Article pubs.acs.org/crt
Photoprotective Effects of Oxyresveratrol and Kuwanon O on DNA Damage Induced by UVA in Human Epidermal Keratinocytes Shuting Hu,† Feng Chen,‡ and Mingfu Wang*,† †
School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong Institute for Food & Bioresource Engineering, College of Engineering, Peking University, Beijing, P. R. China
‡
ABSTRACT: Ultraviolet A not only plays a major part in photoaging and skin tanning but also induces genetic damage and mutation in the epidermal basal layer of human skin. The photoprotective effect of oxyresveratrol and kuwanon O, two phenolic compounds from the root extract of Morus australis, in human primary epidermal keratinocytes was investigated in this study. Both of them were nontoxic to cells at a concentration less than 10 and 0.5 μM, respectively. After pretreatment at the concentrations of 5 and 10 μM, oxyresveratrol increased cell viability, exhibited significant suppressions on UVA- or H2O2induced cellular ROS. UVA-enhanced nitrotyrosine was also reduced by post-treatment with oxyresveratrol at theses concentrations. Kuwanon O presented similar inhibitions on cellular ROS and nitrotyrosine with lower concentrations (0.25 and 0.5 μM), but there is no significant protection on cell survival after UVA irradiation. Their photoprotective effects also involved the enhanced repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and cyclobutane pyrimidine dimers (CPDs) as mediated by the augment of p53 expression after UVA radiation.
1. INTRODUCTION
genetic damage and mutations in the epidermal basal layer, which is consisted of dividing keratinocyte cells. Using active photoprotectives is one crucial approach to protect the skin from the harmful effects of UV irradiation. Recently, naturally occurring compounds such as plant polyphenols have gained considerable attention as protective agents.7 Plant polyphenols are members of a group of natural products also known as phenols or phenolics. They are considered to be safe and largely free of side effects. Some famous phenolic compounds, including green tea polyphenols (GTPs) and grape seed polyphenols (GSPs), are well studied for their photoprotective effects.7 They were also reported in the past to exert their photoprotective effects by accelerating DNA repair, as well as scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS).8 Morus species, which are rich in phenolic compounds, were found to possess beneficial activities such as antioxidant, anti-inflammatory, as well as anticancer potentials.9−11 Among them, Morus australis is one species growing in and commonly distributed in East Asia. According to some previous research works from our laboratory, the root extract of Morus australis is rich in phenolic compounds.12 Twenty-two phenolic compounds were isolated and identified from the extract. Among these phenolic compounds, only oxyresveratrol and kuwanon O (Figure 1)
Sunlight exposure is inevitable for all humans and UV radiation in sunlight usually causes damage to the skin, leading to sunburn, tanning, and even to DNA damage. As most ultraviolet C (UVC) can be absorbed by the ozone layer and does not reach the earth, ultraviolet A (UVA) and ultraviolet B (UVB) are two major types of ultraviolet radiation that can damage the skin, which is a major target organ of UV irradiation. UVA has the longest wavelength of these three at 320−400 nm, and UVB ranges from 290 to 320 nm. During the past decades, UVB was considered to be more genotoxic than UVA.1 It is also considered as the main cause of sunburn and nonmelanoma skin cancer, which comprised mostly basal cell carcinomas and squamous cell carcinomas.2,3 However, recent investigations indicated that UVA was likely to be more important to induce human skin carcinogenesis.4 UVA is more than 20 times abundant in sunlight than UVB and can penetrate the skin deeper.5 It is well known that cancers usually arise from dividing basal layer cells, rather than fully differentiated cells at the human epidermal surface. UVB can effectively generate cyclobutane pyrimidine dimers (CPDs) and 6−4 photoproducts (6−4 PP) in the upper layer, while UVA can induce CPDs predominately in the basal layer. Moreover, UVA also leads to the accumulation of 8-hydroxy-2′ -deoxyguanosine (8-OHdG) or 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG), a ubiquitous biomarker of DNA oxidation in the basal epidermis.6 Therefore, UVA is more likely to induce © XXXX American Chemical Society
Special Issue: Chemical Toxicology in China Received: December 2, 2014
