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Department of Biology, University "Roma Tre," I-00146 Rome, Italy;
* National Institute for Food and Nutrition Research (INRAN), I-00178 Rome, Italy;
School of Medicine, Health Policy & Practice, University of East Anglia, Norwich, NR4 7TJ, UK;
** Department of Bioengineering, Imperial College, London SW7 2AZ, UK; and
Institute of Human Nutrition and Food Science, Christian Albrechts University, D-4111 Kiel, Germany
2To whom correspondence should be addressed. E-mail: m.marino{at}uniroma3.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • daidzein metabolites • 17ß-estradiol • estrogen receptor • gene transcription • apoptotic cascade
The soy isoflavones, daidzein (D)3 and genistein, may have a preventive effect against various cancers (1–7). Among several mechanisms proposed for these effects (e.g., antioxidant activity, kinase inhibition) (8,9), the possibility that isoflavones could hamper cell proliferation by binding to estrogen receptor (ER) isoforms (10,11) is especially intriguing. D acts as an ER agonist, with a greater affinity for the ERß isoform than the ER
isoform (10,11), inducing receptor-mediated transcription of 17ß-estradiol (E2)-sensitive genes (10). Recently, the ability of E2-activated ERß to activate signal transduction pathways, starting from the plasma membrane and specifically to block cancer cell proliferation, was recognized (12–14); similar results were obtained when ER or ERß interacted with flavonoids (i.e., naringenin, quercetin) (15,16). Hence, isoflavones could decrease the risk of degenerative pathologies in postmenopausal women without the side effects associated with E2 replacement therapy.
Nevertheless, isoflavones are only weakly estrogenic in vivo (17). This low potency may be due to rapid biotransformation, among other factors. After ingestion of soy, hydrolysis by intestinal glucosidases releases the aglycones, daidzein, genistein, and glycitein from the glucosides, acetyl-glucosides, and malonyl-glucosides present in the food (18). The aglycones are further metabolized by gut bacteria, the mucosa of the small intestine, and the liver. The physiological actions of such metabolites may therefore hold the key to understanding the protective benefits of this dietary component. However, data regarding the ability of metabolites to retain the biological activity of the aglycone are still scarce (19).
Equol (Eq) [7-hydroxy-3-(4'-hydroxyphenyl)-chroman], a product of gut bacteria, is probably the most studied isoflavone metabolite and has an estrogen-like activity that is actually stronger than the parent isoflavones. However, Eq is not produced in all healthy adults after consumption of soy or purified daidzein. It is now apparent that there are 2 distinct subpopulations of people and that "bacterio-typing" individuals for their ability to make Eq may hold the clue to the effectiveness of isoflavones in the treatment or prevention of hormone-dependent conditions (20).
A large number of other metabolites, resulting from the combined activities of hydroxylation and conjugating enzymes, circulate in the human blood stream. The formation of conjugates converts the aglycones to more water-soluble products (hydroxylated and reduced forms, and sulfuric and glucuronic acid conjugates) and may affect their biological activity. Cells are exposed predominantly to these resultant metabolites rather than to the parent compounds. In male rats, for example, only a small portion of the aglycone was detected free in the blood and urine after oral administration of D; D sulfates were the major excretory products (10-fold more that the aglycone) (18).
It was shown that glucuronidation reduces the biological activities of D and genistein (2,5). Furthermore, it was reported that sulfation of genistein decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion, and chemotaxis (21,22). At present, no information is available regarding the estrogenic and antiproliferative activity of many metabolites of D. Here we examine the ability of well-characterized, chemically synthesized daidzein metabolites to modulate E2-sensitive gene transcription, cell growth, and a proapoptotic cascade in human cancer cells in an ERß-dependent manner.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasmids.
