Royal Jelly Constituents Increase the Expression of Extracellular

Article pubs.acs.org/jnp

Royal Jelly Constituents Increase the Expression of Extracellular Superoxide Dismutase through Histone Acetylation in Monocytic THP‑1 Cells Junya Makino,† Rie Ogasawara,† Tetsuro Kamiya,*,† Hirokazu Hara,† Yukari Mitsugi,‡ Eiji Yamaguchi,‡ Akichika Itoh,‡ and Tetsuo Adachi† †

Department of Biomedical Pharmaceutics, Laboratory of Clinical Pharmaceutics, and ‡Department of Organic and Medicinal Chemistry, Laboratory of Pharmaceutical Synthetic Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan

ABSTRACT: Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

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is secreted into the extracellular space and protects cells from the damaging effects of superoxide in blood vessel walls by binding to the heparan sulfate proteoglycan in the glycocalyx on cell surfaces.13−16 EC-SOD has also been detected in monocytes/macrophages, and its expression is regulated epigenetically by DNA methylation and histone acetylation.17,18 Previous studies revealed that the strong expression of ECSOD in vessel walls contributed to the suppression of cardiovascular injuries; therefore, natural compounds that induce the expression of EC-SOD in monocytes/macrophages may have potential as candidates or lead compounds against atherosclerosis. In the present study, we determined the effects on the expression of EC-SOD of royal jelly constituents such as 1, 2, 3, and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE,

oyal jelly includes some effective compounds such as 10hydroxydecanoic acid (10HDA, 1), 10-hydroxy-2-decenoic acid (10H2DA, 2), and sebacic acid (SA, 3) and has been shown to exert many pharmacological effects against oxidative stress and diabetes.1−3 A previous study reported that 2 has the potential to inhibit histone deacetylase (HDAC) activities.4 Epigenetic modifications are generally referred to as reversible chromatin rearrangements that modulate gene expression in normal cells without changing DNA sequences.5,6 Histone modifications in the N-terminal tail, such as methylation and acetylation at lysine and arginine residues, also change the chromatin structure and induce or suppress gene expression.7−9 Superoxide dismutase (SOD) plays a pivotal role against oxidative stress induced by superoxide in mammals, and its deficiency may lead to various diseases, such as asthma, atherosclerosis, and type 2 diabetes.10−12 Extracellular SOD (EC-SOD) is one of the main SOD isozymes in mammals that © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 21, 2016

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DOI: 10.1021/acs.jnatprod.6b00037 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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royal jelly constituents 1, 2, and 3 on SOD expression were investigated. The treatment with 1 mM 1, 2, or 3 did not affect Cu,Zn-SOD expression (Figure 1A, left). Furthermore, 2 and 3 increased Mn-SOD expression (Figure 1A, middle). On the other hand, EC-SOD mRNA and protein levels were significantly increased by treatment with 1, 2, or 3 (Figure 1A, right, and 1B). Trichostatin A (TSA), an HDAC inhibitor, increased both EC-SOD mRNA and protein levels. These results indicate that 1, 2, and 3 markedly increase the expression of EC-SOD mRNA and protein, but not that of the other SOD isozymes, through their inhibitory effects on HDAC activities. Mn-SOD is mainly expressed in mitochondria and effectively scavenges mitochondria-induced ROS. In addition, functions such as the β-oxidation of fatty acids and tricarboxylic acid (TCA) cycle are included in mitochondrial metabolic mechanisms;23,24 therefore, royal jelly constituents may be involved in the homeostasis of these mitochondrial functions, which may induce Mn-SOD expression. Involvement of Histone Acetylation in Royal Jelly Component-Induced EC-SOD Expression. Epigenetics may be defined as inherited modifications in gene expression that are not encoded in the DNA sequence itself.16 Epigenetic mechanisms include DNA methylation, histone modifications, and miRNA regulation. Histone modifications such as histone acetylation, methylation, and phosphorylation are known to be involved in epigenetic gene regulation.8,25 Histone acetylation at the ε-N-terminal has been shown to induce transcriptional activation, whereas histone lysine methylation causes transcriptional activation or repression depending on the position of the methylated lysine.26 We previously demonstrated that the expression of EC-SOD was regulated by histone H3 and H4 acetylation.27 Therefore, the involvement of histone acetylation was investigated. As shown in Figure 2A, the treatment with 1 or 3 increased histone H3 and H4 acetylation levels to the same extent as that with 2. A chromatin immunoprecipitation (ChIP)

4), which is a derivative of 2-decenoic acid and has a hydroperoxy group in the 4′ position. Compounds 1, 2, and 3 significantly increased the expression of EC-SOD and induced histone H4 acetylation in the EC-SOD promoter region through the inhibition of HDAC. On the other hand, 4 also increased EC-SOD expression and induced histone H3 and H4 acetylation in the EC-SOD promoter region through the inhibition of HDAC gene expression and HDAC activities. Our results confirmed that 4 activated ERK signaling, which played a significant role in the 4-elicited induction of EC-SOD expression. Furthermore, 4 increased the expression of ECSOD at a lower dose than 1, 2, or 3. Therefore, 4 has potential as a candidate or lead compound against atherosclerosis.

