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Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden;
* Division of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, S-751 23 Uppsala, Sweden; and
Department of Biochemistry, National University of Singapore, Singapore 117597
3To whom correspondence should be addressed. E-mail: joseph.rafter{at}mednut.ki.se.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–stimulated HT-29 cells and pure enzymatic activity in a COX-2–catalyzed prostaglandin biosynthesis in vitro assay. The major phenolic compounds identified were phenylpropionic acid, phenylacetic acid, cinnamic acid, and benzoic acid derivatives. Of 13 fecal water samples analyzed, 12 significantly decreased PGE2 production (range 5.4–39.7% inhibition, P-value < 0.05) compared with control cells and 13 of 14 samples analyzed decreased COX-2 protein levels in HT-29 cells (19–63% inhibition). Of the 20 fecal water samples, 2 also weakly inhibited enzymatic activity of purified COX-2 (22–24% inhibition). Three compounds identified in fecal water, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid, decreased the protein level at 250 µmol/L (15–62% inhibition). This study shows for the first time that human fecal water contains components that can affect both the COX-2 protein level and enzymatic activity.
KEY WORDS: • COX-2 inhibitors • colon cancer • fecal water • phenolic compounds • HT-29 cells
Results from epidemiologic and experimental studies strongly support the contention that diet plays an important role in the etiology of colon cancer. A diet high in fruit and vegetables is thought to reduce the risk of developing this tumor form (1–3). Plant foods contain a multiplicity of phytochemicals, which may affect this process (4–6). Phenols comprise one important group of these phytochemicals; thousands have been identified in plants, including edible plants (7). In the human diet, the most abundant groups of polyphenols are flavonoids and phenolic acids (7,8). These compounds have been reported to exhibit a wide variety of biological effects including anti-inflammatory and cancer preventive effects (4).
In recent years, there has been considerable interest in the role of the aqueous phase of human feces (fecal water) in studies examining the mechanisms underlying the dietary etiology of colon cancer; the motivation for this is that components of this fecal fraction are more likely to be able to exert effects on the cells of the colonic epithelium than components bound to food residues and the bacterial mass. It was demonstrated that dietary alterations can influence the biochemical composition of fecal water (9,10) and that components in this fecal fraction can influence several cellular variables of relevance for tumorigenesis, i.e., genotoxicity (11,12), proliferation (13), apoptosis (14), and cell signaling pathways (15). A more recent study showed that components in human fecal water have the capacity to modulate cyclooxygenase-2 (COX-2)4 transcription in human colonic cells (16), indicating that this important signaling molecule may also be a target for diet-related fecal water components. In the latter study, although the majority of fecal water samples tested induced COX-2 activity, some actually inhibited COX-2.
Both isoforms of COX, COX-1 and COX-2, catalyze prostaglandin (PG) biosynthesis. COX-2, in contrast to COX-1, is not expressed in the majority of cells, but it can be induced in response to stimuli such as growth factors, cytokines, phorbol esters, tumor necrosis factor- (TNF-), mitogenic agents, and lipopolysaccharides (17–19). Several studies reported an increased production of PGs and an overexpression of the COX-2 protein in human colonic tumor tissue (20,21). COX-2 is suggested to be involved in many processes in carcinogenesis, such as angiogenesis, apoptosis, and tumor invasiveness (22). Thus, in addition to being an important regulator of inflammation, COX-2 is considered to be an important target for the prevention and treatment of cancer (23,24). Several compounds of plant origin in the human diet have received attention for their effects on COX-2, either on enzymatic activity or protein or mRNA levels (25,26). Several phenolic compounds were reported to exhibit COX-2 inhibitory or stimulatory effects (5,27,28). Thus, in view of the considerable interest in COX-2 inhibitors as cancer preventive agents, we considered it of interest to further investigate the presence of natural COX-2 inhibitors in human colonic contents.
In the present study, we investigated whether fecal water samples from volunteers consuming a vegetarian diet could affect COX-2 enzymatic activity and protein levels. A number of the fecal water samples were also analyzed for their content of phenolic compounds to obtain an initial overview of which phenolic compounds were present and their concentrations. Phenolic compounds may exert important effects in the gastrointestinal tract (29). Finally, a number of pure compounds, both diet-related and identified in fecal water, were investigated for their effects on COX-2.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Materials.
