Epigenetic modulation of mechanisms involved in inflammation: Influence of selected polyphenolic substances on histone acetylation state

Food Chemistry 131 (2012) 1015–1020

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Short Communication

Epigenetic modulation of mechanisms involved in inflammation: Influence of selected polyphenolic substances on histone acetylation state Anna K. Kiss a,⇑, Sebastian Granica a, Magdalena Stolarczyk a, Matthias F. Melzig b a b

Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Poland Pharmaceutical Biology, Institute of Pharmacy, Free University of Berlin, Germany

a r t i c l e

i n f o

Article history: Received 22 February 2011 Received in revised form 11 August 2011 Accepted 26 September 2011 Available online 2 October 2011 Keywords: Epigenetic modulators Polyphenols Inflammation Ellagic acid

a b s t r a c t In the present study, we have investigated the influence of low concentrations of polyphenols (5 lM) on histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity, a monocyte cell system related to the inflammatory process. We selected gallic acid, ellagic acid, oenothein B, valoneic acid dilactone and penta-O-galloyl-b-D-glucose, which are known to affect inflammatory responses, such as IL-6 secretion and/or NF-jB down-regulation and polyphenol metabolites (urolithins A, B and C). TNF-a stimulation resulted in a reduction of THP-1 cell viability by 28.9%, reduced HDAC activity from 25.2 to 13.5 pmol/ lg protein and increased HAT activity from 24.0 to 45.5 pmol/lg protein. The coincubation with ellagic acid and oenothein B restored the viability and reversed the effect of TNF-a on HAT and HDAC activities. Urolithins B and C, gallic acid and penta-O-galloyl-b-D-glucose only showed significant reductions of HAT activity, by 40–50%. Our results prove that polyphenols act as epigenetic modulators. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Over the past decade, epigenetics have been demonstrated to be a major contributor in the pathogenesis of several illnesses, including ageing, inflammatory lung diseases, inflammatory skin disorders, diabetes and cardiovascular diseases (Barnes, Adcock, & Ito, 2005; Kirk, Cefalu, Ribnicky, Liu, & Eilertsen, 2008; Vanden Berghe & Haegeman, 2010). Histone acetylation/deacetylation plays an important role in inflammation. The reversible histone acetylation is controlled by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Two closely related HATs, p300 (adenoviral protein E1A) and CBP (CREB-binding protein), have the function of transcriptional co-factors. Activation of these co-activators by pro-inflammatory transcription factors, such as NF-jB and AP-1, leads to increased gene expression. The effect of HAT on gene expression is counteracted by histone deacetylases (HDACs). HDACs play a critical role in the suppression of gene expression by reversing the hyperacetylation of core histones and by targeting acetylated transcription factors (Barnes et al., 2005; Rahman, Marwick, & Kirkham, 2004). HDAC not only cause the inhibition of gene transcription, but are also directly associated with inactive p65 and play a role in the regulation NF-jB-mediated gene transcription (Barnes et al., 2005; Rahman et al., 2004). HADC1 ⇑ Corresponding author. Address: Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland. Tel.: +48 22 5720942; fax. +48 22 5720985. E-mail address: [email protected] (A.K. Kiss). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.09.109

and HDAC2 are able to deacetylate acetylated NF-jB and promote its association with the inhibitor IjB-a within the nucleus, in order to promote export into the cytoplasm and, thus terminate the activity of NF-jB (Barnes et al., 2005). Additionally, investigation of the co-factor regulation, in TNF-a, IL-6 promoter stimulation, revealed a strong synergism between p65 and CBP/p300, which is highly dependent on its HAT properties (Vanden Berghe, De Bosscher, Boone, Plaisance, & Haegeman, 1999). On the other hand, we should emphasise that histone deacetylase inhibitors (HDIs) may be quite effective in blocking inflammation in inflammatory bowel disease, colonic mucosa inflammation associated with increased risk of malignancies and autoimmune diseases. HDIs also exert strong anti-angiogenic effects (Huang, 2006). Various natural nutritional compounds (curcumin, epicatechin gallate, epigallocatechin gallate, garcinol and isothiocyanate) have been reported to influence HDAC and HAT activities and, by that, modulate metabolic disturbance, inflammatory responses and immunological senescence. Various flavonoids have been identified as activators of class III HDACs (SIRTs). Turmeric and green tea and their constituents have been identified as sources of natural inhibitors of p300/CBP HAT (Szarc vel Szic, Ndlovu, Vanden Berghe, & Haegeman, 2010). It has been also shown that curcumin and theophylline are able to induce HDAC activity in stimulated monocytes and macrophages, respectively (Ito et al., 2002; Yun, Jialal, & Devaraj, 2010). There is still a gap in information about the modulation of HAT and HDAC activities in defined cell culture systems by polyphenols. For our study, we selected polyphenols, as well as some polyphenol

