Polyphenol acertannin prevents TRAIL-induced apoptosis in human keratinocytes by suppressing apoptosis-related protein activation

Chemico-Biological Interactions 189 (2011) 52–59

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Polyphenol acertannin prevents TRAIL-induced apoptosis in human keratinocytes by suppressing apoptosis-related protein activation Chung Soo Lee a,∗ , Eun-Ra Jang a , Yun Jeong Kim a , Seong Jun Seo b , Sun Eun Choi c , Min Won Lee c a b c

Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, South Korea Department of Dermatology, Chung-Ang University Hospital, Seoul 156-755, South Korea Pharmacognosy Laboratory, College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea

a r t i c l e

i n f o

Article history: Received 31 August 2010 Received in revised form 13 October 2010 Accepted 14 October 2010 Available online 4 November 2010 Keywords: TRAIL Acertannin Human keratinocytes Apoptosis-related proteins Cell protection

a b s t r a c t The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been implicated in the inflammatory and immune responses, and apoptosis in skin diseases, such as atopic dermatitis. Dysregulated apoptosis is associated with various pathologic conditions, including inflammation and cancer in skin. Polyphenols, including flavonoids and tannins, have been shown to have anti-oxidant, antiinflammatory and anti-tumor effects. However, the effect of acertannin on TRAIL-induced apoptosis in keratinocytes has not been determined. To assess the preventive effect of acertannin on apoptosismediated skin inflammation, we investigated the effect of acertannin on TRAIL-induced apoptosis in human keratinocytes. TRAIL induced nuclear damage, decreased Bid, Bcl-2, Bcl-xL and survivin protein levels, increased Bax levels, induced cytochrome c release, activated caspases (-8, -9 and -3) and increased tumor suppressor p53 levels. Acertannin prevented the TRAIL-induced formation of reactive oxygen/nitrogen species, apoptosis-related protein activation and cell death. The results suggest that acertannin may reduce apoptotic effect of TRAIL on human keratinocytes by suppressing the activation of the caspase-8- and Bid-pathways and the mitochondria-mediated apoptotic pathway, leading to caspase-3 activation. The preventive effect of acertannin on TRAIL-induced apoptosis may be associated with the inhibitory effect on formation of reactive oxygen/nitrogen species. Acertannin may prevent the TRAIL-induced apoptosis-mediated skin inflammation. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Keratinocytes are considered to play a critical role in the pathogenesis of inflammatory skin disease, such as atopic dermatitis and psoriasis [1,2]. Keratinocytes secrete inflammatory mediators such as chemokines and cytokines in response to a variety of stimuli, including epidermal barrier perturbation, which may be involved in skin inflammation [1]. Inflammatory mediators produced from keratinocytes elicit enhanced recruitments as well as sustained survival and activation of T cells and dendritic cells. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, and has been implicated in the inflammatory and immune responses, and apoptosis in skin diseases, such as atopic dermatitis [3–5]. Increased expression of TRAIL is detected in T cells and monocytes in the peripheral blood and skin lesions in patients with atopic dermatitis [5,6]. Apoptosis of keratinocytes caused by skin-infiltrating T cells may be involved in the formation of eczema in atopic dermatitis

∗ Corresponding author. Tel.: +82 2 820 5659; fax: +82 2 813 5387. E-mail address: [email protected] (C.S. Lee). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.10.009

[3,7]. It has been shown that dysregulated apoptosis is associated with various pathologic conditions, including inflammation and cancer in skin [8]. TRAIL induces apoptosis in transformed or tumor cells but not in normal cells. TRAIL initiates apoptosis via the activation of cell death receptors and mitochondria-mediated apoptotic pathway [9,10]. Polyphenols are found in various plants and are divided into tannins, lignins and flavonoids [11,12]. It has been shown that polyphenols reveal anti-oxidant and anti-inflammatory effects, and anti-tumor effects [11–13]. Acertannin is also demonstrated to exhibit a scavenging action on oxidant superoxide and 1,1diphenyl-2-picrylhydrazyl radicals [14]. However, polyphenol epigallocatechin-3-gallate does not scavenge exogenous hydrogen peroxide, but rather, it synergistically increased hydrogen peroxide-induced oxidative cell damage in pancreatic beta cells [15]. Polyphenols have been shown to promote cell proliferation and survival and cause DNA damage and inflammation [16,17]. Regulation of disrupted apoptosis may confer a benefit in the treatment of inflammatory skin diseases [8]. Polyphenols, including flavonoids and tannins, have been shown to have anti-oxidant and anti-inflammatory effects. However, compared to flavonoids, researches on cytoprotective effect of tannins are rare. Further-

C.S. Lee et al. / Chemico-Biological Interactions 189 (2011) 52–59

Fig. 1. Chemical structure of acertannin.

