS analysis of extracts was performed as described previously (32). ITC and nitrile analysis and quantification of extracts was done as described by Rose et al. (33), with the exception that methanol was used instead of acetonitrile. Glucosinolate analysis and quantification of freeze-dried broccoli was performed as previously described (34). The NMR experimental procedure was as described by Wang et al. (35) (25 mg of freeze-dried extracts were added to 1 mL of phosphate buffer).

    RNA extraction and array hybridization. In 3 separate experiments, Caco-2 cells were treated for 24 h with a 1 in 6 dilution (in media) of each broccoli extract or an equivalent volume of water (control). Total RNA was isolated from 3 biological replicates using a GenElute Mammalian Total RNA Miniprep kit (Sigma). RNA quality was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). Aliquots of the replicate samples from each experiment were pooled and analyzed using Affymetrix Human U133 Plus 2.0 chips. Double-stranded cDNA synthesis and generation of biotin-labeled cRNA were performed according to the manufacturer's instructions (Affymetrix). Hybridization onto a total of 12 arrays and data capture were performed as previously described (36). The 12 CEL files were loaded into GeneSpring GX 7.3.1 (Agilent Technologies) for normalization, creation of expression values, and statistical analysis. Background correction using the Perfect Match model and normalization were performed using the robust multi-chip average method. We conducted an additional per-gene normalization step (normalize to median); this ensures that the expression value for 1 gene across the different conditions is centered on 1, by dividing the expression value by the median value of the expression values for that gene across the conditions. The individual treatments (0, 1, and 4 min) were also loaded separately into GeneSpring, as described above, with the controls. Normalized data were statistically tested (see later section).

We used the GenMAPP 2.1 program (37) to identify significant changes in pathways following treatments with broccoli extracts. All Kyoto encyclopedia of genes and genomes (KEGG), Gene ontology (GO), contributed, and tissue-specific maps and pathways were used. Gene lists were produced for each individually normalized treatment, where each probe was assigned a P-value using Welch modified t test (separate 0-, 1-, and 4-min experiments) or ANOVA (all samples normalized together). These lists were used as input files for the GenMAPP software, the criterion being a change in gene expression P ≤ 0.05.

    Real-time RT-PCR. We chose 15 genes that had >2-fold change in expression relative to the control value in at least 1 of the extract treatments for analysis by RT PCR. Target mRNA was quantified using an ABI 7500 Real Time PCR system (Applied Biosystems). Primers and probes (Supplemental Table 1) were designed using ABI Prism Primer Express software v 2.0 and were purchased from Sigma Genosys. The probes were labeled with a 5' reporter dye (FAM, 6-carboxyfluorescein) and a 3' quencher dye (TAMRA, 6-carboxytetramethylrhodamine). RT-PCR were carried out in a microamp optical 96-well plate in a total volume of 25 µL/well, consisting of Taqman 1-step RT-PCR mix Reagent kit (Applied Biosystems), 10 ng total RNA, 0.25 U/µL Multiscribe, and concentrations of primers and probes ranging from 100 to 500 nmol/L. Reverse transcription was performed for 30 min at 48°C, AmpliTaq gold activation for 10 min at 95°C, followed by 40 PCR cycles of denaturation at 95°C for 15 s and annealing/extension 60°C for 1 min. Reactions were carried out in triplicate and data analyzed by the 7500 system SDS software using a standard curve to quantify mRNA amount. Standard curves were produced for each set of primers and probes using 5, 10, 20, 40, and 80 ng of total RNA per reaction. Results were normalized on the basis of 18s rRNA quantification. Taqman Gene Expression Assays (Applied Biosystems) contained probes labeled with a 5' reporter dye (FAM, 6-carboxyfluorescein) and nonfluorescent quencher. Reactions were conducted in a total volume of 20 µL/well using 1 µL assay mix per reaction. All other conditions were as described above.