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metabolites, which are known to affect inflammatory responses, such as IL-6 secretion and/or NF-jB down-regulation, and we tested their influence on HAT and HDAC activities. We used THP-1 monocyte cells which were stimulated by TFN- a in order to induce the inflammatory process. 2. Material and methods 2.1. Materials Gallic acid ellagic acid and curcumin were purchased from ChromaDex (Santa Ana, USA). TNF-a (tumour necrosis factor), PMA (4b-phorbol-12b-myristate-a13-acetate) and MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), were purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Fibronectine was purchased from Roche (Mannheim, Germany). HAT and HDAC assay kit (fluorimetric) and nuclear extract kit were purchased from Active motif (Carlsbad, USA). RPMI 1640 medium, featal bovine serum (FBS) and L-glutamine were purchased from Biochrom (Berlin, Germany). All other chemicals used were of analytical grade. 2.2. Isolation of polyphenolic compounds Penta-O-galloyl-b-D-glucose was isolated, as described previously (Kiss, Derwin´ska, Dawidowska, & Naruszewicz, 2008) from dried aqueous extract of defatted seeds of Oenothera paradoxa Hudziok containing ca. 45% of phenolic compounds obtained from Agropharm S.A. (Tuszyn, Poland). Oenothein B and valoneic acid dilactone were isolated from the herb of Epilobium angustifolium L. The dried plant material (800 g) was extracted successively with chloroform and 70% methanol. The water residue was extracted with ethyl acetate and with n-butanol saturated with water. The EtOAc fraction (30 g) was then fractionated over polyamide (Carl Roth, Germany, 4  40 cm), using water with increasing contents of methanol, and then with acetone–water (7:3), giving 23 main fractions. Valoneic acid dilactone (50 mg), present in fraction 22, was then cleaned on Sephadex LH-20 (Pharmacia, Sweden, 2  50 cm), using 100% methanol and 80% methanol as eluents. The BuOH fraction (15 g) was fractionated on Sephadex LH-20 (5  50 cm) with 50% methanol. Fractions containing oenothein B were separated with 70% methanol on Sephadex LH-20 (5  50 cm) and with methanol–acetone–water (7:1:2) on toyopearl HW40F (Tosoh, Japan, 2  40 cm), giving 250 mg of oenothein B. The structures of all compounds were confirmed by 1H and 13C NMR in comparison with those reported in the literature. All isolated substances used in the biological assays had purity > 95% (HPLC). The isolated compounds were stored at 4 °C.

J = 2.4 Hz, 1H); 13C NMR (75 MHz, CD3OD) d 163.50 C-6, 161.56 C3, 154.01 C-4a, 137.17 C-10a, 136.48 C-9, 131.20 C-7, 128.69 C-8, 125.63 C-1, 122.58 C-6a, 120.73 C-10, 114.51 C-10b, 111.44 C-2, 104.32 C-4; ESIMS m/z 211 [M H] , 423 [2M H] . Urolithin C: 1H NMR (300 MHz, DMSO) d 7.84 (d, J = 8.7 Hz, 1H), 7.49 (s, 1H), 7.44 (s, 1H), 6.79 (dd, J = 8.6, 2.2 Hz, 1H), 6.69 (d, J = 2.2 Hz, 1H); 13C NMR (75 MHz, DMSO) d 160.24 C-6, 158.53 C3, 153.40 C-9, 151.40 C-4a,.146.08 C-8, 129.12 C-10a, 123.66 C-1, 114.16 C-7, 112.81 C-2, 110.81 C-6a, 109.73 C-10b, 106.80 C-10, 102.73 C-4; ESIMS m/z 243 [M H] , 487 [2M H] . All synthesised substances used in the biological assays had purity > 95% (HPLC). The synthesised compounds were stored at 4 °C. 2.4. Biological assays 2.4.1. Cell culture THP-1, human monocytic cell line, purchased from DSMZ (Braunschweig, Germany), was subcultivated at 37 °C under humidified 5% CO2 in RPMI 1640 medium containing 10% FBS and 2 mM glutamine. For experiments, cells were seeded in fibronectine-coated 24-well plates in a density of 2  106 cells per well and allowed to differentiate for 48 h in the presence of phorbol myristate acetate (PMA, 50 ng/ml) prior to 24 h stimulation with TNF-a (20 ng/ml) in the presence or absence of polyphenols (5 lM). Curcumin was used as a positive control. For MTT assay, cells were seeded in fibronectine-coated 96-well plates, in a density of 1  105 cells per well and then treated as above. 2.4.2. MTT assay Cells were incubated in a 96 well-plate for 2 h at 37 °C with MTT (0.5 mg/ml). The insoluble formazan product was dissolved in 100 ll of DMSO and measured spectrophotometrically at 570 nm, using a microplate reader, Tecan Deutschland GmbH (Crailsheim, Germany). 2.4.3. Preparation of nuclear lysate After treatment, the cell medium was aspirated and the nuclear lysates were prepared using Nuclear extract Kit (Actif motif). The protein content in the lysates was measured by BCA protein assay (Pierce) according to the manufacturer’s protocol. 2.4.4. Measurement of HDAC and HAT activities Following treatment with various polyphenols, cells were harvested and nuclear lysate was prepared. HDAC and HAT activities were determined according to the manufacturer’s protocols, using 10 and 20 lg of nuclear lysates, respectively. Fluorescence was measured with excitation at 360 nm and emission at 450 nm, using a microplate reader, Tecan Deutschland GmbH (Crailsheim, Germany).

