Interactions between sulforaphane and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells

Vanda Svehlíková, Shuran Wang1, Jana Jakubíková2, Gary Williamson3, Richard Mithen and Yongping Bao4

Nutrition Division, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK
Present addresses: 1 Department of Nutrition and Food Hygiene, Harbin Medical University, Harbin 150001, P. R. China, 2 Department of Molecular Immunology, Cancer Research Institute, Vlarska 7, Bratislava, Slovak Republic and 3 Nestlé Research Center, PO Box 44, CH-1000 Lausanne 26, Switzerland

4 To whom correspondence should be addressed Email: yongping.bao{at}bbsrc.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The isothiocyanate, sulforaphane and the flavonoid, apigenin modulate gene expression including phase II detoxifying enzymes, such as glutathione S-transferases (GST) and UDP-glucuronosyltransferases (UGT). Using undifferentiated CaCo-2 cells, we have examined the interactions between sulforaphane and apigenin in the regulation of UGT and GST expression. We show that apigenin induces UGT1A1 transcription (4-fold) but not GSTA1, and that sulforaphane induces both UGT1A1 (3.7-fold) and GSTA1 (2.8-fold) transcription in both dose- and time-dependent manners. The combination of sulforaphane and apigenin resulted in a synergistic induction of UGT1A1 mRNA up to 12-fold, although this interaction was not seen for GSTA1. Nuclear factor kappa B (NF-{kappa}B) mRNA was induced by apigenin and sulforaphane (2.5- and 2-fold, respectively). NF-{kappa}B translocation inhibitor SN50 and phosphatidylinositol 3-kinase (PI3) inhibitor LY294002 decreased the induction of GSTA1 by sulforaphane almost to baseline level. However, the MEK inhibitor PD98059 enhanced significantly the induction of GSTA1 by sulforaphane. This suggests that NF-{kappa}B and PI3-kinase signaling pathways play a role in GSTA1 gene expression. Conversely, the induction of UGT1A1 transcription by sulforaphane was totally abolished by PD98059, although PD98059 slightly enhanced (20%) the induction of UGT1A1 by apigenin implying that the induction of UGT1A1 by sulforaphane is mediated by MAPK/extracellular signal-regulated kinase kinase, whereas UGT1A1 induction by apigenin may be associated with NF-{kappa}B translocation since the NF-{kappa}B translocation inhibitor, SN50 enhanced the induction of UGT by apigenin. The results show that UGT1A1 and GSTA1 are regulated by sulforaphane through different signal transduction pathways and the differences in the mechanisms of modulation of UGT1A1 transcription by sulforaphane and apigenin resulted in a synergistic effect between these two compounds in the induction of UGT1A1.

Abbreviations: ARE, antioxidant responsive element; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; ITCs, isothiocyanates; MAPK, mitogen-activated protein kinase; MEK1, MAPK/extracellular signal-regulated kinase kinase; NF-{kappa}B, nuclear factor kappa B; Nrf2, NF-E2-related factor 2; PI3-kinase, phosphatidylinositol 3-kinase; SFN, sulforaphane; RT–PCR, reverse transcription polymerase chain reaction; SP1, special protein 1; UGT, UDP-glucuronosyltransferase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is considerable and growing evidence suggesting that diets rich in fruits and vegetables are protective against chronic diseases including heart disease and cancer (13). The protective effects are at least partially attributed to dietary phytochemicals, especially isothiocyanates (ITCs) and flavonoids (46). The anticarcinogenic activity of ITCs involves the modulation of carcinogen metabolism by induction of phase II detoxification enzymes (7,8), inhibition of phase I carcinogen-activating enzymes (9), inhibition cell growth by cell cycle arrest, and activation of apoptosis (10). In contrast, flavonoids possess a wide range of antioxidant properties in vitro such as inhibition of lipid and LDL oxidation, anti-proliferation of human cancer cells, and induction of phase II enzymes (11,12). UDP-glucuronosyl transferases (UGT) and glutathione S-transferases (GST) are two major phase II detoxifying enzymes, which protect cells against both endogenous and exogenous carcinogens by glucuronidation and nucleophilic addition of glutathione to a variety of different substrates, respectively (8,1315). Transcriptional regulation of phase II enzymes by ITCs is via Keap1–NF-E2-related factor 2 (Nrf2)–antioxidant responsive element (ARE) mediated pathways (16,17). ITCs have been shown to induce cell cycle arrest and apoptosis in tumor cell lines, associated with the activation of caspase-8, JNK1 and tyrosine phosphorylation (10,1821). The flavonoid apigenin has also been shown to inhibit cell growth by inducing cell cycle arrest in G2/M phase and programmed cell death (2224). There is, however, little information available on the induction of phase II enzymes by apigenin. In this study, we have examined the interaction between sulforaphane (SFN) and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells, a well-characterized human small intestinal undifferentiated cell model (25).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Sulforaphane (4-methylsulfinylbutyl isothiocyanate) (purity, 97%) was purchased from ICN Biomedicals (Basingstoke, UK), and apigenin from Apin Chemicals (Oxon, UK). Digitonin, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), phenylmethylsulfonylfluoride (PMSF), Bradford reagent for protein quantification and secondary antibody and HRP-conjugated goat anti-rabbit IgG were purchased from Sigma (Dorset, UK). Inhibitors nuclear factor kappa B (NF-{kappa}B) SN50, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), 2'-amino-3'methoxyflavone (PD98059), human recombinant GSTA1-1 and primary polyclonal anti-human GSTA1-1 antibody were purchased from Calbiochem (San Diego, CA). Rabbit polyclonal primary antibody to special protein 1 (SP1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). WB-UGT1A1 western blotting kit was obtained from Gentest (Woburn). Rabbit polyclonal antibody to NF-{kappa}B P65 was purchased from Abcam (Cambridge, UK). Electrophoresis, western blotting supplies, and molecular weight marker (MagicMarkTM) were obtained from Invitrogen (Paisley, UK), and chemiluminescence kit was Amersham Bioscience (Little Chalfont, UK).

