Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC-3 cells

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Gallic Acid Provokes DNA Damage and

Suppresses DNA Repair Gene Expression in

Human Prostate Cancer PC-3 Cells

AQ2 Kuo-Ching Liu,1,2Heng-Chien Ho,3An-Cheng Huang,4Bin-Chuan Ji,5Hui-Yi Lin,6Fu-Shin Chueh,7Jai-Sing Yang,8Chi-Cheng Lu,9Jo-Hua Chiang,9Menghsiao Meng,2Jing-Gung Chung10,11

1

AQ2 Department of Medical Laboratory Science and Biotechnology, China Medical University,

Taichung 404, Taiwan 2

Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan

AQ1

3

Department of Biochemistry, China Medical University, Taichung 404, Taiwan 4

Department of Nursing, St. Mary’s Medicine Nursing and Management College, Yilan 266, Taiwan 5

Division of Respiratory Care Center, Department of Internal Medicine, Changhua Christian Hospital, Changhua 500, Taiwan

6

School of Pharmacy, China Medical University, Taichung 404, Taiwan 7

Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan 8

Department of Pharmacology, China Medical University, Taichung 404, Taiwan 9

Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan 10

Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan 11

Department of Biotechnology, Asia University, Taichung 413, Taiwan

Received 31 March 2011; revised 25 May 2011; accepted 4 June 2011

ABSTRACT: Our earlier studies have demonstrated that gallic acid (GA) induced cytotoxic effects includ-ing induction of apoptosis and DNA damage and inhibited the cell migration and invasion in human cancer cells. However, GA-affected DNA damage and repair gene expressions in human prostate cancer cells are still unclear. In this study, we investigated whether or not GA induces DNA damage and inhibits DNA repair gene expression in a human prostate cancer cell line (PC-3). The results from flow cytometric assay indicated that GA decreased the percentage of viable PC-3 cells in a dose- and time-dependent manner. PC-3 cells after exposure to different doses (50, 100, and 200 lM) of GA and various periods of time (12, 24, and 48 h) led to a longer DNA migration smear (comet tail) occurred based on the single cell gel elec-trophoresis (Comet assay). These observations indicated that GA-induced DNA damage in PC-3 cells, which also confirm by 4,6-diamidino-2-phenylindole dihydrochloride staining and DNA agarose gel elec-trophoresis. Alternatively, results from real-time polymerized chain reaction assay also indicated that GA

J_ID:ZRV Customer A_ID:11-121.R1 Cadmus Art:TOX20752 Date:22-JUNE-11 Stage: I Page: 1

ID:nareshrao Date:22/6/11 Time:13:01 Path:N:/3b2/TOX#/Vol00000/110054/APPFile/JW-TOX#110054 Correspondence to: J.-G. Chung or M. Meng e-mail:

[email protected] or [email protected] Contract grant sponsor: China Medical University. Contract grant number: CMU99-ASIA-23.

Contract grant sponsor: Taiwan Department of Health Clinical Trial and Research Center of Excellence.

Contract grant number: DOH100-TD-B-111-004

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20752?

C 2011 Wiley Periodicals, Inc.

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inhibited ataxia telangiectasia mutated, ataxia-telangiectasia and Rad3-related, O6-methylguanine-DNA methyltransferase, DNA-dependent serine/threonine protein kinase, and p53 mRNA expressions in PC-3 cells. Taken together, the present study showed that GA caused DNA damage and inhibited DNA repair genes as well as both effects may be the critical factors for GA-inhibited growth of PC-3 cells in vitro. #_{2011 Wiley Periodicals, Inc. Environ Toxicol 21: 000–000, 2011.}

Keywords: gallic acid; DNA damage; comet assay; DNA repair; human prostate cancer PC-3 cells