Daidzein, genistein, hesperetin, kaempferol, butyric acid (sodium salt), cholic acid (sodium salt), chenodeoxycholic acid (sodium salt), taurocholic acid (sodium salt), benzoic acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4,5-trihydroxybenzoic acid (gallic acid), 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid were obtained from Sigma Chemical. Deoxycholic acid (sodium salt) and lithocholic acid (sodium salt) were obtained from Calbiochem. The reference substances, aspirin and indomethacin, were from Sigma and NS-398 ([N-[2-(cyclohexyloxy)-4-nitrophenyl]methane-sulfonamide] from Cayman Chemical. Rofecoxib was a gift from Merck Research Laboratories. TNF- was obtained from Sigma.
Fecal water sample preparation. Fecal water samples were prepared according to a well-established procedure designed to provide the free water fraction of stool to which the colonic epithelium is exposed in vivo (12). All fecal water extracts were sterile filtered (Millipore, 0.8:0.2 µm) and stored at –20°C until analysis.
Analysis of fecal water samples by GC-MS. Fecal water samples (n = 5; samples 8, 9, 12, 13, and 14) were analyzed for their content of phenolic compounds using the procedure of Jenner at al (30). Briefly, samples were acidified and internal standards added before application to a column containing diatomaceous earth (Isolute HM-N, IST-International Sorbent Technology). The phenolic compounds were eluted with ethylacetate and their trimethylsilylated derivatives prepared before analysis with a Hewlett-Packard 5973 mass selective detector interfaced with a Hewlett-Packard 5890II gas chromatograph, equipped with a fused silica capillary column, 12 m x 0.2 mm i.d. coated with cross-linked 5% phenylmethylsiloxane (film thickness 0.33 µm) (Agilent, J and W). Selected-ion monitoring was performed using the electron-ionization mode at 70 eV with the ion source maintained at 230°C. The characteristic ions used for the analysis of the phenolic derivatives are reported by Jenner et al. (30). Quantitation of phenolic compounds was achieved by relating the peak area of the compound with the internal standard peak area. Each sample was analyzed 3 times. This method enabled good recovery of the analytes and quantitation of phenolics in fecal water (30).
Cell culture and treatment with test compounds. The human colon adenocarcinoma cell line HT-29 (American Type Culture Collection) was cultured in monolayer in DMEM [supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 1% penicillin/streptomycin] at 37°C and 5% CO2. All experiments were carried out in 0.1% DMEM (0.1% FBS).
COX-2 protein in HT-29 cells.
Confluent cells were incubated with TNF- (50 µg/L) in the absence or presence of the fecal water samples (diluted 1:20) or test compounds (25–500 µmol/L) for 5 h. Untreated cells were included in each experiment.
Proteins were isolated essentially as previously described (16). The protein concentration was determined using the Bradford protein assay (31). Cell extracts were prepared and protein concentration was adjusted to 80 µg/sample. Proteins were separated by SDS-PAGE (8%) as described by Laemmli (32) and blotted onto a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham). The membranes were incubated for 1 h with mouse monoclonal antibody against COX-2 and actin (Transduction Laboratories, 1:500 dilution and Santa Cruz Biotechnology, 1:500 dilution, respectively), followed by a 1 h-incubation with anti-mouse IgG conjugated with horseradish peroxidase (Dakopatts, 1:2000 dilution). The ECL+ Western blot detection system was used to detect the bands, and the light emitted was quantified using a FUJI LAS 100. Actin was used as an internal control for equal protein concentration loading per lane. Protein bands were quantified densitometrically and expressed as the percentage inhibition of the TNF-–treated cell band intensity (COX-2:actin). The values are means ± SEM, n = 3. Values were normalized for enabling comparison between different experiments. All fecal water samples were tested for cytotoxicity in the AlamarBlueTM assay to ensure that potential COX-2 inhibitory effects were not due to cell death.
Prostaglandin E2 (PGE2) production in HT-29 cells.
