Conceived and designed the experiments: AD LB RQ. Performed the experiments: SB YD FV. Analyzed the data: SB YD FV. Wrote the paper: SB YD RQ.
¶ These authors also contributed equally to this work.
The funder L'Oreal had no role whatsoever in data collection and analysis or preparation of the manuscript, but Albert Duranton and Lionel Breton (employees of L'Oreal) had some role to play in the study design and publication of the data. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238–245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol.
Using human skin tissue, we report here the presence of specific [3H]-resveratrol binding sites (KD = 180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3–3 mM) resulted in cell death which was reduced by resveratrol (EC50 = 14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC50 = 95 µM) and epigallocatechin gallate (EC50 = 200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP.
Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.
Resveratrol (3,5,4′-trihydroxystilbene) is a naturally occurring polyphenol and is particularly found in grape skin, nuts and pomegranate. It has been hypothesized that resveratrol contributes to the ability of polyphenols rich Mediterranean diet to reduce the incidence of age-related diseases such as coronary heart disease
The characterization of resveratrol as a promising agent with anti-aging properties, in addition to many other health benefits, has prompted interest in investigating its protective effects against factors known to alter the course of normal skin aging
We aim to determine first the presence and distribution of [3H]-resveratrol binding sites using autoradiography. As shown in
Upper panel (A). Human skin sections were incubated during 8 months with [3H]-resveratrol (25, 60 and 130 nM), stained with hematoxylin/eosin and visualized using a microscope. Higher levels of silver grains were observed in the epidermis as compared to the dermis. Total and non-specific binding represents [3H]-resveratrol binding with or without cold resveratrol (100 µM), respectively. Lower panel (B). Photomicrographic distribution of human skin incubated with [3H]-resveratrol (25, 60 and 130 nM) and exposed to liquid emulsion for 8 months. The widespread distribution of silver grains (black dots) in the epidermis suggested that [3H]-resveratrol binding sites are mostly located in keratinocytes, which makes about 90% of skin epidermis. The number of silver grains is similar in undifferentiated (cells in the basal layer) and differentiated (cells at the surface of the skin) keratinocytes.
The affinity (KD) and maximal binding capacity (Bmax) of the saturation isotherms were estimated by Scatchard transformation of 12 sections from 3 human skins.
Brain homogenates and human skin sections were incubated with [3H]-resveratrol in the presence of increasing concentration of cold resveratrol (0.1 nM–100 µM). Each point represents the mean±SEM of data obtained from three to five determinations, each performed in triplicate and expressed as percentage of specific binding.
Binding experiments were performed at 4°C in the presence or the absence of 100 µM unlabelled resveratrol; specific (round points) binding represents the difference between total (square points) and non-specific (triangle points) binding. Insert shows the mathematical transformation as Scatchard plots of the binding isotherms of [3H]-resveratrol. Each point was done in triplicate and repeated 3 times.
[3H]-Resveratrol | Specific binding sites (nCi/mg tissue wet weight) | |
Epidermis | Dermis | |
25 nM | 0.577±0.04 | 0.009±0.011 |
60 nM | 1.156±0.09 | 0.066±0.06 |
130 nM | 2.003±0.15 | 0.133±0.08 |
Levels of [3H]-resveratrol binding were quantified using a MCID image analysis (St. Catherines, ON, Canada). Total [3H]-resveratrol binding was subtracted from adjacent sections incubated in the presence of 100 µM unlabelled resveratrol and expressed as specific [3H]-resveratrol binding sites (nCi/mg tissue, wet weight). Data represent the mean ± SEM of 12 sections obtained from 3 different subjects.
We then performed a functional assay to evaluate the capacity of resveratrol to protect HaCaT cells against the toxicity induced by the NO releasing SNP. Treatment of HaCaT cells with SNP (0.3–3 mM) inhibited the growth of HaCaT cells in a concentration dependent manner, as evaluated by the MTT and calcein assays (
Cells were exposed to SNP (0.3–3 mM) in the presence or absence of resveratrol (1–30 µM). Cell viability was determined 24 hours later using both the MTT and calcein AM assays. Values represent mean ± SEM of at least three separate experiments. *p<0.05, **p<0.01 comp.
The number of SNP-treated cells stained with SYTO 16 increased, indicating that SNP exerted an apoptotic effect on HaCaT cells (
Cells were exposed to SNP (0.3–3 mM) in the presence or absence of resveratrol (1–30 µM). Cell viability was determined 24 hours later using both the SYTO 16 (A) and JC-1 (B) assays. Values represent mean ± SEM of at least three separate experiments. *p<0.05, **p<0.01 compared to groups treated with SNP alone. +p<0.05 compared to control group.