Cell culture and treatments
Human colon adenocarcinoma CaCo-2 cells were obtained from the European Cell Culture Collection (Wiltshire, UK). Cells were seeded in 6-well plates for mRNA assays and in 10 cm dishes for protein extract preparation at the density of 3 x 104 cells/cm2. The cells were used at passage 35–53 and cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 50 µg/ml streptomycin under 5% CO2 in air at 37°C. Cells were treated with SFN and apigenin for 8, 24 and 72 h when they reached ~80% confluence. During the latter incubation, the cell culture medium including treatments was changed every 24 h. The test compounds were dissolved in DMSO, all treatments and controls contained a final concentration of 0.1% DMSO in the media. When CaCo-2 cells were treated with inhibitors, NF-{kappa}B SN50 (50 µg/ml) was added to the medium 30 min prior to the stimulation with apigenin or SFN, PD98059 (50 µM) and LY294002 (10 µM) were added 90 min prior to apigenin and SFN addition and cells were incubated for 6 h.

TaqMan real-time reverse transcription polymerase chain reaction (RT–PCR)
Total RNAs from treated and control CaCo-2 cells were isolated using GenEluteTM Total Mammalian RNA Kit (Sigma, UK) according to the manufacturer's instructions. RNA concentration and purity were determined based on measurement of the absorbance at 260 and 280 nm. After adding RNase inhibitor (20 U) the total RNA was stored at –70°C. Target mRNA was quantified by real-time RT–PCR (TaqMan®) using an ABI PRISMTM 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). Forward and reverse primers and the fluorogenic TaqMan® probes were designed using ABI PRISM Primer Express Software (Applied Biosystems). TaqMan assays for UGT1A1 and GSTA1 have been reported and validated previously (26). All the primer and probe sequences have been listed in Table I.


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Table I. Primer and probe sequences

 
The probes were labeled with a 5' reporter dye FAM (6-carboxyfluorescein) and 3' quencher dye TAMRA (6-carboxytetramethylrhodamine). RT–PCR reactions were carried out in a 96-well plate in a total volume of 25 µl/well consisting of TaqMan one-step RT–PCR master mix reagent (Applied Biosystems), 10 ng of total RNA (5 ng for ß-actin), 100 nM probe, 200 nM forward primer and 200 nM reverse primer to amplify UGT1A1, GSTA1, ß-actin or 300 nM reverse primer for NF-{kappa}B amplification. Reverse transcription was performed for 30 min at 48°C, AmpliTaqTM gold activation for 10 min at 95°C, followed by 40 PCR cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Reactions were carried out in triplicate. Data were analyzed by TaqMan software using a standard curve method as described in User Bulletin No. 2 (ABI PRISM 7700 Sequence Detection System) to quantify mRNA amount. Standard curves were constructed for each amplified gene sequence using 1, 5, 10, 20 and 40 ng of total RNA per reaction in triplicates. ß-actin mRNA was measured as an internal reference.

Preparation of protein extracts
Treated and control CaCo-2 cells were washed twice with PBS and harvested using trypsin–EDTA. After centrifugation for 5 min at 176 g, cell pellets were re-suspended in 0.5 ml 0.1 M Tris–HCl buffer (pH 7.4) containing 0.1% digitonin, 1 mM DTT and 1 mM PMSF. The cells were disrupted by sonication on ice for 20 min. The cell homogenate was centrifuged for 20 min at 12 000 g at 4°C. The supernatant was collected and protein concentration was determined using Bradford assay using bovine serum albumin as standard (27).

Immunoblotting
Protein extracts were heated at 90°C for 5 min in sample treatment buffer and in the presence of reducing agent and loaded on NuPAGE®–Novex Bis-Tris pre-cast gel together with a positive control and a molecular weight marker. Electrophoresis and transfer to nitrocellulose membrane were performed according to the manufacturer's instructions. The membrane was blocked with 5% fat free milk and 1% BSA in 10 mM Tris/150 mM NaCl/0.05% Tween 20 (TBST), pH 7.4, for 2 h and incubated with a specific primary antibody in 1% milk in TBST for 1 h. The blots were washed three times for 5 min with TBST and then incubated with the secondary antibody, HRP-conjugated goat anti-rabbit IgG, diluted 1:2000 with 1% milk in TBST for 1 h. After washing the membrane again three times for 5 min with TBST, the antibody binding was determined by chemiluminescence detection kit.