PGE2 is a major product produced by COX-2 from arachidonic acid and is often used to estimate COX-2 activity in cells (26). Cells were plated in 12-well plates and cultured to 60–80% confluence. To inactivate COX-1, cells were pretreated with 100 µmol/L aspirin for 10 h. At d 3, the cells were incubated with TNF- (50 µg/L) in the absence or presence of fecal water (13 samples, diluted 1:20) or test compounds (250–500 µmol/L) for 5 h. After treatment, the test solutions were removed and replaced with 100 µmol/L arachidonic acid (Sigma) diluted in 0.1% DMEM and left for 1 h. The PGE2 concentration in the cell supernatants was determined by RIA using [3H]PGE2 obtained from Amersham Pharmacia Biotech, and polyclonal antiserum to PGE2 (Sigma). The samples were assayed undiluted and a standard curve with PGE2 was included in each run. Each fecal water sample was tested at least twice in the cell system and later analyzed in duplicate in the RIA and expressed as the percentage inhibition of the TNF-–treated cells. The values are presented as means ± SD, n = 4. The values were normalized for enabling comparison between different experiments.
COX-2 enzymatic activity (purified enzyme).
Effects on COX-2 enzymatic activity were assayed using a radiochemical COX-2 in vitro assay (33), with minor modification (i.e., the test compounds/samples and enzyme were incubated for 3 min at 37°C, and heptane replaced n-hexane in the eluent). Purified COX-2 enzyme (Cayman Chemical) was incubated with the fecal water sample (nondiluted) or 20 µL of the test-compound (100, 300, or 500 µmol/L) for 10 min before addition of [1-14C]-arachidonic acid (Amersham Pharmacia Biotech). Phenolic acids (3-phenylpropionic acid, 3-(4-hydroxyphenyl)-propionic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxycinnamic acid, benzoic acid, 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxybenzoic acid) and flavonoids (daidzein, genistein, hesperetin, kaempferol) were dissolved in 20% dimethyl sulfoxide (DMSO; with the final concentration in the wells being 4% DMSO), bile acids (chenodeoxycholic, cholic, deoxycholic, lithocholic, and taurocholic acids), and fatty acid (butyric acid) in water. NS-398 (8.5 µmol/L) was used as positive control, and solvent vehicle controls were included. The inhibition of COX-2–catalyzed PG biosynthesis was calculated as the relative decrease in radioactivity [disintegrations per minute (dpm)] of the samples containing the test substance compared with the solvent vehicle. In this assay, 
20% inhibition was regarded as the cutoff point for an actual effect. The percentage was calculated as follows:
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where MaxPG is the maximum production of PGs with only solvent present, measured in dpm; background is measured in dpm; PGtest is the amount of PGs produced with test fecal water present, measured in dpm.
Statistical analysis.
All statistical analysis was performed using the statistical software Statistica version 7. PGE2 inhibition in cells for individual fecal waters was compared with TNF-–treated cells with the Kruskal-Wallis test (nonequal variance in the groups). Fecal water samples as a group were compared with TNF-–treated cells with the Mann-Whitney U test. An value of 0.05 was regarded as significant. A correlation analysis was carried out to compare COX-2 protein inhibition and PGE2 production in cells. All data are presented as means ± SEM or range.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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induced HT-29 cells.
Colonic HT-29 cells exhibit a low basal level of COX-2 protein, which can be upregulated by TNF- stimulation. In our experiments, cells treated with TNF- (50 µg/L) potently induced the COX-2 protein, and this concentration was used in the experiments described below. To further evaluate the cell system, a selection of anti-inflammatory compounds was tested for their ability to inhibit the induction of COX-2 by TNF-. The selective COX-2 inhibitors rofecoxib and NS-398 significantly inhibited the induction of the COX-2 protein (25 µmol/L), whereas indomethacin was less active (Fig. 1). Aspirin had no effect at the concentration tested (25 µmol/L).
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and the highest inhibition of COX-2 by fecal water both occurred after a 5-h treatment (Fig. 2). The majority of the fecal water samples tested (14 samples) and evaluated for their effects on COX-2 in stimulated HT-29 cells, decreased COX-2 protein levels (38.8 ± 4.4%, range 19–63% inhibition) (Fig. 3). The effects were not due to toxicity because fecal water samples that reduced cell viability by >10% were not tested (samples 1, 3, 4, 6, 8, and 9).
