(−)-Epigallocatechin gallate induces Nrf2-mediated antioxidant enzyme expression via activation of PI3K and ERK in human mammary epithelial cells

Archives of Biochemistry and Biophysics 476 (2008) 171–177

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi

()-Epigallocatechin gallate induces Nrf2-mediated antioxidant enzyme expression via activation of PI3K and ERK in human mammary epithelial cells Hye-Kyung Na a, Eun-Hee Kim a, Joo-Hee Jung a, Hyun-Hee Lee a,c, Jin-Won Hyun b, Young-Joon Surh a,c,* a

National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea Department of Biochemistry, College of Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju-si 690-756, South Korea c Cancer Research Institute, Seoul National University, Seoul 110-799, South Korea b

a r t i c l e

i n f o

Article history: Received 17 January 2008 and in revised form 31 March 2008 Available online 6 April 2008 Keywords: Antioxidant enzymes EGCG Nrf2 Manganese superoxide dismutase Glutamate–cysteine ligase Heme oxygenase MCF10A cells

a b s t r a c t The chemopreventive and chemoprotective activities of green tea have been attributed to the polyphenolic ingredient ()-epigallocatechin-3-gallate (EGCG). Here, we report that treatment of human breast epithelial (MCF10A) cells with EGCG induces the expression of glutamate–cysteine ligase, manganese superoxide dismutase (MnSOD), and heme oxygenase-1 (HO-1). NF-E2-related factor (Nrf2) has been reported to regulate the antioxidant response element (ARE)-mediated expression of many antioxidant as well as detoxifying enzymes. The nuclear accumulation, ARE binding and transcriptional activity of Nrf2 were increased by EGCG treatment. Silencing of Nrf2 by siRNA gene knockdown rendered the MCF10A cells less sensitive to the EGCG-induced expression of HO-1 and MnSOD. Furthermore, EGCG activated Akt and extracellular signal-regulated protein kinase1/2 (ERK1/2). The pharmacologic inhibition of these kinases abrogated the nuclear translocation of Nrf2 induced by EGCG. These findings suggest that Nrf2 mediates EGCG-induced expression of some representative antioxidant enzymes, possibly via Akt and ERK1/2 signaling, which may provide the cells with acquired antioxidant defense capacity to survive the oxidative stress. Ó 2008 Elsevier Inc. All rights reserved.

Green tea is one of the most popular and widely consumed beverages. Results from many laboratory and epidemiologic studies suggest a protective role of green tea consumption against development of various types of cancers [1]. Thus, tea drinking has been associated with a reduced risk of stomach [2], pancreatic and colorectal cancers [3], and also with decreased recurrence of stage I and II breast cancer [4]. Phase I and II clinical trials were performed with green tea extract as an anticancer regimen [5,6]. Green tea is rich in a variety of catechin polyphenols such as ()-epicatechin, ()-epicatechin-3-gallate, ()-epigallocatechin and ()-epigallocatechin-3-gallate (EGCG)1 [7]. Among these components, EGCG is the most abundant polyphenol with potent antioxidant and chemopreventive activities. EGCG has been shown to be protective against experimentally induced lung [8], forestomach [8], colon [9], breast [10], prostate [11], and skin cancer [12]. Chemopreventive effects of EGCG are attributed to modulation of the intra-

* Corresponding author. Address: National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea. Fax: +82 2 874 9775. E-mail address: [email protected] (Y.-J. Surh). 1 Abbreviations used: EGCG, epigallocatechin-3-gallate; ROS, reactive oxygen species; ARE, antioxidant response element; GCLC, glutamate–cystein ligase catalytic subunit; MnSOD, manganese superoxide dismutase; HO-1, heme oxygenase-1; Nrf2, NF-E2-related factor; MAPK, mitogen-activated protein kinases; ERK1/2, extracellular signal-regulated kinase 1/2; PI3K, phosphatidylinositol 3-kinase. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.04.003

cellular signaling network responsible for carcinogenesis [13,14]. EGCG caused G1 cell cycle arrest by inducing the expression of cyclin-dependent kinase inhibitors and down-regulating hyperphosphorylated Rb protein [15]. It also induces apoptosis in several types of cancer cells by inactivating some transcription factors, such as NF-jB [16], AP-1 [17] and STAT3 [18], stabilizing the tumor suppressor p53 [19], or interacting with 67-kDa laminin receptor [20]. EGCG prevents cancer cell invasion [21], angiogenesis [22], and metastasis [23] by down-regulating the expression of matrix metalloproteinases and by inhibiting the cell adhesion function. In addition, EGCG inhibited the inflammatory events by suppressing over-expression of cyclooxygenase-2 [24,25] and nitric oxide synthase [26]. NF-E2-related factor (Nrf2) is responsible for regulation of antioxidant response element (ARE)–driven expression of genes encoding the majority of phase II detoxification and antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase-1 (NQO1), glutathione S-transferases, glutamate–cysteine ligase, and heme oxygenase-1 (HO-1). The antioxidant/phase II detoxifying enzyme expression was found to be abrogated in the Nrf2-deficient mice [27,28]. Nrf2-null mice developed the much larger number of tumors in the forestomach [27], liver [29], urinary bladder [30] and skin [28] compared with the wild-type mice after treatment with a carcinogen. Therefore, Nrf2 is considered as an important molecular target for cancer prevention [31].

