CoCl₂ Decreases EC-SOD Expression through Histone Deacetylation in COS7 Cells

Abstract

Extracellular-superoxide dismutase (EC-SOD), one of the SOD isozymes, is negatively regulated under hypoxic conditions, and decreases in its expression may exacerbate vascular diseases. Moreover, epigenetics, such as DNA methylation and histone modifications, are known to play a critical role in the progression of cancer, type 2 diabetes, and atherosclerosis. We previously investigated the involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK) in decreases in EC-SOD expression in hypoxic COS7 cells; however, the role of epigenetics in this process currently remains unknown. In the present study, we demonstrated that the hypoxia mimetic cobalt chloride (CoCl₂) decreased histone acetylation levels, and a pretreatment with 4-phenyl butyric acid (PBA), an inhibitor of histone deacetylase, significantly suppressed CoCl₂-elicited histone deacetylation and decreases in EC-SOD. We found that CoCl₂-elicited decreases in EC-SOD were accompanied by reductions in histone H3 acetylation levels within its promoter region. Furthermore, luteolin, a well-known flavonoid, significantly suppressed the CoCl₂-elicited accumulation of ROS, p38-MAPK activation, and histone deacetylation. Collectively, the results of the present study showed for the first time that CoCl₂ decreases the expression of EC-SOD through its deacetylation and luteolin may be one of the seed compounds that maintain redox homeostasis, even under hypoxic conditions.

Reactive oxygen species (ROS) are known to participate in the progression of several kinds of diseases including atherosclerosis, diabetes, and cancer.^1–3) Anti-oxidative enzymes such as superoxide dismutase (SOD) and catalase protect cells and tissues from the damaging effects of ROS.^4,5) Extracellular-SOD (EC-SOD) is one of the SOD isozymes and plays a pivotal role in the regulation of redox homeostasis, particularly in extracellular spaces.^6–8) A previous study reported that the knockdown of EC-SOD exacerbated the progression of atherogenesis through the accumulation of foam cells in the neointima region⁹⁾; on the other hand, the overexpression of EC-SOD has been shown to suppress atherogenesis.¹⁰⁾ Therefore, maintaining the expression of EC-SOD at high levels in vessel walls may prevent the initiation and progression of vascular diseases.

Chronic kidney disease (CKD), defined as kidney injury lasting more than 3 months, is a global public health issue.^11,12) During the progression of CKD, kidney cells are exposed to hypoxic stress due to tissue fibrosis, and this stress exacerbates tubulointerstitial injury.¹³⁾ We previously demonstrated that the expression of EC-SOD is negatively regulated through intracellular ROS signaling in tubular-epithelial COS7 cells.¹⁴⁾ Recent studies reported that epigenetics, such as DNA methylation and histone modifications, play pivotal roles in the progression of CKD, and some inhibitors of histone deacetylase (HDAC) exert beneficial effects against CKD.^15–17) Moreover, the expression of EC-SOD is known to be regulated by epigenetics in human lung cancer cells,^18,19) monocytes/macrophages,²⁰⁾ and retina endothelial cells²¹⁾; however, limited information is currently available on EC-SOD regulation mechanisms in CKD models and the involvement of epigenetics.

Some flavonoids including luteolin and quercetin exert biological effects, such as anti-oxidative and anti-inflammatory activities.^22–24) We previously reported that luteolin significantly suppressed phorbol ester (TPA)-induced human monocytic differentiation into macrophages, and these events were closely associated with the inhibition of intracellular ROS signaling.²⁵⁾ Accordingly, some flavonoids may be seed compounds that maintain redox homeostasis.

In the present study, we demonstrated the involvement of epigenetics in cobalt chloride (CoCl₂)-elicited decreases in EC-SOD, and found that a treatment with CoCl₂ reduced the acetylated levels of histone H3 and H4 through intracellular ROS-p38-mitogen-activated protein kinase (MAPK) signaling. Moreover, luteolin significantly ameliorated CoCl₂-elicited decreases in EC-SOD by inhibiting ROS-p38-MAPK signaling and histone deacetylation. Collectively, these results provide a novel insight into the involvement of epigenetics in the regulation of EC-SOD in the kidney, and the moderate uptake of luteolin may contribute to maintaining good redox homeostasis.

