Green Tea epigallocatechin-3-gallate Protects Against Oxidative Stress-Induced Nuclear Translocation of p 53 and Apoptosis in Retinal Pigment Epithelial Cells , ARPE-19

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenolic flavonoids in green tea has been shown to possess strong antioxidant activities. Oxidative stress causes the defect of retinal pigment epithelial (RPE) cells that contribute to several retinal diseases. Several studies have shown that increasing the body’s defenses against oxidative stress with specific antioxidants and mineral supplements could preserve the vision. Therefore, the purpose of this study was to determine the protective role of EGCG against exogenous reactive oxygen species hydrogen peroxide (H2O2)-induced cell death in ARPE-19 cells, human retinal pigment epithelial cell line. ARPE-19 cells were pretreated with EGCG in the presence/absence of H2O2. The protective effects of EGCG and the underlined mechanisms against H2O2 were evaluated. The present study demonstrated that 400 μM H2O2 significantly decreased cell viability, increased the accumulation of intracellular reactive oxygen species (ROS) and increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and chromatin condensed nuclei. In addition, H2O2 induced p53 nuclear translocation, up-regulated Bax and down-regulated Bcl-2 expression thereby increased Bax/Bcl-2 ratio. These toxic effects of H2O2 were reversed by 100 μM EGCG pretreatment. These studies suggest that EGCG protects H2O2-induced cell death in ARPE-19 cells by its antioxidant property and attenuation of p53 nuclear translocation.

It is well established that free hydroxyl radicals generated from excess H 2 O 2 leading cell apoptosis since H 2 O 2 is a potent DNA damage inducer (Jornot, Petersen, & Junod, 1998).For intrinsic apoptosis pathway, Bcl-2 family proteins are particularly important as they are potent regulators involving in the mitochondria-initiated intrinsic apoptosis (Brunelle & Letai, 2009;Sharpe, Arnoult, & Youle, 2004).This family consists of both pro-and anti-apoptotic members that perform the opposing effects on mitochondria, including the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax (Brunelle & Letai, 2009).Therefore, the ratio of Bax to Bcl-2 serves as a rheostat to determine the susceptibility of cells to apoptosis.translocate from cytosol and accumulate within the nucleus where it enhances the expression of target apoptotic genes, particularly Bax by binding on the promoter of gene (Samuels-Lev et al., 2001).Consequently, Bax forms a homodimer and releases cytochrome c from the mitochondria resulting in caspase-9 activation, which subsequently activates the effectors, caspase-3 and caspase-7, which are responsible for the dismantling of an apoptotic cell (Haupt, Berger, Goldberg, & Haupt, 2003).
Retinal pigment epithelium (RPE) is a single layer of cuboidal cells that forms the outer blood-retinal barrier.It is essential as nutritional or metabolic support for photoreceptor, phagocytosis and degradation of shed photoreceptor outer segments (Strauss, 2005).RPE is particularly susceptible to oxidative stress by reactive oxygen species (ROS), such as superoxide anion, hydroxyl radical, singlet oxygen, and H 2 O 2 , due to its locations and functions (Liang & Godley, 2003).Dysfunction of RPE contributes to retinal diseases such as retinitis pigmentosa (RP) (Strauss, 2005) and age-related macular degeneration (AMD) (Zarbin, 2004) leading to visual impairment.H 2 O 2 has been widely used as the oxidative stress-induced apoptosis model in RPE (Godley, Jin, Guo, & Hurst, 2002;Jin, Hurst, & Godley, 2001), and has been shown to mediate apoptosis through p53 dependent pathway in several circumstances cells such as human fibroblasts (Youn et al., 2007), glioma cells (Datta, Babbar, Srivastava, Sinha, & Chattopadhyay, 2002) and RPE cells (Jin et al., 2001).The present study, therefore, aimed to investigate the protective effects of EGCG against H 2 O 2 -induced apoptosis in ARPE-19 cells focusing on the lineage of p53 dependent pathway.The results help enlighten the underlined protective mechanism of EGCG against oxidative stress-induced RPE apoptotic cell death.

Cell Viability Measurement by MTT Assay
ARPE-19 cells (1×10 5 cells/well) were seeded and grown in 96-well plates for 24 h.At the designated times, cell viability was determined using methylthiazolium bromide (MTT) assay.Briefly, 100 µl of MTT solution (5 mg/ml) was added to each well and incubated for 4 h at 37ºC in the dark.After incubation, 100 µl of dimethylsulfoxide (DMSO) was added to each well to lyse the cells.The absorbance of the sample was measured at 490 nm on a Versamax microplate reader using SoftMax Pro 4.8 analysis Molecular Devices software (Sunnyvale, CA).