Statistical analyses
Two sets of data were compared by Student's t-test. Comparisons of multiple groups were performed by one-way analysis of variance followed by Newman–Keuls test in the case of statistically significant differences among groups. The criterion for statistical significance was set at P < 0.05, P < 0.01 and P < 0.001.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of UGT1A1 and GSTA1 mRNA levels
The effect of SFN and apigenin on the induction of UGT1A1 and GSTA1 in human CaCo-2 cells was measured using real-time RT–PCR assays. Up-regulation of GST and UGT mRNA was initiated at 4–8 h after treatment with SFN (20 µM) and reached a maximum value for UGT1A1 (3.7-fold) and GSTA1 (2.8-fold) at 12 and 24 h, respectively (Figure 1A). When cells were treated with different concentrations of SFN (1–40 µM), the induction of UGT1A1 and GSTA1 mRNA levels were dose-dependent (Figure 1B). Interestingly, SFN at 10 µM induced UGT and GST ~2-fold, this was comparable with a previous study using broccoli/onion extract (containing 11 µM SFN) in a human jejunum in vivo perfusion (28). In contrast, apigenin (10 µM) induced UGT1A1 mRNA up to 4-fold, but, suppressed GSTA1 mRNA at 10–25 µM and had no effect at lower concentrations (0.2–5 µM) (Figure 1C). The level of ß-actin mRNA was not changed after SFN (1–10 µM), apigenin (1–25 µM) and SFN + apigenin (5 + 5, 10 + 10 µM) treatments, but increased 17 and 26% following 20 and 40 µM SFN treatment, respectively (Figure 1D).




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Fig. 1. Induction of UGT1A1 and GSTA1 by sulforaphane and apigenin at the transcriptional level. (A) UGT1A1 and GSTA1 gene transcription in SFN (20 µM) treated CaCo-2 cells. Total RNA was isolated and the relative quantity of UGT1A1 and GSTA1 mRNA was determined by real-time RT–PCR. (B) Dose-dependent activation of UGT1A1 and GSTA1 gene expression in sulforaphane-treated CaCo-2 cells. Cells were incubated with the SFN or DMSO (final concentration 0.1%) for 24 h. UGT1A1 and GSTA1 mRNA were analyzed in the total RNA extract by real-time RT–PCR. (C) Dose-dependent activation of UGT1A1 and GSTA1 gene expression in CaCo-2 cells exposed to apigenin for 24 h. (D) Effects of SFN on housekeeping gene, ß-actin expression. Experiments were performed in triplicate, data shown represent mean ± SD. One-way analysis of variance followed by Newman–Keuls test was used for comparisons of data groups (significant difference compared with control, *P < 0.05, **P < 0.01, ***P < 0.001).

 
Synergy between SFN and apigenin in the induction of UGT1A1 expression
Since both SFN and apigenin are potent inducers of UGT1A1, we investigated possible interactions when the cells were treated with both compounds. CaCo-2 cells were treated with SFN and apigenin at 1, 5 and 10 µM either alone or in combination. The results show a significant synergistic effect (more than additive) between SFN and apigenin in the induction of UGT1A1 mRNA expression (Figure 2). SFN and apigenin at 1 µM alone induced 1.3 ± 0.1 and 1.8 ± 0.2-fold, respectively, whereas in combination, SFN and apigenin induced 3.9 ± 0.6-fold; SFN and apigenin at 5 µM alone induced 1.4 ± 0.1 and 2.6 ± 0.5-fold, respectively, and in combination induced 9.4 ± 0.8-fold. At 10 µM, SFN induced 1.9 ± 0.1-fold and apigenin induced 3.8 ± 0.5-fold; in combination SFN and apigenin induced 12.0 ± 0.4-fold.



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Fig. 2. Synergistic effect of apigenin and sulforaphane on UGT1A1 expression. CaCo-2 cells were incubated with combinations of apigenin and SFN for 24 h. Effect of the treatment on induction of UGT1A1 transcription was analyzed in a real-time RT–PCR assay relative to control (treated with 0.2% DMSO). Experiments were performed in triplicate; data shown represent mean ± SD. One-way analysis of variance followed by Newman–Keuls test was used for comparisons of data groups (significant difference compared with control cells, **P < 0.01, ***P < 0.001).

 
Induction of UGT1A1 and GSTA1 at protein level
It was reported that a maximum induction of UGT1A1 protein in CaCo-2 cells by flavonoids occurred after 72 h following treatment (29). Immunoblot analyses of protein extracts from CaCo-2 cells after treatment with SFN for 72 h, confirmed the dose-dependent induction of UGT1A1 (Figure 3A). GSTA1-1 protein was also increased in the CaCo-2 cells treated with SFN at relatively lower concentration (1 µM) (30), and GST protein increased at up to 20 µM SFN treatment. However, SFN at 40 µM decreased the GST protein expression to the control level (Figure 3B). This may be a specific effect of SFN on GSTA1-1 translation since UGT1A1 and ß-actin protein levels were not affected by 40 µM SFN. However, it may also be due to a cytotoxic effect since the cells are less viable at high concentration of SFN treatment (IC50 = 83 µM, 24 h; IC50 = 31 µM, 48 h; IC50 = 23 µM, 72 h). Apigenin also induced UGT1A1 protein (Figure 3C). Moreover, the co-treatment with both SFN and apigenin induced a significant expression of UGT1A1 protein although it was not as pronounced as the synergy at the transcriptional level (Figure 3D).



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Fig. 3. Induction of UGT1A1 and GSTA1 at protein level-western blotting. CaCo-2 cells were incubated with SFN for 72 h. Control cells were treated with equivalent amount of DMSO (final concentration 0.1%). (A) UGT1A1 protein from CaCo-2 cells after SFN treatment (20 µg total protein/lane). (B) GSTA1-1 proteins in CaCo-2 cells after SFN treatment (10 µg total proteins/lane). (C) UGT1A1 protein in Caco-2 cells after apigenin treatment. (D) UGT1A1 protein in Caco-2 cells after SFN and apigenin co-treatment (40 µg total protein/lane). Lane 1, control; lane 2, SFN (5 µM); lane 3, apigenin (5 µM); lane 4, SFN + apigenin (5 + 5 µM). For ß-actin protein, 20 µg total protein/lane was loaded.

