Chemopreventive Activity of Ellagitannins from Acer pseudosieboldianum (Pax) Komarov Leaves on Prostate Cancer Cells

Several studies have shown that compounds from Acer pseudosieboldianum (Pax) Komarov leaves (APL) display potent anti-oxidative, anti-inflammatory, and anti-proliferative activities. Prostate cancer (PCa) is the most common cancer among older men, and DNA methylation is associated with PCa progression. This study aimed to investigate the chemopreventive activities of the compounds which were isolated from APL on prostate cancer cells and elucidate the mechanisms of these compounds in relation to DNA methylation. One novel ellagitannin [komaniin (14)] and thirteen other known compounds, including glucose derivatives [ethyl-β-D-glucopyranose (3) and (4R)-p-menth-1-ene-7,8-diol 7-O-β-D-glucopyranoside (4)], one phenylpropanoid [junipetrioloside A (5)], three phenolic acid derivatives [ellagic acid-4-β-D-xylopyranoside (1), 4-O-galloyl-quinic acid (2), and gallic acid (8)], two flavonoids [quercetin (11) and kaempferol (12)], and five hydrolysable tannins [geraniin (6), punicafolin (7), granatin B (9), 1,2,3,4,6-penta-galloyl-β-D-glucopyranoside (10), and mallotusinic acid (13)] were isolated from APL. The hydrolyzable tannins (6, 7, 9, 10, 13, and 14) showed potent anti-PCa proliferative and apoptosis-promoting activities. Among the compounds, the ellagitannins in the dehydrohexahydroxydiphenoyl (DHHDP) group (6, 9, 13, and 14), the novel compound 14 showed the most potent inhibitory activity on DNA methyltransferase (DNMT1, 3a and 3b) and glutathione S-transferase P1 methyl removing and re-expression activities. Thus, our results suggested that the ellagitannins (6, 9, 13, and 14) isolated from APL could be a promising treatment option for PCa.


Introduction
Prostate cancer (PCa), the cancer of the prostate gland, is the most common cancer in older men between the age of 54 to 75 years in Western countries. DNA methylation activity occurs in several cancer types, including PCa. DNA methylation is a process where methyl groups are added to the cytosine, creating a methylated cytosine, and DNA methylation can change DNA segment activity without changing the sequence. DNA methylation typically inhibits gene transcription, which is located in the gene promoter regions, and this process is called gene silencing. Due to the activity of gene silencing, a number of key processes occur, including X-chromosome inactivation, inhibition of transposable elements, aging, and carcinogenesis [1][2][3][4][5]. Oxidative stress, inflammation, cytokine release, and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) can catalyze DNA methylation via the upregulation of DNA methyltransferases (DNMTs) in PCa [6][7][8][9][10][11][12][13][14].
DNMTs, including DNMT1, DNMT3a, and DNMT3b, are the family of enzymes that catalyze the transfer of methyl group to DNA [15][16][17]. DNMT1 is the most abundant DNMT Acer species (Acereae) are a genus of trees and shrubs commonly known as maple. There are almost 128 species in the world and 17 species are native to Korea [52]. It is reported that flavonoids, tannins, phenylpropanoids and terpenoids, etc., were isolated from Acer species [53][54][55][56]. About 20 Acer species have been used in Chinese ethnomedical medicine; the expelling of wind, clearing heat, detoxifying the body, relieving rheumatism and lubricating joints, improving eyesight, treating sore eyes, reducing blood pressure, eliminating blood stasis, etc. [57]. The pharmacological activities of Acer species in rheumatoid arthritis, and arthralgia, eliminating stasis to resolve swelling and anti-oxidative, anti-inflammatory, anti-atopic dermatitis activity, anti-bacterial, anti-viral, anti-fungal, anti-obesity, anti-hyperglycemic and anti-tumor activities were reported [58][59][60][61]. It is reported that the Acer pseudosieboldianum (Pax) Komarov leaf (APL) extract shows potent anti-oxidative, anti-inflammatory, and anti-PCa proliferative activities [62,63]. This study aimed to assess the chemopreventive activities and elucidate the mechanisms of those compounds which were isolated from APL in relation to DNA methylation.
Komaniin (14) was obtained as an amorphous brown power. Structural data were as follows:  Figure S34) also showed six glucosyl signals at δ 90. 28, 69.39, 62.51, 65.48, 71.73 and 62.88. Careful analysis of the 1 H-NMR and 13 C-NMR spectra, revealed one galloyl group (δ 7.16 singlet on 1 H-spectrum), one hexahydroxydiphenoyl (HHDP) group (δ 6.62 and 7.08 every singlet on 1 H-NMR) and one DHHDP group (δ 5.82, 6.66, 7.24 every singlet on 1 H-NMR and 186.02 on 13 C-NMR spectra), which is similar to the A form of geraniin (6); 14 established an ellagic acid group at δ 7.51 and 7.84 (every singlet) in the 1 H-NMR spectrum and at δ 108. 3 −6.70) indicated that the absolute configuration of the DHHDP group is R. Generally, there are two forms of DHHDP group as an equilibrium mixture in an aqueous solution (A form and B form), the A form is a six-membered hemiketal form and the B form is a five-membered hemi-ketal form. However, only the A form of geraniin (6) was observed. According to the correlation of geraniin A form and ellagic acid, it is suggested that the geraniin A form was fixed and cannot convert to the B form. This ellagitannin is composed of one galloyl group at H Glc -1, one HHDP group at H Glc -3 and H Glc -6, one DHHDP group at H Glc -2 and H Glc -4, and H EA -3 and H EA -4 of ellagic acid is correlated with C DHHDP -5 and C DHHDP -3 was tentatively named komaniin (14). From these results, the structure of 14 was established in Figure 2.