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RESULTS AND DISCUSSION Structures of Royal Jelly Constituents and Their Effects on SOD Expression. Royal jelly contains medium chain fatty acids (MCFAs) such as 10HDA (1), 10H2DA (2), and SA (3), which have an aliphatic tail of 10 carbon atoms and exhibit several biological activities including antiangiogenesis, insulin secretion, and lipoprotein metabolism.19−22 However, it currently remains unclear whether royal jelly constituents regulate antioxidative enzymes. Therefore, the effects of the

Figure 1. Expression of SOD isozymes by treatment with royal jelly constituents or TSA. (A and B) THP-1 cells were treated with 1 mM 10HDA (1), 10H2DA (2), or SA (3) or 1 μM TSA for 24 h. (A) Real-time RT-PCR was then performed. Real-time RT-PCR data were normalized using 18S rRNA levels (*p < 0.05, **p < 0.01 vs untreated cells). (B) The concentration of EC-SOD protein was determined by ELISA (*p < 0.05, **p < 0.01 vs untreated cells). B



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Figure 3. Involvement of HDACs in the induction of EC-SOD by royal jelly constituents. (A) THP-1 cells were treated with 1 mM 10HDA (1) or SA (3) for 24 h. After the cells had been treated, realtime RT-PCR was carried out. Real-time RT-PCR data were normalized using 18S rRNA levels. (B) Cells were treated with 1 mM 1, 2, or 3 for 1 h. After the cells had been treated, HDAC activities were measured (**p < 0.01 vs untreated cells).

Figure 2. Involvement of histone H3 and H4 acetylation in EC-SOD induction by the treatment with royal jelly constituents or TSA. (A) THP-1 cells were treated with 1 mM 10HDA (1), 10H2DA (2), or SA (3) or 1 μM TSA for 12 h. After cells had been treated, acetylated histone H3 and H4 were determined by Western blotting. The loading of histone was monitored by Coomassie staining. (B) Cells were treated with 1 mM 1 or 3 for 12 h. After the cells had been treated, a ChIP assay was performed. Relative binding to the promoter region was expressed as the percentage amount over input (%) (**p < 0.01 vs untreated cells; N.S., not significant).

CoII(tpp) {5,10,15,20-tetraphenyl-21H,23H-porphine cobalt(II)} (3.4 mg, 5.1 × 10−3 mmol, 0.5 mol %) were dissolved in 8 mL of 2-propanol−dichloromethane (1:1) in a 50 mL flask equipped with three-way stopcock. An oxygen balloon was attached to the flask through the three-way stopcock. After triethylsilane (0.18 mL, 1.1 mmol, 110 mol %) had been added to the solution, and the reaction mixture was stirred for 5 h. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel with n-hexane−ethyl acetate (20:1−10:1) to afford 4 as a colorless oil (22.2 mg, 10%) (Scheme 1). Effects of 4-HPO-DAEE on EC-SOD Expression. Compound 4 was synthesized from 2-decenoic acid by esterification and the addition of a hydroperoxy group at the 4′ position. The cytotoxic effects of 4 in THP-1 cells were investigated. As shown in Figure 4A, since 4 did not affect cell viability, a concentration of 100 μM was used in subsequent experiments. The effects of 4 on the expression of EC-SOD were then examined. The expression of EC-SOD was significantly increased at 100 μM 4, but not by 1 or 10 μM 4 (Figure 4B). Moreover, the expression of EC-SOD was increased in a time-dependent manner by 100 μM 4 (Figure 4B). Similar to the results of 1 and 3, EC-SOD protein level was also increased by 100 μM 4 (Figure 4C). The effects of 4 on histone acetylation were then determined. As shown in Figure 4D, 4 promoted the acetylation of histone H3 and H4, similar to the other royal jelly constituents tested. Moreover, a ChIP assay indicated that 4 significantly promoted the enrichment of acetylated histone H3 and H4 within the ECSOD promoter region (Figure 4E), indicating that 4 increased the expression of EC-SOD through the acetylation of histone H3 and H4. Moreover, ERK activation was also observed after the treatment with 4 (Figure 4F), and an ERK inhibitor partially suppressed the 4-elicited expression of EC-SOD (Figure 4G). This result suggests that ERK activation plays a