 
Induction of NF-{kappa}B expression
NF-{kappa}B is present in the cytoplasm of unstimulated cells in an inactive form bound to its inhibitor I{kappa}B. Upon stimulation, the inhibitor is degraded by proteolysis, and activated NF-{kappa}B can then be translocated to the nucleus and bind to the promoter of a target gene (31). Treatment of CaCo-2 cells with SFN and apigenin resulted in a dose-dependent increase of NF-{kappa}B mRNA (2- and 2.5-fold, respectively) (Figure 4A). A time course for SFN on NF-{kappa}B induction showed that NF-{kappa}B mRNA responded to SFN as early as 4 h after treatment and the induction was up to 3-fold (Figure 4B). The NF-{kappa}B P65 protein levels were increased after treatment with SFN (5, 20 and 40 µM) for 8 h, while a suppression was observed after 24 h treatment by 20 and 40 µM SFN (Figure 4C). In contrast, apigenin (5–25 µM) did not affect NF-{kappa}B P65 protein expression at any of the time points tested up to 72 h (Figure 4D).



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Fig. 4. Induction of NF-{kappa}B mRNA and protein. (A) NF-{kappa}B mRNA in CaCo-2 cells treated with apigenin and SFN for 24 h. (B) Time course of NF-{kappa}B activation at mRNA level in CaCo-2 cells exposed to SFN (20 µM) as determined by real-time RT–PCR. (C) Western blotting of NF-{kappa}B P65 protein in CaCo-2 cells treated with SFN for 8, 24 and 72 h. Control cells were treated with DMSO (0.1%). Total protein loading was 5 µg/lane. (D) Western blotting of NF-{kappa}B P65 protein levels in cells treated with apigenin for 8, 24 and 72 h in comparison with DMSO treated control. Total protein loading was 5 µg/lane. For ß-actin protein, 20 µg total protein/lane was loaded.

 
Effect of inhibitors of NF-{kappa}B, MAPK/extracellular signal-regulated kinase kinase (MEK1) and phosphatidylinositol 3-kinase (PI3-kinase)
As a result of the differences in activation of UGT1A1 and GSTA1 by SFN and apigenin in CaCo-2 cells, we further studied the involvement of NF-{kappa}B, the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade and the PI3-kinase in the induction of UGT and GST. The PI3-kinase Akt and NF-{kappa}B pathways converge at different points as shown in several studies (32,33). The ERK MAPK pathway is reported to be linked to the transcription factor Nrf2, which interacts with ARE, found in the 5' flanking regions of several antioxidant enzyme genes including GSTs, thioredoxin reductase 1 and UGT (18,3436).

To study the significance of NF-{kappa}B in UGT and GST induction we used NF-{kappa}B SN50, a synthetic peptide containing a cell membrane-permeable motif and a nuclear localization sequence of NF-{kappa}B p50, which inhibits nuclear translocation of the transcription factor (37). In SFN treated cells, NF-{kappa}B SN50 decreased the GSTA1 induction by SFN from 1.9- to 1.2-fold (Figure 5A), whereas MEK1 inhibitor PD98059 (a selective inhibitor of MEK1, which is an upstream kinase regulating ERK1/2), increased SFN-induced expression of GST by 30%. This suggests that the ERK pathway might negatively regulate GSTA1 expression. When the cells were incubated with the PI3-kinase inhibitor LY294002 (10 µM), the induction of GST by SFN was abolished (Figure 5A). PD98059 (50 µM) abolished the induction of UGT1A1 mRNA by SFN, whereas SN50 showed no effect (Figure 5B). Upon stimulation of CaCo-2 cells with apigenin, UGT1A1 mRNA expression was induced by pre-treatment with inhibitors SN50 and PD98059. This may play a role in the synergy with SFN in the induction of UGT1A1. PI3-kinase inhibitor LY294002 (10 µM) had no effect on both SFN and apigenin induced UGT mRNA expression.



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Fig. 5. Effect of NF-{kappa}B, MEK1 and PI3-kinase inhibitors on gene expression. CaCo-2 cells were incubated with NF-{kappa}B SN50 peptide (50 µg/ml) for 30 min, PD98059 (50 µM) and LY294002 (10 µM) for 90 min prior to exposure of the cells to 10 µM concentration of apigenin or SFN, respectively, for 6 h. UGT1A1 and GSTA1 mRNA was analysed by real-time RT–PCR. (A) GSTA1 mRNA expression in SFN-treated cells with or without inhibitions. (B) Changes of UGT1A1 mRNA expression in SFN (white bars) and apigenin (black bars) treated cells with or without inhibitors. Experiments were performed twice in triplicate; data represent mean ± SD. Two sets of data were compared by t-test, data groups were compared by one-way analysis of variance followed by Newman–Keuls test (significant difference compared with apigenin and SFN-treated cells, respectively, *P < 0.05, **P < 0.01; significant difference compared with control, {dagger}P < 0.05, {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001).