    Protein extraction and western blotting. Caco-2 cells were treated in triplicate with broccoli extracts and a water control as previously described and/or transforming growth factor (TGF)-β1 (2 µg/L) for 8 h. Protein was extracted using RIPA buffer [50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% (v:v) Triton-X, 0.5% (wt:v) sodium deoxycholate, 0.2% (wt:v) SDS, 1 mmol/L Na3VO4, and 1 50-mg tablet/10 mL complete protease inhibitor cocktail tablet (Roche Diagnostics)]. Protein concentration was estimated using the BCA assay (Sigma). Samples (40 µg) were separated on 10% NUPAGE gels (Invitrogen) and transferred to nitrocellulose membrane (Bio-Rad). Phosphorylated Smad2 (pSmad2) was detected with a monoclonal antibody for phospho Smad2 and anti rabbit horseradish peroxidase, diluted to 1:1000 and 1:2000, respectively (Cell Signaling Technologies). GAPDH was detected with a monoclonal antibody for GAPDH (diluted to 1:4000, Ambion/Applied Biosystems) and anti mouse-horseradish peroxidase (diluted to 1:10,000, Sigma) followed by chemiluminescence substrate (Pierce) and exposed to Hyperfilm ECL (GE Healthcare).

    Polyamine analysis. The polyamine content of treated and untreated cells was determined by precolumn dansylation/reverse-phase HPLC using a Luna 5 µm C18(2) 100A 250- x 4.60-mm column (Phenomenex) with 1,3-diaminopropane as the internal standard (38). Protein concentrations were determined using a commercial protein-assay kit (Pierce) based on the method of Bradford (39) with bovine serum albumin as standard.

    Statistical analysis. Data were expressed as means of the fold change ± SD, calculated using Minitab 15 statistical software. For microarray analysis of all samples, a 1-way ANOVA (Welch modified) adjusted to compensate for multiple testing using Benjamini and Hochberg's false discovery rate (40) was performed in GeneSpring and repeated in R. Changes in gene expression with corrected P ≤ 0.1 were considered significant. The individual treatments were also compared separately with the controls in GeneSpring using a Welch modified t test (P < 0.01). Pearson correlations (r2) between fold changes from array data and RT-PCR were calculated using Minitab 15 statistical software. All statistics measured in the gene ontology analysis were conducted using GenMapp 2.1, which used a hypergeometric distribution to calculate a Z-score and a PermuteP, a nonparametric bootstrapping test based on 2000 permutations of the data. Adjusted P was calculated using the Westfall and Young adjustment for multiple testing (41). Adjusted P < 0.05 was considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Methods and Materials
 Results
Discussion
 LITERATURE CITED
 
    Analysis of broccoli extracts: ITC and glucosinolate content. The glucoraphanin concentration of the unprocessed broccoli was 6.25 µmol/g dry weight. The 0-, 1-, and 1.5-min extracts all contained SF, iberin, and SF-nitrile (SF-CN). The 1-min extract containing the highest amounts of SF and iberin, while the SF-CN levels decreased significantly with 1 min cooking time (Table 1). The 3- and 4-min extracts did not contain ITC or nitriles but did contain glucosinolates. Thus, the 0-, 1-, and 4-min extracts were chosen to treat Caco-2 cells, which after dilution contained 9.5, 25.5, and 0 µmol/L SF, respectively. Cell viability increased when they were treated with the diluted extracts at the concentrations as stated above: 111% ± 1.6, 136% ± 4.8, and 114% ± 5.6 for the 0-, 1-, and 4-min extracts, respectively.


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  		TABLE 1 Concentrations of SF, SF-CN, and glucoraphanin in cooked broccoli extracts1

 
Further analyses of the 0-, 1-, and 4-min extracts by liquid chromatography-MS suggested that despite varying in their glucosinolate/ITC profiles (Supplemental Fig. 1), 1H NMR revealed little changes between the 3 extracts except for the disappearance of fumarate in the 4-min extract and all the nucleobases (uracil and adenine) and nucleosides (uridine, guanosine, and cytidine). Only 1 undetermined nucleoside appears in the 4-min extract.