MATERIALS AND METHODS

Flavonoids

Luteolin (Lut) (>98.0%) and chrysin (Chr) (>98.0%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and CosmoBio Co., Ltd. (Tokyo, Japan), respectively. Apigenin (Ap), diosmetin (Di), chrysoeriol (Chl), 3′,4′-dimethoxy luteolin (Dlu), and tricetin (Tr) were synthesized using previously described methods.²⁶⁾ Their purities were assessed as >95.0% using analytical HPLC. After filtration of flavone solutions (1 mg/mL in MeOH) through 0.20 mm membrane filter (ADVANTEC^®, Japan), samples were analyzed by HPLC (Shimadzu Corp., Japan). Analysis was carried out using a TSKgel ODS-100V analytical column (5 mm, 4.6×250 mm). The isocratic mobile phase employed was 1% AcOH aq./MeCN=1/1. The column eluate was monitored at 330 nm UV absorbance (shown in Supplementary Materials).

Cell Culture

COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37°C in a humidified 5% CO₂ incubator. Culture medium was replaced every 2 d. After 90% confluence, culture medium was replaced with DMEM containing 0.5% calf serum (CS), cultured overnight, and then subjected to experiments.

Western Blotting

COS7 cells (seeded at 1.2×10⁵ cells/35-mm dishes) were treated with CoCl₂ in the presence or absence of 4-phenyl butyric acid (PBA), luteolin, or SB203580 (an inhibitor of p38-MAPK) for the indicated time. After this treatment, histone proteins were collected and purified by the method described in our previous study.²⁰⁾ In order to assess phospho- or total p38-MAPK levels, COS7 cells (seeded at 3.6×10⁵ cells/60-mm dishes) were treated with CoCl₂ in the presence of luteolin for 1 h. Whole cell lysates were then prepared by the method described in our previous study.²⁷⁾ An equal amount of histone (assessed by Coomassie staining) or a whole cell lysate (20 µg) was separated by polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE, respectively. After transferring electrophoretically onto polyvinylidene difluoride (PVDF) membranes, the membranes were incubated with anti-acetyl-histone H3 (06-599, Millipore: 1 : 2000), anti-acetyl-histone H4 (06-598, Millipore: 1 : 2000), or anti-phospho-p38 MAPK (#9215, Cell Signaling: 1 : 1000) overnight. Blots were incubated with a biotin-conjugated goat anti-rabbit or anti-mouse antibody (1 : 1000) followed by ABC reagents (Vector Laboratories, Inc.: 1 : 5000). Bands were detected using SuperSignal West Pico (Thermo Scientific, U.S.A.) or ImmunoStar LD (Wako Pure Chemical Industries, Ltd.) and imaged using an LAS-3000 UV mini apparatus (FUJIFILM, Tokyo, Japan).

Reverse Transcriptional (RT)-PCR Analysis

After cells had been treated, they were lysed in 1 mL TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). cDNA was prepared, and RT-PCR was performed by the methods described in our previous study²⁷⁾ with minor modifications. The primer sequences used were as follows: EC-SOD, sense 5′-AGA AAG CTC TCT TGG AGG AG-3′; antisense 5′-ACC GCG AAG TTG CCG AAG TC-3′: glucose-regulated protein 78 kDa (GRP78), sense 5′-TTT CTG CAT GGT TCT CAC T-3′; antisense 5′-CCC AGA TGA GTA TCT CCA TT-3′: 18S ribosomal RNA (rRNA), sense 5′-CGG CTA CCA CAT CCA AGG AA-3′; antisense 5′-GCT GGA ATT ACC GCG GCT-3′. After amplification, PCR products were loaded onto 2% (w/v) agarose gels for electrophoresis, and a densitometric analysis of PCR products was performed with Multi Gauge V3.0.

Chromatin Immunoprecipitation Assay (ChIP)

ChIP assays were performed as described in our previous study.²⁰⁾ The abundance of EC-SOD promoter regions in ChIP precipitates was quantified using a PCR analysis. The primer sequences for EC-SOD were sense 5′-GTG GAG GCG AAG CAA TTC TA-3′; antisense 5′-CTG TTA GCG CGA GTG CAG GA-3′. After amplification, these PCR products were loaded onto a 1.2% (w/v) agarose gel for electrophoresis and visualized using FLA5100, and a densitometric analysis of the PCR products was then performed with Multi Gauge V3.0.