 
Effect of sulforaphane on SP1 expression
There is a SP1 binding site in the UGT1A1 promoter (38). We examined the effect of apigenin and SFN on SP1 mRNA and protein expression. SFN and apigenin have no effect on SP1 mRNA expression. However, SFN induced a dose-dependent increase in SP1 protein levels (Figure 6), while apigenin had no effect on SP1 protein expression (data not shown). The role of SP1 in SFN induced UGT protein expression requires further investigation.



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Fig. 6. Effect of sulforaphane on SP1 protein expression. SP1 protein (P95) was analyzed by western blotting (20 µg of protein loaded in each lane). Control cells were treated with DMSO (0.1%) only. CaCo-2 cells were treated with SFN (5, 20 and 40 µM) for 8 and 24 h.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The UGT protein family is widely distributed in human hepatic and extrahepatic tissues, but the human colon contains a more diversified spectrum of UGT1A isoforms than the liver. UGT1A1 catalytic activity reaches significant levels in the intestinal microsomes and is responsible for the conversion of substrates to water-soluble products enabling their subsequent elimination in urine, bile and the small intestine itself (15,28,39,40). GSTA1 also is a major detoxifying enzyme in the human gastrointestinal tract and the highest levels are expressed in the duodenum and small intestine, while its expression in the colon is much lower. This would suggest that compared with the duodenum and small intestine, the colon has a low potential for GST-dependent detoxification of chemical carcinogens, and is therefore at a higher risk of genotoxic effects (41). UGT1A1 and GSTA1 can detoxify heterocyclic amines, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (42), which is implicated in the etiology of human colorectal cancer (29,43,44). Increased expression of the phase II detoxifying enzymes in response to phytochemicals is of importance in colon cancer chemoprevention.

SFN is a potent inducer of phase II enzymes (18,36) and an inhibitor of phase I enzymes (9,19), and induces thioredoxin reductase 1 (45,46). Moreover, SFN also induces apoptosis at similar or higher concentrations that lead to the detoxification effect (20,21), and there are common signals controlling increased expression of detoxifying enzymes and activation of apoptosis (19). Apigenin has been shown to have a potent anticarcinogenic effect arising predominantly from its activity in cell cycle inhibition and induction of apoptosis in different cell lines (22,24,47,48). Although UGT expression has been found to be increased by several flavonoids in CaCo-2, HT-29 and HepG2 cells (26,29,49,50), there is only one report on the ability of apigenin to induce UGT1A1 glucuronidation activity in HepG2 cells (51).

This study focused on induction of phase II enzyme gene expression in CaCo-2 cells by SFN and apigenin. SFN induced both UGT1A1 and GSTA1 mRNA, and apigenin induced UGT1A1 but not GSTA1 in CaCo-2 cells. The synergistic effect of SFN and apigenin on UGT1A1 induction at transcriptional level suggests a difference in the mechanism by which both compounds regulate expression of GST and UGT. Therefore, by using inhibitors of specific kinases in signal transduction, we partly dissected some of the signaling pathways leading to induction of these two enzymes.

GST was reported previously to be induced via the activation of ARE by transcription factor Nrf2 (34). SFN was found to induce ARE-mediated reporter gene activity by activation of the ERK MAPK pathway. The MAPK pathway is linked to Nrf2, which is implicated in the binding and transcriptional activation of ARE sequences (18). PD98059, an MEK1 inhibitor, was shown to block SFN-induced ERK activation, and subsequently, inhibition of ERK diminished quinone reductase activity (52). In this study, however, PD98059 did not prevent transcriptional activation of GSTA1 by SFN; on the contrary, PD98059 significantly increased GSTA1 transcription. Kang and coworkers have reported a similar effect of PD98059 on rGSTA2 gene expression in the rat hepatoma cell line H4IIE (53), and demonstrated that PD98059 also did not inhibit the ARE binding activity, suggesting a contribution of a distinct signaling pathway to ARE-mediated phase II gene induction. They have also found PI3-kinase and Akt to be responsible for ARE-mediated rGSTA2 induction by t-BHQ in H4IIE cells. PI3-kinase inhibitors, wortmannin and LY294002, inhibited the binding activity of ARE induced by t-BHQ and subsequently, the inhibitors prevented the increase in rGSTA2 mRNA by t-BHQ. In the present study, inhibition of PI3-kinase by LY294002 led to a decrease of GSTA1 mRNA transcription induced by SFN in CaCo-2 cells. This suggests the existence of a different pathway regulating GST induction, in which PI3-kinase and Akt might be responsible for the phosphorylation step required for the activation of ARE (53).

Heiss and coworkers have demonstrated that SFN inhibited DNA binding to NF-{kappa}B, but did not influence NF-{kappa}B translocation to the nucleus (31). NF-{kappa}B activation and nuclear translocation depends on the intracellular redox conditions, which can be modulated by SFN through the regulation of glutathione (GSH). Very recently, Kim and his colleagues have reported that the cellular level of GSH decreased when cells were exposed to SFN (4 h) and increased to 2.2-fold over control in 24 h (54). In the present study, SFN increased NF-{kappa}B P65 expression in CaCo-2 cells after 8 h exposure, but suppression was observed after 24 h at higher SFN concentrations (Figure 4C), this may be related to the redox conditions such as the GSSG/GSH ratio (31). After prevention of translocation of NF-{kappa}B to the nucleus using SN50, 6 h after SFN treatment, GSTA1 mRNA was down-regulated in comparison with the SFN-only treated cells (Figure 5A). Since LY294002 also abolished SFN induced GST expression, this suggests that NF-{kappa}B is possibly the nuclear factor involved in activation of Akt mediated by a PI3-kinase-dependent mechanism (32).