more, the effect of acertannin on TRAIL-induced apoptosis in keratinocytes has not been determined. To assess the preventive effect of acertannin on apoptosis-mediated skin inflammation, we investigated the effect of acertannin on TRAIL-induced apoptosis in human keratinocytes. 2. Materials and methods 2.1. Materials The TiterTACSTM colorimetric apoptosis detection kit was purchased from Trevigen, Inc. (Gaithersburg, MD, USA), and the Quantikine® M human cytochrome c assay and caspase (-8, -9 and -3) assay kits were purchased from R&D systems (Minneapolis, MN, USA). Antibodies [anti-Bid (5C9), anti-Bax (6A7), anti-Bcl-2 (10C4), anti-Bcl-xL (H-5), anti-survivin (D-8), anti-cytochrome c (A-8), anti-p53 (DO-1) and anti-␤-actin] were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TRAIL (Apo2L; human recombinant), horseradish peroxidase-conjugated anti-mouse IgG, z-Asp-(OMe)-Gln-Met-Asp(OMe) fluoromethyl ketone (z-DQMD.fmk) and z-Ile-Glu-(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (z-IETD.fmk) were all purchased from EMD-Calbiochem (La Jolla, CA, USA). SuperSignal® West Pico chemiluminescence substrate for cytochrome c detection in Western blot was purchased from PIERCE Biotechnology Inc. (Rockford, IL). The Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was purchased from OXIS International Inc. (Portland, OR, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), z-Leu-Glu-(O-ME)-His-Asp(O-Me) fluoromethyl ketone (z-LEHD.fmk), 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), NG -methyl-l-arginine acetate salt (l-NMMA) and other chemicals were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). 2.2. Extraction, isolation and structural identification of acertannin Acertannin was isolated from the bark of Acer ginnala Maxim (Fig. 1). Desiccated barks (5.5 kg) were extracted with 80% aqueous acetone at room temperature. After removal of acetone under vacuum, the extract was dissolved in water and then aqueous solution was filtered through filter paper (Tokyo Roshi Kaisha Ltd., Japan). The solution filtered was concentrated, applied to the column filled with Sephadex LH-20 (10–25 ␮m, 10 cm × 80 cm, GE Healthcare Bio-Science AB, Uppsala, Sweden) and then the solution was eluted with H2 O contained methanol (30–100% gradient). The solution was applied to thin layer chromatography by increasing concentrations of methanol (10–100%), which afforded 8 fractions. In the upper fraction number 8, acertannin (2,6-digalloyl1,5-anhydroglucitol) as crystals yielded (8.37 g). The solution in fraction number 8 was again applied to the columns filled with ODS-B gel (40–60 ␮m, Daiso, Osaka, Japan) with 50% methanol isocratic and Disogel (300 g, 3 cm × 50 cm, SP-120-40/60-ODS-B, Daiso, Osaka, Japan) with 20–100% methanol gradient, and middle

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pressure liquid column chromatography was performed. Column was connected with Waters 650 pump (Waters, Milford, USA) and Gilson 112 UV/VIS detector (254 nm, Gilson, Middleton, WI, USA), and operated under waters 650 system controller (Waters, Milford, USA). From these procedures, acertannin (2.87 g) was yielded. Purity of acertannin was analyzed using a high performance liquid chromatography (Waters 600 system, Milford, USA). The yield had approximately 98% purity. The structural identity of acertannin was characterized by spectral analyses. The 1-D nuclear magnetic resonance (NMR) findings such as 1 H (600 MHz) and 13 C (150 MHz) NMR were recorded with Gemini 2000 and VNS (Varian, Palo Alto, CA, USA). Low resolution fast atom bombardment mass spectrum (LRFAB-MS) was recorded with JMSAX505WA (JEOL, Tokyo, Japan). White amorphous powder, [˛]D : +27.6◦ (c = 0.005, acetone), negative FAB-MS: m/z: 467 [M−H]− . 1 H NMR (600 MHz, DMSOd6 + D2 O): ı 3.24 (1H, t, J = 10.8 Hz, H-1), 3.32 (1H, t, J = 9 Hz, H-3), 3.45 (2H, m, H-3, 5), 3.91 (1H, dd, J = 10.8 Hz, 5.4 Hz, H-1), 4.21 (1H, dd, J = 12.0 Hz, 5.4 Hz, H-6b), 4.44 (1H, br d, J = 12.0 Hz, H-6a), 4.72 (1H, m, H-2), 6.95 (2H × 2, s, galloyl-H). 13 C NMR (150 MHz, DMSOd6 + D2 O): ı 64.0 (C-6), 66.5 (C-1), 70.6 (C-4), 72.0 (C-2), 75.1 (C-3), 75.7 (C-5), 109.0, 109.2 (C-2 ,6 ,2 ,6 ), 119.6, 119.7 (C-1 ,1 ), 138.8 (C-4 ,4 ), 145.8 (C-3 ,5 ,3 ,5 ), 166.9, 166.2 (C-7 ,7 ). 2.3. Keratinocyte culture Human keratinocytes (HEK001, tissue: skin; morphology: epithelial; cell type: human papillomavirus 16 E6/E7 transformed) were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in keratinocyte-SFM supplemented with bovine pituitary extract, recombinant epidermal growth factor, 100 U/ml penicillin and 100 ␮g/ml streptomycin (GIBCO® , Invitrogen Co., Grand Island, NY, USA). 2.4. Cell viability assay with MTT reduction Cell viability was measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases [18]. In this study, the treatment time of TRAIL to keratinocytes was based on previous reports [19,20]. Keratinocytes (3 × 104 cells/200 ␮l) were treated with TRAIL in the presence or absence of acertannin for 24 h at 37 ◦ C. The medium (200 ␮l) was then incubated with 10 ␮l of 10 mg/ml MTT solution for 2 h at 37 ◦ C. After centrifugation at 412 × g for 10 min, the culture medium was removed, and 100 ␮l of dimethyl sulfoxide was added to each well to dissolve the formazan. The absorbance was measured at 570 nm using a microplate reader (Spectra MAX 340, Molecular Devices Co., Sunnyvale, CA, USA). Cell viability was expressed as a percentage of the absorbance value of control cultures. 2.5. Cell viability assay with neutral red uptake Cell viability was determined using the neutral red uptake assay, which is based on the observation that neutral red is accumulated in the lysosomes of live cells [21]. Keratinocytes (3 × 104 ) were treated with 50 ng/ml TRAIL for 24 h at 37 ◦ C. The cell suspension (200 ␮l) was then incubated with 10 ␮l of 1 mg/ml neutral red solution for 3 h at 37 ◦ C. After centrifugation at 412 × g for 10 min, culture medium was removed, and the dye was extracted with 100 ␮l of a 1% acetic acid and 50% ethanol solution for 20 min. The absorbance was measured at 540 nm using a microplate reader. 2.6. Observation of changes in nuclear morphology To clearly define the inhibitory effect of acertannin on TRAIL-induced nuclear damage, we investigated the effects at