INTRODUCTION

Adenocarcinoma of the prostate, the most frequently diag-nosed noncutaneous cancer, is the second leading cause of cancer-related deaths among men in the United States (Wingo et al., 1995; Jemal et al., 2009). In Taiwan, prostate cancer also is the seventh of cancer-related deaths in men and about eight persons per 100,000 die annually from prostate cancer based on 2009 report from the Department of Health, Republic of China (Taiwan) (Liu et al., 2011). So far, the exact molecular mechanisms responsible for the development and progression of prostate cancer in human remain poorly investigated. Currently, there is as yet no effective therapy for human prostate cancer (Grossmann et al., 2001; Nieto et al., 2007). Hence, the understanding of the development in prostate cancer is urgent.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA), a poly-hydroxyphenolic compound and a basic unit of tannic acid, is widely distributed in the natural plants (Atkinson et al., 2004; Ng et al., 2004). GA exhibits various biological prop-erties such as antibacterial (Kang et al., 2008), inflam-matory (Kim et al., 2006), antiviral (Kaur et al., 2009), anti-oxidant (Inoue et al., 2000), and anticancer effects (Kawada et al., 2001; Faried et al., 2007; Ji et al., 2009; Kaur et al., 2009; Lo et al., 2010). Much evidence has shown that GA is able to inhibit proliferation of tumor cells in culture by causing apoptosis and/or cell-cycle arrest (Agarwal et al., 2006; Veluri et al., 2006). In our laboratory, we also found that GA induced apoptosis in human lung cancer NCI-H460 cells via a caspase-3 and mitochondrion-dependent pathway and inhibited the in vivo tumor growth of NCI-H460 cells in xenograft models (Ji et al., 2009). Also, GA-induced DNA damage of lung cancer cells is examined by Comet assay (Ji et al., 2009). GA triggered apoptotic death in human melanoma A375.S2 cells through caspase-de-pendent and -indecaspase-de-pendent pathways (Lo et al., 2010). More-over, GA promoted macrophage phagocytosis in WEHI-3 leukemia mice in vivo (Ho et al., 2009). Recently, it was reported that GA induced cell-cycle arrest at G2/M phase through Chk2-mediated phosphorylation of Cdc25C in a bladder transitional carcinoma cell line (Ou et al., 2010), and this might be a DNA damage response as indicated by Ser-139 phosphorylation of histone H2A.X (Ou et al., 2010).

Until now, there is no available information addressing GA-induced DNA damage in human prostate cancer PC-3 cells. Therefore, in the present study, we focused on thein

vitro GA-induced DNA damage and affected repair gene expression in human prostate cancer PC-3 cells. Results indicated that GA promoted DNA damage and inhibited DNA repair gene expression of PC-3 cellsin vitro.

MATERIALS AND METHODS

Chemicals and Reagents

GA, dimethyl sulfoxide (DMSO), propidium iodide (PI), trypan blue, sodium chloride (NaCl), Tris–HCl, Na2EDTA, Triton X-100, and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich Corp (St. Louis, MO). RPMI-1640 medium, fetal bovine serum (FBS), L

-gluta-mine, penicillin–streptomycin, trypsin-EDTA, and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Invitrogen Life Technologies (Carlsbad, CA). Tris/borate/EDTA (TBE) buffer was purchased from Amresco (Solon, OH). High-Capacity cDNA Reverse Tran-scription Kit and 2X SYBR Green PCR Master Mix was purchased from Applied Biosystems by Life Technologies (Foster City, CA).

Cell Culture

PC-3 human prostate cancer-cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were immediately placed onto 75-cm2 tissue culture flasks and grown at 378C under a humidified 5% CO2atmosphere with RPMI-1640 medium with 2 mML-glutamine were adjusted to contain 10% FBS,

100 U/mL penicillin, and 100 lg/mL streptomycin.

Flow Cytometric Assay and a PI Exclusion

Method for the Percentage of Viable PC-3

Cells In Vitro

Cells (2 3 105 cells/well) maintained in 12-well plates were incubated with GA at final concentrations of 0, 50, 100, and 200 lM, and vehicle (1% DMSO) for 48 h, and exposed to 100 lM of GA for 0, 12, 24, and 48 h. Cells from each treatment were harvested and stained with propi-dium iodide (PI, 5 lg/mL) and then were analyzed with a flow cytometer (Becton-Dickinson FACSCalibur, San Jose, CA) equipped with an argon ion laser at 488 nm wave-length and calculated by using BD CellQuest Pro software

ID:nareshrao Date:22/6/11 Time:13:01 Path:N:/3b2/TOX#/Vol00000/110054/APPFile/JW-TOX#110054

2 LIU ET AL.

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as previously described (Yeh et al., 2000; Chiang et al., 2011).