Cytokine Inhibitory Activities
Inhibition of IL-6 has a role in prostate cancer growth and prostate prevention with high specificity. The six hydrolyzable tannins (6, 7, 9, 10, 13, and 14) and two flavonoids (11 and 12) showed potent anti-oxidative and anti-inflammatory activities. These six compounds were selected for further downstream experiments. The tannins showed stronger IL-6 production inhibitory activity than that of the positive control, Bay 11-7082(BAY) and the efficiency depended on the number of galloyl groups (10 > 7 > 6, 9,13,14). In addition, the (S)-DHHDP group was less potent in inhibiting IL-6 production than that of the (R)-DHHDP group, according to the results of IC50 of 9 < 6, 13, and 14 (Table 3). Table 3. IC50 values of the interleukin (IL)-6 production inhibitory activities of the compounds (6, 7, 9, 10, 11, 12, 13 and 14) from APL. The IC50 values were defined as the concentration required for 50% inhibition of IL-6. a-e indicate different superscript letters that correspond to different significant differences (p < 0.05).

NF-κB Inhibitory Activities of Hydrolysable Tannins
NF-κB inhibition was suggested as a good cancer therapy and prevention target. The four ellagitannins (6, 9, 13, and 14) in the DHHDP group and the two hydrolyzable tannins (7 and 10) not in the DHHDP group were examined for their NF-κB inhibitory activities. The results showed that 6, 9, 13, and 14 inhibit NF-κB activity more potently than 7 and 10, or the positive control, BAY ( Figure 9, Table 4).

Cytokine Inhibitory Activities
Inhibition of IL-6 has a role in prostate cancer growth and prostate prevention with high specificity. The six hydrolyzable tannins (6, 7, 9, 10, 13, and 14) and two flavonoids (11 and 12) showed potent anti-oxidative and anti-inflammatory activities. These six compounds were selected for further downstream experiments. The tannins showed stronger IL-6 production inhibitory activity than that of the positive control, Bay 11-7082(BAY) and the efficiency depended on the number of galloyl groups (10 > 7 > 6, 9,13,14). In addition, the (S)-DHHDP group was less potent in inhibiting IL-6 production than that of the (R)-DHHDP group, according to the results of IC50 of 9 < 6, 13, and 14 (Table 3). Table 3. IC50 values of the interleukin (IL)-6 production inhibitory activities of the compounds (6, 7, 9, 10, 11, 12, 13 and 14) from APL. The IC50 values were defined as the concentration required for 50% inhibition of IL-6. a-e indicate different superscript letters that correspond to different significant differences (p < 0.05).

DNA Methyltransferases Inhibitory Activities of Selected Ellagitannins
High concentrations of ellagitannins potently inhibited DNMT3a and DNMT3b p tein expressions. In particular, the DHHDP group conjugated with ellagic acid group co pound, 14, showed the strongest inhibitory activity for DNMT3; 6 only inhibited DNM minimally, and 13 and 14 in the (R)-DHHDP group showed more potent DNMTs inhi tory activities than that of 9 in the (S)-DHHDP group ( Figure 10).