assay indicated that 1 and 3 increased EC-SOD expression through histone H4, but not H3 acetylation in the EC-SOD promoter (Figure 2B). The histone H3 tail has lysine residues at 4, 9, 27, and 36, and these were shown to be methylated by histone methyltransferase.28 Moreover, several co-repressors, such as the NuRD complex, bind to the histone H3 tail accompanied by HDAC 1 or 2 and repress gene expression.29 On the other hand, the histone H4 tail has only one methylated lysine residue; therefore, histone H4 has fewer processes than H3 to switch methylation into acetylation. Therefore, histone H4 is more easily acetylated. However, higher doses or longer treatment times with royal jelly constituents enhance the induction of histone H3 and H4 acetylation, thereby promoting the expression of EC-SOD. Inhibition of HDAC Activities by 1 or 3. HDACs allow histones to wrap DNA more tightly by removing acetyl groups from the ε-N-acetyl lysine amino acid on a histone and regulate the expression of several genes.30,31 Therefore, the expression and activities of HDACs were determined in THP-1 cells treated with 1 or 3. As shown in Figure 3A, the expression of HDAC classes I and II was not changed by 1 or 3. On the other hand, the treatment with 1 or 3 significantly decreased HDAC activities by 20%, which was similar to that by the treatment with 2 (Figure 3B). These results indicate that 1 and 3 increase the expression of EC-SOD by promoting histone H4 acetylation through the inhibition of HDAC activities. Synthesis of 4-HPO-DAEE (4). The known compound 4 was prepared using adaption of the procedure of Itoh and coworkers.32 trans,trans-2,4-Nonadienal (200 mg, 1.0 mmol) and C



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Scheme 1. Synthesis of 4-Hydroperoxy-2-decenoic Acid Ethyl Ester (4-HPO-DAEE, 4)

region of EC-SOD. Moreover, the expression of several HDAC mRNAs was up- or down-regulated by 4. This result suggests that some HDAC activities may be suppressed by decreases in the expression of HDACs such as HDAC 1, 2, 5, or 6. In THP1 cells, the expression of HDAC 1 and 2 was markedly stronger than that of the other HDACs. Therefore, changes in HDAC 1 or 2 may involve histone deacetylation. As expected, the inhibition of HDAC 1 by royal jelly constituents and 4 may affect histone acetylation and the expression of EC-SOD based on the results shown in Figure 5C. Furthermore, 4 exerted stronger effects than 1, 2, and 3 and may have other effects on the expression of EC-SOD because the hydroperoxy group structure may strongly bind to the amino acids composed of HDAC than royal jelly constituents and keep the inhibition of HDAC activities. In the present study, only 4 promoted ERK phosphorylation (Figure 4F). ERK phosphorylation was previously reported to be promoted by trans-2-decenoic acid ethyl ester (DAEE).45,46 Furthermore, the expression of ECSOD is known to be regulated by MEK/ERK signaling during monocytic differentiation.33,34 Thus, ERK phosphorylation may play a role in the 4-mediated induction of EC-SOD expression. The results of the present study revealed the involvement of ERK phosphorylation in the expression of EC-SOD. Thus, 4 may use another pathway apart from the inhibition of HDAC 1 activity. The results of the present study demonstrated that the increases observed in EC-SOD expression may play a role in the maintenance of redox homeostasis in the vascular system. The effects of 4 at a lower dose than that of 1 or 3 indicate its potential as a new drug candidate for the prevention of some diseases including atherosclerosis; therefore, further studies on the expression of EC-SOD by 4 are warranted in order to elucidate the mechanisms underlying target cell signaling following treatments with 4.

role in the expression of EC-SOD because its expression is regulated by ERK signaling.33−35 Inhibitory Effects of 4 on HDAC Activities. The effects of 4 on the expression and activities of HDACs were investigated. As shown in Figure 5A, the expression of HDAC 4 and 10 was significantly increased by the treatment with 4, whereas that of HDAC 1, 2, 5, and 6 was significantly decreased. In addition, the treatment with 4 significantly suppressed HDAC activities (Figure 5B). Furthermore, 4 and the other royal jelly constituents tested suppressed HDAC 1 activity by approximately 50%, indicating that histone acetylation by the treatment with 4 and the other royal jelly constituents might be due to the inhibition of HDAC 1 activity (Figure 5C). These results suggest that the expression of ECSOD is regulated by histone acetylation within its promoter region through at least the inhibition of HDAC 1 activity. The excess production of reactive oxygen species (ROS) in blood vessel walls contributes to the production of oxLDL, which is one of the lipoproteins oxidized, and oxLDL is then recognized by scavenger receptors. Moreover, ROS induce endothelial dysfunctions and monocytic differentiation into macrophages, which have been implicated in the development of atherosclerosis. OxLDL has a key role during foam cell formation and is detected in the plaques of atherosclerotic lesions. The expression of EC-SOD is known to be very important for the prevention of cardiovascular diseases by accelerating the scavenging reaction of ROS, causing LDL oxidation. The overexpression of EC-SOD has been shown to protect against myocardial infarction caused by atherosclerosis in rabbits, and its administration also reduced the oxidation of LDL.36,37 On the other hand, the knockout of EC-SOD promoted the expansion of atherosclerotic lesions with transverse aortic constriction in mice, and oxLDL accelerated the destabilization of EC-SOD mRNA during foam cell formation in vitro.38,39 Thus, increases in the expression of EC-SOD in blood vessels may contribute to protection against ROS-induced vascular injuries. In the present study, the effects of royal jelly constituents on the expression of EC-SOD were examined. The treatment with 1 mM 1, 2, or 3 increased EC-SOD expression and the enrichment of acetylated histone H4 within the proximal promoter region of EC-SOD, but did not affect the expression of HDAC mRNAs. MCFAs, such as 1, 2, and 3, were expected to have the potential to bind with G protein-coupled receptor 40 (GPR40), GPR84, or GPR120 because they have been reported to stimulate cell signaling such as PKC and MEK/ ERK signaling through these receptors.40−44 Therefore, the induction of EC-SOD expression by royal jelly constituents might be mediated through GPRs. On the other hand, the results of the HDAC 1 activity assay suggest that HDAC 1 activity is directly inhibited by royal jelly constituents (Figure 5C), indicating that the increases induced in the expression of EC-SOD by 1, 2, and 3 are partially attributed to the direct inhibition of the HDAC catalytic domain. The treatment with 100 μM 4 increased the expression of EC-SOD and enrichment of acetylated histones H3 and H4 within the proximal promoter