Measurement of ROS Accumulation

COS7 cells (seeded at 1.2×10⁵ cells/35-mm dishes) were treated with CoCl₂ for 1 h in the presence of luteolin, washed with PBS twice, and incubated with PBS containing 10 µM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) for 30 min in a CO₂ incubator. After cells had been washed with PBS three times, they were visualized under an HS All-In-One fluorescence microscope BZ-9000 (KEYENCE, Osaka, Japan).

Statistical Analysis

Data are expressed as the mean±standard deviation (S.D.) of three independent experiments. A statistical analysis of data was performed using ANOVA followed by post hoc Bonferroni tests. A p value less than 0.05 was considered significant.

RESULTS

Involvement of Histone Deacetylation in CoCl₂-Elicited Decreases in EC-SOD

We previously reported that hypoxia (1% O₂) and the hypoxia mimetic, CoCl₂ decreased the expression of EC-SOD in COS7 cells.¹⁴⁾ Moreover, EC-SOD expression is known to be regulated by epigenetics such as DNA methylation and histone modifications, particularly by histone deacetylation in human lung cancer,²⁸⁾ human endothelial cells,²¹⁾ and monocytes/macrophages.²⁰⁾ Accordingly, decreases in EC-SOD elicited by an exposure to CoCl₂ may be regulated by epigenetics. In order to elucidate the involvement of epigenetics in CoCl₂-elicited decreases in EC-SOD, histone acetylation levels were measured after the exposure to CoCl₂. As shown in Fig. 1A, treatment with CoCl₂ decreased the levels of acetylated histone H3 and H4 in a dose-dependent manner. These data are consistent with our previous finding, in which CoCl₂ did not decrease EC-SOD expression at 100 µM CoCl₂.¹⁴⁾ HDAC has been shown to play a pivotal role in the regulation of gene expression through chromatin remodeling.^29–31) As expected, the pretreatment with PBA, an inhibitor of HDAC, significantly suppressed CoCl₂-elicited decreases in EC-SOD and was accompanied by the inhibition of histone deacetylation (Figs. 1B, C). On the other hand, PBA has the potential to inhibit endoplasmic reticulum (ER) stress, and ER stress has been shown to regulate the expression of EC-SOD in COS7 cells and 3T3-L1 adipocytes.^32,33) Our results demonstrated that CoCl₂ induced GRP78, an ER stress marker³⁴⁾; however, PBA did not affect its induction (Fig. 1C). These results suggest that CoCl₂-elicited decreases in EC-SOD were regulated in an ER stress-derived signal-independent manner. We also found decreases in acetylated histone enrichment within the proximal promoter region of EC-SOD following the exposure to CoCl₂ (Fig. 1D), suggesting that CoCl₂ decreases EC-SOD through histone deacetylation within its promoter region.

Fig. 1. Involvement of Histone Deacetylation in CoCl₂-Elicited Decreases in EC-SOD in COS7 Cells

(A) COS7 cells were treated with the indicated concentrations of CoCl₂ for 24 h. (B, C) COS7 cells were pretreated with (+) or without (−) 1 mM PBA for 1 h, and then treated with (+) or without (−) 300 µM CoCl₂ for 24 h. (D) The cells were treated with 300 µM CoCl₂ for 24 h. After cells had been treated, Western blotting (A, B), RT-PCR (C), and a ChIP assay (D) were performed. (* p<0.05, ** p<0.01 vs. vehicle, ^# p<0.05 vs. CoCl₂-treated cells.)

Involvement of p38-MAPK in CoCl₂-Elicited Histone Deacetylation

MAPK including p38-MAPK, extracellular-regulated kinase (ERK), and c-jun N-terminal kinase (JNK) play pivotal roles in a large number of physiological processes such as apoptosis, proliferation, and differentiation.^35,36) Moreover, we previously reported that p38-MAPK is involved in the regulation of EC-SOD in COS7 cells under hypoxic conditions.¹⁴⁾ Therefore, we investigated the involvement of p38-MAPK in CoCl₂-elicited histone deacetylation. The pretreatment with SB203580, an inhibitor of p38-MAPK, suppressed CoCl₂-elicited histone H3 and H4 deacetylation (Fig. 2), suggesting that histone deacetylation is mediated by p38-MAPK-related signaling in COS7 cells.

