FEBS Letters 585 (2011) 3436–3441

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Reactive oxygen species downregulate catalase expression via of a CpG Island in the Oct-1 ⇑ Xiaoyuan Quan, Seung-Oe Lim, Guhung Jung

The Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, South Korea article info abstract

Article history: (ROS) caused oxidative stress plays a key role in . The POU Received 26 September 2011 domain factor Oct-1 and catalase is closely associated with ROS. However, a correla- Accepted 27 September 2011 tion between these two key proteins has not been demonstrated before. In this report, we show that Available online 6 October 2011 Oct-1 acts as an activator of catalase, by binding to the catalase promoter in hepatocellular carci- noma (HCC) cell lines. In addition, we suggest that Oct-1 is downregulated by ROS via CpG island Edited by Barry Halliwell methylation in its promoter. These findings contribute to a better understanding of the epigenetic changes induced by ROS in the process of carcinogenesis. Keywords: Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Reactive oxygen species Oct-1 Catalase Methylation Hepatocellular carcinoma

1. Introduction tion, controls by interacting with an 8-bp octamer motif (50-ATGCAAAT-30) and related sequences [17]. Many studies Reactive oxygen species (ROS) are involved in the multistage have elucidated the role of Oct-1 in cell cycle regulation [18,19]. process of carcinogenesis and induce both genetic and epigenetic Recently, it has also been reported that Oct-1 acts as a sensor for alterations [1,2]. ROS-induced DNA methylation is the most fre- cellular stress [20]. quent mechanism in the epigenetic regulation of gene expression Approximately 85–95% of liver is hepatocellular carci- by ROS and plays a crucial role in the development of cancer [3]. noma (HCC), and annually this causes approximately half a million In particular, hypermethylation of CpG islands in promoter regions deaths worldwide [21] also. Oxidative stress plays a key role in the of tumor suppressor genes is frequently seen in tumor cells [4,5]. pathogenesis and progression of liver cancer [22]. A correlation be- However, this change is reversible [6]. tween ROS and HCC has been demonstrated and, moreover, certain

Catalase is an antioxidant that catalyzes degradation of H2O2 genes, including E-cadherin and catalase, have been reported to be and plays a crucial role in protecting cells against ROS [7]. How- methylated by ROS in HCC [8,23]. ever, prolonged exposure to ROS downregulates catalase expres- In this study, we analyzed Oct-1 promoter regulation via meth- sion, via hypermethylation of a CpG island in the catalase ylation and identified a link between the Oct-1 promoter [8]. Decreased expression of catalase in [9,10] and catalase expression. In HCC cells, Oct-1 binds to the catalase is associated with tumor formation and metastasis [11–13]. promoter and acts as an activator. We also found that ROS induce Oct-1 is a member of the POU-domain transcription factor fam- methylation of CpG sites in the Oct-1 promoter. Overall, we ily, which is ubiquitously expressed [14]. Oct-1, known to function showed that ROS-induced methylation of the Oct-1 promoter can as both an activator [15] and a suppressor [16] of gene transcrip- be another mechanism by which catalase is downregulated by ROS in HCC.

Abbreviations: 5-aza-dC, 5-aza-20-deoxycytidine; BS, bisulfite sequencing; ChIP, chromatin immunoprecipitation; DNMT, DNA methyltransferase; EMSA, electro- 2. Materials and methods (see also Supplementary data) phoretic mobility shift assay; HCC, hepatocellular carcinoma; MSP, methylation- specific polymerase chain reaction; PMS, phenazine methosulfate; ROS, reactive 2.1. Cell culture and treatments oxygen species ⇑ Corresponding author. Address: Department of Biological Sciences and Seoul Human hepatoma cells, Huh7 and Hep3B, were cultured National University, 504-408 Daehak-dong, Gwanak-gu, Seoul 151-742, South Korea. Fax: +82 2 872 1993. and treated with H2O2, menadione, PMS as previously described E-mail address: [email protected] (G. Jung). [23]. For some experiments, the cells were pretreated with

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.09.035 X. Quan et al. / FEBS Letters 585 (2011) 3436–3441 3437

N-acetylcysteine (Sigma–Aldrich) and 5-aza-20-deoxycytidine by immunoblotting. All other reagents were purchased from (5-aza-dC) (Sigma–Aldrich) as previously described [8]. Cell trans- Sigma. fections were carried out using Fugene 6 reagent (Roche) accord- ing to the manufacturer’s instructions. For shRNA experiments, 2.2. Oligonucleotides cells were transfected with shRNA-Oct-1 (NM_002697) and con- trol shRNA (pLKO.1) (Sigma–Aldrich) using Fugene 6 (Roche). All the oligonucleotides used in this study are listed in 72 h after transfection, Oct-1 protein expression was analyzed Supplementary Tables 1–4.

