Inflammation Research

Inflammation Research Effects of pre- and post-treatment with plant polyphenols on human keratinocyte responses to solar UV --Manuscript Draft-Manuscript Number:

INRE-D-13-00030R1

Full Title:

Effects of pre- and post-treatment with plant polyphenols on human keratinocyte responses to solar UV

Article Type:

Original Research Paper

Corresponding Author:

Vladimir Kostyuk Byelorussian state university Minsk, BELARUS

Corresponding Author Secondary Information: Corresponding Author's Institution:

Byelorussian state university

Corresponding Author's Secondary Institution: First Author:

Alla Potapovich

First Author Secondary Information: Order of Authors:

Alla Potapovich Vladimir Kostyuk Tatyana Kostyuk Chiara de Luca Liudmila Korkina

Order of Authors Secondary Information: Abstract:

BACKGROUND: The understanding of the anti-inflammatory mechanisms of action of plant polyphenols (PPs) and clarification of the relationship between their antiinflammatory and antioxidant properties may result in a new therapeutic approach to skin cancers. OBJECTIVE: To elucidate the underlying mechanism we analyzed the ability of PPs to attenuate inflammatory, metabolic and oxidative cellular responses to UV irradiation. METHODS: Normal human epidermal keratinocytes (NHEK) were exposed to physiologically relevant dose of solar-simulated UV irradiation. Effects of pre- and posttreatment with PPs on the overproduction of peroxides and inflammatory mediators (mRNA and protein) were analyzed using real-time RT-PCR, enzyme-linked immunosorbent and fluorometric techniques. RESULTS: Differences between the effectiveness of pre- and post-treatment with polyphenols was found. In particular, PPs post-treatment, but not pretreatment completely abolished overexpression of Cyp1a1 and Cyp1b1 genes and elevation of intracellular peroxides in NHEK irradiated by UV. Post-treatment with PPs also more efficiently than pretreatment prevented UV-induced overexpression of IL-1 beta, IL-6 and COX2 mRNAs. CONCLUSION: Our data strongly suggest that PPs predominantly affect delayed molecular and cellular events initiated in NHEK by solar UV rather than primary photochemical reactions. PPs may be important component in cosmetic formulations for post-sun skin care.

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Effects of pre- and post-treatment with plant polyphenols on human keratinocyte responses to solar UV

Alla I. Potapovicha,b, Vladimir A. Kostyuka,b*, Tatyana V. Kostyuka, Chiara de Lucab, Liudmila G. Korkinab

a

Biology department, Byelorussian state university, Minsk 220050, Belarus

b

Istituto Dermopatico dell’Immacolata (IDI IRCCS), Rome 00167, Italy

______________________________________________ *Corresponding author at: the Lab. Tissue Engineering And Skin Pathophysiology, Istituto Dermopatico Dell’Immacolata (IDI IRCCS), Via Monti Di Creta 104, I-00167 Rome, Italy; Corresponding author email: [email protected]

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ABSTRACTS BACKGROUND: The understanding of the anti-inflammatory mechanisms of action of plant polyphenols (PPs) and clarification of the relationship between their anti-inflammatory and antioxidant properties may result in a new therapeutic approach to skin cancers. OBJECTIVE: To elucidate the underlying mechanism we analyzed the ability of PPs to attenuate inflammatory, metabolic and oxidative cellular responses to UV irradiation. METHODS: Normal human epidermal keratinocytes (NHEK) were exposed to physiologically relevant dose of solar-simulated UV irradiation. Effects of pre- and post-treatment with PPs on the overproduction of peroxides and inflammatory mediators (mRNA and protein) were analyzed using real-time RT-PCR, enzyme-linked immunosorbent and fluorometric techniques. RESULTS: Differences between the effectiveness of pre- and post-treatment with polyphenols was found. In particular, PPs post-treatment, but not pretreatment completely abolished overexpression of Cyp1a1 and Cyp1b1 genes and elevation of intracellular peroxides in NHEK irradiated by UV. Post-treatment with PPs also more efficiently than pretreatment prevented UVinduced overexpression of IL-1 beta, IL-6 and COX2 mRNAs. CONCLUSION: Our data strongly suggest that PPs predominantly affect delayed molecular and cellular events initiated in NHEK by solar UV rather than primary photochemical reactions. PPs may be important component in cosmetic formulations for post-sun skin care.

Keywords Plant polyphenols; inflammatory mediators; keratinocytes; solar-simulated UV irradiation; skin photoprotection; peroxynitrite.