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
Nuclear DNA fragmentation of apoptotic cells was determined using the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) following the manufacture's instruction.ARPE-19 cells were grown on Lab-Tek chamber slides (Nunc, Thermo Fisher Scientific, NY) until 95-100% confluence and then were performed the experiments.Cells were fixed with 4% methanol-free paraformaldehyde for 25 min at 4°C, washed with PBS, permeabilized with 0.2% Triton X-100 for 5 min and then washed.Fixed monolayer cell cultures were then covered with 100 μl of equilibration buffer for 10 min, followed with 50 μl of TUNEL reaction mixture containing equilibration buffer, nucleotide mix and TdT enzyme for 60 min at 37°C.The reaction was terminated by in 2X saline sodium citrate for 15 min at RT and then washed.The cells were then counterstained with Hoechst 33342 for 10 min in the dark, washed in de-ionized water and mounted with anti-fade solution.TUNEL-positive cells (green fluorescence) were observed by a fluorescent microscope (Nikon E600).Percentage of TUNEL-positive cells was averaged from a total of 500 cells from 3 independent slides.

Western Blot Analysis
Cells were grown in 6 well plates until 95-100% confluence then were performed experiments.Cells were lysed in lysis buffer [3 mM MgCl 2 , 1 mM EGTA, 10 mM Na Orthovanadate, 10 mM Na Pyrophosphate, 50 mM NaF, 1 mM protease inhibitor (Roche)] for 30 min.The lysate was collected in to a 1.5 ml microfuge tube, centrifuged at 12000 g for 15 min.The supernatant was then kept at -80°C until used.Protein concentration was determined using BCA TM Protein Assay Kit (Pierce, Rockford, IL).Twenty micrograms of protein was mixed with loading buffer and run in a 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 100 V.The proteins were then transferred onto a nitrocellulose membrane and blocked with 5% nonfat dry milk in 1X Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 0.1% Tween 20) for 2 h.Membranes were incubated with primary then secondary antibodies and developed using the Amersham Biosciences chemiluminescent ECL kit and Hyperfilm ECL (Piscataway, NJ).Primary antibodies used in this study were rabbit anti-Bcl-2, rabbit anti-Bax, rabbit anti-phospho-p53 (at serine 15) and mouse anti α-tubulin.HRP conjugated anti-rabbit and HRP conjugated anti-mouse IgG were used as secondary antibodies.The band density was normalized by α-tubulin.

Immunocytochemistry
To investigate the nuclear translocation of p53, immunocytochemistry was performed using anti-p53 antibody as a primary antibody.Cells were grown to 95-100% confluence on round coverslips then performed experiments.Cells were fixed in 4% paraformaldehyde for 5 min, blocked with 1% normal goat serum in 0.05% Triton X-100 for 30 min, then incubated overnight at 4°C with the diluted primary antibody: anti-p53 (1:500).After washed with PBS-Tween, cells were incubated with FITC-conjugated secondary antibody for 1 h at 37°C.Nuclei of cells were counterstained with TO-PRO-3.Immunofluorescence was visualized under a confocal laser-scanning microscope (FV 1000, Olympus, Tokyo, Japan).Negative controls were cells omitted anti-p53 antibody.