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EXPERIMENTAL SECTION

General Experimental Procedures. 1H NMR spectra was obtained with a JEOL ECA 500 at room temperature in CDCl3 as a solvent (500 MHz for 1H NMR). Chemical shifts (δ) are expressed in parts per million and are internally referenced [0.00 ppm (tetramethylsilane) for 1H NMR]. Thin-layer chromatography (TLC) was carried out on precoated plates of silica gel (Merck, silica gel F-254, 0.5 mm). Subsequent to elution, plates were visualized using UV radiation (254 nm) on a Handy UV lamp SLUV-4 254 nm (AS ONE Co.). Flash column chromatography was performed with Kanto silica gel 60N (spherical, neutral, 40−50 mm). Reagents. Antiacetyl-histone H3 (cat. no. 06-599) and H4 (cat. no. 06-598) rabbit polyclonal antibodies were purchased from Millipore Co. (Billerica, MA, USA). Biotin-conjugated goat anti-rabbit and -mouse IgG (H+L) antibodies were purchased from Zymed Laboratories (San Francisco, CA, USA). An antiphospho-ERK mouse monoclonal antibody (cat. no. 9106) and anti-ERK rabbit monoclonal antibody (cat. no. 4695) were purchased from Cell Signaling Technology (Danvers, MA, USA). An HDAC cell-based activity assay kit, HDAC1 inhibitor screening assay kit, and trichostatin A were purchased from Cayman Chemical (Ann Arbor, MI, USA). 10Hydroxydecanoic acid (1) (>97%) and sebacic acid (3) (>97%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). D



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Figure 5. Involvement of HDACs in EC-SOD induction by the 4HPO-DAEE treatment. (A) THP-1 cells were treated with 100 μM 4HPO-DAEE (4) for 24 h. After cells had been treated, real-time RTPCR was carried out. Real-time RT-PCR data were normalized using 18S rRNA levels (*p < 0.05, **p < 0.01). (B) Cells were treated with the indicated concentration of 4 for 1 h. After cells had been treated, HDAC activities were measured (**p < 0.01 vs untreated cells). (C) The HDAC1 recombinant was reacted with 1 mM 10HDA (1), 10H2DA (2), or SA (3) or 100 μM 4 for 1 h. HDAC1 activity was then measured (**p < 0.01 vs untreated cells). 10-Hydroxy-2-decenoic acid (2) (≥99%) was purchased from Nagara Science Co., Ltd. (Gifu, Japan). Cell Culture. The human leukemic cell line, THP-1, was cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2 incubator. The cells were seeded at 2.0 × 106 cells/dish on 3.5 cm dishes, and 1, 2, 3, or 4 was added. After being treated, cells were collected and washed with cold phosphatebuffered saline (PBS) followed by an analysis with a real-time reverse transcriptional-polymerase chain reaction (RT-PCR), Western blotting, and HDAC activity assay. Measurement of Cell Viability. A 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was used to estimate the cytotoxicity of 4. Following the treatment of THP-1 cells with 4 in a 24-well plate, culture medium was changed and cells were added to 100 μL of 10% FCS-RPMI 1640 medium containing 0.5 mg/mL MTT (Chemicon Int. Inc., Temecula, CA, USA). After cells were transferred to 96-well plates, they were then incubated at 37 °C for 2 h in a humidified atmosphere of 5% CO2/95% air. After being incubated, cells were added to 100 μL of 2-propanol containing 0.04 N HCl and were then mixed thoroughly to dissolve MTT formazan. MTT formazan was measured at 595 nm with a reference wavelength of 655 nm. Real-Time RT-PCR Analysis. After the cells had been treated, they were lysed in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The preparation of cDNA and RT-PCR was performed by the methods described in our previous study.47 The primer sequences used are shown in Table 1. Enzyme-Linked Immunosorbent Assay (ELISA) Analysis. After THP-1 cells were treated with 1 mM 1, 2, or 3 or 100 μM 4 for 24 h, they were collected in PBS. The content of EC-SOD in THP1 cells was measured by ELISA as described in our previous study,33 with minor modifications. We have already confirmed that the concentration of EC-SOD closely correlated with SOD activity.48