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–stimulated induction of COX-2 protein in cells. Thus, cells were treated with 25–500 µmol/L of 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-phenylpropionic acid, 3-(4-hydroxyphenyl)-propionic acid, and 4-hydroxy-3-methoxycinnamic acid (ferulic acid). Only 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid decreased COX-2 protein levels at the higher concentrations (Table 2).
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(50 µg/L) potently induced PGE2 production, and this concentration was used in the experiments described below. NS-398 inhibited PGE2 production in TNF-–stimulated HT-29 cells by 50 ± 0.79%.
All of the individually tested fecal water samples (13 samples), except fecal water sample 19, inhibited PGE2 production in stimulated HT-29 cells compared with TNF-–treated control cells (Kruskal-Wallis test, P < 0.05) (Fig. 3). The fecal water samples as a group (24.4 ± 3.4, range 5.4–39.7% inhibition) also inhibited PGE2 production in HT-29 cells compared with TNF-–treated control cells, P < 0.05 (Mann-Whitney U test). The effects were not due to toxicity because fecal water samples that reduced cell viability by >10% were not tested (samples 1, 3–6, 8, and 9). Although the majority of the samples inhibited COX-2 at both the protein and PGE2 production levels, they were not strongly correlated (r = 0.27, P >> 0.10).
Of the phenolic compounds identified at relatively high concentrations in the fecal water samples, 5 were evaluated for their ability to inhibit TNF-–stimulated induction of PGE2 production in cells. Thus, cells were treated with 250–500 µmol/L of 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-phenylpropionic acid, 3-(4-hydroxyphenyl)-propionic acid, and 4-hydroxy-3-methoxycinnamic acid (ferulic acid). These experiments showed that none of the tested compounds had any effect on the COX-2 activity in the cells at the doses tested (results not shown).
Inhibition of COX-2 enzymatic activity (purified enzyme). The effects on COX-2 enzymatic activity of the fecal water samples were also investigated using a COX-2–catalyzed PG biosynthesis in vitro assay (Fig. 4). Two of the samples (18 and 20) inhibited the enzyme weakly (22–24%); 2 samples (3 and 8) appeared to stimulate the production of PGs. In addition, the effect on COX-2 activity of 18 pure diet-related compounds was tested: 8 phenolic acids and 4 flavonoids (whose presence was detected in the analyzed fecal waters), 5 bile acids, and 1 fatty acid. However, none of the tested compounds gave > 20% inhibition (results not shown).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phytochemicals such as flavonoids and phenolic compounds, found in fruits, vegetables, spices, and tea, are thought to protect against cancer. Of the polyphenols present in our diet, flavonoids are the most abundant group (7). Only part of the ingested flavonoids is absorbed, suggesting that much of the compound may have a local effect in the lumen of the colon (29,34). A number of studies also showed that phenolic compounds may either stimulate or inhibit the COX-2 enzyme (35,36). Therefore, we considered it of interest to analyze the fecal water fraction from the vegetarians for the presence of phenolic compounds. In the present study, the mean concentration of total phenolic compounds in fecal water samples was 789 ± 66 µmol/L, whereas flavonoids were detected only in minor amounts (<3 µmol/L). The major part of the phenolic compounds consisted of phenylpropionic acid, phenylacetic acid, cinnamic acid, and benzoic acid derivatives, compounds likely to be metabolites of the larger dietary flavonoids. For a comprehensive list of all phenolic compounds identified in fecal water samples, see Jennner et al. (30,34). A number of the identified phenolic compounds were tested in the bioassays discussed below. The high interindividual variation in fecal phenolic compounds is most likely due to differences in dietary intake of fruit and vegetables (phenolic compounds) and to some extent, differences in bacterial metabolism of phenolic compounds. It was reported previously that dietary flavonoids are metabolized into phenylacetic acids and other low-molecular-weight phenolic acids in the colon by the microbial flora (37). The present study did not record dietary intake. A follow-up study with dietary information is required to fully elaborate the effect of dietary intake of phenolic compounds on fecal phenolics.