Signaling pathways leading to the activation of UGT1A1 expression have not been fully elucidated although there are putative AREs in the UGT1A1 promoter. Primary structure analysis revealed the presence of SP1 binding sites in the UGT1A1 promoter (38). This may partly explain the difference between UGT and GST induction by these two compounds. Apigenin is known as a selective inhibitor of CK2 (55,56), and also of I{kappa}B kinase complex, as it was found to inhibit NF-{kappa}B activation through the prevention of I{kappa}B degradation (57). Furthermore, apigenin inhibits the ERK MAPK pathway (47,58) and down-regulates PI3-kinase activity (59) providing protection of cells from programmed death under most circumstances (60). In the present study, after pre-treatment of CaCo-2 cells with a NF-{kappa}B translocation inhibitor, SN50 and PD98059, apigenin significantly increased UGT transcription. In contrast, pre-treatment with PD98059 abolished the SFN-induced increase of UGT1A1 mRNA.

Based on the results presented here, it is clear that UGT1A1 and GSTA1 are up-regulated by SFN by distinct mechanisms. SFN and apigenin modulate ERK and NF-{kappa}B translocation may play a role in the regulation of these two phase II enzymes. Effects of other signal transduction pathways such as JNK and p38 remain to be examined. However, a limitation in applying these kinase inhibitors to determine the mechanisms of the interactions exists as the inhibitors may have multiple effects on cultured cells. It is of interest to use microarray and proteomic approaches to further dissect the mechanisms of the interaction. In summary, the remarkable synergistic effect between SFN and apigenin on UGT1A1 expression suggests that there is a possibility to apply multi-components in cancer chemoprevention in the future based on sound evidence of the mechanisms of their interaction.