The DCF assay indicated that a 5-hour exposure to SNP (1 and 3 mM) causes a significant increase (*P<0.01) in ROS accumulation (106 and 198% above control values, respectively;
Cells were exposed to SNP (1 and 3 mM) in the presence or absence of resveratrol (1–30 µM). ROS and nitrite accumulation was determined 5 hours later using the DCF assay and the Griess reagent respectively. Values represent mean ± SEM of at least four separate experiments. *p<0.05, **p<0.01 compared to groups treated with 1 mM SNP only. #p<0.05, ##p<0.01 compared to groups treated with 3 mM SNP alone. +p<0.05 compared to control group.
We compared the antioxidant properties of various polyphenols [(i.e. resveratrol and its analog piceatannol, epicatechin and its gallate ester epigallocatechin gallate (EGCG)] and their capacity to block the toxicity of SNP in HaCaT cells, using the DCF and calcein assays, respectively. Among the polyphenols tested here, resveratrol was most effective to protect cells against SNP-induced toxicity with an EC50 of 14.7 µM, followed by epicatechin (EC50 = 58.9 µM), while piceatannol and epigallocatechin gallate (EGCG) were less effective (EC50 = 94 and 140 µM, respectively) (
Cells were exposed to SNP (2 mM) in the presence or absence of polyphenols (0.3–30 µM). ROS accumulation was determined 5 hours later using the DCF assay. Values represent mean ± SEM of at least three separate experiments. *p<0.05, **p<0.01 compared to groups treated with 2 mM SNP alone.
ELISA assays were performed to investigate whether the neuroprotective action of resveratrol involved a modulation of the activity of caspases and p38 MAPK, two intracellular effectors that have been reported to be involved in the apoptotic effect of NO production (30). The caspase-3 inhibitor (5 µM) significantly increased cell viability compared to 2 mM SNP-treated cells [59.5±5 vs 26.2±2; p<0.01 (calcein values)], whereas the caspase-9 inhibitor (5 µM) was not effective [36.9±2 vs 32±2; NS (calcein values)]. A role for caspases in NO-induced apoptosis in HaCaT cells was confirmed by ELISA as SNP (0.5–3 mM) increased the activity of both caspases-3 and -9 with a significant effect at 1 mM and 0.5 mM, respectively (
Cells were exposed to SNP (0.12–3 mM) in the presence or absence of resveratrol (1–30 µM). Caspase-3 and -9 activities were measured 24 hours later using colorimetric assay kits. Values represent mean ± SEM of at least three separate experiments. +p<0.05, ++p<0.01 compared to control group. *p<0.05, **p<0.01 compared to groups treated with SNP alone.
Treatment | Relative level ofPhospho-p38 MAPK(% of control) | Relative level oftotal p38 MAPK(% of control) |
Control | 100±1 | 100±2 |
SNP (2 mM) | 125±3 | 118±2 |
+ Resveratrol (20 µM) | 124 ± 4 | 120±3 |
Control | 100±1 | 100±2 |
SNP (2 mM) | 119±3 | 119±3 |
+ SB 203580 (25 µM) | 105 ± 3 |
102±3 |
Values represent mean ± SEM of four separate experiments.
*p<0.01 compared to groups treated with SNP alone.