Fig. 2. Involvement of p38-MAPK in CoCl₂-Elicited Histone Deacetylation in COS7 Cells

COS7 cells were pretreated with (+) or without (−) 1 µM SB203580 for 30 min, and then treated with (+) or without (−) 300 µM CoCl₂ for 24 h. After cells had been treated, Western blotting was performed.

Effects of Flavonoids on CoCl₂-Elicited Decreases in EC-SOD

Previous studies reported that some flavonoids including luteolin exhibit potent anti-oxidative activities, and may be seed compounds against cancer and atherosclerosis.^22–24) In this study, we attempted to clarify whether some flavonoids have the ability to suppress CoCl₂-elicited decreases in EC-SOD. Among the 7 flavonoids examined in the present study (Fig. 3A), chrysin and luteolin significantly suppressed CoCl₂-elicited decreases in EC-SOD (Fig. 3B), and this occurred in a dose-dependent manner (data not shown). Based on these results, we further investigated the inhibitory mechanism of luteolin on decreases in EC-SOD in CoCl₂-treated COS7 cells. Based on our previous findings, in which we showed the involvement of intracellular ROS-derived signaling in CoCl₂-elicited decreases in EC-SOD,¹⁴⁾ we investigated the effects of luteolin on the above process. As expected, luteolin suppressed the CoCl₂-elicited intracellular accumulation of ROS (Fig. 4A) and activation of p38-MAPK (Fig. 4B). Moreover, the pretreatment with luteolin significantly suppressed CoCl₂-elicited histone deacetylation (Fig. 4C), suggesting that CoCl₂ decreases the expression of EC-SOD through intracellular ROS, p38-MAPK activation, and the histone deacetylation axis.

Fig. 3. Effects of Flavonoids on CoCl₂-Elicited Decreases in EC-SOD in COS7 Cells

(A) The structure of flavonoids used in this study. (B) COS7 cells were pretreated with 20 µM of the flavonoids being tested: chrysin (Chn), apigenin (Ap), luteolin (Lut), diosmetin (Di), chrysoeriol (Chl), 3′,4′-dimethoxy luteolin (Dlu), and tricetin (Tri), for 1 h, and then treated with 300 µM CoCl₂ for 24 h. RT-PCR was subsequently performed. (** p<0.01 vs. vehicle, ^# p<0.05, ^## p<0.01 vs. CoCl₂-treated cells.)

Fig. 4. Luteolin Suppresses CoCl₂-Elicited Decreases in EC-SOD by Inhibiting ROS-p38-MAPK Signaling in COS7 Cells

COS7 cells were pretreated with (+) or without (−) 20 µM luteolin for 1 h, and then treated with (+) or without (−) 300 µM CoCl₂ for 1 h (A, B) or 24 h (C). Measurements of intracellular ROS accumulation (A) and Western blotting (B, C) were then performed. Scale bar shows 50 µm. 

DISCUSSION

Epigenetics, such as DNA methylation and histone modifications, play important roles in cell differentiation and cellular development.^29,37,38) DNA methylation, which is mediated by DNA methyltransferase (DNMT) with S-adenosyl-methionine as a methyl donor, has been shown to regulate gene expression and genomic imprinting.^39–41) Recent studies revealed that global DNA hypomethylation and hypermethylation are both involved in several physiological processes, including tumorigenesis and CKD.^42–44) Based on a vast amount of findings, genes, which may be regulated by DNA methylation, are known to possess CpG islands within proximal promoter regions. We previously reported that the expression of EC-SOD in basal human monocytic THP-1 cells was silenced, and attributed this to CpG hypermethylation within its promoter region.²⁰⁾ As noted above, DNA methylation processes may be associated with CoCl₂-elicited decreases in EC-SOD in COS7 cells; however, a treatment with 5-azacytidine, an inhibitor of DNMT, did not suppress these decreases (data not shown). These results indicate that CoCl₂-elicited decreases in EC-SOD are regulated in a DNA methylation-independent manner.