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a16 h-incubation. Keratinocytes (1 × 106 cells/ml) were treated with TRAIL for 16 h at 37 ◦ C, and the change in nuclear morphology was assessed using Hoechst dye 33258 [22]. Cells were incubated with 1 ␮g/ml Hoechst 33258 for 3 min at room temperature and nuclei were visualized using an Olympus Microscope with a WU excitation filter (Tokyo, Japan). 2.7. Measurement of oligonucleosomal DNA fragmentation To clearly define the inhibitory effect of acertannin on TRAILinduced DNA fragmentation, we investigated the effects at a 16 h-incubation. DNA fragmentation due to the activation of endonucleases was assessed by gel electrophoresis. Keratinocytes (4 × 106 cells/ml) were treated with TRAIL for 16 h at 37 ◦ C and then washed with PBS. DNA was isolated with the DNA purification kit, according to the manufacturer’s directions (Wizard® Genomic, Promega Co., WI, USA). DNA pellets were loaded onto a 1.5% agarose gel in Tris-acetate buffer (pH 8.0) and 1 mM EDTA, and separated at 100 V for 2 h. DNA fragments were stained with ethidium bromide and visualized using a UV transilluminator after. 2.8. Quantitative analysis of DNA fragmentation DNA fragmentation during apoptosis was assessed by performing a solid-phase enzyme-linked immunosorbent assay (ELISA). Keratinocytes (3 × 104 ) were treated with TRAIL for 16 h at 37 ◦ C, washed with phosphate-buffered saline (PBS) and fixed with formaldehyde solution. Deoxynucleotides (dNTPs) were incorporated at the 3 -ends of DNA fragments using terminal deoxynucleotidyl transferase (TdT) and the nucleotides were detected using streptavidin-horseradish peroxidase and TACSSapphire according to the TiterTACS protocol. Data were expressed as absorbance at 450 nm. 2.9. Western blot analysis It has been shown that cells start to show signs of cell death post 6 h treatment of TRAIL [19]. From this report, to assess the inhibitory effect of acertannin on TRAIL-induced apoptosis-related protein action as an early phenomenon, we investigated the effects at a 6 h-incubation. The cytosolic levels of Bid, Bax, Bcl-2, Bcl-xL, survivin, cytochrome c and p53 were assessed by Western blotting. Keratinocytes (5 × 106 cells/ml) were harvested by centrifugation at 412 × g for 10 min, washed twice with PBS and suspended in lysis buffer A (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin and 20 mM HEPES-KOH, pH 7.5). The lysates were homogenized further by successive passages through a 26-gauge hypodermic needle. The homogenates were centrifuged at 100,000 × g for 5–30 min depending on the protein that was being detected, and the supernatant was used for Western blotting analysis and ELISA. The protein concentration was determined by the Bradford method, according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). Cell mitochondrial fraction was isolated using digitonin lysis method [19,23]. Keratinocytes (5 × 106 ) were incubated with icecold digitonin lysis buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM PMSF, 190 ␮g/ml digitonin and 20 mM HEPES-KOH, pH 7.5) for 10 min. The cell suspension was centrifuged at 2500 × g for 10 min and the supernatant was re-centrifuged at 15,000 × g for 15 min. The subsequent pellet, taken as the mitochondrial fraction, was suspended in lysis buffer A. Cytosolic and mitochondrial fractions were mixed with sodium dodecyl sulfate-polyacrylamide gel electrphoresis (SDS-PAGE) sample buffer and boiled for 5 min. Samples (30 ␮g protein/well)

