Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line

Stepwise accumulations of genetic mutations and epigenetic alterations are vital events in the carcinogenic process. The main epigenetic change in cancer is the deregulation of DNA methylation, and both hypomethylated and hypermethylated DNA regions are observed throughout the cancer genome (Ehrlich and Lacey, 2013). Bladder cancer, which is more prevalent in men, is the ninth most common cancer globally (Ploeg et al., 2009). Beside genetic mutations and epigenetic alterations, oxidative stress is also critically involved in the bladder cancer carcinogenesis. Increase in oxidative stress in patients with bladder cancer has been reported (Akcay et al., 2003, Opanuraks et al., 2010). Reactive oxygen species (ROS) directly damage the cellular DNA and promote tumor development not only through genetic mutations, but also through epigenetic alterations (Wachsman, 1997). Global hypomethylation and regional (site-specific CpG island promoter) hypermethylation of


Introduction
Stepwise accumulations of genetic mutations and epigenetic alterations are vital events in the carcinogenic process. The main epigenetic change in cancer is the deregulation of DNA methylation, and both hypomethylated and hypermethylated DNA regions are observed throughout the cancer genome (Ehrlich and Lacey, 2013).
Bladder cancer, which is more prevalent in men, is the ninth most common cancer globally (Ploeg et al., 2009). Beside genetic mutations and epigenetic alterations, oxidative stress is also critically involved in the bladder cancer carcinogenesis. Increase in oxidative stress in patients with bladder cancer has been reported (Akcay et al., 2003, Opanuraks et al., 2010. Reactive oxygen species (ROS) directly damage the cellular DNA and promote tumor development not only through genetic mutations, but also through epigenetic alterations (Wachsman, 1997). Global hypomethylation and regional (site-specific CpG island promoter) hypermethylation of

Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line
Wikrom Wongpaiboonwattana 1 , Piyaratana Tosukhowong 1 , Thasinas Dissayabutra 1 , Apiwat Mutirangura 2 , Chanchai Boonla 1 * the tumor suppressor genes are demonstrated in bladder cancer (Sanchez-Carbayo, 2012). Methylation in repetitive DNA region reduces genomic instability by inactivation of transposons around the genome (Kazazian and Goodier, 2002), while methylation in the promoter decreases expression of the gene. Long interspersed nuclear element-1 (LINE-1), a tremendous transposon of human genome, has been considered as a good representative for measuring global methylation (Kazazian and Goodier, 2002). Hypomethylation of LINE-1 in bladder cancer has been demonstrated by various groups (Jurgens et al., 1996, Chalitchagorn et al., 2004, Wilhelm et al., 2010. Recently, we reported that patients with bladder cancer had increased oxidative stress and LINE-1 hypomethylation relative to the healthy controls (Patchsung et al., 2012). Furthermore, increased oxidative stress was found to correlate with decreased LINE-1 methylation both in patients and control subjects, suggested that hypomethylation of LINE-1 was in fact associated with increased oxidative stress (not cancer status per se). However, whether LINE-1 hypomethylation is a cause or effect of oxidative stress 3774 is remained to be elucidated. Although inflammationinduced hypomethylation of LINE-1 (by IL-6) was demonstrated (Gasche et al., 2011), oxidative stressinduced LINE-1 hypomethylation has not been explored.
Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene located at 1p36. RUNX3 promoter is frequently hypermethylated in many cancers , Li et al., 2004, Lau et al., 2006. In bladder cancer, hypermethylation of RUNX3 promoter in the tumor tissues was firstly reported by Kim et al. (2005). Later, they demonstrated that promoter hypermethylation of RUNX3 was associated with the shorter survival suggested as a potential prognostic marker for bladder cancer (Kim et al., 2008). Wolff et al suggested that promoter hypermethylation of RUNX3 could be a molecular clock of bladder tumor, as it increased with age, was not present in the normal urothelial cells and occurred early in the tumorigenesis (Wolff et al., 2008). Histone deacetylase inhibitor, nicotinamide was capable of re-expressing RUNX3 gene and preventing bladder tumorigenesis in mouse (Kim et al., 2011). Recently, Yan and colleagues demonstrated that RUNX3 methylation status could predict the risk of progression in patients with non-muscle-invasive bladder cancer (Yan et al., 2012). Moreover the RUNX3 methylation status in histologically normal surrounding urothelium was associated with bladder tumor number and progression (Jeong et al., 2012). These studies underline the causal relationship between RUNX3 promoter hypermethylation and bladder cancer development. Oxidative stress coincides with dysregulation of DNA methylation, however, the causal relationship between these two events is not well understood. Oxidative stress-induced RUNX3 promoter hypermethylation in human colorectal cancer cell line was demonstrated (Kang et al., 2012), but induction of RUNX3 promoter hypermethylation by ROS in bladder cancer cell line has not been investigated.
We hypothesize that oxidative stress may contribute to the carcinogenesis of bladder cancer via modification of DNA methylation in both genes and non-gene repetitive elements. We investigated herein changes of methylation patterns of LINE-1 and RUNX3 promoter in bladder cancer cell line under the oxidative stress condition