Comet Assay for DNA Damage in GA-Treated

PC-3 Cells

Cells at a density of 2 3 105cells/well seeded in 12-well plates were exposed to GA (0, 50, 100, and 200 lM) for 48 h and treated with final concentration of 100 lM GA for 12, 24, and 48 h, or vehicle (1% DMSO) in RPMI 1640 me-dium grown at 378C in 5% CO2 and 95% air. Cells were harvested for the examination of DNA damage by using the Comet assay (single-cell gel electrophoresis) according to the procedures of Chen et al. (2003) and Wang et al. (2006) with slight modifications. Briefly, the glass slides were pre-coated with 70 lL containing 0.5% (w/v) of normal melting point (NMP) agarose (Sigma-Aldrich Corp.) and 0.5% (w/ v) low melting point (LMP) agarose (Sigma-Aldrich Corp.) allowed drying on a flat surface of the slides at room tem-perature. Subsequently, about 1 3 104 cells per sample from each treatment were gently mixed with 75 lL of 0.5% (w/v) LMP and rapidly layered onto the slides precoated with the mixtures [0.5% (w/v) of NMP agarose and 0.5% (w/v) LMP agarose] before a coverslip was covered at 48C for 5 min. The coverslip was removed, and cells placed onto a glass slide were immersed in the lysis buffer contain-ing 2.5 M NaCl, 10 mM Tris–HCl, 100 mM Na2EDTA, and 1% (v/v) Triton X-100 and adjusted to pH 10 with NaOH at 48C for 1 h. These slides were washed twice with ice-cold deionized water and transferred to an electrophoresis tank with alkalin buffer (300 mM NaOH and 1 mM Na2EDTA at pH 13) at 48C for 20 min. Thereafter, the electrophoresis was carried out at 30 V and 300 mA for 20 min, before slides were removed and flooded with neutralization buffer (0.4 M Tris–HCl at pH 7.5) at 48C for 15 min. Slides were dried in methanol (Sigma-Aldrich Corp.) for 5 min before staining with 50 lL of PI (2.5 lg/mL), and comets were visualized and photographed by using a fluorescence micro-scope at 2003 magnification as previously described (Chen et al., 2009b; Lu et al., 2010). For the quantification of DNA damage, PI-stained DNA tails were quantified by using CometScore software (Tritek Corp, Sumerduck, VA). It is shown that the comet tail that tends to increase rapidly with the levels of damage calculated from the head center. The data from comet tail length were expressed (fold of control) in mean 6 SD at least three independent samples as described elsewhere (Chiang et al., 2011; Yu et al., 2011).

DAPI Staining in Apoptotic PC-3 Cells After

Exposure to GA

Approximately 2 3 105PC-3 cells/well onto 12-well plates were exposed to 0, 50, 100, and 200 lM of GA were

incu-bated for 48 h under 5% CO2and 95% air at 378C. Cells in each treatment were individually fixed with 3.7% (v/v) formaldehyde (Sigma-Aldrich Corp.) for 15 min and then stained by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) dye for determining cell chromatin condensation. All samples were examined and photographed by using flu-orescence microscopy as described elsewhere (Chiang et al., 2011; Yu et al., 2011).

DNA Agarose Gel Electrophoresis for

Examining the DNA Damage in PC-3 Cells

After GA Treatment

Cells (1 3 106cells/well) in six-well plates were incubated with 0, 50, 100, 150, and 200 lM of GA for 24-h exposure. At the end of incubation, cells were centrifuged, and DNA was collected by using a Genomic DNA Purification kit (Genemark Technology Co., Tainan, Taiwan) as followed according to the manufacturer’s protocol. The extracted DNA from each treatment was resuspended with 50 lL TBE buffer (0.045 M Tris, 0.045 M boric acid, 1 mM Na2EDTA, and pH8.3 at 258C). Approximately 1 lg/lL (20 lL) of genomic DNA was loaded in each well, and DNA agarose gel electrophoresis was performed using 1.8% agarose (Sigma-Aldrich Corp.). After ethidium bro-mide (EtBr, Sigma-Aldrich Corp.) staining, the DNA was photographed under UV light as described previously (Lai et al., 2010; Chiang et al., 2011).