2.3. Synthesis of urolithins A, B and C Urolithins were synthesised by the condensation of resorcinol with the appropriately substituted benzoic acids, as described by Bialonska, Kasimsetty, Khan, and Ferreira (2009). The identities of urolithins were confirmed by their molecular mass and 1H and 13 C NMR spectra. Urolithin A: 1H NMR (300 MHz, DMSO) d 8.11 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.32 (dd, J = 8.7, 2.6 Hz, 1H), 6.81 (dd, J = 8.7, 2.3 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H); 13 C NMR (75 MHz, DMSO) d 160.53 C-6, 158.50 C-3, 156.90 C-8, 150.85 C-4a, 126.89 C-10a, 124.09 C-9, 123.74 C-10, 123.52 C-1, 120.12 C-6a, 113.48 C-2, 112.96 C-7, 109.78 C-10b, 102.79 C-4; ESIMS m/z 227 [M H] , 455 [2M H] . Urolithin B: 1H NMR (300 MHz, CD3OD) d 8.21 (dd, J = 8.0, 1.0 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.83–7.74 (m, 1H), 7.52–7.42 (m, 1H), 6.81 (dd, J = 8.7, 2.4 Hz, 1H), 6.69 (d,

2.4.5. Measurement of HDAC and HAT activities in cell-free system HDAC and HAT activities were determined according to the manufacturer’s protocols, using HeLa nuclear extract and recombinant p300 catalytic domain, respectively. Fluorescence was measured, with excitation at 360 nm and emission at 450 nm, using a microplate reader, Tecan Deutschland GmbH (Crailsheim, Germany). Trichostatin A and anacardic acid were used as positive controls. 2.5. Statistical analysis The results were expressed as means ± SEM of three experiments performed in duplicate. Statistical significance of differences between means was established by ANOVA with Tukey post hoc test. P values below 0.05 were considered statistically significant. All analyses were performed using Statistica 8.

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Fig. 1. Chemical structure of tested compounds: gallic acid (1); ellagic acid (2); oenothein B (3), valoneic acid dilactone (4), pentagaloiloglucose (5), urolithin A (6), urolithin B (7) and urolithin C (8).

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and polyphenol-enriched extracts have frequently been demonstrated in in vitro experiments at concentrations varying from 20 to 200 lM, which can never be achieved in vivo and, thus are irrelevant in terms of health benefit in vivo effects (Espin, GarciaConesa, & Tomás-Barberán, 2007; Kim et al., 2004). That is why, as suggested by Szarc vel Szic, Ndlovu, Vanden Berghe, and Haegeman (2010), ‘‘epigenetics’’ sheds a more realistic light on dietary studies, as long-life exposure to physiological concentrations can remodel the epigenome. In the present study, we have

3. Results and discussion Polyphenols, such as flavonoids, but also phenolic acids, catechins and compounds with higher molecular weight, inter alia gallotannins and ellagitannins, are well known for their antioxidant and anti-inflammatory activities, even at low concentrations for some specific structures (Fraga, Galleano, Verstraeten, & Oteiza, 2010; Kim, Son, Chang, & Kang, 2004; Quideau, 2009). However, the health-protecting effects of dietary polyphenols

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Fig. 2. Effect of selected polyphenols at concentration of 5 lM on TNF-a stimulated THP-1 cells viability in MTT assay. Results are shown as means ± SEM of three experiments performed in duplicate. ap < 0.005 compared to not treated control; ⁄p < 0.05 compared to TNF-a. Curcumin (CUR) was used as positive control. Tested polyphenols: gallic acid (GA), ellagic acid (EA), oenothein B (OeB), valoneic acid dilactone (VAD), pentagaloyloglucose (PGG), urolithins A, B and C (UA, UB and UC).