172

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177

Some chemopreventive phytochemicals, such as curcumin, sulforaphane, caffeic acid phenethyl ester, resveratrol, have been reported to activate Nrf2 and induce expression of phase II detoxifying/antioxidant enzymes [31–34]. Recent studies have demonstrated that EGCG induces a distinct set of antioxidant enzymes in different organs or cultured cells [35]. The molecular mechanisms underlying antioxidant enzyme induction by EGCG has been the subject of extensive investigations. In the present study, we have investigated the Nrf2-mediated induction of antioxidant enzymes by EGCG in human mammary epithelial MCF10A cells. Materials and methods EGCG was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM)/F-12, heat-inactivated horse serum, L-glutamine, and penicillin/streptomycin/fungizone mixture were products of GIBCO BRL (Grand Island, NY, USA). Insulin, Cholera toxin, hydrocortisone, recombinant epidermal growth factor, and actin antibody were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against HO-1 and manganese superoxide dismutase (MnSOD) were from Stressgen (Ann Arbor, MI, USA). Glutamate-cystein ligase catalytic subunit (GCLC) antibody was purchased from Lab Vision Corp. (Fremont, CA, USA). Antibodies against Nrf-2, ERK1/2, and pERK1/2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Akt and anti-phospho-Akt were from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies were obtained from Zymed Laboratories Inc. (San Francisco, CA, USA). The ECL chemiluminescent detection reagent was purchased from Amersham Co. (Arlington Heights, IL, USA). The human NQO1 (hNQO1)-ARE luciferase reporter gene construct and GC mutant ARE construct were kindly provided by Dr. Jeffrey A. Johnson (University of Wisconsin, Madison, USA) [36]. The oligonucleotide sequence for Nrf2 siRNA was selected to knockdown Nrf2 expression using siRNA Target Finder software at www.invitrogen.com. The human specific Nrf2 siRNA (sense 50 AAGAGUAUGAGCUGGAAAAACTT-30 ; antisense 50 -GUUUUUCCAGCUCAUACUCUUTT-30 ) and StealthTM RNAi negative control duplexes were provided by Invitrogen (Carlsbad, CA, USA). LY294002 and U0126 were purchased from Calbiochem (San Diago, CA, USA) and TOCRIS (Ellisville, MO, USA), respectively. [c-32P]ATP was the product of NEN Life Science (Boston, MA, USA). EGCG and other substances were dissolved in DMSO and were further diluted with culture medium. Cell culture MCF10A cells was cultured as described previously [25] in DMEM/F-12 medium supplemented with 5% heat-inactivated horse serum, 10 lg/ml insulin, 100 ng/ml Cholera toxin, 0.5 lg/ml hydrocortisone, 20 ng/ml recombinant EGF, 2 mM L-glutamine, 100 lg/ml penicillin/streptomycin/fungi zone mixture at 37 °C in a 5% CO2 atmosphere. Western blot analysis Treated MCF10A cells were washed with PBS and harvested after treatment with lysis buffer (Cell signaling Technology, Beverly, MA, USA). Following centrifugation at 23,000g for 15 min at 4 °C, the supernatant was collected and stored at 70 °C until used. The protein concentration was determined by using the BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). After addition of sample loading buffer, proteins were resolved by 12.5% SDS–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes at 300 mA for 3 h. The membranes were blocked in 5% dry milk reconstituted in 0.1% Tween 20 in PBS (PBST). The blots were then incubated with primary antibodies in 3% dry milk/PBST, washed three times with PBST, and incubated with horseradish peroxidase-conjugated secondary antibodies in 3% dry milk/PBST for 1 h. The blots were washed again three times with PBST, and immunoreactive protein complexes were detected by the ECL detection reagent according to the manufacturer’s instructions and visualized with X-ray film. Reverse-transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from MCF10A cells using TRIzolÒ (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was reverse-transcribed with murine leukemia virus reverse-transcriptase (Promega, Madison, WI, USA) at 42 °C for 50 min and 72 °C for 15 min. The cycling conditions were as follows: 3 min at 95 °C followed by 35 cycles of 95 °C, 30 s; 55 °C, 1 min; 72 °C, 1 min of MnSOD; 30 cycles of 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min of GCLC; 30 cycles of 94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min of HO-1; 26 cycles of 94 °C, 1 min; 63 °C, 2 min; 72 °C, 2 min of the house keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by a final extension at 72 °C for 10 min. The primer pairs and the size of the expected products were as follows (forward and reverse, respectively): HO-1, 50 -CAGGCAGAGAATGCTGAGTTC-30 and 50 -GATGTTGAGCAGGAACGCAGT-30 , 555