Methyl Removing and Re-Expression Activities of Glutathione S-Transferase P1 of Ellagitannins from APL
According to previous research, [29] clustering of CpG sites in and near the transcr tion start site (−216 to +7) were selected to design the primers for methyl specific (M polymerase chain reaction (PCR) to detect DNA methyl removing activity. On the mol ular level, the ellagitannins (6, 9, 13, and 14) in the DHHDP group showed potent hancement of glutathione S-transferase P1 (GSTP1) mRNA and protein expressions. particular, the (R)-DHHDP group ellagitannins (6, 13, and 14) increased the mRNA a protein expressions of GSTP1 more than that of the (S)-DHHDP group ellagitannin (Figures 11 and 12). The mRNA and protein expressions of the unmethylated GSTP1 w enhanced, while that of the methylated GSTP1 was inhibited by the ellagitannins fro APL. In particular, 13 and 14 showed more potent demethylation activity of GSTP1 (F ure 13). These results suggest that, although the DHHDP group can generally enhan GSTP1 expression, the (R)-DHHDP group, compared with the (S)-DHHDP group, mig be better in PCa prevention.

Methyl Removing and Re-Expression Activities of Glutathione S-Transferase P1 of Ellagitannins from APL
According to previous research, [29] clustering of CpG sites in and near the transcription start site (−216 to +7) were selected to design the primers for methyl specific (MS)-polymerase chain reaction (PCR) to detect DNA methyl removing activity. On the molecular level, the ellagitannins (6, 9, 13, and 14) in the DHHDP group showed potent enhancement of glutathione S-transferase P1 (GSTP1) mRNA and protein expressions. In particular, the (R)-DHHDP group ellagitannins (6, 13, and 14) increased the mRNA and protein expressions of GSTP1 more than that of the (S)-DHHDP group ellagitannin (9) (Figures 11 and 12). The mRNA and protein expressions of the unmethylated GSTP1 were enhanced, while that of the methylated GSTP1 was inhibited by the ellagitannins from APL. In particular, 13 and 14 showed more potent demethylation activity of GSTP1 ( Figure 13). These results suggest that, although the DHHDP group can generally enhance GSTP1 expression, the (R)-DHHDP group, compared with the (S)-DHHDP group, might be better in PCa prevention.

Discussion
Phytochemicals are naturally occurring plant-based compounds. Dietary phytochemicals include polyphenols, carotenoids, glucosinolates, organic sulfides and terpenoids and have potential as chemical treatments. They show improved anticancer activity due to the regulation of several cell death pathways, such as apoptosis, inhibition of cell proliferation and invasion and migration [88,89]. The compounds we isolate from APL also include flavonoids and ellagitannins. Thus, compounds from APL may be good agents for prostate cancer prevention.
The compounds we isolate from APL also have components such as ellagic acid, gallic acid and quercetin. Thus, compounds from APL may be good agents for prostate cancer prevention. We isolated the 14 compounds; glucose derivatives (3, 4), one phenylpro-

Discussion
Phytochemicals are naturally occurring plant-based compounds. Dietary phytochemicals include polyphenols, carotenoids, glucosinolates, organic sulfides and terpenoids and have potential as chemical treatments. They show improved anticancer activity due to the regulation of several cell death pathways, such as apoptosis, inhibition of cell proliferation and invasion and migration [88,89]. The compounds we isolate from APL also include flavonoids and ellagitannins. Thus, compounds from APL may be good agents for prostate cancer prevention.
The compounds we isolate from APL also have components such as ellagic acid, gallic acid and quercetin. Thus, compounds from APL may be good agents for prostate cancer prevention. We isolated the 14 compounds; glucose derivatives (3, 4), one phenylpro-

Discussion
Phytochemicals are naturally occurring plant-based compounds. Dietary chemicals include polyphenols, carotenoids, glucosinolates, organic sulfides and noids and have potential as chemical treatments. They show improved anticancer due to the regulation of several cell death pathways, such as apoptosis, inhibition proliferation and invasion and migration [88,89]. The compounds we isolate fro also include flavonoids and ellagitannins. Thus, compounds from APL may b agents for prostate cancer prevention.
The compounds we isolate from APL also have components such as ellagic ac lic acid and quercetin. Thus, compounds from APL may be good agents for prost cer prevention. We isolated the 14 compounds; glucose derivatives (3, 4), one phe