The plasmid containing the promoter of complement component 3 gene, retaining a natural estrogen responsive element (ERE), linked to the gene of luciferase (pC3-luc), the expression vectors for pCR3.1-ß-galactosidase (23), and human pCNX2-hERß (24), were used. An empty plasmid, pCMV5 (23), was used as a control. Plasmids were purified for transfection using a plasmid preparation kit according to the manufacturer’s instructions. A luciferase dose-response curve showed that the maximum effect was present when 1 µg of plasmid was transfected together with 1 µg of pCR3.1-ß-galactosidase to normalize for transfection efficiency (
55–65%).
Cell culture and proliferation assay.
This study was conducted on human cervix epitheloid carcinoma cells (HeLa), devoid of ERs and rendered E2 sensitive by transient transfection with a human ERß expression vector. This model permits the detection of ERß-mediated effects of flavonoids without interference from ER or from ERß splice variants. HeLa cells were grown routinely in modified, phenol red–free, DMEM containing 10% (v:v) charcoal-stripped fetal calf serum, L-glutamine (2 mmol/L), gentamicin (0.01 g/L), and penicillin (100 kU/L) under air containing 5% CO2. Cells were split and the medium changed every 2 d. Cells were grown to 70% confluence in 6-well plates, then transfected with human pCNX2-hERß or pCMV5; 24 h later, they were stimulated with different concentrations of D and D metabolites for 30 h in the presence or absence of the ER inhibitor ICI (1 µmol/L). After treatment, cells were harvested with trypsin, centrifuged at 100 x g for 3 min, stained with trypan blue solution, and counted in a hemocytometer (improved Neubauer chamber) in quadruplicate. D and D metabolites were solubilized in dimethyl sulfoxide (DMSO) to obtain a stock solution (1 mmol/L). Different aliquots from this solution were diluted 1:5 (v:v) in PBS; 10 µL of DMSO:PBS 1:5 (v:v) was used as a vehicle. The final concentration of DMSO in the cell medium was <0.1%. The cell viability, evaluated by Trypan blue exclusion test, was 90–95% in cells stimulated with concentrations of D and D metabolites up to 50 µmol/L.
Transfection and luciferase assay.
Cells were grown to 70% confluence, then transfected using Lipofectamine Reagent according to the manufacturer’s instructions. At 6 h after transfection, the medium was changed; 24 h later, cells were stimulated with different concentrations of D or its metabolites for 6 h. When indicated, 1 µmol/L of the ER inhibitor ICI was added 15 min before treatment with D or its metabolites. The cell lysis procedure and the subsequent measurement of luciferase gene expression were performed using the luciferase kit according to the manufacturer’s instructions with an EC & G Berthold luminometer.
Electrophoresis and immunoblotting. When indicated, 1 µmol/L of the ER inhibitor ICI was added 15 min before treatment with E2 (10 nmol/L), D, or its metabolites (1 µmol/L) for different times. Transfected HeLa cells were lysed as previously described (14), solubilized in 0.125 mol/L Tris HCl (pH 6.8) containing 10% (wt:v) SDS, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.05 g/L leupeptin, and boiled for 2 min. Proteins were quantified using the Bradford Protein Assay. Solubilized proteins (20 µg) were resolved using 10% SDS-PAGE at 100 V for 1 h. The proteins were then electrophoretically transferred to nitrocellulose for 45 min at 150 V and 4°C. The nitrocellulose was treated with 3% bovine serum albumin in 138 mmol/L NaCl, 26.8 mmol/L KCl, 25 mmol/L Tris HCl (pH 8.0), 0.05% Tween-20, 0.1% bovine serum albumin, and then probed at 4°C overnight with one of anti-ERß, anti-phospho-p38, anti-caspase-3, or anti-PARP antibodies. The nitrocellulose was stripped by Restore Western Blot Stripping Buffer (Pierce Chemical) for 10 min at room temperature and then probed with anti-p38 (1 mg/L). Anti-ß-actin antibody (1 mg/L) was used to normalize the sample loading. Antibody reaction was visualized by chemiluminescence.