Fig. 1. Oct-1 acts as a transcriptional activator of catalase (A) Huh7 and Hep3B cells were co-transfected with pGL3-CAT and pCMV/HA-Oct-1, or pCMV/HA and a Renilla luciferase expression vector (pRL-SV40), as a control for transfection efficiency and analyzed for luciferase activity. HA, pCMV/HA; Oct-1, pCMV/HA-Oct-1. (B) Oct-1 mRNA expression was assessed by real-time RT-PCR. (C) Expression of Oct-1 was assessed by immunoblot analysis of cell lysates. b-Actin served as an internal control. (D) Huh7 cells were transfected with shRNA-Oct-1 and assessed by immunoblot. b-Actin served as an internal control. Catalase activity was measured using the cell lysate. C, control.

Fig. 2. Oct-1 binds to the catalase promoter (A) ChIP analysis of Oct-1 on the catalase promoter was performed using an antibody to Oct-1. SM, DNA size marker. (B) EMSA was performed using nuclear protein extract prepared from Huh7 cells. Normal mouse IgG was used as a positive control. (C) Oct-1-induced catalase promoter activities with or without in the Oct-1 binding site. Huh7 cells were transfected with wild-type catalase promoter (CAT) or its derivative containing a mutation in the Oct-1 binding site (Mut). The cells were cotransfected with pCMV/HA-Oct-1 or pCMV/HA control vector, as indicated. 3438 X. Quan et al. / FEBS Letters 585 (2011) 3436–3441

2.3. Methylation-specific polymerase chain reaction Huh7 cells, catalase promoter activity in cells overexpressing Oct-1 protein was approximately 2.5-fold greater than that in the Methylation-specific polymerase chain reaction (MSP) was control cells (Fig. 1A). The patterns of change in Oct-1 mRNA and performed as previously described [8]. Oct-1 primers were de- protein levels, analyzed by real-time PCR and immunoblotting, signed using Methyl Primer Express software (Applied Biosystems). respectively, were similar to the pattern as promoter activity in Oct-1-overexpressing cells (Fig. 1B and C). We performed addi- 2.4. Statistical analysis tional immunoblot analyses and assay for catalase activity in shRNA-Oct-1-transfected cells to confirm the activator effect of Data, represented as bar graphs, are expressed as the mean and Oct-1 and found that both catalase protein levels and catalase standard deviation of 3 independent experiments. All results in the activity were decreased in Huh7 cells treated with Oct-1 shRNA bar graphs are expressed as the fold change relative to untreated or (Fig. 1D). control cells. 3.2. Oct-1 binds to the catalase promoter

3. Results Previous studies have shown that Oct-1 binds to octamer motif- related sequences [17]. We used a ChIP assay to determine 3.1. Transcription factor Oct-1 acts as an activator of catalase whether Oct-1 protein binds to the catalase promoter. The data re- vealed that Oct-1 binds to the catalase promoter (Fig. 2A). To fur- To analyze the effect of transcription factor Oct-1 on catalase ther clarify the binding of Oct-1 to the sequences (50-ATTAAATA- expression, Hep3B and Huh7 cells were transfected with HA-Oct- 30) located at -583 -576 within the catalase promoter, we also 1 and a dual luciferase assay was carried out. In both Hep3B and performed EMSA (Fig. 2B). Oct-1 protein shifted the biotin-labeled

Fig. 3. Reactive oxygen species downregulate Oct-1 expression in hepatocellular carcinoma (HCC) cell lines. (A) Huh7 and Hep3B cells were treated with 300 lMH2O2,20lM menadione and 2 lM PMS (phenazine methosulfate) for 2 and 4 days, after which Oct-1 expression was assessed by immunoblot analysis. Catalase and b-actin were used as controls. SM, DNA size marker; Mena, menadione. (B) Huh7 and Hep3B cells were treated as described in (A). Oct-1 expression was assessed by real-time RT-PCR. (C) Bisulfite-modified DNA was amplified with sodium bisulfite genomic sequencing (BS) primers. h, unmethylated CpG site; j, methylated CpG site. (D) Huh7 and Hep3B cells were treated as described in (A) and methylation-specific PCR (MSP) was performed. SM, DNA size marker; U, unmethylated DNA; M, methylated DNA. (E) Huh7 cells were treated with 300 lMH2O2 for 4 days and then incubated for a further 4 days following withdrawal of H2O2. MSP was performed on genomic DNA isolated from these cells. And Oct-1 expression was assessed by real-time RT-PCR and immunoblotting. X. Quan et al. / FEBS Letters 585 (2011) 3436–3441 3439