A quantitative real-time RT-PCR was performed to evaluate if resveratrol was able to modulate the expression of various genes known to be involved in NO signalling pathway. Among all the genes investigated here, those that display fold change higher than threshold are indicated in
In the present study we describe for the first time, the existence of [3H]-resveratrol specific binding in human skin and human keratinocytes cell line (HaCaT). Receptor autoradiography revealed that specific [3H]-resveratrol binding is present in human skin tissue with the highest level of labelling seen in the epidermis and apparent affinity (KD) of 180±50 nM. The presence of resveratrol binding sites has been also observed in HaCaT cells, and isotherm saturation binding experiments revealed the presence of high affinity sites (KD of 240 nM). Interestingly, we have previously demonstrated the existence of a high affinity polyphenols binding in the brain, with an apparent affinity (KD) of 220 nM
Numerous studies have demonstrated that resveratrol show potent protective effects in various models of toxicity
In addition to their antioxidant properties, polyphenols such as resveratrol have been reported to modulate the properties of markers of apoptosis
It has been reported that p38 MAPK is one of the most prominent protein kinase involved in NO-induced apoptosis upstream of caspase activation
Finally, results from quantitative RT-PCR indicate that resveratrol (10 µM) reversed SNP-altered expression of genes that either protect cells against oxidative stress (e.g. catalase, glutathione peroxidase) or promote oxidative stress (e.g. NADPH dehydrogenase quinone 1) and inflammation (e.g. interleukin 8 and NOS). Moreover, resveratrol by itself upregulated the expression of genes that protect against oxidative damage (i.e. glutathione peroxidase, oxidation resistance 1) or were involved in keratinocyte differentiation [i.e. keratin 1,
In summary, here we show that resveratrol possibly exerts its protective effect on epidermal cells by binding on specific polyphenol binding sites. Intracellular mechanisms underlying the protective effect of resveratrol are not solely due to its antioxidant activities but also involve its inhibitory action on apoptosis, a downstream mitochondrial dysfunction, as revealed by the ability of resveratrol to reduce caspase-3 activity as well as the number of apoptotic HaCaT cells and to prevent mitochondrial dysfunctions. It will be of interest to confirm these results using primary keratinocytes from human samples. Considering that NO is a key mediator implicated in a broad range of age-related skin damages, these findings suggest that resveratrol could delay and even prevent the normal course of skin aging by blocking apoptotic events and mitochondrial dysfunctions. These data also suggest that it could be possible to develop analogues of resveratrol with a higher affinity towards polyphenols binding sites, leading to pertinent effects in reducing skin aging.
Human immortalized HaCaT keratinocytes cells were purchased from CLS Cell Lines Service (Eppelheim, Germany). Materials used for cell cultures were obtained from Invitrogen (Burlington, ON, Canada). Fluorescent dyes (calcein, SYTO16 green, 2′,7′-dichlorofluorescein diacetate, dye 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide) and Griess reagent kit were purchased from Invitrogen-Molecular probes (Burlington, ON, Canada ). Caspases-3 and -9 inhibitors were purchased from Calbiochem (La Jolla, CA, USA). Caspases-3 and -9 colorimetric assay kits were obtained from Chemicon International (Temecula, CA, U.S.A.), whereas the RayBio® Cell-Based p38 MAPK (Thr180/Tyr182) ELISA Kit was from RayBiotech (Norcross, GA, U.S.A.). [3H]-Resveratrol (specific activity 0.3 to 0.6 Ci/mmole) was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Materials for quantitative real-time RT-PCR including the human NO signalling pathway RT2 profiler PCR array were purchased from SABiosciences (Frederick, MD, U.S.A.). Unless otherwise stated, all other compounds were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Stock solutions (10−1M) of resveratrol and other drugs were dissolved in dimethylsulfoxide (DMSO). This solvent at 0.03% (v/v) has no effect by itself on cell survival (data not shown).
Human skin tissue was obtained from patients (female; n = 3; 50, 60 and 65 years old) who were going to have plastic surgery (lifting). Before surgical operation, patients were asked for their consent, if the tissue removed during operation could be used for resveratrol research studies. All human skin tissues used in this study were from patients who had given their written consent. Additionally, the study protocol and the consent form were approved by the Douglas Institute Ethic committee. Informed written consent of patients was obtained before tissue resection and the study was conducted according to the Declaration of Helsinki Principles.
Immediately after surgical removal, skin tissues were kept in humidified (0.9% NaCl solution) pad at 4°C and transported to the Douglas Mental Health University Institute. Tissues were immediately frozen in 2-methylbutane at −40°C for 15 sec and then kept at −80°C until needed. Human skin sections (16 µm) were obtained using a cryomicrotome at −17°C, mounted on Fisher Superfrost slides, dried overnight in a desiccator at 4°C, and then kept at −80°C until use. Receptor autoradiography was performed as described in detail elsewhere
At the end of the incubation period, human skin sections were washed 4 times, one min each in cold Krebs-Ringer phosphate buffer (KRP), rapidly dipped in cold water, fixed in 2.5% formalin and 2.5% glutaraldehyde in phosphate buffered saline (PBS) for 10 min, washed in PBS and dehydrated in successive baths of 70%, 90% and 100% ethanol (5 min each). Sections were apposed against Kodak Biomax MR films (PerkinElmer, Woodbridge, ON, Canada) or liquid emulsion type NTB (Eastman Kodak, Rochester, NY, USA) for eight months alongside with [3H]-radioactive standards (GE Healthcare Canada, Baie d'Urfe, QC, Canada).