were loaded into each lane of a 12% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (GE Healthcare Chalfont St. Giles, Buckinghamshire, UK). Membranes were blocked for 2 h in TBS (50 mM Tris–HCl, pH 7.5 and 150 mM NaCl) containing 0.1% Tween 20 and 5% non-fat dried milk. The membranes were labeled with their specific antibodies overnight at 4 ◦ C with gentle agitation. After four washes in TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG for 2 h at room temperature. The membranes were incubated with SuperSignal® West Pico chemiluminescence substrate, and the proteins were detected using enhanced chemiluminescence in a Luminescent image analyzer (Lite for Las-1000 plus version 1.1, Fuji Photo Film Co., Tokyo, Japan). 2.10. Measurement of cytochrome c amount and caspase activity For the solid phase-ELISA detection of cytochrome c, cells (5 × 105 cells/ml) were suspended in lysis buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin and 20 mM HEPES-KOH, pH 7.5) to harvest whole cell lysates. The supernatants and cytochrome c conjugate were added to the 96well microplates coated with monoclonal antibody specific for human cytochrome c. The procedure was performed, according to the manufacturer’s instructions (R&D systems, Minneapolis, MN, USA). The absorbance of samples was measured at 450 nm in a microplate reader. A standard curve was constructed by plotting the absorbance values of diluted solutions of a cytochrome c standard. The amount was expressed as ng/ml. For quantitative analysis of caspase (-8, -9 and -3) activity, cells (2 × 106 cells/ml) were treated with TRAIL for 6 h at 37 ◦ C and activities of caspases were determined using the caspase assay kits, according to the manufacturer’s directions. The supernatant obtained from centrifugation of lysed cells was added to the reaction mixture containing dithiothreitol and caspase substrates (for -8, -9 and -3) and was incubated for 1 h at 37 ◦ C. The absorbance of the chromophore developed was measured at 405 nm. Data are expressed as arbitrary units. 2.11. Measurement of intracellular reactive oxygen species formation To clearly define the inhibitory effect of acertannin on TRAIL-induced formation of reactive oxygen species as an early phenomenon, we investigated the effects at a 6 h-incubation. The dye DCFH2 -DA, which is oxidized to fluorescent dichlorofluorescin (DCF) by hydroperoxides, was used to measure relative levels of cellular peroxides [24]. Keratinocytes (1 × 105 cells/400 ␮l in 24well plate) were treated with 50 ng/ml TRAIL for 6 h at 37 ◦ C. Cells were washed, suspended in fetal bovine serum-free RPMI-1640, incubated with 50 ␮M dye for 30 min at 37 ◦ C and washed with phosphate buffered saline. The cell suspensions were centrifuged at 412 × g for 10 min and medium was removed. Cells were dissolved with 1% Triton X-100 and fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (SPECTRAFLUOR, TECAN, Salzburg, Austria). 2.12. Measurement of nitrite/nitrate production To clearly define the inhibitory effect of acertannin on TRAILinduced formation of nitric oxide as an early phenomenon, we investigated the effects at a 6 h-incubation. Nitric oxide liberated from keratinocytes was measured by assaying nitric oxide metabolites, nitrite and nitrate (NOx ). Keratinocytes (1 × 105 cells/400 ␮l in

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24-well plate) were treated with 50 ng/ml TRAIL for 6 h at 37 ◦ C. The nitrate in the medium was reduced to nitrite by incubation with nitrate reductase (500 mU/ml), 160 ␮M NADPH and 4 ␮M flavin adenine dinucleotide at room temperature for 2 h. The medium was mixed with an equal amount of Griess reagent (Sigma–Aldrich Inc.). Absorbance was measured at 540 nm and the amount of nitrite was determined using sodium nitrite as the standard. The results were expressed as total nitrite equivalents (NOx ). 2.13. Statistical analysis Data are expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance. When significance was detected, the Duncan’s test for multiple comparisons was performed on the data from experimental groups. A probability value of p < 0.05 was considered statistically significant. 3. Results 3.1. Acertannin reduces TRAIL-induced cell death and DNA damage This study was designated to assess the effect of acertannin on TRAIL-induced cell death in keratinocytes. We first examined the effect of TRAIL on cell viability in keratinocytes. When keratinocytes were treated with 1–100 ng/ml TRAIL for 24 h, cell viability decreased in a concentration-dependent manner (Fig. 2A). The incidence of cell death after the treatment with 50 ng/ml TRAIL for 24 h was approximately 47%. We next examined the effect of acertannin on the TRAIL (50 ng/ml)-induced cell death in keratinocytes. Acertannin significantly reduced TRAIL-induced cell death in a concentration-dependent manner and at 50 ␮M it exhibited approximately 47% inhibition (Fig. 2B). Up to 100 ␮M acertannin alone did not induce cell death significantly. Using the neutral red uptake viability assay, we confirmed the inhibitory effect of acertannin on TRAIL-induced cell death. As shown in MTT reduction assay, treatment with 10–100 ␮M acertannin significantly reduced TRAIL-induced cell death in a concentration-dependent manner (Fig. 3). Acertannin alone did not induce cell death significantly. To clearly define the inhibitory effect of acertannin on apoptotic effect of TRAIL, we examined the effects at fixed concentrations (50 ng/ml TRAIL and 50 ␮M acertannin). To examine the effect of acertannin on TRAIL-induced nuclear damage, we investigated the changes in nuclear morphology in keratinocytes. Nuclear staining with Hoechst 33258 revealed that control cells had regular and round-shaped nuclei (a in Fig. 4A). In contrast, the condensation and fragmentation of nuclei characteristic of apoptotic cells were observed in cells treated with 50 ng/ml TRAIL (b in Fig. 4A). Acertannin (50 ␮M) prevented TRAIL-induced nuclear damage (d in Fig. 4A), while the nuclear morphology in cells treated with acertannin alone was similar to that in control cells (c in Fig. 4A). During apoptosis, DNA fragmentation is caused by activation of endonucleases. The effect of TRAIL and acertannin on the DNA fragmentation as nuclear damage was assessed by agarose gel electrophoresis. DNA extracted from keratinocytes displayed a small increase in the oligonucleosomal cleavage of DNA (lane 1 in Fig. 4B). In contrast, incubation with 50 ng/ml TRAIL for 16 h increased the DNA laddering in keratinocytes (lane 2 in Fig. 4B). Acertannin (50 ␮M) prevented the TRAIL-induced DNA laddering, which was greater than TRAIL alone (lane 4 in Fig. 4B). Acertannin alone did not affect the DNA laddering, which was similar to the finding in control cells (lane 3 in Fig. 4B). We further assessed the inhibitory effect of acertannin on TRAILinduced DNA damage by performing the quantitative analysis of

Fig. 2. Effect of acertannin on TRAIL-induced cell death in keratinocytes. (A) Keratinocytes were treated with 1–100 ng/ml TRAIL for 24 h and then cell viability was determined using a MTT reduction assay. (B) Keratinocytes were treated with 50 ng/ml TRAIL in the presence of 1–100 ␮M acertannin for 24 h. Data represent mean ± S.E.M. (n = 6). + p < 0.05 compared to control (percentage of control); *p < 0.05 compared to TRAIL alone.