0 Fig. 4. N-Acetylcysteine and 5-aza-2 -deoxycytidine restore Oct-1 expression in ROS-treated HCC cell lines. (A) Huh7 and Hep3B cells were treated with 300 lMH2O2,20lM menadione and 2 lM PMS and/or 5 mM N-acetylcysteine and 2 lM 5-aza-dC for 4 days, after which MSP was performed on genomic DNA isolated from these cells. SM, DNA size marker; U, unmethylated DNA; M, methylated DNA; Mena, menadione. (B) Oct-1 expression was assessed by real-time RT-PCR. (C) Oct-1 expression was assessed by immunoblotting. oligonucleotides containing the octamer motif-related sequences HCT116 (Colorectal carcinoma cell line) cells (Supplementary (Fig. 2B, Lane 1) compared to the control (Fig. 2B, Lane 5) and the Fig. S2), as analyzed by immunoblotting and real-time RT-PCR, specificity of Oct-1 binding was confirmed by competitive inhibi- respectively. To address the possibility of methylation of Oct-1 tion with a 200-fold excess of unlabeled oligonucleotides (Fig. 2B, promoter, we treated the cells with ROS sources and analyzed Lane 4). We further confirmed that it was the Oct-1 protein in the the Oct-1 promoter using BS (bisulfite sequencing) and MSP shifted complex by performing a supershift with an antibody spe- (methylation specific PCR) (Fig. 3C and D). Methylation of the cific to Oct-1 using the nuclear extracts (Fig. 2B, Lane 2), or mouse Oct-1 CpG island was observed 4 days after treatment with ROS, normal IgG, as a control (Fig. 2B, Lane 3). In addition, we performed as determined by BS and MSP. These data showed that prolonged a dual luciferase assay using a catalase promoter with a point ROS exposure results in the methylation of the CpG island in the mutation (50-ATTAAATA-30 ? 50-ATTACATA-30) within the Oct-1 Oct-1 promoter and simultaneously downregulates Oct-1 expres- binding site. Compared to the control, decreased promoter activity sion in HCC cell lines. was observed with this mutated promoter, even in Oct-1-over- To determine whether Oct-1 downregulation by ROS was expressing cells (Fig. 2C). reversible, we treated Huh7 cells with H2O2 for 4 days, then re- placed the media with fresh media lacking H2O2, and thereafter 3.3. ROS downregulate Oct-1 expression and this downregulation is incubated the cells for an additional 4 days. MSP analysis was per- reversible in HCC cells formed to determine the methylation status of the CpG island in the Oct-1 promoter. The results showed that the CpG island was ROS downregulate catalase expression via hypermethylation of demethylated and that Oct-1 mRNA levels and protein expression a CpG island in its promoter [8] and Oct-1-deficient cells are hyper- recovered after the removal of ROS (Fig. 3E). These data indicate sensitive to hydrogen peroxide [20]. To determine the effects of that ROS-induced methylation of the Oct-1 promoter and that prolonged exposure to ROS on Oct-1, we treated HCC cell lines with downregulation of Oct-1 expression is reversible in HCC cell lines.

ROS sources (H2O2 and menadione) for 4 days. In H2O2 treated cells, cell proliferation decreased, while the number of apoptotic 3.4. N-Acetylcysteine and 5-aza-dC restore Oct-1 expression cells slightly increased (Supplementary Fig. S1). Treatment of Hep3B and Huh7 cells with ROS sources resulted in reduction of ROS-induced methylation of the Oct-1 promoter was inhibited Oct-1 protein (Fig. 3A) and messenger RNA (mRNA) (Fig. 3B), and following the treatment with N-acetylcysteine, an antioxidant, or so did in Hela (Human epithelial carcinoma cell line) and with 5-aza-dC, a DNA methyltransferase 1 (DNMT1) inhibitor 3440 X. Quan et al. / FEBS Letters 585 (2011) 3436–3441

Fig. 5. Expression of Oct-1 can inhibit invasiveness induced by ROS. (A) Oct-1 overexpressing and control Huh7 and Hep3B cells were tretaed with 300 lMH2O2 for 4 days.