Histone modifications in the N-terminal tail, including acetylation, methylation, and phosphorylation, play critical roles in gene regulation as well as DNA methylation. Histone acetylation at lysine residues is closely related to gene induction, and these phenomena are regulated by histone acetyltransferase (HAT) and HDAC, two opposing groups of enzymes involved in chromatin remodeling.^31,45,46) Histone acetylation neutralizes the positive charge and facilitates the binding of transcription factors to nucleosomal DNA, thereby inducing its transcription. Acetylation-dependent gene induction has been shown to play a pivotal role in the progression of cancer invasion, metastasis, and kidney fibrosis.^47–49) We previously reported that the excessive production of ROS induced epithelial-mesenchymal transition (EMT), which is considered to be critically involved in cancer metastasis and fibrosis and is mediated by histone acetylation in the human breast cancer cell line MCF-7.²⁷⁾ Therefore, obtaining a deeper understanding of the role of ROS and regulation of redox homeostasis may lead to novel CKD therapies. In the present study, we examined the involvement of histone H3 deacetylation in CoCl₂-elicited decreased in EC-SOD, which is closely associated with ROS-p38-MAPK signaling. It has been reported that p38-MAPK induces serine phosphorylation of p300.⁵⁰⁾ Phosphorylated-p300 will be targeted for degradation by the proteasome, leading to the suppression of p300-dependent transcription. Moreover, it is well known that suppressed p300 activity is correlated with the activation of HDAC; therefore, we conclude that p38-MAPK-mediated histone de/acetylation plays a pivotal role in the regulation of EC-SOD.

Flavonoids commonly found in fruits, vegetables, and tea have beneficial effects including anti-oxidative properties. Luteolin functions as an inhibitor of TPA-induced monocytic differentiation²⁵⁾ and oxidized low-density lipoprotein-elicited EC-SOD decreases in human vascular smooth cells.⁵¹⁾ In the present study, we tested whether some flavonoids suppress CoCl₂-elicited decreases in EC-SOD, and found that luteolin exerted a potent inhibitory effect against this phenomenon. These results are consistent with our previous findings, in which luteolin exhibited potent radical scavenger activity and significantly suppressed monocytic differentiation into macrophages.²⁵⁾ These results indicate that luteolin maintains good redox homeostasis, particularly in extracellular spaces.

In the present study, we identified a significant role for histone de/acetylation in the regulation of EC-SOD in COS7 cells. However, unfortunately, we could not determine a critical role of histone de/acetylation in the regulation of EC-SOD under hypoxic condition (1% O₂, data not shown). Accordingly, we consider that additional studies will be necessary to determine the exact molecular mechanisms governing the regulation of EC-SOD under hypoxia. On the other hand, we demonstrated that the knockdown of EC-SOD increased sensitivity against ROS and induced cell death in human fibroblasts (data not shown); therefore, our results provide evidence for luteolin contributing to the maintenance of redox homeostasis.