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Fig. 3. (A) Modulation of HDAC activity by selected polyphenols at concentration of 5 lM on TNF-a stimulated THP-1 cells. Curcumin (CUR) was used as positive control. Results are shown as mean ± SEM of three experiments performed in duplicate. ap < 0.005 compared to not treated control; ⁄p < 0.02; ⁄⁄p < 0.005 compared to TNF-a. (B) Direct HDAC activity inhibition by selected polyphenols at concentration of 5 lM. Trichostatin A (TSA) was used as a selective HDAC inhibitor. Results are shown as means ± SEM of three experiments performed in duplicate; ⁄⁄p < 0.005 compared to control Tested polyphenols: gallic acid (GA); ellagic acid (EA), oenothein B (OeB), valoneic acid dilactone (VAD), pentagaloyloglucose (PGG), urolithins A, B and C (UA, UB and UC).

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investigated the influence of low concentrations of 5 lM polyphenols on HAT and HDAC activities in a monocyte cell system mimicking the inflammatory process by PMA and TNF-a. We selected polyphenols (gallic acid, ellagic acid, oenothein B, valoneic acid dilactone, pentagaloyloglucose) which are known to affect the inflammatory responses such as IL-6 secretion and/or NF-jB down-regulation (Chen, Yang, & Lee, 2000; Kuppan, Balasubramanyam, Monickaraj, Srinivasan, Mohan & Balasubramanyam, 2010; Lee et al., 2007; Umesalma & Sudhandiran, 2010), as well as some polyphenol metabolites (urolithins A, B and C) (Fig. 1). We compared our results with curcumin, which was shown, in low concentrations (1.5–6 lM), to modulate the HAT and HDAC activities in high glucose-induced monocytes (Yun et al., 2010). We first examined the effect of TNF-a (20 ng/ml) stimulation on cell viability and HAT and HDAC activities (Figs. 2, 3A and 4A). Under stimulated condition, the viability of THP-1 cells, in comparison to the untreated cells, was reduced by 28.9%. The stimulated cells had a significantly (p < 0.005) reduced HDAC activity, from 25.2 to 13.5 pmol/lg protein, and an increased HAT activity (p < 0.05) from 24.0 to 45.5 pmol/lg protein. The co-incubation of stimulated cells with curcumin, at a concentration of 5 lM, restored the cells viability to 91.3% of the control (Fig. 2). Curcumin, similarly to high glucose-induced monocytes (Yun et al., 2010), significantly reversed the effect of TNF-a on HAT and HDAC, the activities of which achieved the level of 32.4 and 20.1 pmol/lg protein, respectively (Figs. 3A and 4A). Among polyphenols tested at a concentration of 5 lM, ellagic acid showed the most statistically significant effects on both

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enzyme activities, as well as the cell viability. The activity of ellagic acid at this concentration was even more statistically significant in comparison with curcumin, especially in reduction of HAT activity by 40% at a level of 18.3 pmol/lg protein (Figs. 2, 3A and 4A). Interestingly, valoneic acid dilactone, showing a quite similar structure, proved to be inactive. Ellagic acid (EA) is not only the constituent of some medicinal plants rich in ellagitannins (Eucaliptus globulus Labill., Myrtus communis L., Epilobium sp., Terminalia sp., and Geranium sp.), but it is also present in various food products containing raspberry fruits, strawberry fruits, and other berries, pomegranate juice and different nuts (Vattem & Shetty, 2005). A few bioavailability studies indicate a rather poor absorption and rapid elimination of EA, whose plasma concentration varies around 0.06–0.2 lM after consumption of food products containing about 25 mg of free EA (Larrosa, Garcia-Conesa, Espin, & Tomás-Barberán, 2010). However, its bioavailability might be enhanced by using nanoparticles or phospholipids (Murugan, Mukherjee, Maiti, & Mukherjee, 2009; Ratman, Chandraiah, Meena, Ramarao, & Kumar, 2009). In our study, we used the concentration of 5 lM of EA which showed a significant biological effect. It is probable that supplementation with lower doses of ellagic acid for a longer time may well show a health benefit in vivo. In contrast to ellagic acid, urolithins (mainly A and B), the ellagitannins and ellagic acid are microbialderived metabolites. They could be detected in plasma at concentrations ranging from 0.5 to 18.6 lM (Larrosa et al., 2010). In our investigation, only urolithins B and C seem to be active by reducing the HAT activity by more than 50% at a concentration of 5 lM. The