Statistical analysis. Data were analyzed by 1-way ANOVA and post hoc Bonferroni test (INSTAT software system for Windows). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To determine further the involvement of ERß in pC3-luc promoter activity, HeLa cells were pretreated with the pure antiestrogen, ICI. When added alone, ICI did not affect pC3 promoter activity, whereas its addition before E2, D, or the active D metabolites completely blocked the induction (Fig. 1D). These results indicate that some D metabolites (i.e., Eq and D-7S) can trigger the ERß-mediated genomic mechanism mimicking the effects of E2, whereas others (i.e., O-DMA, D-4'S, and D-diS) cannot.
We recently reported that if ERß is expressed, E2 or the flavanone naringenin can drive cells out of cell cycle by rapid activation of p38/mitogen-activated protein kinase (MAPK) which, in turn, initiates an apoptotic cascade (i.e., caspase-3 activation and PARP cleavage) (14,15). We therefore evaluated the ability of D and its metabolites to modulate these ERß-induced activities.
The growth of HeLa cells transfected with either empty plasmid or the ERß expression vector was examined 30 h after stimulation with different concentrations of E2 or D and its metabolites. Only the highest concentration of D (100 µmol/L) decreased the growth of HeLa cells transfected with empty plasmid, whereas there was no effect of D metabolites even at this concentration (Table 1). In ERß-transfected HeLa cells, however, E2, D, Eq, and D-7S (but not D-diS, D-4'S, or O-DMA) decreased growth with respect to untreated cells (Fig. 2A and B) in a time- and dose-dependent manner within the range used (0.1–10 µmol/L), suggesting that the presence of ERß is necessary for the isoflavone effects at physiological concentrations. The pretreatment of ERß-transfected HeLa cells with the ER inhibitor ICI completely blocked the effects on cell growth of 1 µmol/L D, Eq, and D-7S, respectively (Fig. 2C), further confirming the ER dependence of the antiproliferative effects induced by lower isoflavone concentrations.
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2 times greater than D-7S or E2 (Fig. 3E and F). None of the test compounds induced p38/MAPK phosphorylation or caspase-3 and PARP cleavage at any concentration in the ERß-negative HeLa cells (data not shown). Furthermore, proapoptotic protein activations were prevented by the specific ER inhibitor ICI in ERß-transfected HeLa cells (Fig. 3G). Together these results demonstrate that D and its metabolites, Eq and D-7S, specifically activate an apoptotic cascade involving caspase-3 and its downstream substrate PARP in the presence of ERß.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isoflavones, once consumed either as aglycone or glycosides, enter a complex pathway of biotransformation and the presence of the original molecule becomes almost negligible. The relative concentration of different metabolites in both plasma and tissues is determined by the specific contribution of intestinal microflora and by deconjugation/conjugation processes within the human body. In fact, available data on the estrogenic activity of D metabolites are restricted largely to Eq, whose production depends on the individual’s ability to host specific intestinal microflora (19). Once absorbed, daidzein is efficiently reconjugated, either with glucuronic acid or, to a lesser extent, sulfate. Conjugation with sulfonic acid also takes place in the liver with hepatic and intestinal sulfotransferase enzymes. As a consequence, isoflavones are present in the circulation predominantly in their glucuronide and sulfate forms (27). Our study addressed the estrogen-like activity of the sulfates only.
Our data indicate that daidzein treatment is associated with a superinduction of luciferase activity, i.e., an induction of the reporter gene product above the level maximally inducible by E2. This phenomenon of superinduction was observed in a cell context–specific fashion, for a number of compounds including resveratrol, genistein, diphenyl esters, and others (11,28–32). The underlying mechanism remains obscure. Resveratrol and some flavonoids were recently characterized as antagonists of the aromatic-hydrocarbon receptor, which in turn can interfere with estrogenic signaling at the DNA level (33,34). In addition, binding of daidzein to ER-ß might induce conformational changes in the receptor structure that result in greater recruiting of transcriptional coactivators to the C3 promoter than is seen with estradiol. This activity has already been suggested for other flavonoids binding to ER- (35). Of the D metabolites considered here, Eq and D-7S increased ERß-dependent luciferase induction. They reached their maximal effect at 100 µmol/L, but due to the cytotoxic effects, this concentration was not considered. At 0.1 µmol/L, these D metabolites had already increased promoter induction to the same extent as 0.01 µmol/L E2. Daidzein-4' and -4',7 sulfuric acid conjugates and O-DMA were inactive.