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1. INTRODUCTION Epidemiological and experimental studies provide strong evidence for a critical link between solar UV radiation and skin cancers [1]. Cellular responses after exposure to solar UV are mediated by reactive oxygen species (ROS), mainly superoxide anion and singlet oxygen, which are produced during irradiation, as a result of the type I and type II reactions following light absorption by cellular photosensitizers (e.g. riboflavin or nicotinamide coenzyme). Extended production and enhanced levels of ROS and nitric oxide (NO) were also found after UV-irradiation [2,3]. Reactive oxygen and nitrogen species may directly damage chromatin and promote mutagenesis via DNA single-strand breaks, oxidative DNA adducts and DNA-protein cross-links [4] or initiate signal transduction processes leading to inflammation. Keratinocytes play a crucial role in the regulation of skin inflammation, responding to solar UV. After exposing to solar UV they produce inflammatory mediators including prostaglandins, chemokines, cytokines and surface adhesion molecules [3,5-7] that control the recruitment and functions of the immune cells [8,9]. Upon activation, these cells involved in inflammatory skin reactions releasing, among other factors, a large amount of nitric oxide, superoxide, H2O2, and hypochlorite [10,11]. However, if mild inflammation is normally adaptive in nature [12] in the case of severe inflammation, ROS can cause tissue damage and promote chronic inflammation which in turn may be a cause of carcinogenesis and tumor progression in a variety of cancers [13,14]. Therefore, pharmacological control of the inflammatory response of the skin to solar radiation seems to be beneficial in reducing the incidence of skin cancer.

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There is increasing interest in systemic and topical applications of plant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

polyphenols (PPs) for sun protection of skin. Initially, much of this interest has been due to their antioxidant properties. However, many findings from recent cellular studies have led to new insights into the molecular mechanisms underlying beneficial effects of PPs skin application. At present, the ability of PPs to protect skin against harmful UV radiation through altering signal transduction and epigenetic regulation of gene expression is widely discussed [9,15-17]. A number of publications have demonstrated anti-inflammatory effect associated with modulation of signal transduction for many PPs, such as green tea polyphenols [18,19], quercetin [20-22] resveratrol [21-23]. However, although the influence of these compounds on signal transduction and gene expression is well established, the further understanding of the anti-inflammatory mechanisms of action of PPs and clarification of the relationship between their anti-inflammatory and antioxidant properties may result in a new therapeutic approach to skin cancers. In the present work, we have analyzed the ability of PPs, namely the glycosylated phenylpropanoid verbascoside (Vb), the stilbenoid resveratrol (Rv) and the flavonoid quercetin (Qr) to attenuate inflammatory, metabolic and oxidative responses of normal human epidermal keratinocytes (NHEK) exposed to physiologically relevant dose of solar-simulated UV irradiation. Two approaches for the treatment of NHEK with PPs were utilized in order to distinguish between the effects of pre- and post-exposure administration on cellular responses to solar UV.

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2. MATERIALS AND METHODS 2.1. Chemicals and biochemicals Resveratrol was purchased from Biomol (Research Lab, Plymouth, MA, USA), verbascoside (97% purity; HPLC grade) isolated from Syringa vulgaris plant cell cultures was a kind gift from Dr. Roberto Dal Toso (IRB S.r.l., Altavilla Vicentina, Italy). Quercetin dihydrate, 4,5-diaminofluorescein diacetate (DAF2DA), dihydrorhodamine 123 (DHR), salts and solvents were purchased from Sigma-Aldrich (Milan, Italy). Other reagents, primers, and cell culture mediums are mentioned herein below in the appropriate subsections.

2.2. Cell cultures and treatments Primary cultures of NHEK were obtained from skin biopsies of healthy volunteers after their informed consent approved by the local Ethical Committee (IDI IRCCS - San Carlo Hospital) as previously reported [22]. NHEK were grown in a 5 % CO2 humidified atmosphere in keratinocyte growth medium containing growth factors (KGM-Gold; Lonza, Walkersville, MD). For 24 h before experiments, NHEK were starved in a medium deprived of all supplements (KBM). Plant polyphenols (PPs) were added to NHEK at the indicated concentrations and time intervals. Equal volumes of DMSO (a vehicle for PPs at 0.1 % [v/v] final concentration in the medium) were added to control cultures.