base pair;GCLC, 50 -GAGGCTATGTGTCAGACATTGATTGTCG-30 and 50 GTGACTCCTCTGCAAGCTCCGTG-30 , 680 base pair; MnSOD, 50 -CCTCAACGTCACCGAGGAGAAG-30 and 50 -CTCCCAGTTGATTACATTAGT-30 , 451 base pair; GAPDH, 50 TGAAGGTCGGTGTCAACGGATTTGGC-30 and 50 -CATGTAGGCCATGAGGTCCACCAC-30 , 983 base pair. Amplification products were analyzed on 1.2% agarose gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light. Measurement of MnSOD activity MnSOD activity was measured as described previously [37]. Briefly, MCF10A cells treated with various concentrations of EGCG were suspended in 10 mM potassium phosphate buffer (pH 7.5) and lysed using a sonicator (15 s pulses) on ice. Triton X-100 (final 0.1%) was added to the lysates and incubated for 10 min on ice. The lysates were centrifuged at 5000g for 10 min at 4 °C and KCN (1 mM) was added to supernatant in order to inhibit CuZnSOD. After quantification, the proteins (50 lg) were added to 50 mM sodium phosphate buffer (pH 10.2) containing 1 mM epinephrine and 0.1 mM EDTA. Epinephrine rapidly undergoes auto-oxidation at pH 10 to form pink-colored adrenochrome, which can be measured at 480 nm using a UV/VIS spectrophotometer. The amount of the enzyme required to produce 50% inhibition of epinephrine auto-oxidation was defined as 1 U of SOD activity. Nrf2-siRNA transient transfection For Nrf2-siRNA transfection, the cells were transfected with Nrf2-siRNA or Nrf2negative control siRNA for 48 h upon plating the cells (1  105/well) in a 6-well dish. The transfected cells were treated with EGCG for 12 h followed by lysis buffer (Cell signaling Technology, Beverly, MA, USA) for Western blot analysis. ARE luciferase assay MCF10A cells seeded at a density of 2  104/well in a 24-well dish were grown to 60–70% confluence in complete growth media. The cells were co-transfected with 2 lg of the luciferase reporter gene fusion construct (pTi-luciferase), wild-type ARE, or GC mutant ARE and 0.5 lg of pCMV-b-galactosidase control vector with WelFect-MTM Gold transfection reagent (WelGENE Inc. Seoul, Korea) according to the manufacturer’s instructions. After 24-h transfection, the cells were treated with EGCG for additional 6 h and then washed with PBS and lysed in 1 reporter lysis buffer (Promega, Madison, WI). The activities of firefly luciferase in the cell lysates were measured using the luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA) by Luminoskan luminometer (Thermo Labsystems, Helsinki, Finland). b-Galactosidase activity was measured by using the commercially available assay kit (Promega, Madison, WI, USA). The relative luciferase activity was obtained by normalizing the firefly luciferase activity against the b-galactosidase activity. Preparation of nuclear extracts MCF10A cells were cultured in 100-mm dishes in the absence or presence of EGCG. Cells were gently washed with cold PBS, scraped, and centrifuged at 1300g for 5 min. Pellets were suspended in cold hypotonic buffer [10 mM HEPES, pH 7.9, 1.5 mM MgCl2,0.3 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. The lysates were incubated for 10 min on ice and then centrifuged at 20,200g for 15 min at 4 °C. The pellets were washed with hypotonic buffer and resuspended in hypertonic buffer (30 mM HEPES, 1.5 mM MgCl2, 0.3 mM EDTA, 10% glycerol, 450 mM NaCl, 0.1 mM PMSF) in ice for 30 min during rocking followed by centrifugation at 20,200g for 15 min. After determination of the protein concentration, the supernatant was stored at 80 °C before use. Electrophoretic mobility shift assay (EMSA) The oligonucleotides harboring the ARE consensus sequence were end-labeled with [c-32P]ATP using T4 polynucleotide kinase (Takara, Japan). The nuclear protein (7–10 lg) was mixed with 8.5 ll of incubation buffer (30 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.3 mM EDTA, pH 8.0, 10% glycerol) and 2.5 ll polydI-dC (0.5 lg/ll), and the hypertonic buffer was added to make the final volume 20 ll. After pre-incubation at room temperature for 15 min, the labeled oligonucleotide (50,000– 100,000 cpm) was added and incubation continued for additional 30 min at room temperature. To insure the specificity of the binding, a competition assay was carried out with the excess unlabeled oligonucleotide. Samples were separated on the 6% acrylamide gels at 150 mA in 0.25  Tris–borate–EDTA buffer (pH 8.3). After vacuum-dried, the gel was exposed to X-ray film for autoradiography at 70 °C. Immunofluorescence staining of Nrf2 Localization of Nrf2 in the cells was assessed by immunofluorescent staining as described previously [38]. MCF10A cells (8-well chamber slide) were treated with EGCG in the presence or absence of pharmacological inhibitors of MEK or Akt for 12 h. The cells were fixed in 10% neutral-buffered formalin solution for 30 min at room temperature. After a rinse with PBS, fixed cells were incubated in a fresh