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Fig. 4. (A) Modulation of HAT activity by selected polyphenols at concentration of 5 lM on TNF-a stimulated THP-1 cells. Curcumin (CUR) was used as positive control. Results are shown as means ± SEM of three experiments performed in duplicate. ap < 0.005 compared to not treated control; ⁄p < 0.05; ⁄⁄p < 0.02 compared to TNF-a. (B) Direct HAT activity inhibition by selected polyphenols at concentration of 5 lM. Anacardic acid (AA) was used as a selective HAT inhibitor. Results are shown as means ± SEM of three experiments performed in duplicate; ⁄p < 0.02; ⁄⁄p < 0.005 compared to control Tested polyphenols: gallic acid (GA); ellagic acid (EA), oenothein B (OeB), valoneic acid dilactone (VAD), pentagaloyloglucose (PGG), urolithins A, B and C (UA, UB and UC).

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tested ellagitannin, oenothein B, showed statistically significant reduction of the TNF-a effect in monocytic cells (Figs. 2, 3A and 4A). The in vivo anti-inflammatory effect of this compound might be probable only when applied externally because it is rather poorly absorbed from the gut. The effects of gallic acid (GA) and the gallotannin, penta-O-galloyl-b-D-glucose (PGG), were only significant in the reduction of HAT activity, app. 43% for both compounds. The HAT inhibitory activity of gallic acid has been shown previously, but only in an assay using isolated enzymes, not in a cell culture system directly (Choi et al., 2009). The plasma level of gallic acid and its 4-O-methylated metabolite reach 4.7 lM after consumption of black tea beverage containing 50 mg of gallic acid, and 1.57 lM after red wine containing 4 mg of GA (Lafay & Gil-Izquierdo, 2008). The pharmacokinetics of PGG, one of the main constituents of Rhus chinensis Mill., Paeonia suffruticosa Andrews and O. paradoxa Hudziok, are not well established. However, the injection of PGG in a dose of 0.5 mg per mouse resulted in a free plasma concentration of 3–4 lM PGG (Li et al., 2011). A large part of the screening tests for HDAC and HAT inhibitors are performed in commercial nuclear extracts or with recombinant enzymes. Because of this fact, we also investigated the direct influence of tested polyphenols on HDAC and HAT enzyme activities. None of the tested compounds inhibited HDAC activity at concentration of 5 lM, in comparison to the selective inhibitor, trichostatin (TSA) (Fig. 3B). Curcumin, ellagic acid, and PGG showed weaker (12.5%, 14.7% and 25.6%) and statistically less significant inhibition of HAT activity at tested concentration in comparison to anacardic acid (Fig. 4B). Gallic acid, oenothein B and valoneic acid dilactone (VAD) reduced the enzyme activities by 32.5%, 46.7% and 51.3%, respectively (Fig. 4B). Interestingly, VAD showed no activity in the monocytic cell system, while oenothein B is probably too big and too hydrophilic to reach the nucleus, and more likely acts at the cell surface by signalling pathways and/or specific receptors. Therefore, we think that the results obtained in assays using isolated enzymes should be discussed carefully, and it seems to be necessary to recheck active compounds in a relevant cell system. Our study has demonstrated that polyphenols, as well as some polyphenol metabolites, are able to regulate the HAT and HDAC activities in a monocyte cell system, mimicking the inflammatory process. Ellagic acid and ellagitannin oenothein B modulated both enzymes, while gallic acid and gallotannin penta-O-galloyl-b-Dglucose appeared to more specifically down-regulate HAT activity. However, more studies are needed to investigate the effect of even lower concentrations for long-term supplementation. References Barnes, P. J., Adcock, I. M., & Ito, K. (2005). Histone acetylation and deacetylation: Importance in inflammatory lung diseases. European Respiratory Journal, 25, 552–563. Bialonska, D., Kasimsetty, S. G., Khan, S. I., & Ferreira, D. (2009). Urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay. Journal of Agricultural and Food Chemistry, 57, 10181–10186. Chen, Y.-C., Yang, L.-L., & Lee, T. J.-F. (2000). Oroxylin A inhibition of lipoolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-jB activation. Biochemical Pharmacology, 59, 1445–1457. Choi, K.-C., Lee, Y.-H., Jung, M. G., Kwon, S. H., Kim, M.-J., Jun, W. J., et al. (2009). Gallic acid suppresses lipopolysaccharide-induced nuclear factor-jB signalling

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