Discussion
Phytochemicals are naturally occurring plant-based compounds. Dietary phytochemicals include polyphenols, carotenoids, glucosinolates, organic sulfides and terpenoids and have potential as chemical treatments. They show improved anticancer activity due to the regulation of several cell death pathways, such as apoptosis, inhibition of cell proliferation and invasion and migration [88,89]. The compounds we isolate from APL also include flavonoids and ellagitannins. Thus, compounds from APL may be good agents for prostate cancer prevention.
The compounds we isolate from APL also have components such as ellagic acid, gallic acid and quercetin. Thus, compounds from APL may be good agents for prostate cancer prevention. We isolated the 14 compounds; glucose derivatives (3,4), one phenylpropanoid (5), three phenolic acid derivatives (1, 2, 8), two flavonoids (11,12) and five hydrolyzable tannins (6, 7, 9, 10 and 13) including a novel ellagitannin 14 from APL. The hydrolyzable tannins (6, 7, 9, 10, 13 and 14) isolated from APL have potent anti-proliferative and apoptosis-promoting activity, especially the ellagitannins (6, 9, 13 and 14) in the DHHDP group. In particular, the novel compound 14, which has a structure of ellagic acid conjugating with geraniin A form, showed the strongest chemopreventive activities among the other compounds. Ellagic acid has four hydroxyls and two lactone functional groups, which allow the removal of various ROS and reactive nitrogen species. The antioxidant properties of ellagic acid are related to anti-inflammatory action, neuroprotection, diabetes, cardiovascular disease, and protection against prostate cancer [90,91]. In our study, when comparing geraniin (6) with a novel compound (14), both compounds showed potent anti-proliferative and apoptosis-promoting activity. However, 14 showed the most potent inhibitory activity on DNA methyltransferase (DNMT1, 3a and 3b).
In this study, several hydrolyzable tannins (6, 7, 9, 10, 13 and 14) which were isolated from APL showed potent anti-proliferative and apoptosis-promoting activity, compared with other compounds, especially the ellagitannins (6, 9, 13, and 14). In addition, the ellagitannins (6, 9, 13, and 14) which have a DHHDP group in their structure, showed potent IL-6 and NF-κB inhibitory activities. Among the DHHDP compounds, the novel compound 14, showed the most potent inhibitory activity. So the following experiments were conducted mainly on ellagitannins (6, 9, 13, and 14) in the DHHDP group. The ellagitannins (6, 9, 13, and 14) displayed demethylation activity as measured by methyl removal, GSTP1 re-expression, and DNMT inhibitory activities. In particular, compounds 13 and 14 showed more potent activity on the demethylation activity of GSTP1 and potent inhibitory activity on DNA methyltransferase (DNMT1, 3a and 3b) and GSTP1 methyl removing and re-expression activities. In addition, DNA methylation-inducing factors, IL-6 and NF-κB, which are associated with the regulation of DNMTs, were inhibited by the ellagitannins (6, 9, 13, and 14). According to the results of demethylation activity, it appeared that methylation of GSTP1 is associated with DNMT1. These results suggest that the ellagitannins in the DHHDP group, especially the novel compound 14, from APL can be developed as a new agent for PCa chemoprevention. This study showed the possibility that ellagitannins have effective preventive activity against prostate cancer. Further, additional research will be tried to verify whether chemopreventive effects are also exhibited in vivo.

Isolation of the Compounds from APL
The leaves of AP (1.62 kg) were extracted with 80% prethanol A (ethyl alcohol) at room temperature (25 • C) three times to obtain the extract (595.61 g). The extract was dissolved in water and filtered through Celite (Duksan Pure Chemical, Ansan, Korea), the water-soluble fraction was applied to a Sephadex LH-20 column equilibrated with water and eluted with a water-methanol gradient system and washed by 60% acetone, eleven fractions (Fr.) were yielded. Fr. 2 (10.53 g) was applied to the MCI CHP20P column with a water-methanol gradient system; Fr. 2-26 was dissolved with methanol and filtered to yield 1 (30.9 mg). Repeated column chromatography of Fr. 2-2 (1544.5 mg) on an ODS-B column with a water-methanol gradient system was conducted to yield 2 (121.9 mg). Similarly, Fr. 2-4 was subjected to an ODS semi-prep column with an MPLC system with a water-methanol gradient system to yield 3 (37 mg). Fr. 2-22 (294.9 mg) was subjected to an ODS semi-prep column with an MPLC system with a water-methanol gradient system to yield 4 (71.7 mg). Following the chromatography, Fr.2 yielded 5 (17.9 mg). Fr.7 (94.59 g) was subjected to an MCI CHP20P column with a water-methanol gradient system to yield 6 (45.53 g). Repeated chromatography of Fr. 7-2 (281.9 mg) on an ODS semi-prep column with MPLC with 25% methanol yielded 8 (24.2 mg), 9 (23.9 mg) and 13 (46.2 mg). Fr. 7-13 (2.1 g) was subjected to an ODS semi-prep column with an MPLC system and a water-methanol gradient system to yield 11 (210.1 mg). Fr. 10 (1.99 g) was applied to a Sephadex LH-20 column with a water-methanol gradient system to yield 7 (236.6 mg). Repeated chromatography of Fr. 10-7 (417 mg) on an ODS semi-prep column with an MPLC system with 30% methanol yielded 10 (49.7 mg). Fr. 7-15 (2.1 g) was subjected to an ODS semi-prep column with an MPLC system with a water-methanol gradient system to yield 12 (32.4 mg). Fr. 11 (11.30 g) was applied on MCI CHP20P and a Sephadex LH-20 column with a water-methanol gradient system to yield 14 (192.7 mg).