Total RNA Extraction and Reverse

Transcription Extracted from PC-3 Cells After

Incubation with GA

Cells (1 3 106 cells/well) maintained in six-well plates were maintained in RPMI 1640 medium contained with or without 100 lM of GA and then were incubated for 24 h. Cells were washed twice with PBS and trypsinized, and, subsequently, the cell pellets were collected by centrifuga-tion at 1000 3 g for 5 min at 48C. The total RNA from each treatment was extracted by using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) as previously described (Mozaffarieh et al., 2010; Chiang et al., 2011). The RNA purity was measured the ratio of the absorbance at 260 and 280 nm (A260/A280), where a ratio ranging from 1.8 to 2.0 was considered to be pure for further experiment (Mozaf-farieh et al., 2010). RNA samples from each treatment were then individually reverse-transcribed for 30 min at 428C with High Capacity cDNA Reverse Transcription Kit to made first-strand cDNA according to the standard protocol of the supplier (Applied Biosystems, Carlsbad, CA). The product was aliquoted in equal volumes and stored at 2208C for real-time polymerized chain reaction (PCR) analysis.

3 AQ1

GA AFFECTS DNA DAMAGE AND REPAIR GENE IN PC-3 CELLS

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Real-Time PCR for Gene Expressions of ATM,

ATR, MGMT, DNA-PK, and p53 in PC-3 Cells

After GA Exposure

Quantitative PCR from each sample was carried out for amplifications included by the condition: 2 min at 508C, 10 min at 958C, 40 cycles of 15 s at 958C, and 1 min at 608C using 1 lL of the cDNA reverse-transcribed as described earlier, 2X SYBR Green PCR Master Mix (Applied Biosys-tems), and 200 nM forward (F) and reverse (R) primers for each gene as shown in Table

T1 I. Finally, each assay was run

on an Applied Biosystems 7300 Real-time PCR system in triplicates, and expression fold-changes were derived using the comparative threshold cycles (CT) method (Heid et al., 1996). Values were shown to normalize the human GAPDH mRNA expression as an endogenous/internal control gene as described elsewhere (Lu et al., 2009; Chiang et al., 2011).

Statistical Analysis

All data were presented as the means (S.D.) and one-way ANOVA followed by Dunnett’s test was used to analyze differences between GA-treated and untreated (control) groups. All the statistical analyses were performed *P \ 0.05, which was considered significant.

RESULTS

GA Decreased the Percentage of Viable

PC-3 Cells

The PC-3 cells were incubated with GA at 0, 50, 100, and 200 lM for 48 h and exposed to 100 lM of GA for 0, 12, 24, and 48 h. The cells were harvested for measuring the

percentage of viable PC-3 cells by flow cytometry, and results are shown in Figure 1, which indicated that GA _F1 decreased the viability of PC-3 cells, and these effects are in a dose- and time-dependent manner [Fig. 1(A,B)].

GA-Triggered DNA Damage in PC-3 Cells Was

Examined by Comet Assay

It is shown that GA induced cytotoxic effects (decrease the percentage of viable cells) on PC-3 cells (Fig. 1) to confirm GA whether or not affects DNA damage in PC-3 cells. Thus, the comet assay was selected for determining the DNA dam-age in examined PC-3 cells. The results are shown in Figure

F2 2(A,B), which indicated that GA-induced DNA damage of PC-3 cells in a dose-dependent effect [Fig. 2(A,B)]. The higher concentrations (100–200 lM) of GA led to a longer

TABLE I. The DNA sequence was evaluated using the primer express software

Primer Name Primer Sequence (50–30)

HumanATM-F TTTACCTAACTGTGAGCTGTCTCCAT

HumanATM-R ACTTCCGTAAGGCATCGTAACAC

HumanATR-F GGGAATCACGACTCGCTGAA

HumanATR-R CTAGTAGCATAGCTCGACCATGGA

HumanMGMT-F CCTGGCTGAATGCCTATTTCC

HumanMGMT-R TGTCTGGTGAACGACTCTTGCT

HumanDNA-PK-F CCAGCTCTCACGCTCTGATATG

HumanDNA-PK-R CAAACGCATGCCCAAAGTC

Humanp53-F GGGTTAGTTTACAATCAGCCACATT

Humanp53-R GGGCCTTGAAGTTAGAGAAAATTCA

HumanGAPDH-F ACACCCACTCCTCCACCTTT

HumanGAPDH-R TAGCCAAATTCGTTGTCATACC

ATM, ataxia telangiectasia mutated; ATR, ataxia-telangiectasia and Rad3-related; MGMT,O6-methylguanine-DNA methyltransferase; DNA-PK, DNA-dependent serine/threonine protein kinase and GAPDH, glycer-aldehydes-3-phosphate dehydrogenase. Each assay was conducted at least twice to ensure reproducibility.