Figure 4. Effects of 4-HPO-DAEE on EC-SOD expression and histone H3/H4 acetylation. (A) THP-1 cells were treated with the indicated concentration of 4-HPO-DAEE (4) for 24 h, and cell viability was measured. Data are shown as the mean ± SD (n = 3). (B) Cells were treated with the indicated concentrations of 4 for 24 h or 100 μM 4 for the indicated time. After the cells had been treated, real-time RT-PCR was carried out. Real-time RT-PCR data were normalized using 18S rRNA levels (**p < 0.01 vs untreated cells). (C) Cells were treated with 100 μM 4 for 24 h. The concentration of EC-SOD protein was then determined by ELISA (*p < 0.05, **p < 0.01 vs untreated cells). (D) Cells were treated with the indicated concentration of 4 for 12 h. After the cells had been treated, acetylated histone H3 and H4 were determined by Western blotting. The loading of histone was monitored by Coomassie staining. (E) Cells were treated with 100 μM 4 for 12 h. After cells had been treated, a ChIP assay was performed. Relative binding to the promoter region was expressed as the percentage amount over input (%) (**p < 0.01 vs untreated cells). (F) Cells were treated with the indicated concentration of 4 for 3 h. After cells had been treated, ERK phosphorylation was detected. (G) Cells were pretreated with 5 μM U0126 for 30 min. After cells were treated with 100 μM 4 for 24 h, real-time RT-PCR was carried out. Real-time RT-PCR data were normalized using 18S rRNA levels (*p < 0.05, **p < 0.01 vs untreated cells, ##p < 0.01 vs 4-treated cells). E



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buffer I (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% deoxycholic acid, and proteinase inhibitors), RIPA buffer II (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% deoxycholic acid, and proteinase inhibitors), and Tris-Hcl, pH 8.0, containing 1 mM EDTA (TE) buffer and then incubated in ChIP elution buffer (10 mM Tris-HCl, pH 8.0, containing 300 mM NaCl, 5 mM EDTA, and 0.5% SDS) with RNase A at 37 °C for 30 min and proteinase K at 65 °C for 2 h. After phenol−chloroform extraction and ethanol precipitation, genomic DNA was eluted in 20 μL of TE buffer. The abundance of EC-SOD promoter regions in ChIP precipitation was quantified using a PCR analysis. The primer sequences used for EC-SOD were sense 5′-GTG GAG GCG AAG CAA TTC TA-3′ (−175 base upstream from transcription start site (TSS)); antisense 5′-CTG TTA GCG CGA GTG CAG GA-3′ (−49 base upstream from TSS). 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 performed with Multi Gauge V3.0. HDAC Cell-Based Activity Assay. THP-1 cells were seeded in 90 μL of culture medium on a 96-well plate (5.0 × 104 cells/well) and then treated with 1 mM 1, 2, or 3 or 100 μM 4 for 1 h. As a positive control, cells were seeded in 80 μL of culture medium on a 96-well plate (5.0 × 104 cells/well), and 10 μL of 2 μM TSA was added for 1 h. After cells had been treated, 10 μL of diluted HDAC substrate was added to each well in order to initiate HDAC reactions, and the plate was incubated at 37 °C for 2 h for optimal development. Fifty microliters of the Lysis/Developer Solution was then added to each well, and the plate was shaken on a plate shaker for 1 min. The plate was then incubated at 37 °C for 15 min. The fluorescent intensity (excitation 365 nm, emission 410−460 nm) of each well was read using the GloMax-Multi Detection System (Promega, Madison, WI, USA). In Vitro HDAC1 Activity Assay. A total of 140 μL of assay buffer, 10 μL of diluted HDAC1, and 10 μL of 1 mM 1, 2, or 3 or 100 μM 4 was added to the wells on the black plate. Reactions were initiated by adding 10 μL of HDAC substrate to all the wells, and the plate was incubated at 37 °C for 30 min in the dark. Forty microliters of developer was added, and the plate was incubated at room temperature for 15 min. The fluorescence intensity (excitation 365 nm, emission 410−460 nm) of each well was read using the GloMax-Multi Detection System (Promega). 4-HPO-DAEE (4): 1H NMR (CDCl3, 500 MHz) δ 7.99 (1H, brs, −OOH), 6.88 (1H, dd, J = 16.0, 6.9 Hz), 6.04 (1H, d, J = 16.0 Hz), 4.51 (1H, q, J = 6.9 Hz, CHOOH), 4.22 (2H, q, J = 6.9 Hz, COOCH2CH3), 1.68−1,24 (13H, m), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (CDCl3, 125 MHz) δ 166.4 (C), 146.7 (CH), 123.4 (CH), 84.8 (CH), 60.8 (CH2), 32.2 (CH2), 31.7 (CH2), 29.2 (CH2), 25.2 (CH2), 22.6 (CH2), 14.3 (CH3), 14.1 (CH3); anal. C 62.39, H 9.63%, calcd for C12H22O4: C 62.58, H 9.63%. The chemical purity of prepared 4 was determined by combustion elemental analysis (>95%). Statistical Analysis. Data are expressed as the means ± SD of three independent experiments. Statistical evaluations of the data were performed using ANOVA followed by post hoc Bonferroni tests. A p value less than 0.05 was considered significant.