Cell Proliferative Inhibition Assay
The anti-proliferative activities of compounds in PC-3 and LNCaP cell lines were measured by the MTT assay. PC-3 and LNCaP cells (1 × 105 cells / well) were pre-cultured in a 96-well plate for 4 h and treated with 20 µL samples (final concentration of 100, 50, 25, 12.5 and 6.25 µM for compound level samples) and 180 µL medium for the sample group or 200 µL medium for the control group. After incubating for 48 h at 37 • C, the medium was replaced with 1 mg/mL MTT and incubated for a further 4 h at 37 • C. Then, the suspension was removed and the produced formazan was dissolved in 100 µL DMSO. After mixing gently, the optical density was measured at 540 nm using the FlexStation 3 Multi Mode Microplate Reader (San Jose, CA, USA). IL-6 production was measured using a mouse IL-6 ELISA kit (Young In Frontier, Seoul, Republic of Korea). After 100 µL of samples or standard were added to the 96 well plate of the kit for 2 h at 37 • C, the suspension was removed and the wells were washed three times with 100 µL/well of washing buffer. Then they were incubated for 1 h at 37 • C after adding the diluted secondary antibody. The suspension was removed and each well was washed three times again; 100 µL of working streptavidin HRP was added. After incubating for 30 min at 37 • C, the suspension was removed and each well was washed three times. After 100 µL of the substrate was added into each well, the liquid began to turn blue. Incubating the plate at room temperature for 5 min, 100 µL stop solution was added and the absorbance was measured at 450 nm by an ELISA reader (Tecan, Salzburg, Austria). Based on the standard curve, the inhibitory effect of IL-6 production and IC50 value, which is the concentration required for 50% IL-6 production, were calculated. Bay 11-7082 (BAY) (Sigma, St. Louis, MO, USA) was used as the positive control group.

Western Blotting Assay
After the cells were treated with compounds (final concentration of 100, 50, and 25 µM for compound level samples), the cells were collected and solubilized in ice-cold lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease (Thermo Scientific, Waltham, MA, USA). The extracted total proteins were separated by SDS-PAGE on 10 % polyacrylamide gels and electrophoretically transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in a blocking buffer for 1 h and subsequently incubated in a blocking buffer containing primary antibodies at room temperature for 2 h. After washing the membranes with tris-buffered saline containing 0.5 % Tween-20, the secondary antibodies were incubated for 1 h at room temperature. Then, the proteins were visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA), and the band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The IC50 value, which is the concentration required for inhibition of 50% of the protein concentration, was calculated. Bay 11-7082 was used as the positive control group.