A structural comparison of D and its metabolites with E2 indicates that the phenolic hydroxyl groups in positions 4' and 7 can be considered equivalent to the position 3 and 17 hydroxyl groups of E2. The main role in the E2-like activity of D is played by the 4'-position hydroxyl group because the presence of a 4'-position sulfate group abrogates the estrogenic activity of D. The 7-position hydroxyl group on the A ring is less important: comparing data from D and D-7S shows that when this substituent was conjugated with sulfuric acid, the isoflavone-induced luciferase activity decreased but was still significant. These results, together with data for nonphysiologically relevant compounds obtained by methylation of the hydroxy substituent in the 7 and/or 4' position (36), suggest that the structural conformation of the metabolites plays an important role in their estrogenic activity although it remains to be determined whether structural changes exert their effects by directly influencing ER-isoflavone interactions, or through influences upstream of ER (for example by affecting cellular uptake).
A more complex structure-activity relation was found for the antiproliferative effects of D and its metabolites. At a high concentration (100 µmol/L), D inhibited cell proliferation even in absence of ERß (Table 1), and this effect was independent of the proapoptotic cascade (data not shown). An ER-independent cytotoxic effect of high concentrations of D was reported earlier in different cell lines (37,38) and probably involves nonspecific actions such as the inhibition of tyrosine kinases, or antioxidant effects (8,9,37). Notably, neither equol nor D-7S induced antiproliferative activity in the absence of ERß, suggesting that the spatial conformation is fundamental to the ER-independent activity of D. A similar impairing effect of sulfation was recently reported for the antioxidant activity of daidzein (21,22).
In the presence of ERß, D had antiproliferative effects at lower, more physiological concentrations (0.1–10 µmol/L) that were dependent on the proapoptotic pathway. The current study indicates for the first time that some D metabolites but not others retain this potentially beneficial antitumor activity, again at physiological concentrations. The results highlight the need to use physiologically relevant metabolites when investigating the putative beneficial properties of isoflavones. Furthermore, because the intestinal microflora plays a key role in the metabolism and bioavailability of isoflavones (39), factors influencing the microflora also require consideration. For example, a high-carbohydrate milieu causes increased intestinal fermentation and hence more extensive biotransformation, greatly enhancing equol formation. In addition, a Clostridium sp. strain that converts D principally to O-DMA was identified from intestinal microflora; this could render isoflavones less active (40). Among individuals who regularly consume soy products, those who produce equol or D-7S may have longer exposure to potent estrogenic compounds. Premenopausal women who excrete equol have plasma hormone profiles associated with a lower risk of breast cancer (40). Therefore, not only biotransformation but also the interindividual and gender variation in isoflavone metabolism should be taken into account when examining the disease preventative activity of dietary isoflavones.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: D, daidzein; D-4'S, daidzein-4' sulfate; D-7S, daidzein-7 sulfate; D-diS, daidzein-4',7 disulfate; DMSO, dimethyl sulfoxide; E2, 17ß-estradiol; EC50, 50% agonist effective concentration; Eq, equol; ER, estrogen receptor; ERE, estrogen responsive element; ICI, ICI 182,780; MAPK, mitogen-activated protein kinase; O-DMA, O-desmethylangiolensin; PARP, poly(ADP-ribose)polymerase.
Manuscript received 22 March 2005. Initial review completed 8 June 2005. Revision accepted 29 July 2005.
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