Table 1. Primer Sequences of SODs and HDACs Used in Real-Time RT-PCR gene EC-SOD Cu,Zn-SOD Mn-SOD HDAC1

HDAC2

HDAC3

HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC9 HDAC10 18S rRNA

sequence

amplicon size

the primer was purchased from QIAGEN (cat. no. QT01664327) S; 5′-GCGACGAAGGCCGTGTGCGTG-3′ 348 bp AS; 5′-TGTGCGGCCAATGATGCAATG-3′ S; 5′-CGACCTGCCCTACGACTACGG-3′ 365 bp AS; 5′-CCAGCCAACCCCAACCTGAGC-3′ S; 5′-CCTGAGGAGAGTGGCGATGA-3′ 69 bp AS; 5′-GTTTGTCAGAGGAGCAGATCGA3′ S; 5′-GCTCTCAATGGCGGTTCAG-3′ 75 bp AS; 5′-AGCCCAATTAACAGCCATATCAG3′ S; 5′-CCCAGACTTCACACTTCATCCA-3′ 70 bp AS; 5′-GGTCCAGATACTGGCGTGAGTT3′ S; 5′-GGGAGAGGATCAAGCTCGTTT-3′ 73 bp AS; 5′-GGGAGAGGATCAAGCTCGTTT-3′ S; 5′-CAACGAGTCGGATGGGATGT-3′ 74 bp AS; 5′-GGGATGCTGTGCAGAGAAGTC-3′ S; 5′-TGCCTCTGGGATGACAGCTT-3′ 69 bp AS; 5′-CCTGGATCAGTTGCTCCTTGA-3′ S; 5′-AGCAGCTTTTTGCCTCCTGTT-3′ 66 bp AS; 5′-TCTTGCGCAGAGGGAAGTG-3′ S; 5′-CGGCCAGACCGCAATG-3′ 56 bp AS; 5′-CACATGCTTCAGATTCCCTTT-3′ S; 5′-AGGCTCTCCTGCAGCATTTATT-3′ 75 bp AS; 5′-AAGGGAACTCCACCAGCTACAA-3′ S; 5′-ATGACCCCAGCGTCCTTTACT-3′ 66 bp AS; 5′ - CGCAGGAAAGGCCAGAAG-3′ S; 5′-CGGCTACCACATCCAAGGAA-3′ 187 bp AS; 5′-GCTGGAATTACCGCGGCT-3′

Western Blotting. Whole cell extracts were prepared in lysis buffer as described in our previous study.32,33 Extracts containing 20 μg of protein were boiled with sample buffer for 5 min and separated by SDS-PAGE on 12% (w/v) polyacrylamide gels. After being transferred electrophoretically onto PVDF membranes, nonspecific binding sites were blocked with PBS containing 1% BSA. After the membranes were incubated with the respective specific primary antibodies (1:1000), the blots were incubated with the biotin-conjugated goat anti-rabbit or -mouse antibody (1:1000). After the membranes had been washed three times with PBS containing 0.1% Tween 20, the blots were incubated with ABC reagent (Vector Laboratories, Inc., Burlingame, CA, USA; 1:5000). The bands were then detected using SuperSignal West Pico (Thermo Scientific, Rockford, IL, USA) and imaged using an LAS-3000 UV mini (Fuji Film). ChIP Assay. The ChIP assay was performed as described below. After cells (5 × 106 cells) had been treated, protein−DNA complexes were cross-linked using formaldehyde at room temperature for 5 min. After centrifugation at 1000g for 3 min, the pellets were sequentially washed with PBS and NP-40 buffer (10 mM Tris-HCl, pH 8.0, containing 10 mM NaCl and 0.5% NP-40), dissolved in 100 μL of SDS buffer (50 mM Tris-HCl, pH 8.0, containing 1% SDS and 10 mM EDTA), and added to 400 μL of ChIP dilution buffer (50 mM TrisHCl, pH 8.0, containing 167 mM NaCl, 1.1% Triton X-100, 0.11% deoxycholic acid, 10 mM NaF, 1 mM Na3VO4, 20 mM βglycerophosphate, 1 mM DTT, and 1 mM PMSF). Genomic DNA was sheared using a Vivracell VC100 ultrasonic homogenizer (Sonic & Materials, Danbury, CT, USA) to archive an estimated DNA size range of 150 to 800 bp, and 500 μL of ChIP dilution buffer was added. Sheared genomic DNA was incubated with Dynabeads Protein G (Invitrogen), which binds the respective primary antibodies overnight. After being incubated, the beads were sequentially washed with RIPA