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion for Science (T.K., No. 26460070) and a Grant for the encouragement of young scientists from Gifu Pharmaceutical University (T.K.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Cave AC, Brewer AC, Narayanapanicker A, Ray R, Grieve DJ, Walker S, Shah AM. NADPH oxidases in cardiovascular health and disease. Antioxid. Redox Signal., 8, 691–728 (2006).
• 2) Vachier I, Chanez P, Le Doucen C, Damon M, Descomps B, Godard P. Enhancement of reactive oxygen species formation in stable and unstable asthmatic patients. Eur. Respir. J., 7, 1585–1592 (1994).
• 3) Wright E Jr, Scism-Bacon JL, Glass LC. Oxidative stress in type 2 diabetes: the role of fasting and postprandial glycaemia. Int. J. Clin. Pract., 60, 308–314 (2006).
• 4) Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stress in obesity and its impact on metabolic syndrome. J. Clin. Invest., 114, 1752–1761 (2004).
• 5) Roberts CK, Barnard RJ, Sindhu RK, Jurczak M, Ehdaie A, Vaziri ND. Oxidative stress and dysregulation of NAD(P)H oxidase and antioxidant enzymes in diet-induced metabolic syndrome. Metabolism, 55, 928–934 (2006).
• 6) Adachi T, Marklund SL. Interactions between human extracellular superoxide dismutase C and sulfated polysaccharides. J. Biol. Chem., 264, 8537–8541 (1989).
• 7) Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. U.S.A., 79, 7634–7638 (1982).
• 8) Ookawara T, Imazeki N, Matsubara O, Kizaki T, Oh-Ishi S, Nakao C, Sato Y, Ohno H. Tissue distribution of immunoreactive mouse extracellular superoxide dismutase. Am. J. Physiol., 275, C840–C847 (1998).
• 9) Lu Z, Xu X, Hu X, Zhu G, Zhang P, van Deel ED, French JP, Fassett JT, Oury TD, Bache RJ, Chen Y. Extracellular superoxide dismutase deficiency exacerbates pressure overload-induced left ventricular hypertrophy and dysfunction. Hypertension, 51, 19–25 (2008).
• 10) Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, French BA. Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation, 103, 1893–1898 (2001).
• 11) Bohle A, Wehrmann M, Bogenschütz O, Batz C, Vog W, Schmitt H, Müller CA, Müller GA. The long-term prognosis of the primary glomerulonephritides. A morphological and clinical analysis of 1747 cases. Pathol. Res. Pract., 188, 908–924 (1992).
• 12) Lee PJ, Jiang BH, Chin BY, Iyer NV, Alam J, Semenza GL, Choi AM. Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J. Biol. Chem., 272, 5375–5381 (1997).
• 13) Mimura I, Nangaku M. The suffocating kidney: tubulointerstitial hypoxia in end-stage renal disease. Nat. Rev. Nephrol., 6, 667–678 (2010).
• 14) Kamiya T, Hara H, Yamada H, Imai H, Inagaki N, Adachi T. Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells. Free Radic. Res., 42, 949–956 (2008).
• 15) Huang J, Wan D, Li J, Chen H, Huang K, Zheng L. Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury. Epigenetics, 10, 62–71 (2015).
• 16) Smyth LJ, McKay GJ, Maxwell AP, McKnight AJ. DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease. Epigenetics, 9, 366–376 (2014).
• 17) Van Beneden K, Geers C, Pauwels M, Mannaerts I, Verbeelen D, van Grunsven LA, Van den Branden C. Valproic acid attenuates proteinuria and kidney injury. J. Am. Soc. Nephrol., 22, 1863–1875 (2011).
• 18) Zelko IN, Mueller MR, Folz RJ. CpG methylation attenuates Sp1 and Sp3 binding to the human extracellular superoxide dismutase promoter and regulates its cell-specific expression. Free Radic. Biol. Med., 48, 895–904 (2010).
• 19) Zelko IN, Stepp MW, Vorst AL, Folz RJ. Histone acetylation regulates the cell-specific and interferon-gamma-inducible expression of extracellular superoxide dismutase in human pulmonary arteries. Am. J. Respir. Cell Mol. Biol., 45, 953–961 (2011).
• 20) Kamiya T, Machiura M, Makino J, Hara H, Hozumi I, Adachi T. Epigenetic regulation of extracellular-superoxide dismutase in human monocytes. Free Radic. Biol. Med., 61, 197–205 (2013).
• 21) Yamamoto T, Sakaguchi N, Hachiya M, Nakayama F, Yamakawa M, Akashi M. Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger. Leukemia, 23, 761–769 (2009).
• 22) Seelinger G, Merfort I, Schempp CM. Anti-oxidant, anti-inflammatory and anti-allergic activities of luteolin. Planta Med., 74, 1667–1677 (2008).
• 23) Yi L, Chen CY, Jin X, Zhang T, Zhou Y, Zhang QY, Zhu JD, Mi MT. Differential suppression of intracellular reactive oxygen species-mediated signaling pathway in vascular endothelial cells by several subclasses of flavonoids. Biochimie, 94, 2035–2044 (2012).