    Analysis of gene expression after exposure to broccoli extracts. Affymetrix U133A PLUS 2.0 oligonucleotide arrays, which contain 54,682 probes, were used to detect changes in gene expression in response to treatment with differentially cooked broccoli extracts. Analysis of the normalized samples by a 1-way ANOVA identified 639 genes changing across all samples relative to the control (Supplemental Table 2). Whereas a Welch modified t test on samples normalized separately identified 999, 2910, and 659 genes changed in expression between control and 0, 1, and 4 min, respectively (P < 0.01), of which 381, 1017, and 101 exhibited >2-fold change (Table 2). Changes in the gene expression of 13 genes [Aldo-keto reductase 1C1 (AKR1C1), Cytochrome P450 1A1 (CYP1A1), Glutamate-cysteine ligase, modifier subunit (GCLM), Notch homolog 2 (NOTCH2), Retinoic acid induced 3 (RAI3), Alpha-methylacyl-CoA racemase (AMACR), cyclin-dependent kinase inhibitor 1C (p57), sirtuin 1 (SIRT1), spermidine/spermine N1-acetyltransferase 1 (SSAT), tumor necrosis factor receptor superfamily, member 10b (TNFRSF10b), Kruppel-like factor 4 (KLF4), cyclin-dependent kinase inhibitor 1A (p21), thioredoxin reductase 1 (TR1)] were further quantified by RT-PCR. With the exception of SIRT1 (r2 = 0.462; P = 0.562), there was a high degree of correlation between the array and RT-PCR results (r2 = 0.943–0.998; P = 0.057–0.002) (Supplemental Table 3).


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  		TABLE 2 Genes changing 24 h after treatment of Caco-2 cells with broccoli extracts cooked for 0, 1, and 4 min relative to the control (water) treatment1

 
    Effect of broccoli treatment on polyamine content of Caco-2 cells. Results from microarray and RT-PCR analysis of RNA extracted from Caco-2 cells treated with broccoli extracts for 24 h showed there were changes in the gene expression of SSAT, a gene involved in polyamine catabolism. To explore whether this change in gene expression was associated with changes in polyamine metabolism, Caco-2 cells were treated with all 3 broccoli extracts and 50 µmol/L SF as a comparison. Cell extracts were analyzed for polyamine content and expressed as nmol polyamine/mg protein. Treatment with all broccoli extracts resulted in an increase in putrescine (PUT) and N-acetyl-spermine (NAcSPM) levels but did not affect spermidine (SPD) or spermine (SPM) (Fig. 1). In contrast, SF treatment resulted in decreased levels of PUT and did not affect NAcSPM.


Figure 1
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  		FIGURE 1  Effects of SF, broccoli extract, and control (water) treatments on the polyamine profile in Caco-2 cells. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

 
    Pathway analysis. Pathways in which genes that were significantly changed in expression occurred more frequently than would be expected by chance (Supplemental Table 4). As expected, there were more changes after treatment with the 1-min extract than with the other extracts (13 compared with 7 for the 0-min and 2 for 4-min extract). Insulin signaling and sphingoglycolipid metabolism were both significantly changed by the 0- and 1-min extracts. The 1-min extract also affected the FAS pathway and stress induction of HSP regulation, apoptosis mechanisms, and apoptosis modulation by HSP70, all of which show considerable overlap. The TGFβ receptor pathway was significantly perturbed by 0- and 1-min extracts and to a certain extent by the 4 min, which also affected the TGFβ signaling pathway.

    Effect of broccoli extract treatment on TGFβ signaling pathway. Pathway analysis of Microarray data suggested that the TGFβ receptor/signaling pathway was significantly perturbed by treatment with all 3 broccoli extracts. To further investigate the effects of broccoli extracts on TGFβ signaling, the level of pSmad2 was quantified by western blotting. Caco-2 cells constitutively expressed pSmad2 in culture, but the level was enhanced following exposure to TGFβ1 (2 µg/L). Exposure to the 3 broccoli extracts led to decreased pSmad2 relative to the control, with the greatest decrease observed with the 1-min extract (Fig. 2). Coexposure to TGFβ1 and the 1-min extract resulted in enhanced pSmad2 expression, but to a lesser extent than with TGFβ1 alone.