A commonly used model to study effects on COX-2 is the human colonic epithelial carcinoma cell line HT-29 (16,38,39). COX-2 is expressed in these cells, and protein levels can be upregulated by stimulatory agents such as TNF- and bile acids (16,19,40,41). TNF- is a cytokine naturally present in the colon; it induces COX-2 via activation of the nuclear factor (NF)-
B transcription factor (42). We showed that the 2 specific COX-2 inhibitors, rofecoxib and NS-398, inhibited the induction of the COX-2 protein by TNF-. Also, the nonselective COX-2 inhibitor, indomethacin, inhibited COX-2 protein levels, whereas aspirin (a more specific COX-1 inhibitor) had no effect. Inhibition of COX-2 expression by NSAIDs was reported previously in monocytes, colon, prostate, and lung cells (43–46). However, to our knowledge, rofecoxib and NS-398 were not reported previously to inhibit COX-2 protein expression in colon cells. We then assessed the effects of the fecal water samples (diluted 1:20), which were not cytotoxic, on protein levels in this cell system. Dilution of the fecal water was justified because it was suggested that the stem cells at the bottom of the crypt are exposed to lower concentrations of luminal components than the concentrations in the lumen itself (13). The majority of the fecal water samples tested had a marked capacity to decrease COX-2 protein levels, whereas 2 of the samples (11 and 18) had weaker effects. Although the mechanism for this inhibitory effect has yet to be elucidated, one possibility is through effects on the NF-B signaling pathway (47,48). All fecal water samples tested also inhibited PGE2 production in cells. Thus, 13 of 14 fecal water samples tested inhibited both COX-2 protein expression and PGE2 production in cells. Fecal water sample 11 both stimulated COX-2 protein expression and inhibited enzyme activity, indicating a composition that diverged from the other samples. The effects of all fecal water samples on pure COX-2 enzymatic activity were also evaluated using a radiochemical COX-2 in vitro assay, presently in use for the identification of natural products as inhibitors of PG biosynthesis (33). Of the 20 fecal water samples, 2 weakly inhibited COX-2–catalyzed PG biosynthesis (mean of inhibition for all samples 8.2 ± 27.5%), whereas 2 samples appeared to exert a stimulating effect. The stimulatory effects were not a focus of the present paper, but may be relevant to investigate further in future studies. The 2 fecal water samples that inhibited COX-2 enzyme activity using purified enzyme also inhibited the enzyme in the cell system. This may indicate that the main inhibitory effect on PGE2 production in cells is due to inhibition of protein expression rather than direct inhibition of the enzyme. However, it cannot be ruled out that some fecal water samples contain compounds that directly inhibit enzymatic activity.
In an initial attempt to screen for the compounds in fecal water samples responsible for the above inhibitory effects, we investigated whether 5 of the phenolic compounds identified at high concentrations in fecal water samples (i.e., 4-hydroxy-3-methoxycinnamic acid, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxy-phenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid) could modify COX-2 protein levels or enzymatic activity in the colonic cells. Interestingly, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid, at concentrations of 250 and 500 µmol/L, decreased COX-2 protein levels. None of the tested phenolic compounds, however, inhibited PGE2 production in cells. Of the 5 fecal water samples analyzed for phenolic compounds by GC-MS, only 3 (12, 13, and 14) could be analyzed for COX-2 inhibition in the cell system. These samples strongly inhibited COX-2 protein expression in cells. One of the phenolics, 3-phenylpropionic acid, was present at concentrations > 250 µmol/L in 2 of these samples (Table 1). We speculate that this phenolic contributed to the COX-2 protein inhibitory effect of these fecal water samples in the colonic cells. However, work will continue to determine whether additional phenolic compounds are responsible for the COX-2 protein inhibitory effects and will address synergistic effects. It is noteworthy that none of the tested COX-2 selective inhibitors, phenylacetic and phenylpropionic acid derivatives, 4-hydroxy-3-methoxycinnamic acid (ferulic acid), or fecal water samples had any effects on the basal COX-2 level in the cell system (i.e., in unstimulated cells). These 5 phenolic above-mentioned compounds and 13 other pure compounds were also screened for their effects on enzyme activity using purified enzyme: 8 phenolic acids and 4 flavonoids, which were present in the fecal water samples, and 5 bile acids and 1 fatty acid [bile and fatty acids were reported previously to be present in fecal water (13)]. However, none of the tested compounds affected COX-2 enzymatic activity using pure enzyme, indicating that the observed inhibitory effects of fecal water samples are due to hitherto untested phenolic compounds, synergistic effects of more than 1 compound or hitherto unidentified compounds in the fecal water.