Fig. 3. Acertannin reduces TRAIL-induced cell death. Keratinocytes were treated with 50 ng/ml TRAIL in the presence of 10–100 ␮M acertannin for 24 h and then cell viability was determined using neutral red uptake assay. Data represent mean ± S.E.M. (n = 6). + p < 0.05 compared to control (percentage of control); *p < 0.05 compared to TRAIL alone.

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Fig. 4. Effect of acertannin on TRAIL-induced nuclear damage. Keratinocytes were pre-treated with 50 ␮M acertannin for 20 min and then exposed to 50 ng/ml TRAIL in combination with acertannin for 16 h. (A) Cells were observed by fluorescence microscopy after staining of the nuclei with Hoechst 33258. The figure represents microscopic morphology of the control cells (a); cells treated with TRAIL (b); cells treated with acertannin alone (c) and cells treated with TRAIL plus acertannin (d). (a)–(d) are representative of four different experiments. (B) DNA was extracted, separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, untreated cells; lane 2, cells treated with TRAIL; lane 3, cells treated with acertannin alone and lane 4; cells treated with TRAIL plus acertannin. Data are representative of three independent experiments. (C) The 3 -ends of DNA fragments were detected as described in Section 2. Data are expressed as absorbance and represent mean ± S.E.M. (n = 6). + p < 0.05 compared to control; *p < 0.05 compared to TRAIL alone.

DNA fragmentation. The amount of fragmented DNA was measured by monitoring the binding of dNTP to the 3 -ends of DNA fragments and detected by a quantitative colorimetric assay. Control keratinocytes showed absorbance of 0.115 ± 0.009 (mean ± S.E.M., n = 6), while exposure to 50 ng/ml TRAIL alone for 16 h increased the absorbance approximately 2.5-fold. Combination of 50 ␮M acertannin markedly reduced the TRAIL-induced DNA fragmentation, while acertannin alone did not cause DNA damage (Fig. 4C).

3.2. Acertannin reduces activation of apoptosis-related proteins We examined whether TRAIL-induced apoptosis was mediated by caspase activation using specific caspase inhibitors. As shown in Fig. 5, treatment with 30 ␮M z-IETD.fmk, z-LEHD.fmk or z-DQMD.fmk (cell permeable inhibitors of caspase-8, -9 or -3, respectively) reduced the 50 ng/ml TRAIL-induced cell death. Caspase inhibitors alone did not affect cell viability. We assessed the effect of acertannin on the TRAIL-induced cell death process by measuring the activation of apoptosisrelated proteins in keratinocytes. Treatment with 50 ng/ml TRAIL

Fig. 5. Effect of caspase inhibitors on TRAIL-induced cell death. Keratinocytes were pre-treated with 30 ␮M caspase inhibitors (z-IETD.fmk, z-LEHD.fmk or zDQMD.fmk) for 20 min and exposed to 50 ng/ml TRAIL in the presence of caspase inhibitors for 24 h. Cell viability was then determined. Data represent mean ± S.E.M. (n = 6). + p < 0.05, compared to control; and *p < 0.05, compared to TRAIL alone.

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Fig. 6. Effect of acertannin on TRAIL-induced change in apoptosis-related protein levels. (A) Keratinocytes were pre-treated with 50 ␮M acertannin for 20 min and then exposed to 50 ng/ml TRAIL for 6 h. The levels of Bid, Bax, Bcl-2, Bcl-xL, survivin, cytochrome c and p53 were analyzed by Western blotting with their specific antibodies. Data are representative of three independent experiments. (B–D) Keratinocytes were pre-treated with 50 ␮M acertannin or the scavengers (1 mM N-acetylcysteine (NAC), 30 ␮M trolox or 30 ␮M Mn-TBAP (TBAP)) for 20 min and then exposed to 50 ng/ml TRAIL in combination with acertannin or scavengers for 6 h. The amount of released cytochrome c and activities of caspases (-8, -9 and -3) were measured using analysis kits. Data are expressed as ng/ml for cytochrome c and arbitrary units (a.u.) for activities of caspases. Values represent mean ± S.E.M. (n = 6). + p < 0.05 compared to control; *p < 0.05 compared to TRAIL alone.

caused a decrease in cytosolic Bid, Bcl-2, Bcl-xL and survivin levels, a decrease in mitochondrial cytochrome c levels but an increase in cytosolic Bax and cytochrome c levels (Fig. 6A). Treatment with 50 ␮M acertannin prevented TRAIL-induced changes in the Bid, Bax, Bcl-2, Bcl-xL, survivin and cytochrome c levels. We further examined whether inhibitory effect of acertannin on TRAIL-induced apoptosis was mediated by changes in tumor suppressor p53 levels. Treatment with 50 ng/ml TRAIL induced an increase in p53 levels in keratinocytes (Fig. 6A). Acertannin (50 ␮M) prevented TRAIL-induced alteration of p53 levels. Acertannin alone did not induce alterations of apoptosis-related protein levels. We confirmed the inhibitory effect of acertannin on the TRAIL-induced cytochrome c release and caspase-3 activation by performing quantitative analysis. Treatment with 50 ng/ml TRAIL induced release of cytochrome c and activation of caspases (-8, -9 and -3) in keratinocytes (Fig. 6B–D). Acertannin (50 ␮M) reduced TRAIL-induced cytochrome c release and activation of caspases (-8, -9 and -3). Acertannin alone did not induce cytochrome c release and activation of caspases. 3.3. Acertannin inhibits formation of reactive oxygen species and nitric oxide To examine whether TRAIL-induced apoptosis is mediated by actions of reactive oxygen and nitrogen species, we assessed the oxidant scavenger effect on cytochrome c release and caspase3 activation. Treatment with 1 mM N-acetylcysteine, 30 ␮M trolox (a scavenger of hydroxyl radicals and peroxynitrite) or