Real-Time Reverse Transcription Polymerase Chain Reaction
PC-3 and LNCaP cell lines (2 × 10 6 cells/well) were pre-cultured in a 6-well plate for 4 h and treated with 200 µL of compounds from the APL (final concentration of 100, 50, 25 µM for compound level) and 1800 µL of the medium. The normal control group was only treated with 2000 µL of the medium. The experimental and control groups were then incubated for 48 h at 37 • C. Total RNA was extracted from the cells using the TRIzol reagent (Invitrogen, Waltham, MA, USA). Chloroform was added to the TRIzol reagent, and the tubes were briefly shaken. The mixtures were centrifuged at 13,500 rpm and 4 • C for 15 min. The upper phase was then transferred to a new tube containing isopropanol. After the mixtures were incubated at room temperature for 10 min, they were centrifuged at 13,500 rpm and 4 • C for 10 min. The supernatant was removed and washed with 70% ethanol. After centrifuging at 11,500 rpm and 4 • C for 10 min, the RNA pellet was briefly dried. The purified RNA was dissolved in Diethyl Pyrocarbonate (DEPC)-treated water. 1 µg of isolated RNA underwent reverse transcription with the First-Stand Synthesis System kit (Thermo Scientific, Waltham, MA, USA). After adding oligonucleotides and dNTP to the 1 µg of RNA, the mixture was incubated at 65 • C for 5 min. Following incubation, SSIV buffer, dithiothreitol, and reverse transcriptase (RT) enzyme provided in the kit were added, and the mixture was incubated at 55 • C for 10 min. The RT reaction was inactivated by incubating the solution at 80 • C for 10 min to obtain cDNA samples; 1 µL of the cDNA samples were added to 10 µL of the Universal SYBR Green Supermix (BioRAD, Hercules, CA, USA), 2 µL of 10 pM primers (1 µL each of 10 pM upstream and downstream primers), and 7 µL DEPC-treated deionized water. The realtime PCR conditions were: denaturation at 95 • C for 5 min for the first cycle, 30 s for the second cycle, 30 s of annealing at 59 • C, and 30 s of extension at 72 • C. The final extension was performed at 72 • C for 10 min. The following primer sequences were used for βactin (187, 5 -ACCATGGATGATGATATCGC-3 ; 5 -ACAGGCTGGGGTGTTGAAG-3 ) and GSTP1 (5 -TCACTCAAAGCCTCCTGCCTAT-3 ; 5 -CAGTGCCTTCACATAGTCATCC-3 ).

Methyl Specific-Polymerase Chain Reaction
PC-3 and LNCaP cell lines (2 × 10 6 cells/well) were pre-cultured in a 6-well plate for 4 h and treated with 200 µL samples (final concentration of 25, 50, and 100 µM for compound level) and 1800 µL of the medium. The normal control group was only treated with 2000 µL of the medium and then incubated for 48 h at 37 • C. The cells were collected and washed in ice-cold PBS, and the DNA was extracted using the QIAamp DNA Mini Kit (QI-AGEN, Hilden, Germany). PBS, QIAGEN protease, and AL buffer from the kit were added to the collected cells. After incubating at 56 • C for 10 min, the mixture was centrifuged, and ethanol was added. Then, the mixture was centrifuged at 8000 rpm, and the collection tubes were discarded. After washing twice with AW1 buffer and AW2 buffer, AE buffer was added to elute the DNA samples. DNA bisulfite modification was performed using the MethylEasy Xceed kit (Genetic Signatures, NSW, Australia); 3M NaOH was added to the collected DNA, and the mixture was incubated for 15 min at 37 • C. After adding the combined Reagent 1 and Reagent 2 from the kit, the mixture was incubated at 80 • C for 45 min. After adding Reagent 3, the DNA was washed twice using Reagent 4. Finally, Reagent 5 was added, and the solution was incubated at 95 • C for 20 min. After DNA bisulfite modification, PCR was carried out; 2 µL of the bisulfite-modified DNA samples were added to 10 µL of the Universal SYBR Green Supermix (BioRAD, Hercules, CA, USA), 2 µL of 10 pM primers (1 µL each of 10 pM upstream and downstream primers), and 6 µL DEPC-treated deionized water. The real-time PCR conditions were denaturation at 95 • C for 5 min for the first cycle and 30 s starting from the second cycle, 30 s of annealing at 63.7 • C, and 30 s of extension at 72 • C. The final extension was performed at 72 • C for 10 min. The primer sequences for unmethylated GSTP1 were (5 -AAAGAGGGAAAGGTTTTTTTGGTTAGTTGTGTGGTG-3 ; 5 -AAACTCCAACAAAAACCTCACAACCTCCA-3 ) and methylated GSTP1 were (5 -GGTTTTTTTCGGTTAGTTGCGCGGCG-3 ; 5 -CCAACGAAAACCTCGCGACCTCCG-3 ).

Statistical Analysis
All data were calculated as mean ± S.D of a triplicate experiment. Data were analyzed by ANOVA (one-way analysis of variance), and the post hoc analysis was conducted by the Student-Newman-Keuls (S-N-K) test (p < 0.05). Data were tested by t-test. * indicate p < 0.05, ** indicate p < 0.01,*** indicate p < 0.001.