Fig. 1. GA decreased the percentage of viable PC-3 cells. Cells (2 3 105cells/well) seeded in 12-well plates were incu-bated with GA at final concentrations of 0, 50, 100, and 200 lM and vehicle (1% DMSO) for 48 h (A). Cells were treated with 100 lM of GA for 0, 12, 24, and 48 h (B). Cells from each treatment were stained with PI (5 lg/mL) and analyzed by flow cytometry as described in the Materials and Meth-ods section. *P \ 0.05 was considered significant.

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DNA comet tail (migration smear). Figure 2(B) indicates that more DNA was damaged in PC-3 cells when compared with the control sample. Alternatively, GA also increased DNA damage in PC-3 cells after exposure to 100 lM of GA for various intervals of time (12–48 h), and there is a time-dependent response as can be seen in Figure 2(C,D).

GA Induced DNA Condensation and

Fragmentation in PC-3 Cells

Results from DAPI-staining analysis indicated that GA increased DNA condensation and break in PC-3 cells [Fig. F3 3(A,B)], and this effect is a dose-dependent response. To con-firm the induction of DNA damage and condensation in GA-treated PC-3 cells, we also used DNA agarose gel

electropho-resis for examining the DNA fragmentation. DNA was isolated from each treatment of PC-3 cells for 24 h, and then DNA fragments were determined by DNA agarose gel electrophore-sis. Results shown in Figure 3(C) indicated that DNA damage, condensation, and fragments were carried out in GA-treated PC-3 cells [Fig. 3(C)]. The higher doses of GA (100–150 lM) led to more DNA damage and fragments in PC-3 cells than that of low dose (50 lM) incubation in the examined cells.

GA Influenced DNA Damage and Repair

Genes Expression on PC-3 Cells by Real-Time

PCR

Results from comet assay, DAPI staining, and DNA aga-rose gel electrophoresis have shown that GA induced

Fig. 2. GA-induced DNA damage in PC-3 cells was examined by Comet assay. Cells (2 3 105cells/well) were incubated with 0, 50, 100, and 200 lM of GA for 48 h and exposure to 100 lM of GA for 12, 24, and 48 h in PC-3 cells. DNA damage was determined by Comet assay as described in the Materials and Methods section. Representative images of Comet assay for dose-dependent effect (A) and quantification for comet length (fold of control) (B); representative pictures of Comet assay for a time-dependent response (C) and quantifica-tion (D). *P \ 0.05 shows a significant difference between control and treated cells. [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] _AQ2

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DNA damage, condensation, and fragments in PC-3 cells. To investigate whether or not GA affects DNA damage and repair genes expressions, PC-3 cells were treated without (untreated control) or with 100 lM GA for 24 h. Thereafter, total RNA was isolated from each treatment, and the associated genes expressions were examined by real-time PCR. The results shown in Figure

F4 4 revealed

that genes expression-associated with DNA damage and repair including ataxia telangiectasia mutated (ATM), ATR, methylguanine DNA methyltransferase (MGMT), DNA-dependent serine/threonine protein kinase (DNA-PK), and p53 mRNA from PC-3 cells after 24-h treatment of GA were suppressed (Fig. 4) when compared with the untreated control group.

DISCUSSION

It is well documented that agents induced DNA damage, which is associated with cytotoxic effects including cell death and inhibition of cell growth (Kondo et al., 2010; Lou et al., 2010; Namdar et al., 2010). Previous studies have shown that GA induced cytotoxic effects in many types of human cancer cell lines (Kawada et al., 2001; Agarwal et al., 2006; Veluri et al., 2006; Faried et al., 2007; Ji et al., 2009; Kaur et al., 2009). However, regarding the effects of GA on DNA damage and repair gene expression in human prostate cancer cells is not well investigated. Thus, in the present study, we found that a dose- and time-dependent increase in DNA damage was observed in PC-3

Fig. 3. GA-induced DNA condensation and damage in PC-3 cells was examined by DAPI staining and DNA agarose gel electrophoresis. Cells were incubated with 0, 50, 100, and 200 lM of GA for 48 h and then were harvested for DAPI staining (A) and quantification (B). The arrow shows the DNA condensation in PC-3 cells. MFI means mean fluorescence in-tensity. Cells were exposed to 0, 50, 100, 150, and 200 lM of GA for 24 h, and DNA were isolated from each treatment for gel electrophoresis (C) as described in the Materials and Methods section. M, marker. [Color figure can be viewed in the online issue, which is avail-able at wileyonlinelibrary.com.]