Cell and culture conditions
Human bladder cancer cell line, UM-UC-3, was purchased from ATCC and cultured in Eagle's minimal essential medium (EMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) under 37 o C, 5% CO 2 and 95% humidity. The cells were challenged with H 2 O 2 (10 and 50 µM) for oxidative stress stimulation. Tocopheryl acetate (TA) was used as antioxidant for neutralizing oxidative stress. Before each experiment, cells were seeded in 24-well plate with density of 10,000 cells/cm 2 and cultured for 24 h. In each treatment condition, serum-free EMEM was used, and the conditioned medium was changed every 24 h. For TA treatment, the cells were pre-incubated with 50 µM or 300 µM of TA for 1 h prior to addition of H 2 O 2 .

MTT assay and protein carbonyl determination
To assess the cytotoxicity of H 2 O 2 , MTT assay was carried out. UM-UC-3 cells were seeded in 96-well plate with 2x10 5 cells/well and cultured for 24 h. Cells were treated with varied concentrations of H 2 O 2 (10, 20, 50, 100 and 500 µM) for 72 h with change of conditioned media every 24 h. Solution of 0.1 M 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT was added and incubated with the cells for 2 h at 37 o C. The cells were washed with PBS and lyzed with dimethyl sulfoxide (100 µl/well). Absorbance of the formazan solution was measured at OD 570 . The obtained purple signal is proportionate to the amount of viable cells. The untreated cells were served as control and calculated as 100% viability.
The protein carbonyl content in whole cell lysate was used as an indicator of protein oxidation or oxidative protein damage. Cells in each condition were lyzed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris-Cl, pH 7.4). Procedure for protein carbonyl measurement was fully described in our previous report (Patchsung et al., 2012). The content of protein carbonyl in cell lysate was expressed per mg of total proteins (measured by the dye-binding method).

DNA extraction and bisulfite treatment
DNA was extracted using the high-pure PCR template preparation kit (Roche, Basel, Switzerland) according to manufacturer's protocol. In brief, 40 ng/µl of extracted DNA was incubated with 2 M NaOH (5.5 µl) at 37 o C for 10 min. After that, 10 mM hydroquinone, pH 5.0 (30 µl) (Sigma-Aldrich, MO, USA) and 3 M sodium bisulfite (520 µl) (Sigma-Aldrich, MO, USA) were added and incubated at 50 o C for 16 h. Bisulfite-treated DNA was purified using Wizard DNA clean-up system (Promega, WI, USA). DNA was precipitated with absolute isopropanol (220 µl) containing 10 mM ammonium acetate (17 µl) and 20 mg/ ml glycogen (1 µl) at -20 o C for 2 h. DNA was harvested by centrifuge at 14,000 rpm (4 o C) for 15 min. The DNA pellet was air dried and re-suspended with H 2 O (20 µl).

Statistical analysis
Data presented as mean±SEM. One-way ANOVA and Bonferroni multiple comparison tests were used for testing the differences among groups. GraphPad Prism 5 and STATA version 10 were employed for graphs and statistical analyses. P value<0.05 was considered as statistically significant.