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81 58 230 8100. Fax: +81 58 230 8105. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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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), a grant for the encouragement of young scientists from Gifu Pharmaceutical University (T.K.), and a grant from Api Co., Ltd. (Gifu, Japan). F



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(33) Kamiya, T.; Makino, J.; Hara, H.; Inagaki, N.; Adachi, T. J. Cell. Biochem. 2011, 112, 244−255. (34) Makino, J.; Kamiya, T.; Hara, H.; Adachi, T. Free Radical Res. 2012, 46, 637−644. (35) Laurila, J. P.; Castellone, M. D.; Curcio, A.; Laatilainen, L. E.; Haaparanta-Solin, M.; Gronroos, T. J.; Marjamaki, P.; Martikainen, S.; Santro, M.; Laukkanen, M. O. Mol. Ther. 2009, 17, 448−454. (36) Li, Q.; Bolli, R.; Qui, Y.; Tang, X. L.; Guo, Y.; French, B. A. Circulation 2001, 103, 1893−1898. (37) Laukkanen, M. O.; Lehtolainen, P.; Turunen, P.; Aittomaki, S.; Oikari, P.; Marklund, S. L.; Ylä-Herttuala, S. Gene 2000, 254, 173−179. (38) Lu, Z.; Xu, X.; Hu, X.; Zhu, G.; Zhang, P.; van Deel, E. D.; French, J. P.; Fassett, J. T.; Oury, T. D.; Bache, R. J.; Chen, Y. Hypertension 2008, 51, 19−25. (39) Makino, J.; Nii, M.; Kamiya, T.; Hara, H.; Adachi, T. Arch. Biochem. Biophys. 2015, 575, 54−60. (40) Hara, T.; Hirasawa, A.; Ichimura, A.; Kimura, I.; Tsujimoto, G. J. Pharm. Sci. 2011, 100, 3594−3601. (41) Miyamoto, J.; Mizukure, T.; Park, S. B.; Kishino, S.; Kimura, I.; Hirano, K.; Bergamo, P.; Rossi, M.; Suzuki, T.; Arita, M.; Ogawa, J.; Tanabe, S. J. Biol. Chem. 2015, 290, 2902−2918. (42) Zamarbide, M.; Etayo-Labiano, I.; Ricobaraza, A.; MartínezPinilla, E.; Aymerich, M. S.; Luis, L. J.; Pérez-Mediavilla, A.; Franco, R. Hippocampus 2014, 24, 733−739. (43) Ou, H. Y.; Wu, H. T.; Hung, H. C.; Yang, Y. C.; Wu, J. S.; Chang, C. J. Am. J. Physiol. Endocrinol. Metab. 2013, 304, E668−676. (44) Suzuki, M.; Takaishi, S.; Nagasaki, M.; Onozawa, Y.; Iino, I.; Maeda, H.; Komai, T.; Oda, T. J. Biol. Chem. 2013, 288, 10684−10691. (45) Makino, A.; Iinuma, M.; Fukumitsu, H.; Soumiya, H.; Furukawa, Y.; Furukawa, S. Biomed. Res. 2013, 34, 259−267. (46) Makino, A.; Iinuma, M.; Fukumitsu, H.; Soumiya, H.; Furukawa, Y.; Furukawa, S. Biomed. Res. 2010, 31, 379−386. (47) Yamamoto, M.; Hara, H.; Adachi, T. Atherosclerosis 2002, 163, 223−228. (48) Adachi, T.; Ohta, H.; Yamada, H.; Futenma, A.; Kato, K. Clin. Chim. Acta 1992, 212, 89−102.