Figure 2
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  		FIGURE 2  Western-blot analysis of Caco-2 cells for pSMAD2 after treatment with water (control), broccoli extracts, and/or TGFβ1. (A) pSMAD2. (B) GAPDH.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Methods and Materials
 Results
 Discussion
LITERATURE CITED
 
Exposure of Caco-2 cells to crude broccoli extracts led to changes in gene expression. There were more changes following exposure to the 0- and 1-min extract, compared with the 4-min extract. These extracts possess degradation products from both aliphatic and indole glucosinolates. There were fewer changes in gene expression following exposure to the 4-min extract, in which only intact glucosinolates were found. Bioinformatic analyses identified several pathways that were associated with these changes in gene expression. Some of the pathways were associated with processes previously associated with the activity of ITC, such as mitogen-activated protein kinase and epidermal growth factor signaling and apoptosis (21,28,42–44), but the majority of pathways were not those that would have been expected to have been perturbed on the basis of existing knowledge of the activity of glucosinolate degradation products. These may be due to our lack of knowledge of these compounds and their interaction with other phytochemicals, or these changes may be due to nonglucosinolate-derived phytochemicals. The biochemical profile of the 4-min extract showed the major difference from the 0- and 1-min extracts was the lack of glucosinolate degradation products (Supplemental Fig. 1). Cooking broccoli has shown to change the phytochemical profile, increasing carotenoids and some polyphenols and decreasing other phenolic compounds and vitamin C (13,45). There were, however, fewer changes in gene expression in cells exposed to 4 min and these changes were only associated with perturbation of 2 pathways (focal adhesion and TGFβ1 signaling). This suggests that the majority of changes in gene expression in cells exposed to the 0- or 1-min extracts were predominantly due to glucosinolate degradation products, even if the mechanistic links are yet to be elucidated.

However, despite the majority of changes in gene expression being associated with extracts that contain glucosinolate degradation products, 72 genes exhibited similar changes in expression after treatment by all 3 extracts, including genes involved in polyamine metabolism. Because the 4-min extract contained no glucosinolate degradation products, these results suggest that other phytochemicals in broccoli must have contributed to this response. However, exactly which group or groups of compounds are responsible has yet to be identified.

SSAT gene expression was significantly upregulated by all 3 treatments. SSAT is involved in polyamine catabolism, acetylating SPD and SPM and leading to increased levels of PUT. All these compounds play a role in cell growth and cell cycle progression of normal and cancerous cells, and roles in the control of cell death and apoptosis have been suggested. Decreasing polyamine levels has been shown to lead to a loss of cellular proliferation capacity (46). Studies in TRAMP mice showed that activating polyamine catabolism through overexpression of SSAT depleted acetyl-CoA pools and suppressed prostate tumor growth (47). Conversely, deletion of SSAT in APCmin mice led to reduced gastrointestinal tumorigenesis (48). The exact effect of broccoli extract on polyamine catabolism is not clear; as well as an increase in SSAT expression, SPM oxidase gene expression is also induced by the 0- and 1-min extracts. This catabolic enzyme cleaves acetylated SPM and SPD to SPM and PUT, respectively (49). However, treating Caco-2 cells with broccoli extracts and comparing to pure SF (50 µmol/L) results in differently altered polyamine profiles (Fig. 1). All 3 extracts caused increased PUT and NAcSPM, but no significant change in SPM and SPD levels, whereas SF treatment resulted in decreased PUT, SPM, and SPD. These preliminarily results suggest that broccoli is promoting polyamine catabolism, possibly via increased SSAT and SPM oxidase activity, and that this effect is independent of SF. There is little published evidence of the effect of polyphenols on polyamine metabolism. Although flavonols and procyanidins in cocoa have been shown to inhibit growth and polyamine biosynthesis in Caco-2 cells by inhibiting the polyamine biosynthetic enzymes (50), a similar affect has not yet been shown by polyphenols from broccoli.