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cells after exposure to various concentrations of GA (Fig. 2), which was associated with a loss of cell viability (Fig. 1). These results and conclusions showed a significant increase in the tail moment of the comets of PC-3 cells from comet assay (single-cell gel electrophoresis) (Fig. 2). The longer of comet tail meant that the higher DNA dam-age and DNA condensation and fragments from DAPI staining [Fig. 3(A,B)] and DNA agarose gel electrophoresis [Fig. 3(C)], respectively, in GA-treated PC-3 cells.

Numerous studies have been demonstrated that comet assay is a high sensitive technique for DNA damage exami-nation (Pool-Zobel et al., 1994; Ashby et al., 1995). Fur-thermore, Comet assay can be used as a measurement for trend-break formation during the process of excision repair of DNA may also cause DNA migration (Tice et al., 1990). It is well known that some of the major characteristic of ap-optosis are DNA condensation (Chiang et al., 2011) and DNA ladder (DNA fragmentation) of nuclei (Bakshi et al., 2010; Ramachandran et al., 2011). Herein, our results from DAPI staining and DNA agarose gel electrophoresis demonstrated that GA induced DNA condensation and frag-mentation in PC-3 cells (Fig. 3). It was reported that GA induction of apoptosis also part through the reactive oxygen species (ROS) production in mammalian cells (Chen et al., 2009a; You and Park, 2011). Thus, GA-induced DNA dam-age might be mediated through the production of ROS in PC-3 cells. Apparently, further studies are needed to estab-lish the role of the interaction of GA with DNA in carcinogenesis.

It was also reported that in mammalian cells, the DNA damage can be reduced by DNA repair through eliminating DNA lesions (Moeller et al., 2010; Kryston et al., 2011). These ROS, produced either directly by tumors or indi-rectly via inflammatory responses, can cause DNA damage

in healthy neighboring cells as well as distant sites (Kryston et al., 2011). Agents may cause DNA damage by an indirect pathway through promoting oxidative stress and inflamma-tory responses through dysfunction of mitochondria or inflammasomes (Kryston et al., 2011; Nabeshi et al., 2011). It was reported that ROS interacted with the biological mol-ecules and disrupt the normal synthesis and repair of DNA, and this disruption is primarily associated with inhibition/ inactivation of antioxidant key proteins as well as DNA repair enzymes induced by ROS-damage to these biomole-cules (Gillard et al., 2004; Eiberger et al., 2008).

In response to genotoxic agents, cells remained homeo-static via activation of signaling pathways that turn on spe-cific gene expressions; one of crucial guardian of genomic integrity is the checkpoint kinase ATM (Cuadrado et al., 2006). ATM and ATR are two master checkpoint kinases activated by double-stranded DNA breaks (DSBs) (Lavin, 2007). Our results showed that GA inhibited DNA repair genes expression including ATM, ATR, DNA-PK, MGMT, and p53 in PC-3 cells (Fig. 4). In response to DSBs, the ATM kinase phosphorylates and regulates a cascade of downstream effectors such as checkpoint kinase Chk2 and other components of the DNA repair pathways and the cell-cycle check points, in order to minimize the risk of genetic damage (Lavin, 2007; Matsuoka et al., 2007). DNA-PK plays a critical role in DNA damage repair (Mi et al., 2009). TheO6-MGMT reduces cytotoxicity of therapeutic or environmental alkylating agents (Jesien-Lewandowicz et al., 2009).

In conclusion, GA induced DNA damage in PC-3 cells and then followed by inhibiting DNA repair-associated gene expressions including ATM, ATR, MGMT, DNA-PK, andp53 and thereafter led to DNA damage maintain as the

proposed model can be shown in Figure5. _F5

Fig. 5. The possible flow chart for GA-inhibited gene expression of DNA damage and repair in human prostate cancer PC-3 cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 4. GA-affected DNA damage and -inhibited repair genes expression in PC-3 cells was determined by real-time PCR. The total RNA was extracted from the PC-3 cells after treatment without (control) and with 100 lM GA for 24 h, and RNA samples were reverse-transcribed cDNA then for real-time PCR as described in the Materials and Methods sec-tion. The ratios of ATM, ATR, MGMT, DNA-PK, and p53 mRNA/GAPDH are shown in panel. Data represent mean 6 SD of three experiments. *P \ 0.05 is considered significant when compared with the control sample.

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