A
DOI: 10.1021/tx500497u Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
H2O2 treatment as described before.13 Briefly, keratinocytes were plated at a density of 15,000 cells per well in 96-well plates 24 h before use. HEK cells were pretreated for 24 h with different concentrations of agents and then washed three times with PBS before UVA irradiation or H2O2 treatment. After irradiation by 4.32 J/cm2 UVA or treatment with 200 μM of H2O2 for 2 h, fluorometric determination of intracellular ROS was estimated by loading the cells with 100 μL of DCFA (25 uM) in PBS for 30 min at 37 °C. Plates were placed in Victor X4Multilabel Plate Reader (PerkinElmer, MA, USA). Fluorescence was monitored using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Individual absorbance values were corrected with cell viability with the CCK-8 assay before pooling. Each experimental point was performed in triplicate. 2.5. Measurement of nitrotyrosine. The detection of nitrotyrosine was performed using reagents provided in a competitive ELISA kit ab113848 (Abcam, Cambridge, MA, USA). 3NT BSA was used as a standard positive control to validate the assays. In brief, 2 × 105/well keratinocytes were seeded in 24-well plate 24 h before use. Collection of adherent cells by scraping occurred at 16 h after UVA irradiation and subsequent treatment by phenolic agents.14 The cell pellet was solubilized in extraction buffer for 20 min on ice. After centrifuging at 4 °C for 20 min, the supernatants were collected to clean tubes. Each sample was diluted and adjusted to approximately the same protein concentration by a protein assay (Bio-Rad, Hercules, CA). Fifty microliters of each diluted standard or sample, together with 50 μL of 2× HRP detector antibody, was added to each well of nitrotyrosine coated 96-well microplates and incubated for 2 h at room temperature. After washing four times, 100 μL of HRP development solution was added to each well. OD values were measured according to the manufacturer’s instructions. The concentrations of nitrotyrosine were calculated by taking into account the standard curve created by 3NT BSA. 2.6. Measurement of 8-OHdG. As one of the oxidative DNA damage byproducts, 8-OHdG is a ubiquitous maker of oxidative stress.15 The measurement of 8-OHdG in DNA samples was achieved using OxiSelect Oxidative DNA Damage ELISA Kit (8-OHdG Quantitation) (Cell Biolabs, San Diego, USA). Six separate experiments were performed for each group (UVA; UVA + oxyresveratrol; UVA + kuwanon O; no UVA vehicle; oxyresveratrol only; and kuwanon O only). Keratinocytes were seeded in a 6-well plate at a density of 8 × 105/well. Following 24 h of incubation, three groups of cells were exposed to 4.32 J/cm2 UVA in a thin layer of PBS. Immediately after irradiation, cells were further incubated with or without treatment by oxyresveratrol and kuwanon O for 60 min at 37 °C before detachment. Subsequently, DNA samples were extracted as described above. Each DNA sample was diluted to 40 μg/mL in cold PBS. DNA samples were then converted to singlestranded DNA by incubating the DNA sample at 95 °C for 10 min and rapidly chilling on ice for 10 min. Denatured DNA samples were digested to nucleosides after incubating with 10 units of nuclease P1 for 2 h at 37 °C in 20 mM sodium acetate (pH 5.2), and followed with treatment with 5 units of alkaline phosphatase for 1 h at 37 °C in 100 mM of Tris (pH 7.5). The reaction mixture was then centrifuged for 5 min at 6000g, and the supernatant was used for the 8-OHdG ELISA assay. Fifty microliters of sample or 8-OHdG standard was added to the 8-OHdG conjugate coated plate and incubated at room temperature for 10 min on an orbital shaker. Fifty microliters of the diluted anti-8-OHdG antibody was then added to each well and incubated at room temperature for 1 h in an orbital shaker. Subsequently, each well was washed 3 times with 250 μL of 1X wash buffer and followed by incubation with 100 μL of the diluted secondary antibody−enzyme conjugate for 1 h. After washing three times as described, 100 μL of substrate solution was immediately added to each well. The enzyme reaction was stopped by adding 100 μL of stop solution into each well, and the results were read immediately on Victor X4Multilabel Plate Reader (PerkinElmer, MA, USA) at 450 nm. 2.7. Measurement of CPDs. The detection and quantitation of CPDs in DNA samples were achieved using OxiSelect Oxidative UVInduced DNA Damage ELISA Kit (Cell Biolabs, San Diego, USA). Six
Figure 1. Chemical structures of oxyresveratrol and kuwanon O.