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177

173

blocking buffer (0.5% Tween-20 in PBS, pH 7.4, containing 10% normal goat serum) for 1 h at room temperature. Cells were then incubated overnight at 4 °C upon addition of anti-Nrf2 primary antibody solution (diluted 1:100 in PBS with 1% bovine serum albumin). Afterwards, the cells were washed three times with PBST (PBS containing 0.1% Tween-20) and then incubated for 1 h at room temperature upon addition of FITC-goat anti-rabbit IgG secondary antibody diluted (1:1000) in PBST with 3% bovine serum albumin. Cells were then rinsed with PBS and the stained cells were analyzed under a confocal microscope. Statistical analysis When necessary, data were expressed as mean ± SEM. The data were first analyzed using one-way analysis of variance (ANOVA). Significant differences between control cells and EGCG-treated cells were evaluated using Tukey test. Statistical analyses were performed using a commercial statistical analysis package (SigmaStat 3.11: Jandel Scientific Software, San Rafael, CA, USA). The level of statistical significance was set at p < 0.05.

Results EGCG induced expression of antioxidant enzymes in MCF10A cells Protecting cells from oxidative stress can be achieved either by directly scavenging reactive oxygen species (ROS) or more fundamentally by fortifying the body’s antioxidant defenses through induction of antioxidant gene expression. Treatment of MCF10A cells with EGCG induced mRNA (Fig. 1A) and protein (Fig. 1B) expression of HO-1, an important stress-responsive enzyme which catalyzes the conversion of heme to biliverdin and carbon monoxide, and GCLC, the rate-limiting enzyme in the synthesis of GSH, in a concentration dependent fashion. In addition, EGCG induced expression

Fig. 2. Time-dependent changes in the expression of MnSOD in EGCG-treated MCF10A cells. (A) The time-related expression of MnSOD and its mRNA transcript in MCF10A cells treated with EGCG (100 lM). (B) The catalytic activity of MnSOD in MCF10A cells treated with EGCG (25, 50 and 100 lM). Bars represent means ± SEM. (n = 3). *Significantly different from the vehicle control (p < 0.05). The experimental details are described in Materials and methods.

of MnSOD in concentration- (Fig. 1) and time- (Fig. 2A) dependent manners. The catalytic activity of MnSOD was also elevated by EGCG treatment (Fig. 2B). EGCG activated the ARE elements in MCF10A cells Nrf2 is a redox-sensitive basic-leucine zipper transcription factor that translocates into the nucleus upon oxidative or electrophilic stress and is involved in regulating the ARE-mediated expression of several detoxifying and antioxidant enzymes. To determine whether EGCG could induce the activation of the ARE-driven antioxidant gene expression, we performed the electrophoretic mobility shifty assay using the radio-labeled ARE element. EGCG induced ARE binding activities of Nrf2 in concentration- (Fig. 3A) and time(Fig. 3B) dependent manners. Moreover, MCF10A cells transfected with the luciferase reporter gene under the control of ARE-driven promoter exhibited a marked transcriptional activity following exposure to EGCG (Fig. 3C). In contrast, when a reporter construct containing the ARE sequence was mutated in the GC core box, the EGCG-mediated increase in ARE-luciferase activity was abolished. EGCG treatment also led to a transient increase in the levels of Nrf2 protein localized in the nuclear fraction (Fig. 3D). EGCG-induced expression of HO-1 and MnSOD was mediated by Nrf2 Fig. 1. EGCG-induced up-regulation of antioxidant or detoxifying enzymes. (A) Expression of mRNA transcripts of GCLC, HO-1 and MnSOD in MCF10A cells treated with EGCG (100 lM) for 12 h was measured by RT-PCR. (B) Protein expression levels of GCLC, HO-1 and MnSOD in MCF10A cells treated with EGCG (100 lM) for 24 h were determined by Western blot analysis. Actin was included to ensure equal protein loading.

To determine whether up-regulation of HO-1 and MnSOD expression is mediated by Nrf2, we examined the MnSOD and HO-1 expression after siRNA knockdown of Nrf2. The EGCG-induced up-regulation of MnSOD and HO-1 in MCF10A cells were

174

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177

Fig. 3. Enhancement of the nuclear translocation, ARE-DNA binding, and transcriptional activity of Nrf2 in MCF10A cells treated with EGCG (100 lM). (A and B) ARE-DNA binding activity in MCF10A cells treated with various concentrations of EGCG (25, 50 and 100 lM) for 6 h or EGCG (100 lM) for the indicated time. The nuclear extract isolated from EGCGtreated cells was used for electrophoretic mobility shift assay as described in Materials and methods. (C) EGCG-mediated transcriptional activation of ARE was measured by the luciferase reporter assay. MCF10A cells were transfected with pTi-luciferase, wild-type ARE-luciferase, and GC mutant ARE constructs and 0.5 lg of pCMV-b-galactosidase control vector with WelFect-MTM Gold transfection reagent. EGCG (100 lM) were treated with tranfected cells for 6 h. Experimental conditions and other details for the cell transfection and the reporter gene assay are described in Materials and methods. (D) Translocation of Nrf2 in the nuclear fraction of MCF10A cells treated with EGCG for 6 h.