The observed effects of fecal water samples on pure COX-2 enzymatic activity were less pronounced compared with the effects on the protein level and PGE2 production in cells. It is noteworthy that 2 fecal water samples (18 and 20) inhibited COX-2 at all tested levels, i.e., protein expression, PGE2 production in cells, and pure enzyme activity. The 3 phenyl derivatives, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid, that decreased COX-2 protein levels in HT-29 cells had no effect on PGE2 production in cells. We speculate that the lack of effect on PGE2 production in cells by these 3 phenolic compounds is due to a simultaneous activation of COX-2 enzymatic sites (cyclooxygenase and peroxidase). The lack of correlation between the effects of the fecal water samples on COX-2 expression and activity most likely indicates that different compounds (hitherto unidentified) in the fecal water samples are responsible for these 2 effects, which is not surprising given the complex biodiversity of this fecal fraction. From our work with pure compounds, we can presently state that certain phenolics may be contributing to the effect of fecal water on protein expression.
In conclusion, the majority of the tested fecal water samples from the human volunteers potently decreased COX-2 protein levels and PGE2 production in colon cancer cells. Work will now continue to further identify the agents in fecal water samples that are responsible and address possible dietary effects (e.g., vegetarian vs. control diet). Finally, in view of the knowledge that the amount of COX-2 protein is important because there is a correlation between its level of expression and both the size of the colorectal tumors and their propensity to invade underlying tissue (49), even small effects over time on the protein levels and/or activity of this enzyme by dietary components may be important for tumor development in the colon.
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FOOTNOTES |
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2 These authors contributed equally.
4 Abbreviations used: COX-2, cyclooxygenase-2; DMSO, dimethyl sulfoxide; dpm, disintegrations per minute; FBS, fetal bovine serum; NF, nuclear factor; NS-398, [N-[2-(cyclohexyloxy)-4-nitrophenyl]methane-sulfonamide]; NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin; PGHS-2, prostaglandin endoperoxidase H synthase-2; TNF-, tumor necrosis factor .
Manuscript received 16 April 2005. Initial review completed 22 May 2005. Revision accepted 15 July 2005.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Almendingen K., Trygg K., Vatn M. Kostholdets innvirkning på celleproliferasjonen i tykk- og endetarm. Reduserer et strengt vegetarisk kosthold risikoen for å utvikle tarmkreft? Norwegian. [Influence of the diet on cell proliferation in the large bowel and the rectum. Does a strict vegetarian diet reduce the risk of intestinal cancer?]Tidsskr. Nor. Laegeforen. 1995;115:2252-2256.
2. Frentzel-Beyme R., Chang-Claude J. Vegetarian diets and colon cancer: the German experience. Am. J. Clin. Nutr. 1994;59:1143S-1152S.
3. Howe G. R., Benito E., Castelleto R., Cornee J., Esteve J., Gallagher R. P., Iscovich J. M., Deng-ao J., Kaaks R., et al. Dietary intake of fiber and decreased risk of cancers of the colon and rectum: evidence from the combined analysis of 13 case-control studies. J. Natl. Cancer Inst. 1992;84:1887-1896.
4. Jang M., Cai L., Udeani G. O., Slowing K. V., Thomas C. F., Beecher C. W., Fong H. H., Farnsworth N. R., Kinghorn A. D., et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science (Washington, DC). 1997;275:218-220.
5. Goel A., Boland C. R., Chauhan D. P. Specific inhibition of cyclooxygenase-2 (COX-2) expression by dietary curcumin in HT-29 human colon cancer cells. Cancer Lett. 2001;172:111-118.