    Acknowledgments
 
We thank the Biotechnology and Biological Sciences Research Council, UK for funding and EC Marie Curie Training Site Fellowships (QLK5-1999-50510) to V.S. and J.J. We would also like to thank Dr Andrew Goldson and Mr Geoff Plumb for cell culture; Mr Jim Bacon and Ms Wei Wang for TaqMan assays.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Department of Health (1994) Nutritional Aspects of Cardiovascular Disease. Report of the Cardiovascular Review Group of the Committee on Medical Aspects of Food Policy. London: HM Stationery Office.
  2. Department of Health (1998) Nutritional Aspects of the Development of Cancer. Report of the Working Group on Diet and Cancer of the Committee on Medical Aspects of Food and Nutrition Policy. HM Stationery Office, London.
  3. Kris-Etherton,P.M., Hecker,K.D., Bonanome,A., Coval,S.M., Binkoski,A.E., Hilpert,K.F., Griel,A.E. and Etherton,T.D. (2002) Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am. J. Med., 113, 71S–88S.[CrossRef][Medline]
  4. Kelloff,G.J., Crowell,J.A., Steele,V.E. et al. (2000) Progress in cancer chemoprevention: development of diet-derived chemopreventive agents. J. Nutr., 130, 467S–471S.[ISI][Medline]
  5. Hecht,S.S. (1999) Chemoprevention of cancer by isothiocyanates, modifiers of carcinogen metabolism. J. Nutr., 129, 768S–774S.[ISI][Medline]
  6. Sesso,H.D., Gaziano,J.M., Liu,S. and Buring,J.E. (2003) Flavonoid intake and the risk of cardiovascular disease in women. Am. J. Clin. Nutr., 77, 1400–1408.[Abstract/Free Full Text]
  7. Shapiro,T.A., Fahey,J.W., Wade,K.L., Stephenson,K.K. and Talalay,P. (1998) Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables. Cancer Epidemiol. Biomarkers Prev., 7, 1091–1100.[Abstract]
  8. Talalay,P. and Fahey,J.W. (2001) Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J. Nutr., 131, 3027S–3033S.[Abstract/Free Full Text]
  9. Maheo,K., Morel,F., Langouet,S., Kramer,H., Le Ferrec,E., Ketterer,B. and Guillouzo,A. (1997) Inhibition of cytochromes P-450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Res., 57, 3649–3652.[Abstract]
  10. Chiao,J.W., Chung,F.L., Kancherla,R., Ahmed,T., Mittelman,A. and Conaway,C.C. (2002) Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. Int. J. Oncol., 20, 631–636.[ISI][Medline]
  11. Kuntz,S., Wenzel,U. and Daniel,H. (1999) Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur. J. Nutr., 38, 133–142.[CrossRef][ISI][Medline]
  12. Ross,J.A. and Kasum,C.M. (2002) Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu. Rev. Nutr., 22, 19–34.[CrossRef][ISI][Medline]
  13. Mannervik,B. and Danielson,U.H. (1998) Glutathione transferases-structure and catalytic activity. CRC Crit. Rev. Biochem., 23, 283–337.
  14. Tukey,R.H. and Strassburg,C.P. (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Ann. Rev. Phar. Toxicol., 40, 581–616.[CrossRef][ISI]
  15. Fisher,M.B., Paine,M.F., Strelevitz,T.J. and Wrighton,S.A. (2001) The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab. Rev., 33, 273–297.[CrossRef][ISI][Medline]
  16. McMahon,M., Itoh,K., Yamamoto,M. and Hayes,J.D. (2003) Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J. Biol. Chem., 278, 21592–21600.[Abstract/Free Full Text]
  17. Dinkova-Kostova,A.T., Holtzclaw,W.D., Cole,R.N., Itoh,K., Wakabayashi,N., Katoh,Y., Yamamoto,M. and Talalay,P. (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl Acad. Sci. USA, 99, 11908–11913.[Abstract/Free Full Text]
  18. Kong,A.N.T., Yu,R., Hebbar,V., Chen,C., Owuor,E., Hu,R., Ee,R. and Mandlekar,S. (2001) Signal transduction events elicited by cancer prevention compounds. Mutat. Res., 480, 231–241.[ISI]
  19. Thornalley,P.J. (2002) Isothiocyanates: mechanism of cancer chemopreventive action. Anticancer Drugs, 13, 331–338.[CrossRef][ISI][Medline]
  20. Fimognari,C., Nusse,M., Cesari,R., Iori,R., Cantelli-Forti,G. and Hrelia,P. (2002) Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane. Carcinogenesis, 23, 581–586.[Abstract/Free Full Text]
  21. Gamet-Payrastre,L., Li,P., Lumeau,S., Cassar,G., Dupont,M.A., Chevolleau,S., Gasc,N., Tulliez,J. and Terce,F. (2000) Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res., 60, 1426–1433.[Abstract/Free Full Text]
  22. Yin,F., Giuliano,A.E., Law,R.E. and Van Herle,A.J. (2001) Apigenin inhibits growth and induces G2/M arrest by modulating cyclin-CDK regulators and ERK MAP kinase activation in breast carcinoma cells. Anticancer Res., 21, 413–420.[ISI][Medline]
  23. McVean,M., Weinberg,W.C. and Pelling,J.C. (2002) A p21waf1-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34cdc2 and concomitant G2/M arrest by the chemopreventive flavonoid apigenin. Mol. Carcinogen., 33, 36–43.[CrossRef][ISI][Medline]
  24. Wang,W., Heideman,L., Chung,C.S., Pelling,J.C., Koehler,K.J. and Birt,D.F. (2000) Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines. Mol. Carcinogen., 28, 102–110.[CrossRef][ISI][Medline]
  25. Delie,F. and Rubas,W. (1997) A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. Crit. Rev. Ther. Drug Carrier Syst., 14, 221–286.[ISI][Medline]
  26. Basten,G.P., Bao,Y.P. and Williamson,G. (2002) Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) in HepG2 and HT29 cells. Carcinogenesis, 23, 1399–1404.[Abstract/Free Full Text]
  27. Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[CrossRef][ISI][Medline]
  28. Petri,N., Tannergren,C., Holst,B. et al. (2003) Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo. Drug Metab. Dispos., 31, 805–813.[Abstract/Free Full Text]
  29. Galijatovic,A., Otake,Y., Walle,U.K. and Walle,T. (2001) Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in Caco-2 cells-potential role in carcinogen bioinactivation. Pharm. Res., 18, 374–379.[CrossRef][ISI][Medline]
  30. Ye,L., Dinkova-Kostova,A.T., Wade,K.L., Zhang,Y., Shapiro,T.A. and Talalay,P. (2002) Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin. Chim. Acta, 316, 43–53.[CrossRef][ISI][Medline]
  31. Heiss,E., Herhaus,C., Klimo,K., Bartsch,H. and Gerhauser,C. (2001) Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. J. Biol. Chem., 276, 32008–32015.[Abstract/Free Full Text]