abolished by silencing Nrf2 expression with specific siRNA, whereas transfection of the cells with the same amount of nonspecific control siRNA was not effective (Fig. 4). EGCG induced phosphorylation of ERK1/2 and Akt It has been reported that several kinases such as phosphoinositol 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs)

are involved in the activation of Nrf2 and up-regulation of several antioxidant enzyme gene expression in various types of cells [39]. Phosphorylation of serine and threonine residues of Nrf2 by these kinases is known to facilitate the nuclear translocation of Nrf2 and its subsequent binding to the coactivator CBP/p300. A transient increase in the phosphorylation of Akt and ERK1/2 was observed in the EGCG-treated MCF10A cells, whereas the level of unphosphorylated Akt and ERK1/2 remained unchanged (Fig. 5A). To determine whether these kinases are involved in EGCG-driven activation of Nrf2, U0126 and LY294002, the pharmacological inhibitors of MEK–ERK1/2 and PI3K–Akt signaling, respectively, were used. EGCG-induced Nrf2 nuclear translocation was effectively inhibited by U0126 and LY294002 (Fig. 5B) suggesting that ERK1/2 and PI3K play important roles in the activation of Nrf2 in MCF10A cells. Discussion

Fig. 4. Abrogation of HO-1 and MnSOD expressions induced by EGCG in MCF10A cells transiently transfected with Nrf2-siRNA. MCF10A cells were transfected with Nrf2-siRNA or Nrf2-negative control siRNA for 48 h. The transfected cells were treated with EGCG (100 lM) for 12 h and expression of antioxidant enzymes was measured by Western blot analysis with indicated specific antibodies. The experiments were repeated at least three times and a representative blot is shown.

Oxidative and inflammatory injuries are closely linked each other in the process of multi-stage carcinogenesis. Thus, compounds with anti-inflammatory activities are anticipated to inhibit oxidative stress, and vice versa. In this context, it is interesting to note that the anti-inflammatory and antioxidant enzyme-inducing potencies of a series of synthetic triterpenoids are closely correlated (r2 = 0.91) [40]. Likewise, many chemopreventive dietary phenolic substances are capable of regulating both inflammation

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177

175

Fig. 5. Effects of EGCG on phosphorylation of ERK1/2 and Akt and effects of their pharmacological inhibition on the translocation of Nrf2 in the MCF10A cells treated with EGCG. (A) MCF10A cells were treated with EGCG for indicated time periods and expression levels of both phosphorylated and total ERK1/2 and Akt were measured by Western blot analysis. (B) MCF10A cells were treated with EGCG in the presence or absence of pharmacological inhibitors of MEK (U0126, 25 lM) and PI3K/Akt (LY294002, 20 lM) for 12 h. The immunofluoresence staining of Nrf2 was conducted as described under Materials and methods.

and redox signaling [41]. EGCG was found to inhibit the phorbol ester-induced expression of cyclooxygenase-2, a key proinflammatory enzyme, in MCF10A cells [25]. In the present study, we observed that EGCG at concentrations which confer antiinflammatory effects also induced expression of some antioxidant enzymes in MCF10A cells. Blockage of the DNA damage induced by electrophilic carcinogens or reactive oxygen species (ROS) during the initiation stage of carcinogenesis represents an important strategy for cancer chemoprevention [42]. Katiyar et al. [43] reported that topical application of EGCG to human skin prior to UV irradiation attenuated the production of hydrogen peroxide and nitric oxide in both dermis and epidermis. The protective effect of EGCG against UV-induced oxidative injuries in human skin appears to be attributable to its restoration of epidermal glutathione levels and glutathione peroxidase activity that are prone to be reduced by UV irradiation. EGCG pretreatment also inhibited the UV-induced infiltration of inflammatory leukocytes, particularly CD11b (+) cells into the skin, which are considered to be the major producers of ROS [43]. EGCG-induced expression of phase II detoxifying or antioxidant enzymes may hence contribute to the suppression of oxidative stress- or inflammation-induced carcinogenesis. Nrf2 plays an important role in the up-regulation of antioxidant and detoxifying enzymes. Recent studies have shown that failure

of the Nrf2 function predisposes the cells to develop the carcinogen-induced DNA damage and tumor formation [27,29,44]. Therefore, Nrf2 is considered as an important molecular target for cancer prevention [31]. EGCG has been known as the most potent Nrf2 activator among the green tea polyphenols, as evidenced by its pronounced ability to induce ARE-luciferase reporter gene transactivation [45]. It has been reported that EGCG activated Nrf2 and induced expression of HO-1 in endothelial cells [46] and B-lymphoblasts [47]. Our present study demonstrates that EGCG induces Nrf2-mediated expression of MnSOD and HO-1 through activation of ERK1/2 and PI3K/Akt in MCF10A cells. The molecular mechanism underlying antioxidant enzyme induction by EGCG has been the subject of extensive investigations. One of the most plausible mechanisms responsible for activation of Nrf2 involves phosphorylation of serine/threonine residues of Nrf2 by protein kinases, which facilitates enhanced nuclear translocation of Nrf2 and subsequent ARE binding. EGCG-induced HO-1 expression was attributed to activation of Akt and ERK1/2 in endothelial cells [46]. In another study, EGCG-induced HO-1 expression as well as Nrf2 nuclear translocation was shown to be mediated via Akt and p38 MAPK signaling in B lympoblasts [47]. We also observed that the EGCG induced activation of ERK1/2 and Akt through phosphorylation. The pharmacological inhibitors of MEK and PI3K/Akt, which are upstream kinases