HaCaT cells were prepared as described previously, with minor modifications
Binding assays were performed in HaCaT cells, once reached to confluence in 6-well plates and each point was done in triplicate. Cells were washed twice with cold KRP buffer and binding assays were performed at 4°C by adding 2.5 ml of KRP buffer containing [3H]-resveratrol (0.5 to 600 nM) in the presence and absence of 100 µM cold resveratrol (non-specific binding). The binding reaction was stopped by two rapid washes of 5 ml cold Krebs buffer. Cells were lysed with 0.5 ml of 1N NaOH containing 1% TritonX100 for 30 min and neutralized with 0.5 ml of 1N HCl. Radioactivity was quantified using a beta counter with 45% efficiency (Beckman Instruments, Meriden, CT, USA).
SNP-induced toxicity was performed in HaCaT cells plated in 96 wells. On the day of the experiment, the medium was removed and replaced with similar medium with SNP (0.3–3.0 mM) and without FBS in the presence or absence of different drugs. Cell viability was determined 24 hours later using both the calcein AM and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, as described previously
Nuclear staining was performed using the fluorescent nuclear dye SYTO 16. This method has been validated as an indicator of apoptotic cell death
The mitochondrial inner membrane potential in immortalized HaCaT cells was measured using the fluorescent dye 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a cationic dye which is used as an indicator of mitochondrial potential in cells
Intracellular ROS levels were estimated in parallel to cell survival using the 2′,7′-dichlorofluorescein diacetate (DCF-DA, 5 µ
Accumulation of nitrite (NO2−) was measured in culture medium by the Griess reaction using Invitrogen NO colorimetric assay Kit, as described previously
Caspase-3 and -9 activities were detected using the caspase-3 and -9 colorimetric assay kits, respectively. Briefly, cells were exposed to either vehicle or SNP (0.25–3 mM) alone during 24 hrs or in the presence of resveratrol (1–30 µM). Cells were then lysed and briefly centrifuged (10,000 g). Cytosolic extract was exposed to an assay buffer in the presence of either the caspase-3 (Ac-DEVD-pNA) or capase-9 (Ac-LEHD-CHO) substrate. The optical density at 405 nm was measured 1 hour later with a micro-plate reader. Caspase-3 and -9 concentrations were calculated by comparison with the optical density of a pNA standard prepared in buffer.
The relative amount of p38 MAPK kinase was determined according to manufacturer's protocol using either anti-Phospho-p38 MAPK (Thr180/Tyr182) or anti-p38-MAPK. HaCaT cells were seeded into a 96 well tissue culture plate and exposed for different times to SNP alone or in the presence of resveratrol. Cells were then fixed and incubated with either anti-Phospho-p38 MAPK (Thr180/Tyr182) or anti-p38-MAPK. The wells were then washed, and HRP-conjugated anti-mouse IgG was added to the wells. The wells were washed again and a tetramethylbenzidine (TMB) substrate solution was added to the wells and color developed for 30 min in proportion to the amount of phosphorylated form of p38 MAPK and total p38 MAPK. A stop solution was finally added and changes in the optical density at 450 nm were immediately measured using a micro-plate reader.
Expression of genes involved in NO signalling pathway was measured using the two-step quantitative real-time human NO signalling pathway RT2 profiler™ PCR array (PAHS-062A), according to SABiosciences guidelines. Briefly, cells were exposed to either vehicle or SNP (1 mM) in the presence or absence of resveratrol (10 µM) for 24 hours. Total RNA from each sample was isolated using the RNeasy Mini kit (Qiagen (ONT, Canada). The TURBO™ DNase from (Ambion, Applied Biosystems, ONT, Canada) was then used to remove trace quantities of DNA contamination from RNA samples prior to RT-PCR. Reverse transcription of total RNA was carried out with RT2 first strand kit components and cDNA generated was quantified with the RT2 real-time PCR SYBR® Green/ROX master using the Applied Biosystems 7500 Real Time PCR System (CA, USA). Six housekeeping genes including β-actin were used to normalize quantification of mRNA target, and non-specific amplifications were verified by dissociation curve. Each value represents the mean of triplicate samples from two independent experiments.
All binding experiments were repeated 3–5 times (each in triplicate) and binding parameters were determined using GraphPad Prism program (version 4.03) (GraphPad Software Inc., San Diego, CA, USA). Survival of vehicle-treated control groups, not exposed to either SNP or various drugs was defined as 100%. A one-way ANOVA followed by Newman Keuls' multiple comparisons or unpaired Student's
Fold change and 95% confidence interval (95% CI) of genes differentially expressed in HaCaT cells treated with resveratrol (RSV, 10 µM alone and SNP (1 mM) alone or in presence of RSV (10 µM). Fold change was compared to gene expression of the control group.
(0.07 MB DOC)
The authors wish to thank Mira Thakur for proofreading the manuscript.