• 24) Zhang X, Wang G, Gurley EC, Zhou H. Flavonoid apigenin inhibits lipopolysaccharide-induced inflammatory response through multiple mechanisms in macrophages. PLoS ONE, 9, e107072 (2014).
• 25) Makino J, Nakanishi R, Kamiya T, Hara H, Ninomiya M, Koketsu M, Adachi T. Luteolin suppresses the differentiation of THP-1 cells through the Inhibition of NOX2 mRNA expression and the membrane translocation of p47^phox. J. Nat. Prod., 76, 1285–1290 (2013).
• 26) Ninomiya M, Tanaka K, Tsuchida Y, Muto Y, Koketsu M, Watanabe K. Increased bioavailability of tricin-amino acid derivatives via a prodrug approach. J. Med. Chem., 54, 1529–1536 (2011).
• 27) Kamiya T, Goto A, Kurokawa E, Hara H, Adachi T. Cross talk mechanism among EMT, ROS, and histone acetylation in phorbol ester-treated human breast cancer MCF-7 cells. Oxid. Med. Cell. Longev., 2016, 1284372 (2016).
• 28) Yasuda H, Mizukami K, Hayashi M, Kamiya T, Hara H, Adachi T. Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation. J. Clin. Biochem. Nutr., 58, 34–39 (2016).
• 29) Bernstein BE, Meissner A, Lander ES. The mammalian epigenome. Cell, 128, 669–681 (2007).
• 30) Esteller M. Epigenetics in cancer. N. Engl. J. Med., 358, 1148–1159 (2008).
• 31) Gao L, Cueto MA, Asselbergs F, Atadja P. Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family. J. Biol. Chem., 277, 25748–25755 (2002).
• 32) Kamiya T, Hara H, Adachi T. Effect of endoplasmic reticulum (ER) stress inducer thapsigargin on the expression of extracellular-superoxide dismutase in mouse 3T3-L1 adipocytes. J. Clin. Biochem. Nutr., 52, 101–105 (2013).
• 33) Kamiya T, Obara A, Hara H, Inagaki N, Adachi T. ER stress inducer, thapsigargin, decreases extracellular-superoxide dismutase through MEK/ERK signalling cascades in COS7 cells. Free Radic. Res., 45, 692–698 (2011).
• 34) Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell, 101, 451–454 (2000).
• 35) Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev., 81, 807–869 (2001).
• 36) Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev., 22, 153–183 (2001).
• 37) Kristensen LS, Nielsen HM, Hansen LL. Epigenetics and cancer treatment. Eur. J. Pharmacol., 625, 131–142 (2009).
• 38) Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature, 447, 425–432 (2007).
• 39) Delaval K, Feil R. Epigenetic regulation of mammalian genomic imprinting. Curr. Opin. Genet. Dev., 14, 188–195 (2004).
• 40) Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, Spector DL. Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell, 107, 727–738 (2001).
• 41) Holliday R. DNA methylation and epigenetic inheritance. Philos. Trans. R. Soc. Lond. B Biol. Sci., 326, 329–338 (1990).
• 42) Agirre X, Vizmanos JL, Calasanz MJ, Garcia-Delgado M, Larrayoz MJ, Novo FJ. Methylation of CpG dinucleotides and/or CCWGG motifs at the promoter of TP53 correlates with decreased gene expression in a subset of acute lymphoblastic leukemia patients. Oncogene, 22, 1070–1072 (2003).
• 43) Chmelarova M, Krepinska E, Spacek J, Laco J, Beranek M, Palicka V. Methylation in the p53 promoter in epithelial ovarian cancer. Clin. Transl. Oncol., 15, 160–163 (2013).
• 44) Kang JH, Kim SJ, Noh DY, Park IA, Choe KJ, Yoo OJ, Kang HS. Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma. Lab. Invest., 81, 573–579 (2001).
• 45) Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell, 87, 953–959 (1996).
• 46) Roth SY, Denu JM, Allis CD. Histone acetyltransferases. Annu. Rev. Biochem., 70, 81–120 (2001).
• 47) Albrengues J, Bertero T, Grasset E, Bonan S, Maiel M, Bourget I, Philippe C, Herraiz Serrano C, Benamar S, Croce O, Sanz-Moreno V, Meneguzzi G, Feral CC, Cristofari G, Gaggioli C. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat. Commun., 6, 10204 (2015).
• 48) Marumo T, Hishikawa K, Yoshikawa M, Hirahashi J, Kawachi S, Fujita T. Histone deacetylase modulates the proinflammatory and -fibrotic changes in tubulointerstitial injury. Am. J. Physiol. Renal Physiol., 298, F133–F141 (2010).
• 49) Yeh CM, Lin CW, Yang JS, Yang WE, Su SC, Yang SF. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation. Oncotarget, 7, 21952–21967 (2016).
• 50) Poizat C, Puri PL, Bai Y, Kedes L. Phosphorylation-dependent degradation of p300 by doxorubicin-activated p38 mitogen-activated protein kinase in cardiac cells. Mol. Cell. Biol., 25, 2673–2687 (2005).
• 51) Makino J, Nii M, Kamiya T, Hara H, Adachi T. Oxidized low-density lipoprotein accelerates the destabilization of extracellular-superoxide dismutase mRNA during foam cell formation. Arch. Biochem. Biophys., 575, 54–60 (2015).