176

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177

responsible for phosphorylation of ERK1/2 and Akt, attenuated the nuclear localization of Nrf2 induced by EGCG. This finding suggests that MEK–ERK1/2 and PI3K–Akt signaling pathways are required in EGCG-induced Nrf2 activation and subsequent expression of HO-1 and MnSOD in MCF10A cells. Constitutively active forms of the participants in MEK–ERK1/2 and PI3K signaling pathways would be worthwhile being tested to see whether and to what extent they induce translocation of Nrf2 and subsequent activation of the antioxidant enzyme genes. The activity of Nrf2 is normally suppressed in the cytosol by specific binding to cytoskeleton associated cytoplasmic ‘Kelchlike ECH-associated protein 1 (Keap1)’ which hampers the nuclear translocation of Nrf2. However, upon stimulation by electrophilic agents or compounds that possess ability to oxidize or modify specific cysteine thiol groups present in Keap1, Keap1 repression of Nrf2 activity is abrogated, allowing Nrf2 to translocate into the nucleus [48]. It has been reported that flavonoids with a higher intrinsic potential to generate ROS and redox cycling are the more potent inducers of ARE-mediated gene expression [49]. EGCG was found to produce substantial amounts of H2O2 under cell culture conditions [24,50] and can also be oxidized to form semiquinone radical and then o-quinone at the B ring or the gallate ring [51]. These quinones would react with the sulfhydryl group of cysteine or GSH to form the 20 - or 200 -cysteinyl EGCG or 20 - or 200 -glutathionyl EGCG, respectively [51]. Therefore, EGCG to some extent can act as an electrophiles as well as an pro-oxidant [52], which may account for its ability to activate Nrf2–ARE signaling by stimulating the dissociation of Nrf2 from its repressor, Keap1 [53]. However, further studies will be necessary to clarify the physiological relevance of such alternative mechanism. Upon nuclear translocation, Nrf2 also binds to other transacting factors such as small Maf-F/G/K [54] as well as the coac-

Fig. 6. Proposed mechanisms of EGCG-induced activation of Nrf2 and subsequent expression of antioxidant enzymes in MCF10A cells. EGCG can activate ERK or Akt, which in turn phosphorylates Nrf2. This will facilitate the nuclear translocation of Nrf2. Alternatively, EGCG may modify the some critical cysteine thiols present in Keap1, a repressor of Nrf2, facilitating their dissociation. This would be likely to be achieved by some reactive species formed during auto-oxidation or enzymatic conversion of EGCG, rather than EGCG itself. The free Nrf2 then translocates to nucleus for ARE binding. Bach1 forms heterodimers with small Maf proteins and hence competes with Nrf2 for ARE binding. EGCG may repress Bach1, thereby stimulating Nrf2–ARE binding.