Cytotoxicity of hydrogen peroxide and induction of oxidative protein damage
The concentration of H 2 O 2 for methylation experiments was chosen to ensure that the H 2 O 2 treatment was not toxic to the UM-UC-3 cells. MTT assay was carried out to measure the viability of the cells treated with 10-500 µM of H 2 O 2 for 72 h. H 2 O 2 concentrations of 10, 20 and 50 µM did not significantly alter viability of the cells whereas higher concentrations caused significant decrease in cell viability (Figure 2a). H 2 O 2 concentrations of 100 µM and 500 µM significantly reduced cell viability to 58% and 5%, respectively (P<0.001 for both). Therefore, we decided to use 10 and 50 µM of H 2 O 2 for further investigation of DNA methylation.
Induction of oxidative stress in UM-UC-3 cells exposed to H 2 O 2 was investigated. Protein carbonyl content was measured as an indicator of oxidative protein damage. Level of protein carbonyl in H 2 O 2 -treated UM-UC-3 cells was significantly increased relative to untreated control cells (Figure 2b). Pretreatment with 300 µM TA was capable of preventing oxidative protein damage in H 2 O 2 -treated UM-UC-3 cells. This indicated that exposure of UM-UC-3 cells to H 2 O 2 caused increase in oxidative stress.
The percent of hypermethylated loci ( m C 1 m C 2 ) trended to decrease in H 2 O 2 -treated cells and increase in TA-pretreated/H 2 O 2 -treated cells, although statistical significance were not observed. However, the % m C 1 m C 2 in 300 µM TA-pretreated/50 µM H 2 O 2 -treated cells was significantly increased compared to the 10 µM H 2 O 2 -treated cells (P=0.028) (Figure 3b). The percent of hypomethylated loci ( u C 1 u C 2 ) trended to increase in H 2 O 2 -treated cells and decrease in the TA-pretreated/H 2 O 2treated cells, although there was no statistical significance ( Figure 3c). Likewise, the percent of partial methylated loci ( m C 1 u C 2 ) trended to increase in H 2 O 2 -treated cells and decrease in the TA-pretreated/H 2 O 2 -treated cells, although they were not significantly different (Figure 3d).

H 2 O 2 induces RUNX3 promoter hypermethylation
UM-UC-3 cells treated with 10 and 50 µM H 2 O 2 for 72 h showed increase in methylation level of RUNX3 promoter (Figure 4a). The ratio between methylated RUNX3/unmethylated RUNX3 was increased from 6.7 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 3777 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3773 LINE-1 Hypomethylation and RUNX3 Hypermethylation by ROS in m C 1 m C 2 loci, decrease in m C 1 u C 2 and slight decrease in u C 1 u C 2 loci were found in TA pretreatment condition. Based on the current findings we hypothesize that the decreased LINE-1 methylation in H 2 O 2 -treated cells may be due to stepwise demethylation of m C 1 m C 2 to form m C 1 u C 2 and subsequently to u C 1 u C 2 . Further kinetic experiment is required to warrant this.
Although it is not precisely known, several mechanisms of how oxidative stress reduces DNA methylation have been proposed. DNA methyltransferase (DNMT) requires S-adenosylmethionine (SAM) as a methyl donor for methylation reaction, and SAM is synthesized from methionine. In oxidative stress, homocysteine is used for synthesizing glutathione (GSH) to scavenge ROS, and the synthesis of homocysteine requires SAM. Increase in GSH synthesis under oxidative stress condition, therefore, leads to depletion of SAM to be used in the DNA methylation reaction (Hitchler and Domann, 2007). Reduction of GSH that causes reduction of SAM and subsequently DNA methylation is demonstrated (Lertratanangkoon et al., 1996). Another mechanism of ROS-induced loss of DNA methylation involves DNA repairing process. An increase in NAD+/NADH ratio in oxidative environment upregulates the ten eleven translocation (TET), a hydroxylase enzyme that hydroxylates the 5-methylcytosine to form the 5-hydroxymethylcytosine (Chia et al., 2011). The 5-hydroxymethylcytosine is subsequently fixed by the base excision repair to substitute the 5-hydroxymethylcytosine with a new unmethylated cytosine, hence resulting in reduction of DNA methylation (Cedar and Bergman, 2012). In addition, ROS oxidizes the guanine base to yield 8-hydroxyguanine or 8-oxoguanine. This oxidative lesion in CpG dinucleotides strongly inhibits the methylation activity of DNMT (Weitzman et al., 1994). In sum, these three mechanisms are theoretically possible, and in cancer cells all of these pathways may act in concert to cause DNA hypomethylation. The precise mechanisms of ROS-induced LINE-1 hypomethylation in bladder cancer remain to be elucidated.
RUNX3 is a tumor suppressor gene that is strongly associated with bladder cancer carcinogenesis and tumor progression (Kim et al., 2005;, Jeong et al., 2012Yan et al., 2012). We found that ROS caused increase in methylation of RUNX3 promoter, and this hypermethylation was prevented by tocopheryl acetate. Kang et al found that RUNX3 promoter in colon cancer cells was hypermethylated under the oxidative stress condition, possibly due to increases in DNMT and histone deacetylase activity (Kang et al., 2012). O'Hagan et al. (2011) proposed that increase in NAD+/NADH ratio in oxidative condition induces the formation of a large silencing complexes containing DNMTs and polycomb repressive complex 4 at CpG-rich promoter (O'Hagan et al., 2011), and the high activity of DNMTs in this polycomb complex cause hypermethylated promoter. However, ROS-induced RUNX3 promoter hypermethylation requires further experimental proof.
Limitations of the present study should be mentioned. We did not have the transcript expression data of the hypomethylated LINE-1 and hypermethylated RUNX3. Expressions of DNMTs and histone deacetylase did not (control) to 9.7 (10 µM H 2 O 2 ) and 13.7 (50 µM H 2 O 2 ) ( Figure 4b). The methylation/unmethylation (M/U) ratio was significantly increased in 50 µM H 2 O 2 -treated condition compared to the untreated control. On the contrary, pretreatment with 50 µM and 300 µM TA caused decrease in M/U ratio from 13.7 to 9.4 and 3.4, respectively. Significant reduction of M/U ratio after pretreatment with 300 µM TA was revealed (Figure 4b).