presented significant antioxidant effects. They are potential photoprotective agents. In this study, their effects on cell survival rate, as well as cellular ROS and nitrotyrosine after UVA irradiation using human primary epidermal keratinocyte, were investigated. In addition, the enhancement of DNA repair was also evaluated by measuring UVA-induced CPDs.
2. MATERIALS AND METHODS 2.1. Reagents. Oxyresveratrol and kuwanon O were purified from the roots of Morus australis as described before.12 They were dissolved at a concentration of 100 mM in DMSO solution to make the stock solution. The stock solution was diluted to the desired final concentration with growth medium just before use. The final DMSO concentration did not exceed 0.1%. Dermal cell basal media and the keratinocyte growth kit were purchased from American Type Culture Collection (Rockville, MD, USA). Trypsin-EDTA was purchased from Invitrogen (Carlsbad, CA, USA); CCK-8 was purchased from Dojindo China Co., Ltd. (Shanghai, China); DMSO and DCFA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The nitrotyrosine ELISA kit was purchased from Abcam (Cambridge, MA, USA), CPD and 8-OHdG ELISA kits were purchased from Cell Biolabs (San Diego, CA, USA). 2.2. Cell Culture, Phenolic Compound Treatment, and UVA Irradiation. Human primary epidermal keratinocytes were purchased from American Type Culture Collection (Rockville, MD, USA). Keratinocyte at passages 1−5 were cultured in dermal cell basal medium at 37 °C in 5% CO2. Culture medium was supplemented with the keratinocyte growth kit, which contained 0.4% bovine pituitary extract (BPE), 0.5 ng/mL rh TGF-α, 6 mM L-glutamine, 100 ng/mL hydrocortisone, 5 μg/mL insulin, 1.0 μM epinephrine, and 5 μg/mL apo-transferrin. Oxyresveratrol and kuwanon O were diluted to different concentrations and added to the cell medium before or after UV irradiation according to the different experimental setups. Semiconfluent cells (70%) were irradiated using a UVA fluorescent lamp (Vilber Lourmat, France), with a total of 4.32 J/cm2 UVA. All cells were treated identically throughout the procedure; the sham plate was shielded during irradiation. At the time of irradiation, cells were washed with PBS, covered with a thin layer of PBS, and irradiated with UVA without a plastic lid. The intensity of UVA irradiation was measured using a UVA-365 radiometer (Lutron Co, Taiwan). 2.3. Cell Viability Measurement. The cell survival rate was measured by the CCK-8 assay according to the manufacturer’s instructions. After the indicated treatment by chemical agents or UVA irradiation, cells were washed three times with PBS. Subsequently, 10% of CCK-8 solution in culture medium was added to each well. After incubation for 2 h, absorbance was measured at 450 nm using Victor X4Multilabel Plate Reader (PerkinElmer, MA, USA). Values are expressed as the mean cell viability as a percentage of that of the vehicle DMSO (0.1% final volume) − treated cultures. 2.4. Cellular Oxidative Stress (ROS) Measurement. The formation of ROS in cells was evaluated by means of DCFA corrected with the CCK-8 assay to account for cell loss after UVA radiation or B
DOI: 10.1021/tx500497u Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
Figure 2. Dose influence of oxyresveratrol (a) and kuwanon O (b). Cells were treated with oxyresveratrol and kuwanon O at different concentrations and cultured for 24 h. Cell viability was determined by the CCK-8 assay. Each value is presented as the mean ± SD from triplicate independent experiments. separate experiments were performed for each group (UVA; UVA + oxyresveratrol; UVA + kuwanon O; no UVA Vehicle; oxyresveratrol only; and kuwanon O only). Keratinocytes were seeded in 12-well plates at a density of 4 × 105 /well. Following 24 h of incubation, three groups of cells were exposed