Oct-1 and catalase expression were assessed by immunoblotting. b-Actin served as an internal control. C, non-treated control cells; H2O2,H2O2 treated control cells; Oct-1,

Oct-1 overexpressing cells treated with H2O2. (B) Oct-1 overexpressing and control Huh7 and Hep3B cells were treated with 300 lMH2O2 for 4 days. A matrigel invasion assay was performed as described in the Materials and Methods. C, non-treated control cells; H2O2,H2O2 treated control cells; Oct-1, Oct-1 overexpressing cells treated with

H2O2.

(Fig. 4A). Concomitantly, Oct-1 mRNA (Fig. 4B) and protein (Fig. 4C) sure to ROS, the expression of catalase shows the same pattern of were restored in these cells. And the results showed that Oct-1 change as Oct-1 expression (Supplementary Fig. S3). Our data, Oct- expression in the 5-aza-dC and ROS treated cells showed approxi- 1 binds to the catalase promoter to activate the expression of cat- mately 75% recovery of 5-aza-dC treated cells compared to the alase, and data from other studies, suggest another mechanism for control and ROS treated cells, which means approximately 75% of the regulation of catalase expression in HCC. Oct-1 reduction is caused by ROS-induced promoter methylation. Oct-1 decreased after exposure to ROS for 24 h, which is consis- These results indicate a correlation between ROS and downregula- tent with the change in mRNA [24]. However, to our knowledge, tion of Oct-1 and methylation of the Oct-1 promoter. thus far, no study has addressed the epigenetic regulation of the Oct-1 promoter by ROS. In this study, we show that prolonged 3.5. Expression of Oct-1 can inhibit invasiveness induced by ROS exposure to ROS decreases Oct-1 expression, due to methylation of the Oct-1 promoter. On the basis of the correlation between Although ROS increase invasiveness of the cancer cells, this pro- the expression of Oct-1 and catalase, our findings imply that ROS cess can be inhibited by the expression of catalase [23]. In order to regulates catalase not only through a direct mechanism, but also study the effect of Oct-1 on cell invasiveness as Oct-1 activates cat- through an indirect one—downregulation of the transcriptional alase expression, we performed the invasion assay using Oct-1 activator Oct-1. This would further weaken the ability of the cells overexpressing Huh7 and Hep3B, which were transfected with to defend against ROS and increases their sensitivity to ROS. Mean- pRC/CMV-HA-Oct-1 and selected by G418. First, we confirmed that while, Oct-1 expression inhibits invasiveness induced by ROS, thus, Oct-1 overexpressing cells showed increase in both Oct-1 and cat- Oct-1 dowenregulation by ROS can increase the cancer cells alase expression compared to the controls (Fig. 5A). Despite the re- invasion. sults of invasion assay demonstrated the increase in cell invasion In this study, we identified a new role of ROS: triggering DNA by H2O2, Oct-1 expression inhibited the invasiveness (Fig. 5B). methylation of transcription factor Oct-1. Although our investiga- These data indicate that the inhibition of invasiveness of the cancer tion did not uncover a specific functional pathway by which ROS cells induced by ROS might be resulted from catalase upregulation induce CpG island methylation in the Oct-1 promoter, our results caused by Oct-1 expression. strongly suggest that ROS regulate the methylation status of the Oct-1 promoter in HCC cells. Understanding the roles of ROS, 4. Discussion Oct-1, and catalase could lead to new therapeutic approaches for targeting HCC. The studies we have described here provide novel insights into the role of ROS and Oct-1 in HCC cell lines. First, Oct-1 binds to the Funding catalase promoter and acts as a transcriptional activator of the cat- alase gene. Second, ROS downregulate Oct-1 expression via meth- This study was supported by the Korea Healthcare Technology ylation of a CpG island in the Oct-1 promoter. These results suggest Research and Development Project, Ministry of Health and Wel- that prolonged ROS exposure induces methylation of the Oct-1 fare, Republic of Korea (A101530). promoter to downregulate Oct-1, which contributes to the down- regulation of catalase expression. Acknowledgments Transcription factor Oct-1 is a stress sensor [20], and DNA dam- age induces Oct-1 through a posttranscriptional mechanism and The Overseas Koreans Foundation was awarded to Xiaoyuan increases Oct-1 DNA binding affinity [24]. Under short-term expo- Quan. X. Quan et al. / FEBS Letters 585 (2011) 3436–3441 3441

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