Discussion
Increase in ROS production and alterations of DNA methylation are well recognized to vitally involve in the carcinogenic process. However, the causal link between these two events is unknown. We investigated the effect of H 2 O 2 , a representative of ROS, on methylation changes of LINE-1 and RUNX3 promoter in bladder cancer UM-UC-3 cell line. Dose of H 2 O 2 lower than 50 µM was proved to be non-toxic to the UM-UC-3 cells. Thus, changes of methylation levels observed in the present experiments were not caused by cell death, but the degree of oxidative stress. We decided to use 10 and 50 µM H 2 O 2 for investigating the DNA methylation change. Increase in protein carbonyl content in H 2 O 2 -treated cells was demonstrated to ensure an induction of oxidative stress. Many studies reported H 2 O 2 concentrations in the urine, ranged between 1 and 25 µM in normal volunteers (except for a few circumstances that up to 100 µM was reported) (Halliwell et al., 2004). Therefore, doses of H 2 O 2 used in our experiments were not extremely higher than the physiological dose, and these doses were capable of inducing cellular oxidative stress.
We demonstrated for the first time that LINE-1 was hypomethylated in bladder cancer cells exposed to H 2 O 2 , although Gashe et al had demonstrated that the LINE-1 hypomethylation was induced by IL-6 in oral cancer cells (Gasche et al., 2011). The current result warranted that oxidative stress experimentally caused LINE-1 hypomethylation. It might, at least in part, explain our previous clinical finding that showed that bladder cancer patients who had enhanced oxidative stress coincidentally had LINE-1 hypomethylation (Patchsung et al., 2012). In addition to H 2 O 2 , we had tried to experimentally induce LINE-1 hypomethylation in bladder cancer cell by inflammatory cytokine (TNF-α) similar to the study by Gashe et al. (2011). Unfortunately, we found no change of LINE-1 methylation in UM-UC-3 cells exposed to TNF-α (data not shown). It is known that hypomethylation of LINE-1 causes overexpression of LINE-1 transposons leading to induction of genomic instability and eventually malignancy transformation (Kazazian and Goodier, 2002). We showed that LINE-1 hypomethylation was capable of counteracting by an antioxidant, tocopheryl acetate. This data suggest that hypomethylation of LINE-1 by ROS is a reversible and preventable process. Antioxidant might have an additional anti-carcinogenic effect through prevention of LINE-1 hypomethylation.
Change of patterns of LINE-1 methylation under oxidative stress was also investigated. We found that the m C 1 m C 2 loci were decreased, while m C 1 u C 2 and u C 1 u C 2 loci were increased in H 2 O 2 -treated cells. Reversely, increased investigate. Additionally, only one cell line was tested. Further study should be accomplished to warrant our present novel findings.
In conclusion, we firstly demonstrated that bladder cancer cells manifested LINE-1 hypomethylation and RUNX3 promoter hypermethylation under the oxidative stress condition. The present findings support the hypothesis that oxidative stress promotes carcinogenesis through global DNA hypomethylation and promoter hypermethylation of tumor suppressor genes. Attenuation of cellular oxidative stress by antioxidants may be beneficial for preventing alterations of DNA methylation and hence malignant transformation. Our current data also implied that mechanistic pathways of ROS-induced alteration of DNA methylation in gene promoters and repetitive DNA elements might be different.