to 4.32 J/cm2 UVA in a thin layer of PBS. Immediately after irradiation, cells were further incubated with or without treatment by oxyresveratrol and kuwanon O for 60, 90, and 120 min at 37 °C before detachment. Subsequently, cells were harvested using 0.05% Trypsin-EDTA solution to detach the cells gently, avoiding unwanted DNA damage. DNA samples were extracted using a DNeasy blood and tissue kit (Qiagen, CA, USA). Each DNA sample was diluted to 2 μg/mL in cold PBS. DNA samples and the CPD-DNA standard were then converted to single-stranded DNA by incubating the DNA sample at 95 °C for 10 min and rapidly chilling on ice for 10 min. One hundred microliters of dilute denatured DNA sample in cold PBS or CPD-DNA standards was added to the wells of the DNA high-binding plate. After incubation at 37 °C for 2 h, DNA solutions were removed and washed twice by PBS, followed by the addition of 150 μL of assay diluent and blocking for 1 h at room temperature. Subsequently, each well was probed with 100 μL of the diluted anti-CPD antibody at room temperature for 1 h on an orbital shaker. Afterward, the plate was washed with 250 μL 1× wash buffer per well three times, and then 150 μL of prediluted 1× blocking reagent was added to each well and incubated for 60 min at room temperature in an orbital shaker. After washing three times as indicated before, 100 μL of diluted secondary antibody−enzyme conjugate was added to each well for 1 h. Color development was initiated by the addition of 100 μL of substrate solution after thoroughly washing three times with 1× wash buffer. The substrate was allowed to develop for 30−40 min at room temperature before being stopped by the addition of 100 μL of stop solution into each well. Binding of the CPD antibody was determined by reading the absorbance at 450 nm on Victor X4Multilabel Plate Reader (PerkinElmer, MA, USA). The quantity of CPDs in each sample was then determined by comparing its absorbance with that of a known CPD-DNA standard curve. 2.8. Measurement of p53 Expression. The measurement of p53 expression was performed using reagents provided in p53 Human ELISA Kit ab117995 (Abcam, Cambridge, MA, USA). Keratinocytes (2 × 105/well) were seeded in 24-well plates 24 h before use. Collection of adherent cells by scraping occurred at 16 h after UVA irradiation and subsequent treatment by phenolic agents.14 The cell pellet was solubilized in extraction buffer for 20 min on ice. After centrifuging at 4 °C for 20 min, the supernatants were collected to clean tubes. Each sample was diluted and adjusted to approximately the same protein concentration by a protein assay (Bio-Rad, Hercules, CA). Fifty microliters of each diluted standard or sample was added to each well of nitrotyrosine coated 96-well microplates and incubated for 2 h at room temperature. After washing four times, 50 μL of 1× detector antibody was added to each well and incubated for 1 h at room temperature. After washing as described above again, 50 μL of
the HRP label was added to each well for 1 h of incubation. Followed by washing three times, 100 μL of TMB development solution was added to each well. The results were read immediately after adding 100 μL of stop solution on Victor X4Multilabel Plate Reader (PerkinElmer, MA, USA) at 450 nm. 2.9. Statistical Analysis. Data were presented as normalized results from a minimum of three independent experiments. Significant differences of cell survival rate, ROS, and nitrotyrosine were determined by one-way ANOVA followed by the Tukey’s multiple comparisons test using the Graphpad Prism 5 software package (GraphPad Software, San Diego, CA). For time course studies of CPDs, significant differences between each treatment groups were determined by two-way RM ANOVA followed by the Newman−Keuls multiple comparisons test. A p value