30 ␮M Mn-TBAP (a scavenger of peroxynitrite and cell-permeable metalloporphyrin that mimics superoxide dismutase) reduced TRAIL-induced cytochrome c release and activation of caspases (-8, -9 and -3) (Fig. 6B–D). We further examined whether TRAIL-induced cell death was mediated by actions of reactive oxygen and nitrogen species using oxidant scavengers. Cells were treated with 50 ng/ml TRAIL in the presence of various scavengers for 24 h. Treatment with 1 mM Nacetylcysteine, 30 ␮M trolox, 30 ␮M carboxy-PTIO (a scavenger of nitric oxide) and 30 ␮M Mn-TBAP reduced cell death caused by TRAIL (Fig. 7A). We then assessed the formation of reactive oxygen species as the response of stimulated keratinocytes. The formation of reactive oxygen species within cells was determined by monitoring a conversion of DCFH2 -DA to DCF. Keratinocytes treated with 50 ng/ml TRAIL showed a significant increase in DCF fluorescence. The TRAILinduced formation of reactive oxygen species was confirmed by the inhibitory effect of 1 mM N-acetylcysteine or 30 ␮M trolox. Treatment with 50 ␮M acertannin inhibited TRAIL-induced increase in DCF fluorescence (Fig. 7B). We examined the production of nitric oxide in keratinocytes exposed to lipopolysaccharide. Keratinocytes treated with 50 ng/ml TRAIL liberated 4.72 ± 0.29 ␮M NOx (nitric oxide metabolites nitrite and nitrate; mean ± S.E.M., n = 6), which was inhibited by the addition of 1 mM N-acetylcysteine, 30 ␮M carboxy-PTIO or l-NMMA (500 ␮M, an inhibitor of nitric oxide synthase). Acertannin (50 ␮M) significantly inhibited TRAIL-induced formation of NOx (Fig. 7C). These results suggest that acertannin inhibits the formation of reactive oxygen species and nitric oxide.

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4. Discussion

Fig. 7. Effect of acertannin on TRAIL-induced formation of reactive oxygen species and nitric oxide. In experiment (A), keratinocytes were treated with 50 ng/ml TRAIL in the presence of 50 ␮M acertannin or the scavengers [1 mM N-acetylcysteine (NAC), 30 ␮M trolox, 30 ␮M carboxy-PTIO (PTIO) and 30 ␮M Mn-TBAP (TBAP)] for 24 h and then cell viability was determined. Data represent mean ± S.E.M. (n = 6). + p < 0.05 compared to control; *p < 0.05 compared to TRAIL alone. (B) Keratinocytes were 50 ␮g/ml TRAIL in the presence of compounds [50 ␮M acertannin, 1 mM Nacetylcysteine (NAC) or 30 ␮M trolox] for 6 h. Changes in DCF fluorescence were determined. Data are expressed as arbitrary units (a.u.) of fluorescence and represent mean ± S.E.M. (n = 6). (C) Keratinocytes were pre-treated with compounds [50 ␮M acertannin, 1 mM N-acetylcysteine (NAC), 500 ␮M NMMA or 30 ␮M carboxy-PTIO] for 20 min and then exposed to 50 ng/ml TRAIL in combination with compounds for 6 h. Reaction mixtures were mixed with Griess reagent and absorbance was measured. Data represent the amounts of NOx (nitric oxide metabolites nitrite and nitrate) and are mean ± S.E.M. (n = 5). + p < 0.05 compared to control; *p < 0.05 compared to TRAIL alone.