6. Liang Y. C., Huang Y. T., Tsai S. H., Lin-Shiau S. Y., Chen C. F., Lin J. K. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis. 1999;20:1945-1952.
7. Scalbert A., Williamson G. Dietary intake and bioavailability of polyphenols. J. Nutr. 2000;130:2073S-2085S.
8. Andlauer W., Stehle P., Furst P. Chemoprevention—a novel approach in dietetics. Curr. Opin. Clin. Nutr. Metab. Care. 1998;1:539-547.
9. Allinger U. G., Johansson G. K., Gustafsson J. A., Rafter J. J. Shift from a mixed to a lactovegetarian diet: influence on acidic lipids in fecal water—a potential risk factor for colon cancer. Am. J. Clin. Nutr. 1989;50:992-996.
10. Rafter J. J., Child P., Anderson A. M., Alder R., Eng V., Bruce W. R. Cellular toxicity of fecal water depends on diet. Am. J. Clin. Nutr. 1987;45:559-563.
11. Rieger M. A., Parlesak A., Pool-Zobel B. L., Rechkemmer G., Bode C. A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of ’faecal water’. Carcinogenesis. 1999;20:2311-2316.
12. Venturi M., Hambly R. J., Glinghammar B., Rafter J. J., Rowland I. R. Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. Carcinogenesis. 1997;18:2353-2359.
13. Glinghammar B., Holmberg K., Rafter J. Effects of colonic luminal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells. Carcinogenesis. 1999;20:969-976.
14. Haza A. I., Glinghammar B., Grandien A., Rafter J. Effect of colonic luminal components on induction of apoptosis in human colonic cell lines. Nutr. Cancer. 2000;36:79-89.
15. Glinghammar B., Inoue H., Rafter J. J. Deoxycholic acid causes DNA damage in colonic cells with subsequent induction of caspases, COX-2 promoter activity and the transcription factors NF-B and AP-1. Carcinogenesis. 2002;23:839-845.
16. Glinghammar B., Rafter J. Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells. Gastroenterology. 2001;120:401-410.
17. Herschman H. R. Prostaglandin synthase 2. Biochim. Biophys. Acta. 1996;1299:125-140.
18. Smith W. L., Garavito R. M., DeWitt D. L. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem. 1996;271:33157-33160.
19. Weaver S. A., Russo M. P., Wright K. L., Kolios G., Jobin C., Robertson D. A., Ward S. G. Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells. Gastroenterology. 2001;120:1117-1127.
20. Kargman S. L., O’Neill G. P., Vickers P. J., Evans J. F., Mancini J. A., Jothy S. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res. 1995;55:2556-2559.
21. Dimberg J., Samuelsson A., Hugander A., Soderkvist P. Differential expression of cyclooxygenase 2 in human colorectal cancer. Gut. 1999;45:730-732.
22. Subbaramaiah K., Dannenberg A. J. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol. Sci. 2003;24:96-102.
23. Steinbach G., Lynch P. M., Phillips R. K., Wallace M. H., Hawk E., Gordon G. B., Wakabayashi N., Saunders B., Shen Y., et al. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med. 2000;342:1946-1952.
24. Thun M. J., Henley S. J., Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J. Natl. Cancer Inst. 2002;94:252-266.
25. Hong J., Smith T. J., Ho C. T., August D. A., Yang C. S. Effects of purified green and black tea polyphenols on cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissues. Biochem. Pharmacol. 2001;62:1175-1183.
26. Calviello G., Di Nicuolo F., Gragnoli S., Piccioni E., Serini S., Maggiano N., Tringali G., Navarra P., Ranelletti F. O., Palozza P. n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alpha induction pathway. Carcinogenesis. 2004;25:2303-2310.
27. Subbaramaiah K., Chung W. J., Michaluart P., Telang N., Tanabe T., Inoue H., Jang M., Pezzuto J. M., Dannenberg A. J. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J. Biol. Chem. 1998;273:21875-21882.