Statistical Analysis
Data was presented as means ± SEM from three or more independent experiments.Significance was accessed by one way analysis of variance (ANOVA) followed by a Tukey multiple comparison test in the GraphPad Prism program version 5 (GraphPad software, San Diego, CA).Differences with p-values of less than 0.05 were considered statistically significant.
ROS is known as a second messenger that can activate the transduction pathways leading to apoptosis (Son et al., 2011).EGCG is regarded as a potent scavenger of free radicals including singlet oxygen, superoxide anions, hydroxyl radicals, and peroxy radicals based on stereochemistry, a trihydroxyl group on the B ring and the gallate moiety at the 3′ position in the C ring (Nanjo et al., 1996).In this current study, EGCG pretreatment decreased an intracellular accumulation of ROS in ARPE-19 cells injured by oxidant stress H 2 O 2 thus the internalized EGCG may act as a scavenger of intracellular ROS.
We also demonstrated that ARPE-19 cells treated with H 2 O 2 underwent apoptotic cell death and the caspase 3 inhibitor, Ac-DEVD-CHO, attenuated H 2 O 2 -induced the decrease of cell viability.These findings were consistent with the previous studies that H 2 O 2 -induced apoptosis in rat lens epithelial cell (Yao et al., 2003), chicken osteocytes (Kikuyama et al., 2002) and in human RPE cells (Cai, Wu, Nelson, Sternberg, & Jones, 1999;Jin et al., 2001).Pretreating the ARPE-19 cells with EGCG prior to H 2 O 2 showed the significant reduction of the number of cells containing condensed nuclei and TUNEL positive cells.These findings demonstrated that EGCG showed a protective role in H 2 O 2 -induced apoptosis in ARPE-19 cells.Previous studies were reported that EGCG also has protective effects against apoptosis induced by the pro-parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells (Guo, Bezard, & Zhao, 2005).It has also been reported that certain flavanoids including EGCG protected RPE cells from oxidative stress induce cell death (Hanneken et al., 2006) by inducing the expression of transcription factor Nrf2 (Nuclear factor-erythroid 2-related factor-2) and phase 2 enzyme heme-oxygenase, suggesting that EGCG afforded protection via its antioxidant properties.However, a direct action of EGCG on the apoptotic pathway in ARPE-19 cells has not been demonstrated.Therefore, our study determined the anti-apoptotic property of EGCG against H 2 O 2 -induced ARPE-19 cell death.Previous study reported that H 2 O 2 caused mitochondrial DNA damage and promoted apoptosis through p53 dependent pathway with increased caspase-3 activity, decreased expression of Bcl-2 and increased expression of p53 protein in RPE (Jin et al., 2001).
In the present study, we showed that treatment of ARPE-19 cells with H 2 O 2 induced the p53 tumor suppressor protein migration into the cell nuclei, then followed by cell apoptosis, which supported the previous evidence that p53 may play a pivotal role in H 2 O 2 induced apoptosis of ARPE-19.Moreover, pretreatment cells with EGCG prior to exposure with H 2 O 2 could prevent p53 nuclear translocation.This finding suggested that EGCG may protect the ARPE-19 cell from apoptosis by interfering p53 pathway.Recently, EGCG also protected H9c2 cardiomyoblasts via the inhibition of p53 (Sheng, Gu, Xie, Zhou, & Guo, 2010).Moreover, oxidized LDL-induced apoptosis in endothelial cells by increasing p53 activity, the increase of which was strikingly downregulated by EGCG (Choi et al., 2008).EGCG treatment also decreased UVB-induced keratinocyte cytotoxicity and apoptosis via inhibition the mRNA expressions of apoptosis-regulatory gene p53 (Luo et al., 2006).
The tumor suppressor protein p53 is considered to be a major player in the apoptotic response to genotoxins.Therefore, we were interested in elucidating the role of EGCG on p53-dependent pathway for the induction of apoptosis.The phosphorylation of p-53 plays a critical role in the activation and up-regulation of p53 during cellular stress (Haupt et al., 2003).During cell stress, p53 translocates to the nucleus and induces pro-apoptotic gene expression and blocks anti-apoptotic gene expression (Miyashita & Reed, 1995).This leads to the disruption of the balance between pro-and anti-apoptotic proteins.Members of the Bcl-2 family are the mediators of cell survival and apoptosis (Brunelle & Letai, 2009).The interaction between the pro-apoptotic and anti-apoptotic Bcl-2 family members can alter the permeability of mitochondrial membrane and release cytochrome c then activate caspase cascade (Brunelle & Letai, 2009).To further investigate the downstream involvement of apoptotic proteins in p53 signaling pathway, the present study also investigated the expression of Bcl-2 family proteins; one was a pro-apoptotic protein, Bax and the other was anti-apoptotic protein, Bcl-2.Our study showed that H 2 O 2 up-regulated Bax and down-regulated Bcl-2 expression thereby increase in Bax: Bcl-2 ratio which would lead to the caspase 3 activation (Jin et al., 2001) Figure 1.T different H various A -induced ARPE cell death.These findings might support the previous studies about cytoprotective effect of EGCG against H 2 O 2 -induced oxidative stress in PC12 cells The effect of H H 2 O 2 concentra s times (0.5-6 h ANOVA was p ent the cells w tly increased c with 250 µM ity to 55.9 ± 1.
. These results agreed with the previous studies that H 2 O 2 induced