tivators of ARE including cAMP response element binding protein (CREB)-binding protein (CBP)/p300 [55] that can coordinately regulate the ARE-derived antioxidant gene transcription. The mammalian transcription factors Bach1 and Bach2 belong to the cap’n’collar (CNC), b-Zip family of proteins like Nrf2. Bach 1 forms heterodimers with small Maf for ARE binding [56]. As Bach1 can be replaced by Nrf2, it is reasonable to postulate that Bach1 and Nrf2 compete with each other for the same ARE binding site. Thus, a balance of Nrf2 versus Bach1 inside the nucleus influences up- or down-regulation of ARE-mediated gene expression. Overexpression of Bach 1 repressed NQO1 gene expression, corroborating the notion that Bach1 in association with small Maf proteins plays a role in repression of ARE-driven antioxidant gene expression [56]. Recently, it has been reported that EGCG down-regulates the expression of Bach1 in cultured A549 cells [57]. It would be worthwhile examining whether Bach1 and Nrf2 could be reciprocally modulated by EGCG in its inducing antioxidant enzymes in MCF10A cells. In conclusion, EGCG activated the ERK1/2 and Akt, facilitating the release of Nrf2 for nuclear translocation, thereby inducing expression of some representative antioxidant enzymes in MCF10A cells as schematically represented in Fig. 6. Acknowledgments This study was supported by the MOEHRD, Basic Research Promotion Fund (KRF-2006-E00002) from Korea Research Foundation and the Grant (B050007) from the Ministry of Health and Welfare, Republic of Korea. References [1] C.S. Yang, S. Prabhu, J. Landau, Drug Metab. Rev. 33 (2001) 237–253. [2] M. Inoue, K. Tajima, K. Hirose, N. Hamajima, T. Takezaki, T. Kuroishi, S. Tominaga, Cancer Causes Control 9 (1998) 209–216. [3] B.T. Ji, W.H. Chow, A.W. Hsing, J.K. McLaughlin, Q. Dai, Y.T. Gao, W.J. Blot, J.F. Fraumeni Jr., Int. J. Cancer 70 (1997) 255–258. [4] K. Nakachi, K. Suemasu, K. Suga, T. Takeo, K. Imai, Y. Higashi, Jpn. J. Cancer Res 89 (1998) 254–261. [5] A. Jatoi, N. Ellison, P.A. Burch, J.A. Sloan, S.R. Dakhil, P. Novotny, W. Tan, T.R. Fitch, K.M. Rowland, C.Y. Young, P.J. Flynn, Cancer 97 (2003) 1442–1446. [6] K.M. Pisters, R.A. Newman, B. Coldman, D.M. Shin, F.R. Khuri, W.K. Hong, B.S. Glisson, J.S. Lee, J. Clin. Oncol. 19 (2001) 1830–1838. [7] H. Mukhtar, N. Ahmad, Proc. Soc. Exp. Biol. Med. 220 (1999) 234–238. [8] S.K. Katiyar, R. Agarwal, M.T. Zaim, H. Mukhtar, Carcinogenesis 14 (1993) 849– 855. [9] C. Han, Y. Xu, Biomed. Environ. Sci. 3 (1990) 35–42. [10] T. Yamane, N. Hagiwara, M. Tateishi, S. Akachi, M. Kim, J. Okuzumi, Y. Kitao, M. Inagake, K. Kuwata, T. Takahashi, Jpn. J. Cancer Res. 82 (1991) 1336–1339. [11] E.C. Stuart, M.J. Scandlyn, R.J. Rosengren, Life Sci. 79 (2006) 2329–2336. [12] S.K. Katiyar, R. Agarwal, G.S. Wood, H. Mukhtar, Cancer Res. 52 (1992) 6890– 6897. [13] C.S. Yang, J.D. Lambert, Z. Hou, J. Ju, G. Lu, X. Hao, Mol. Carcinog. 45 (2006) 431–435. [14] H.-K. Na, Y.-J. Surh, Mol. Nutr. Food Res. 50 (2006) 152–159. [15] N. Ahmad, V.M. Adhami, S. Gupta, P. Cheng, H. Mukhtar, Arch. Biochem. Biophys. 398 (2002) 125–131. [16] S. Okabe, N. Fujimoto, N. Sueoka, M. Suganuma, H. Fujiki, Biol. Pharm. Bull. 24 (2001) 883–886. [17] J.Y. Chung, C. Huang, X. Meng, Z. Dong, C.S. Yang, Cancer Res. 59 (1999) 4610– 4617. [18] S. Bhattacharya, R.M. Ray, L.R. Johnson, Biochem. J. 392 (2005) 335–344. [19] K. Hastak, S. Gupta, N. Ahmad, M.K. Agarwal, M.L. Agarwal, H. Mukhtar, Oncogene 22 (2003) 4851–4859. [20] M.A. Shammas, P. Neri, H. Koley, R.B. Batchu, R.C. Bertheau, V. Munshi, R. Prabhala, M. Fulciniti, Y.T. Tai, S.P. Treon, R.K. Goyal, K.C. Anderson, N.C. Munshi, Blood 108 (2006) 2804–2810. [21] K. Maeda, M. Kuzuya, X.W. Cheng, T. Asai, S. Kanda, N. Tamaya-Mori, T. Sasaki, T. Shibata, A. Iguchi, Atherosclerosis 166 (2003) 23–30. [22] G. Fassina, R. Vene, M. Morini, S. Minghelli, R. Benelli, D.M. Noonan, A. Albini, Clin. Cancer Res. 10 (2004) 4865–4873. [23] J.D. Liu, S.H. Chen, C.L. Lin, S.H. Tsai, Y.C. Liang, J. Cell. Biochem. 83 (2001) 631– 642. [24] J. Hong, T.J. Smith, C.T. Ho, D.A. August, C.S. Yang, Biochem. Pharmacol. 62 (2001) 1175–1183.