REFERENCES

(1) Shidfar, F.; Jazayeri, S.; Mousavi, S. N.; Malek, M.; Hosseini, A. F.; Khoshpey, B. Iran J. Public Health 2015, 44, 797−803. (2) Azab, K. S.; Bashandy, M.; Salem, M.; Ahmed, O.; Tawfik, Z.; Helal, H. N. Am. J. Med. Sci. 2011, 3, 268−276. (3) Karaca, T.; Demirtaş, S.; Karaboğa, I.̇ ; Ayvazz, S. Turk. J. Med. Sci. 2015, 45, 27−32. (4) Wang, J.; Zhang, W.; Zhou, H.; Lin, Y.; Lin, K.; Zhou, Z.; Qiang, J.; Lin, J.; Chuka, C. M.; Ge, R.; Zhao, S.; Yang, X. Int. Immunopharmacol. 2015, 28, 97−104. (5) Kristensen, L. S.; Nielsen, H. M.; Hansen, L. L. Eur. J. Pharmacol. 2009, 625, 131−142. (6) Santini, V.; Gozzini, A.; Ferrari, G. Curr. Drug Metab. 2007, 8, 383−393. (7) Bernstein, B. E.; Meissner, A.; Lander, E. S. Cell 2007, 128, 669− 681. (8) Esteller, M. N. Engl. J. Med. 2008, 358, 1148−1159. (9) Kondo, Y.; Shen, L.; Cheng, A. S.; Ahmed, S.; Boumber, Y.; Charo, C.; Yamochi, T.; Urano, T.; Furukawa, K.; Kwabi-Addo, B.; Gold, D. L.; Sekido, Y.; Huang, T. H.; Issa, J. P. Nat. Genet. 2008, 40, 741−750. (10) Cave, A. C.; Brewer, A. C.; Narayanapanicker, A.; Ray, R.; Grieve, D. J.; Walker, S.; Shah, A. M. Antioxid. Redox Signaling 2006, 8, 691−728. (11) Vachier, I.; Chanez, P.; Le Doucen, C.; Damon, M.; Descomps, B.; Godard, P. Eur. Respir. J. 1994, 7, 1585−1592. (12) Wright, E., Jr; Scism-Bacon, J. L.; Glass, L. C. Int. J. Clin. Pract. 2006, 60, 308−314. (13) Marklund, S. L. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 7634− 7638. (14) Adachi, T.; Marklund, S. L. J. Biol. Chem. 1989, 264, 8537− 8541. (15) Ookawara, T.; Imazeki, N.; Matsubara, O.; Kizaki, T.; Oh-Ishi, S.; Nakao, C.; Sato, Y.; Ohno, H. Am. J. Physiol. 1998, 275, C840−847. (16) Whayne, T. F. Mol. Biol. Rep. 2015, 42, 765−776. (17) Zelko, I. N.; Mueller, M. R.; Folz, R. J. Free Radical Biol. Med. 2010, 48, 895−904. (18) Zelko, I. N.; Stepp, M. W.; Vorst, A. L.; Folz, R. J. Am. J. Respir. Cell Mol. Biol. 2011, 45, 953−961. (19) Izuta, H.; Chikaraishi, Y.; Shimazawa, M.; Mishima, S.; Hara, H. Evid. Based Complement Alternat. Med. 2009, 6, 489−494. (20) Nomura, M.; Maruo, N.; Zamami, Y.; Takatori, S.; Doi, S.; Kawasaki, H. Yakugaku Zasshi 2007, 127, 1877−1882. (21) Zamami, Y.; Takatori, S.; Goda, M.; Koyama, T.; Iwatani, Y.; Jin, X.; Takai-Doi, S.; Kawasaki, H. Biol. Pharm. Bull. 2008, 31, 2103− 2107. (22) Guo, H.; Saiga, A.; Sato, M.; Miyazawa, I.; Shibata, M.; Takahata, Y.; Morimatsu, F. J. Nutr. Sci. Vitaminol. 2007, 53, 345−348. (23) Houten, S. M.; Wanders, R. J. J. Inherited Metab. Dis. 2010, 33, 469−477. (24) Brière, J. J.; Favier, J.; Gimenez-Roqueplo, A. P.; Rustin, P. Am. J. Physiol. Cell Physiol. 2006, 291, C1114−1120. (25) Bernstein, B. E.; Meissner, A.; Lander, E. S. Cell 2007, 128, 669−681. (26) Schiffgen, M.; Schmidt, D. H.; von Rücker, A.; Müller, S. C.; Ellinger, J. Biochem. Biophys. Res. Commun. 2013, 436, 79−84. (27) Kamiya, T.; Machiura, M.; Makino, J.; Hara, H.; Acachi, T. Free Radical Biol. Med. 2013, 61, 197−205. (28) Zhang, Y.; Reinberg, D. Genes Dev. 2001, 15, 2343−2360. (29) Nishioka, K.; Chuikov, S.; Sarma, K.; Erdjument-Bromage, H.; Allis, C. D.; Tempst, P.; Reinberg, D. Genes Dev. 2002, 16, 479−489. (30) Ma, Y.; Yue, Y.; Pan, M.; Sun, J.; Chu, J.; Lin, X.; Xu, W.; Feng, L.; Chen, Y.; Chen, D.; Shin, V. Y.; Wang, X.; Jin, H. Am. J. Cancer Res. 2015, 5, 663−673. (31) Ropero, S.; Esteller, M. Mol. Oncol. 2007, 1, 19−25. (32) Iinuma, M.; Furukawa, S.; Itoh, A. 4-Hydroxy-trans-2-decenoic acid derivatives and medicine containing same. WO2014142276A1, September 18, 2014. G


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