Activation of TRAIL receptor recruits the adapter proteins, such as Fas-associated protein with death domain and a pro-caspase-8, to form a death-inducing signaling complex (DISC), which causes the autoproteolytic activation and release of caspases [9,10,25]. Activation of caspase-8 induces activation of caspase-3 and -7, and activates the apoptosis initiator Bid protein, which causes mitochondrial cytochrome c release and caspase-9 activation, leading to the caspase-3 activation and apoptosis [26]. Caspase-9 induces activation of caspase-3 through formation of an apoptosome complex with cytochrome c released from mitochondria [27,28]. With respect to apoptosis-related proteins, the activation of pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 family protein levels causes the release of cytochrome c and apoptosis [29]. Survivin, a member of the inhibitor of apoptosis family, functions to inhibit caspase activation [26]. In this study, TRAIL treatment decreased Bid, Bcl-2, Bcl-xL and survivin levels, increased Bax levels, induced cytochrome c release and activated caspases (-8, -9 and -3) in keratinocytes. TRAIL-induced apoptosis in keratinocytes was demonstrated by the condensation and fragmentation of nuclei and an increase in both the release of cytochrome c and the caspase-3 activation. Therefore, changes in apoptosis-related proteins and the preventive effect of caspase inhibitors suggest that TRAIL induces apoptosis in keratinocytes by activating the caspase-8- and Bid-dependent pathways as well as the mitochondria-mediated cell-death pathway, which leads to activation of caspase-3. Regulation of disrupted apoptosis in keratinocytes has been suggested to confer a benefit in the treatment of inflammatory skin diseases [8]. Acertannin, one of tannins, may have an antioxidant effect [14]. However, the effect of acertannin on apoptosis in keratinocytes has not been studied. In this aspect, we examined the effect of acertannin on TRAIL-induced apoptosis in human keratinocytes. Acertannin prevented the TRAIL-induced changes in apoptosis-related protein levels followed by the mitochondrial cytochrome c release and caspase activation. The results suggest that acertannin may prevent TRAIL-induced apoptosis in keratinocytes by suppressing the caspase-8- and Bid-dependent pathways as well as the mitochondria-mediated cell death pathway, leading to activation of caspases, such as -9 and -3. The tumor suppressor and transcription factor p53 modulates cellular stress responses, and the activation of p53 can trigger apoptosis [30,31]. p53 stimulates either the mitochondria-mediated cell death or the death receptor pathway and mediates apoptosis induced by various insults, including DNA damage and oxidative stress [31–33]. p53 acts as a direct transcriptional activator of the Bax gene. We examined whether the inhibitory effect of acertannin on the TRAIL-induced cell death was mediated by its effect on p53 expression. In the present study, acertannin inhibited TRAIL-induced increase in p53 levels in keratinocytes. Therefore, acertannin may reduce apoptotic effect of TRAIL on keratinocytes by suppressing increase in p53 levels. Reactive oxygen species and nitrogen species, including nitric oxide, play a critical role in the physiological regulation of cellular functions and are involved in pathologic conditions, such as chronic inflammatory diseases and airway disease [34,35]. Reactive oxygen species have been shown to be involved in the cytokine production and inflammation in skin [36,37]. Oxidants provoke amplification of inflammatory processes in the airways and lung parenchyma, causing protein dysfunction and cell damage [34,35]. Reactive oxygen species act upon mitochondria, causing changes in the mitochondrial membrane permeability and cytochrome c release [38]. Reactive oxygen species cause changes in the levels of intracellular antioxidants such as GSH, NADH or NADPH, which results in impairment of mitochondrial function [38]. Apop-

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tosis induced by TNF and its family members is mediated via the production of reactive oxygen species [39]. The formation of reactive oxygen species in keratinocytes treated with TRAIL and the inhibitory effect of antioxidants, including N-acetylcysteine and carboxy-PTIO, on cell death, suggest that TRAIL induces the formation of reactive oxygen species and nitrogen species, which is involved in mitochondrial dysfunction and cell death. We examined whether the preventive effect of acertannin on TRAIL-induced apoptosis was linked to the inhibitory effect on oxidative stress. As shown in the data, acertannin significantly reduced the TRAILinduced formation of reactive oxygen species and nitric oxide. Therefore, acertannin may prevent TRAIL-induced activation of the apoptosis-related proteins and changes in the mitochondrial membrane permeability by suppressing the formation of reactive oxygen and nitrogen species. Overall, the results suggested that acertannin may prevent the apoptotic effect of TRAIL on keratinocytes by suppressing the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated cell death pathway, leading to the activation of caspase-3. The preventive effect of acertannin on TRAIL-induced apoptosis may be associated with the inhibitory effect on formation of reactive oxygen/nitrogen species. Acertannin may confer a benefit in the treatment of apoptosis-mediated skin inflammatory diseases. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A091121). References [1] S. Pastore, F. Mascia, G. Girolomoni, The contribution of keratinocytes to the pathogenesis of atopic dermatitis, Eur. J. Dermatol. 16 (2006) 125–131. [2] D. Tsuruta, NF-␬B links keratinocytes and lymphocytes in the pathogenesis of psoriasis, Recent Pat. Inflamm. Allergy Drug Discov. 3 (2009) 40–48. [3] A. Trautmann, M. Akdis, P. Schmid-Grendelmeier, R. Disch, E.B. Brocker, K. Blaser, C.A. Akdis, Targeting keratinocyte apoptosis in the treatment of atopic dermatitis and allergic contact dermatitis, J. Allergy Clin. Immunol. 108 (2001) 839–846. [4] T.J. Kimberley, G.R. Screaton, Following a TRAIL: update on a ligand and its five receptors, Cell Res. 14 (2004) 359–372. [5] E. Vassina, M. Leverkus, S. Yousefi, L.R. Braathen, H.U. Simon, D. Simon, Increased expression and a potential anti-inflammatory role of TRAIL in atopic dermatitis, J. Invest. Dermatol. 125 (2005) 746–752. [6] P. Warnnissorn, A. Nakao, H. Suto, H. Ushio, N. Yamaguchi, H. Yagita, K. Okumura, H. Ogawa, Tumour necrosis factor-related apoptosis-inducing ligand expression in atopic dermatitis, Brit. J. Dermatol. 148 (2003) 829–831. [7] A. Trautmann, M. Akdis, D. Kleemann, F. Altznauer, H.U. Simon, T. Graeva, M. Noll, E.B. Brocker, K. Blaser, C.A. Akdis, T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis, J. Clin. Invest. 106 (2000) 25–35. [8] I. Boehm, Apoptosis in physiology and pathologic skin: implications for therapy, Curr. Mol. Med. 6 (2006) 375–394. [9] G.S. Wu, TRAIL as a target in anti-cancer therapy, Cancer Lett. 285 (2009) 1–5. [10] Z. Mahmood, Y. Shukla, Death receptors: targets for cancer therapy, Exp. Cell Res. 316 (2010) 887–899. [11] W. Guo, E. Kong, M. Meydani, Dietary polyphenols, inflammation, and cancer, Nutr. Cancer 61 (2009) 807–810.