  32. Meng,F., Liu,L., Chin,P.C. and D'Mello,S.R. (2002) Akt is a downstream target of NF-{kappa}B. J. Biol. Chem., 277, 29674–29680.[Abstract/Free Full Text]
  33. Shah,S.A., Potter,M.W., Hedeshian,M.H., Kim,R.D., Chari,R.S. and Callery,M.P. (2001) PI-3' kinase and NF-kappaB cross-signaling in human pancreatic cancer cells. J. Gastrointest. Surg., 5, 603–612.[CrossRef][ISI][Medline]
  34. McMahon,M., Itoh,K., Yamamoto,M., Chanas,S.A., Henderson,C.J., McLellan,L.I., Wolf,C.R., Cavin,C. and Hayes,J.D. (2001) The Cap‘n’Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res., 61, 3299–3307.[Abstract/Free Full Text]
  35. Hintze,K.J., Wald,K.A., Zeng,H., Jeffery,E.H. and Finley,J.W. (2003) Thioredoxin reductase in human hepatoma cells is transcriptionally regulated by sulforaphane and other electrophiles via an antioxidant response element. J. Nutr., 133, 2721–2727.[Abstract/Free Full Text]
  36. Thimmulappa,R.K., Mai,K.H., Srisuma,S., Kensler,T.W., Yamamoto,M. and Biswal,S. (2002) Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res., 62, 5196–5203.[Abstract/Free Full Text]
  37. Lin,Y.Z., Yao,S.Y., Veach,R.A., Torgerson,T.R. and Hawiger,J. (1995) Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem., 270, 14255–14258.[Abstract/Free Full Text]
  38. Bernard,P., Goudonnet,H., Artur,Y., Desvergne,B. and Wahli,W. (1999) Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1. Mol. Pharmacol., 56, 526–536.[Abstract/Free Full Text]
  39. Strassburg,C.P., Nguyen,N., Manns,M.P. and Tukey,R.H. (1999) UDP-glucuronosyl transferase activity in human liver and colon. Gastroenterology, 116, 149–160.[ISI][Medline]
  40. Paine,M.F. and Fisher,M.B. (2000) Immunochemical identification of UGT isoforms in human small bowel and in caco-2 cell monolayers. Biochem. Biophys. Res. Commun., 273, 1053–1057.[CrossRef][ISI][Medline]
  41. Coles,B.F., Chen,G., Kadlubar,F.F. and Radominska-Pandya,A. (2002) Interindividual variation and organ-specific patterns of glutathione S-transferase alpha, mu, and pi expression in gastrointestinal tract mucosa of normal individuals. Arch. Biochem. Biophys., 403, 270–276.[CrossRef][ISI][Medline]
  42. Bacon,J.R., Williamson,G., Garner,R.C., Lappin,G., Langouet,S. and Bao,Y.P. (2003) Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes. Carcinogenesis, 24, 1903–1911.[Abstract/Free Full Text]
  43. Coles,B., Nowell,S.A., MacLeod,S.L., Sweeney,C., Lang,N.P. and Kadlubar,F.F. (2001) The role of human glutathione S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the effect of a polymorphism in hGSTA1 on colorectal cancer risk. Mutat. Res., 482, 3–10.[ISI][Medline]
  44. Malfatti,M.A. and Felton,J.S. (2001) N-glucuronidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N-hydroxy-PhIP by specific human UDP-glucuronosyltransferases. Carcinogenesis, 22, 1087–1093.[Abstract/Free Full Text]
  45. Hintze,K.J., Keck,A.S., Finley,J.W. and Jeffery,E.H. (2003) Induction of hepatic thioredoxin reductase activity by sulforaphane, both in Hepa1c1c7 cells and in male Fisher 344 rats. J. Nutr. Biochem., 14, 173–179.[CrossRef][ISI][Medline]
  46. Zhang,J.S., Svehlikova,V., Bao,Y.P., Howie,A.F., Beckett,G.J. and Williamson,G. (2003) Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation. Carcinogenesis, 24, 497–503.[Abstract/Free Full Text]
  47. Yin,F., Giuliano,A.E. and Van Herle,A.J. (1999) Growth inhibitory effects of flavonoids in human thyroid cancer cell lines. Thyroid, 9, 369–376.[ISI][Medline]
  48. McVean,M., Xiao,H., Isobe,K. and Pelling,J.C. (2000) Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes. Carcinogenesis, 21, 633–639.[Abstract/Free Full Text]
  49. Galijatovic,A., Walle,U.K. and Walle,T. (2000) Induction of UDP-glucuronosyltransferase by the flavonoids chrysin and quercetin in Caco-2 cells. Pharm. Res., 17, 21–26.[CrossRef][ISI][Medline]
  50. Walle,T., Otake,Y., Galijatovic,A., Ritter,J.K. and Walle,U.K. (2000) Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line hep G2. Drug Metab. Dispos., 28, 1077–1082.[Abstract/Free Full Text]
  51. Walle,U.K. and Walle,T. (2002) Induction of human UDP-glucuronosyltransferase UGT1A1 by flavonoids—structural requirements. Drug Metab. Dispos., 30, 564–569.[Abstract/Free Full Text]
  52. Yu,R., Lei,W., Mandlekar,S., Weber,M.J., Der,C.J., Wu,J. and Kong,A.T. (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem., 274, 27545–27552.[Abstract/Free Full Text]
  53. Kang,K.W., Cho,M.K., Lee,C.H. and Kim,S.G. (2001) Activation of phosphatidylinositol 3-kinase and Akt by tert-butylhydroquinone is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol. Pharmacol., 59, 1147–1156.[Abstract/Free Full Text]
  54. Kim,B.R., Hu,R., Keum,Y.S., Hebbar,V., Shen,G., Nair,S.S. and Kong,A.N. (2003) Effects of glutathione on antioxidant response element-mediated gene expression and apoptosis elicited by sulforaphane. Cancer Res., 63, 7520–7525.[Abstract/Free Full Text]
  55. Romieu-Mourez,R., Landesman-Bollag,E., Seldin,D.C., Traish,A.M., Mercurio,F. and Sonenshein,G.E. (2001) Roles of IKK kinases and protein kinase CK2 in activation of nuclear factor-kappaB in breast cancer. Cancer Res., 61, 3810–3818.[Abstract/Free Full Text]
  56. Shen,J., Channavajhala,P., Seldin,D.C. and Sonenshein,G.E. (2001) Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha. J. Immunol., 167, 4919–4925.[Abstract/Free Full Text]
  57. Liang,Y.C., Huang,Y.T., Tsai,S.H., Lin-Shiau,S.Y., Chen,C.F. and Lin,J.K. (1999) Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis, 20, 1945–1952.[Abstract/Free Full Text]
  58. Kim,T.J., Zhang,Y.H., Kim,Y., Lee,C.K., Lee,M.K., Hong,J.T. and Yun,Y.P. (2002) Effects of apigenin on the serum- and platelet derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells. Planta Med., 68, 605–609.[CrossRef][ISI][Medline]
  59. Agullo,G., Gamet-Payrastre,L., Manenti,S., Viala,C., Remesy,C., Chap,H. and Payrastre,B. (1997) Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochem. Pharmacol., 53, 1649–1657.[CrossRef][ISI][Medline]
  60. Downward,J. (1998) Ras signalling and apoptosis. Curr. Opin. Genet. Dev., 8, 49–54.[CrossRef][ISI][Medline]
Received December 16, 2003; revised April 2, 2004; accepted April 9, 2004.