H.-K. Na et al. / Archives of Biochemistry and Biophysics 476 (2008) 171–177 [25] J.K. Kundu, H.-K. Na, K.-S. Chun, Y.K. Kim, S.J. Lee, S.S. Lee, O.S. Lee, Y.C. Sim, Y.J. Surh, J. Nutr. 133 (2003) 3805S–3810S. [26] Y.L. Lin, J.K. Lin, Mol. Pharmacol. 52 (1997) 465–472. [27] M. Ramos-Gomez, M.K. Kwak, P.M. Dolan, K. Itoh, M. Yamamoto, P. Talalay, T.W. Kensler, Proc. Natl. Acad. Sci. USA 98 (2001) 3410–3415. [28] C. Xu, M.T. Huang, G. Shen, X. Yuan, W. Lin, T.O. Khor, A.H. Conney, A.N. Tony Kong, Cancer Res. 66 (2006) 8293–8296. [29] Y. Kitamura, T. Umemura, K. Kanki, Y. Kodama, S. Kitamoto, K. Saito, K. Itoh, M. Yamamoto, T. Masegi, A. Nishikawa, M. Hirose, Cancer Sci. 98 (2007) 19–24. [30] K. Iida, K. Itoh, J.M. Maher, Y. Kumagai, R. Oyasu, Y. Mori, T. Shimazui, H. Akaza, M. Yamamoto, Carcinogenesis (2007). [31] J.-S. Lee, Y.-J. Surh, Cancer Lett. 224 (2005) 171–184. [32] E. Balogun, M. Hoque, P. Gong, E. Killeen, C.J. Green, R. Foresti, J. Alam, R. Motterlini, Biochem. J. 371 (2003) 887–895. [33] T.W. Kensler, T.J. Curphey, Y. Maxiutenko, B.D. Roebuck, Drug Metabol. Drug Interact. 17 (2000) 3–22. [34] P. Talalay, J.W. Fahey, J. Nutr. 131 (2001) 3027S–3033S. [35] F.P. Chou, Y.C. Chu, J.D. Hsu, H.C. Chiang, C.J. Wang, Biochem. Pharmacol. 60 (2000) 643–650. [36] J.M. Lee, M.J. Calkins, K. Chan, Y.W. Kan, J.A. Johnson, J. Biol. Chem. 278 (2003) 12029–12038. [37] K.A. Kang, K.H. Lee, S. Chae, Y.S. Koh, B.S. Yoo, J.H. Kim, Y.M. Ham, J.S. Baik, N.H. Lee, J.W. Hyun, Free Radic. Res. 39 (2005) 883–892. [38] E.J. Joung, M.H. Li, H.G. Lee, N. Somparn, Y.S. Jung, H.K. Na, S.H. Kim, Y.N. Cha, Y.J. Surh, Antioxid. Redox Signal. 9 (2007) 2087–2098. [39] R. Yu, C. Chen, Y.Y. Mo, V. Hebbar, E.D. Owuor, T.H. Tan, A.N. Kong, J. Biol. Chem. 275 (2000) 39907–39913. [40] A.T. Dinkova-Kostova, K.T. Liby, K.K. Stephenson, W.D. Holtzclaw, X. Gao, N. Suh, C. Williams, R. Risingsong, T. Honda, G.W. Gribble, M.B. Sporn, P. Talalay, Proc. Natl. Acad. Sci. USA 102 (2005) 4584–4589.

177

[41] I. Rahman, S.K. Biswas, P.A. Kirkham, Biochem. Pharmacol. 72 (2006) 1439– 1452. [42] A.T. Dinkova-Kostova, W.D. Holtzclaw, R.N. Cole, K. Itoh, N. Wakabayashi, Y. Katoh, M. Yamamoto, P. Talalay, Proc. Natl. Acad. Sci. USA 99 (2002) 11908– 11913. [43] S.K. Katiyar, F. Afaq, A. Perez, H. Mukhtar, Carcinogenesis 22 (2001) 287–294. [44] C. Xu, M.T. Huang, G. Shen, X. Yuan, W. Lin, T.O. Khor, A.H. Conney, A.N. Kong, Cancer Res. 66 (2006) 8293–8296. [45] C. Chen, R. Yu, E.D. Owuor, A.N. Kong, Arch. Pharm. Res. 23 (2000) 605–612. [46] C.C. Wu, M.C. Hsu, C.W. Hsieh, J.B. Lin, P.H. Lai, B.S. Wung, Life Sci. 78 (2006) 2889–2897. [47] C.K. Andreadi, L.M. Howells, P.A. Atherfold, M.M. Manson, Mol. Pharmacol. 69 (2006) 1033–1040. [48] A.T. Dinkova-Kostova, W.D. Holtzclaw, N. Wakabayashi, Biochemistry 44 (2005) 6889–6899. [49] Y.Y. Lee-Hilz, A.M. Boerboom, A.H. Westphal, W.J. Berkel, J.M. Aarts, I.M. Rietjens, Chem. Res. Toxicol. 19 (2006) 1499–1505. [50] L.H. Long, M.V. Clement, B. Halliwell, Biochem. Biophys. Res. Commun. 273 (2000) 50–53. [51] S. Sang, J.D. Lambert, J. Hong, S. Tian, M.J. Lee, R.E. Stark, C.T. Ho, C.S. Yang, Chem. Res. Toxicol. 18 (2005) 1762–1769. [52] C. Xu, C.Y. Li, A.N. Kong, Arch. Pharm. Res. 28 (2005) 249–268. [53] H.K. Na, Y.J. Surh, Food Chem. Toxicol. (2007). [54] S. Dhakshinamoorthy, A.K. Jaiswal, J. Biol. Chem. 275 (2000) 40134–40141. [55] G. Shen, V. Hebbar, S. Nair, C. Xu, W. Li, W. Lin, Y.S. Keum, J. Han, M.A. Gallo, A.N. Kong, J. Biol. Chem. 279 (2004) 23052–23060. [56] S. Dhakshinamoorthy, A.K. Jain, D.A. Bloom, A.K. Jaiswal, J. Biol. Chem. 280 (2005) 16891–16900. [57] M.-H. Kweon, V.M. Adhami, J.-S. Lee, H. Mukhtar. J. Biol. Chem. 281 (2006) 33761–33772.