59

[12] B.W. Bolling, D.L. McKay, J.B. Blumberg, The phytochemical composition and antioxidant actions of tree nuts, Asia Pac. J. Clin. Nutr. 19 (2010) 117–123. [13] J.A. Nichols, S.K. Katiyar, Skin photoprotection by natural polyphenols: antiinflammatory, antioxidant and DNA repair mechanisms, Arch. Dermatol. Res. 302 (2010) 71–83. [14] S.S. Han, S.C. Lo, Y.W. Choi, J.H. Kim, S.H. Bae, AT Antioxidant activity of crude extract and pure compounds of Acer ginnala Max, Bull. Korean Chem. Soc. 25 (2004) 389–391. [15] K.S. Suh, S. Chon, S. Oh, S.W. Kim, J.W. Kim, Y.S. Kim, J.T. Woo, Prooxidative effects of green tea polyphenol (−)-epigallocatechin-3-gallate on the HIT-T15 pancreatic beta cell line, Cell Biol. Toxicol. 26 (2010) 189–199. [16] Y. Sakihama, M.F. Cohen, S.C. Grace, H. Yamasaki, Plant phenolic antioxidant and prooxidant activities: phenolics-induced oxidative damage mediated by metals in plants, Toxicology 177 (2002) 67–80. [17] P. Fresco, F. Borges, C. Diniz, M.P. Marques, New insights on the anticancer properties of dietary polyphenols, Med. Res. Rev. 26 (2006) 46–66. [18] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [19] Y. Dai, M. Liu, W. Tang, Y. Li, J. Lian, T.S. Lawrence, L. Xu, A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB, BMC Cancer 9 (2009) 392. [20] S. Huang, K. Okumura, F.A. Sinicrope, BH3 mimetic obatoclax enhances TRAILmediated apoptosis in human pancreatic cancer cells, Clin. Cancer Res. 15 (2009) 150–159. [21] V. Andrisano, R. Ballardini, P. Hrelia, N. Cameli, A. Tosti, R. Gotti, V. Cavrini, Studies on the photostability and in vitro phototoxicity of Labetalol, Eur. J. Pharm. Sci. 12 (2001) 495–504. [22] F.A. Oberhammer, M. Pavelka, S. Sharma, R. Tiefenbacher, A.F. Purchio, W. Bursch, R. Schulte-Hermann, Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor ␤1, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 5408–5412. [23] H. Wu, G.N. Rao, B. Dai, P. Singh, Autocrine gastrins in colon cancer cells upregulate cytochrome c oxidase Vb and down-regulate efflux of cytochrome c and activation of caspase-8, J. Biol. Chem. 275 (2000) 32491–32498. [24] W. Fu, H. Luo, S. Parthasarathy, M.P. Mattson, Catecholamines potentiate amyloid ␤-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis, Neurobiol. Dis. 5 (1998) 229–243. [25] M. MacFarlane, TRAIL-induced signaling and apoptosis, Toxicol. Lett. 139 (2003) 89–97. [26] Z. Jin, W.S. El-Deiry, Overview of cell death signaling pathways, Cancer Biol. Ther. 4 (2005) 139–163. [27] W. Hu, J.J. Kavanagh, Anticancer therapy targeting the apoptotic pathway, Lancet Oncol. 4 (2003) 721–729. [28] A. Camins, M. Pallas, J.S. Silvestre, Apoptotic mechanisms involved in neurodegenerative diseases: experimental and therapeutic approaches, Methods Find. Exp. Clin. Pharmacol. 30 (2008) 43–65. [29] P.E. Czabotar, P.M. Colman, D.C. Huang, Bax activation by Bim? Cell Death Differ. 16 (2009) 1187–1191. [30] J.E. Chipuk, D.R. Green, Dissecting p53-dependent apoptosis, Cell Death Differ. 13 (2006) 994–1002. [31] K.G. Wiman, Strategies for therapeutic targeting of the p53 pathway in cancer, Cell Death Differ. 13 (2006) 921–926. [32] F. Chen, F.W. Wang, W.S. El-Deiry, Current strategies to target p53 in cancer, Biochem. Pharmacol. 80 (2010) 724–730. [33] B. Zhivotovsky, S. Orrenius, Cell death mechanisms: cross-talk and role in disease, Exp. Cell Res. 316 (2010) 1374–1383. [34] G. Caramori, A. Papi, Oxidants and asthma, Thorax 59 (2004) 170–173. [35] A.E. Redington, Modulation of nitric oxide pathways: therapeutic potential in asthma and chronic obstructive pulmonary disease, Eur. J. Pharmacol. 533 (2006) 263–276. [36] H.B. Köhler, B. Huchzermeyer, M. Martin, A. De Bruin, B. Meier, I. Nolte, TNF-␣ dependent NF-␬B activation in cultured canine keratinocytes is partly mediated by reactive oxygen species, Vet. Dermatol. 12 (2001) 129–137. [37] C.N. Young, J.I. Koepke, L.J. Terlecky, M.S. Borkin, S.L. Boyd, S.R. Terlecky, Reactive oxygen species in tumor necrosis factor-␣-activated primary human keratinocytes: implications for psoriasis and inflammatory skin disease, J. Invest. Dermatol. 128 (2008) 2606–2614. [38] B. Mignotte, J.L. Vayssière, Mitochondria and apoptosis, Eur. J. Biochem. 252 (1998) 1–15. [39] M. Shakibaei, G. Schulz-Tanzil, Y. Takeda, B.B. Aggarwal, Redox regulation of apoptosis by members of the TNF superfamily, Antioxid. Redox Signal. 7 (2005) 482–496.