In addition to observing changes in the expression of individual genes in each of the treatments, pathway analyses indicated that all the broccoli extracts perturbed TGFβ signaling. While quantification of gene expression and pathway analyses provides information concerning which pathways may be modified by time or diet, it can provide little information about the precise nature of how these pathways are perturbed. This requires further analysis of mRNA and protein turnover, and post-translational protein modifications such as phosphorylation, associated with both components of the signal transduction pathway and downstream targets. The TGFβ1 ligand binds to the TGFβII receptor, which then phosphorylates the TGFβI receptor that activates intracellular serine/threonine kinase activity, resulting in multiple intracellular signaling cascades. Most attention has focused on Smad-mediated signaling. Phosphorylation of the Type I receptor results in phosphorylation of the Smad2 and Smad 3 proteins that bind to the Smad 4 protein. The pSmad complex translocates into the nucleus resulting in enhanced transcription of genes associated with cell homoeostasis and suppression of genes associated with proliferation. Although TGFβ1 signaling is primarily concerned with the maintenance of cell homoeostasis and tumor suppression, it can switch to enhancing tumor growth associated with aggressive and metastatic cancers. This may be due to an imbalance between Smad signaling and other TGFβ1-mediated signaling pathways.

Growth of Caco-2 cells result in constitutive pSmad 2 expression, probably due to the presence of TGFβ1 in the fetal calf serum within the culture media. Exposure to additional TGFβ1 resulted, as expected, in enhanced pSmad2 expression, whereas exposure to all the broccoli extracts resulted in inhibition of pSmad2 expression, suggesting downregulation of Smad-mediated signaling. As this occurred after cells were exposed to all the extracts, including the 4-min ITC-free extract, it is unlikely to be mediated by glucosinolate degradation products.

Thus, in conclusion, exposing the Caco-2 cell lines to crude broccoli extracts resulted in many changes in gene expression that are associated with a variety of pathways. The greatest number of changes occurred in extracts in which the level of ITC were maximized and the fewest changes occurred in extracts in which there were no glucosinolate degradation products. Changes in polyamine associated gene expression and TGFβ1 signaling occurred as a result of exposure to all extracts suggesting that factors other than glucosinolate degradation products were important. Thus, it is likely that there are bioactive compounds within crude broccoli extracts that can perturb both polyamine catabolism and TGFβ signaling. In addition, it is more than likely the biological activity of broccoli is due to the combined effects of these phytochemicals, no individual group of compounds being solely responsible. Although it is not known whether these can be absorbed and have systemic effects, it is possible that they may be important in direct exposure of cells of the gastrointestinal tract following broccoli consumption.


   ACKNOWLEDGMENTS
 
We thank Rob Foxall (IFR) for assistance with statistical analysis.


   FOOTNOTES
 
1 Supported by the Biotechnology and Biological Sciences Research Council. Back

2 Authors disclosure: C. S. M. Furniss, R. N. Bennett, J. R. Bacon, G. LeGall, R. F. Mithen, no conflicts of interest. Back

3 Supplemental Tables 1–4 and Supplemental Figure 1 are available with the online posting of this paper at jn.nutrition.org. Back

4 Microarray data cited in this study were deposited with the public repository Array Express under Experiment Accession No: E-MEXP-1372 (http://www.ebi.ac.uk/arrayexpress/). Back

5 0 min extract = 100 g/150 mL broccoli microwaved at 800 W for 0 min (raw); 1 min extract = 100 g/150 mL broccoli microwaved at 800 W for 1 min; 4 min extract = 100 g/150 mL broccoli microwaved at 800 W for 4 min; Control = water. Back

8 Abbreviations used: ESP, epithiospecifier protein; ITC, isothiocyanate; NAcSPM, N-acetyl-spermine; pSMAD2, phosphorylated SMAD2; PUT, putrescine; SF, sulforaphane; SF-CN, sulforaphane nitrile; SPD, spermidine; SPM, spermine; SSAT, spermine spermidine N-acetyl transferase; TGF, transforming growth factor. Back

Manuscript received 12 February 2008. Initial review completed 30 April 2008. Revision accepted 9 July 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Methods and Materials
 Results
 Discussion
 LITERATURE CITED
 

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