28. Luceri C., Caderni G., Sanna A., Dolara P. Red wine and black tea polyphenols modulate the expression of cycloxygenase-2, inducible nitric oxide synthase and glutathione-related enzymes in azoxymethane-induced f344 rat colon tumors. J. Nutr. 2002;132:1376-1379.
29. Halliwell B., Zhao K., Whiteman M. The gastrointestinal tract: a major site of antioxidant action?. Free Radic. Res. 2001;33:819-830.
30. Jenner A. M., Rafter J., Halliwell B. Human fecal water content of phenolics: The extent of colonic exposure to aromatic compounds. Free Radic. Biol. Med. 2005;38:763-772.
31. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248-254.
32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.). 1970;227:680-685.
33. Noreen Y., Ringbom T., Perera P., Danielson H., Bohlin L. Development of a radiochemical cyclooxygenase-1 and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis. J. Nat. Prod. 1998;61:2-7.
34. Halliwell B., Rafter J., Jenner A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not?. Am. J. Clin. Nutr. 2005;81:268S-276S.
35. Huss U., Ringbom T., Perera P., Bohlin L., Vasänge M. Screening of ubiquitous plant constituents for COX-2 inhibition with a scintillation proximity based assay. J. Nat. Prod. 2002;65:1517-1521.
36. Wang H., Nair M. G., Strasburg G. M., Chang Y. C., Booren A. M., Gray J. I., DeWitt D. L. Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. J. Nat. Prod. 1999;62:292-296.
37. Aura A. M., O’Leary K. A., Williamson G., Ojala M., Bailey M., Puupponen-Pimia R., Nuutila A. M., Oksman-Caldentey K. M., Poutanen K. Quercetin derivatives are deconjugated and converted to hydroxyphenylacetic acids but not methylated by human fecal flora in vitro. J. Agric. Food Chem. 2002;50:1725-1730.
38. Yamazaki R., Kusunoki N., Matsuzaki T., Hashimoto S., Kawai S. Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells. FEBS Lett. 2002;531:278-284.
39. Yang W. L., Frucht H. Cholinergic receptor up-regulates COX-2 expression and prostaglandin E(2) production in colon cancer cells. Carcinogenesis. 2000;21:1789-1793.
40. Arbabi S., Rosengart M. R., Garcia I., Jelacic S., Maier R. V. Epithelial cyclooxygenase-2 expression: a model for pathogenesis of colon cancer. J. Surg. Res. 2001;97:60-64.
41. Cianchi F., Cortesini C., Fantappie O., Messerini L., Sardi I., Lasagna N., Perna F., Fabbroni V., Di Felice A. et al. Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer. Clin. Cancer Res. 2004;10:2694-2704.
42. Jobin C., Morteau O., Han D. S., Balfour Sartor R. Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells. Immunology. 1998;95:537-543.
43. Takada Y., Bhardwaj A., Potdar P., Aggarwal B. B. Nonsteroidal anti-inflammatory agents differ in their ability to suppress NF-kappaB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene. 2004;23:9247-9258.
44. Shishodia S., Koul D., Aggarwal B. B. Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis. J. Immunol. 2004;173:2011-2022.
45. Pham H., Banerjee T., Ziboh V. A. Suppression of cyclooxygenase-2 overexpression by 15S-hydroxyeicosatrienoic acid in androgen-dependent prostatic adenocarcinoma cells. Int. J. Cancer. 2004;111:192-197.
46. Agarwal B., Swaroop P., Protiva P., Raj S. V., Shirin H., Holt P. R. Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells. Apoptosis. 2003;8:649-654.
47. Kojima M., Morisaki T., Izuhara K., Uchiyama A., Matsunari Y., Katano M., Tanaka M. Lipopolysaccharide increases cyclo-oxygenase-2 expression in a colon carcinoma cell line through nuclear factor-kappa B activation. Oncogene. 2000;19:1225-1231.
48. Wenzel U., Kuntz S., Brendel M. D., Daniel H. Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer Res. 2000;60:3823-3831.
49. Fujita T., Matsui M., Takaku K., Uetake H., Ichikawa W., Taketo M. M., Sugihara K. Size- and invasion-dependent increase in cyclooxygenase 2 levels in human colorectal carcinomas. Cancer Res. 1998;58:4823-4826.
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