2.3. UV irradiation NHEK were seeded in 6-well plates, grown to 80 % confluence. Two protocols for the treatment of NHEK with PPs were utilized. (1) Cells were pretreated with PPs or vehicle (DMSO) for 30 min. Just before irradiation the medium was replaced by PBS, which has no sunscreen effect. The cells were 5

exposed to 40 mW/cm2 UVA+UVB light produced by Solar Simulator 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(Dermalight Vario Dr. Hoehnle AG, UV Technology, Planegg, Germany) with filter А2 (emission spectrum from 300 nm, emission peak at 375 nm). If not mentioned otherwise, the time of irradiation was 30 s, distance from cells 20 cm, the total dose was 1.1 J/cm2 (UVA 1.0 J/cm2 + UVB 0.1 J/cm2). Immediately after UV irradiation PBS was replaced with KBM without PPs. Sham-irradiated cells were also kept in PBS for equal amount of time. (2) Cells were irradiated in PBS without pretreatment with PPs, immediately after UV irradiation, PBS was replaced KBM with PPs or vehicle. In all experiments Vb was applied at a concentration 50 µmol/L, the concentration of the other compounds was 10 µmol/L. Solutions of 10 µM PPs in PBS were exposed to 40 mW/cm2 solar UV light (Solar Simulator) in 6-well plates, the length of the exposure was 30 s, distance from cells 20 cm, the total dose was 1.1 J/cm2 (UVA 1.0 J/cm2 + UVB 0.1 J/cm2).

2.4. Measurement of PPs absorption and binding by keratinocytes PPs were added to the confluent monolayer of keratinocytes in 6-well plates or to the same volume of culture medium in wells without cells (control). Absorption and binding of PPs by cells were measured by monitoring their disappearance from the culture medium covering the cells for 30 min using a UVVis absorption spectrometer. Data are expressed as percentage of baseline values.

2.5. NO and peroxide assays Cells were grown to confluence in 96-well culture plates. After UV irradiation, intracellular NO and superoxide production was detected using 6

specific fluorescent probes. NO was measured using 2.5 µmol/L DAF-2DA [24]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Formation of intracellular peroxides was detected using 10 µmol/L DHR [25]. Two protocols for the treatment of NHEK with PPs were utilized: (1) cells were pretreated with PPs or vehicle for 30 min, the medium was replaced by PBS and immediately after UV irradiation, PBS was replaced by fresh medium with DHR or DAF-2DA; (2) cells were irradiated in PBS without pretreatment with PPs, immediately after UV irradiation, PBS was replaced with fresh medium containing PPs and DHR or PPs and DAF-2DA. In accordance with both protocols cells were exposed to UV irradiation produced by Solar Simulator without filters (emission spectrum from 280 nm, emission peak at 375 nm) at the total dose of 3.0 J/cm2 (UVA 2.5 J/cm2 + UVB 0.5 J/cm2). Sham-irradiated cells were also kept in PBS for equal amount of time. NHEK were incubated with DAF-2DA or DHR for 60 min at 37°C before measurement. Then the medium was replaced with PBS and fluorescence of adhered cells was measured at excitation and emission wavelengths of 485 and 535 nm, respectively using a microplate reader.

2.6. Treatment of PPs with peroxynitrite Peroxynitrite was synthesized as previously described [26]. Briefly, an aqueous solution of 0.6 mol/L sodium nitrite was rapidly mixed with an equal volume of acidified (0.6 mol/L HCl) 0.7 mol/L hydrogen peroxide. The reaction was immediately quenched by 1.5 mol/L NaOH and residual hydrogen peroxide was removed by treatment with manganese dioxide. The concentration of peroxynitrite was determined spectrophotometrically by the measurement of absorption at 302 nm in 1 mol/L NaOH (ε = 1670M −1 cm−1). The resulting peroxynitrite was stored frozen at -80ºC. Dilutions in 0.1 mol/L NaOH were made 7

immediately before use to achieve the desired concentrations. 10 µl of 50 mmol/L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

peroxynitrite (final concentration 500 µmol/L) was added to 1 ml 100 µmol/L solution of PPs in PBS and the UV-Vis absorption spectra were recorded immediately after the addition. As a control, PPs were added 3 min after peroxynitrite had been added to buffer alone. 2.7. Quantitative RT-PCR Gene expression was analyzed at 6 h after treating cells. The cell culture medium was removed, adherent cells were washed by PBS twice and then immediately frozen. Samples kept at -80°C until the time of the analysis. Total RNA was isolated from frozen NHEK using the GenElute Mammalian Total RNA Kit (Sigma-Aldrich, Milan, Italy) in accordance to manufacturer's instructions. The amount of RNA was determined by absorbance at 260 nm. Total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) at 42°C for 30 min. cDNA was amplified with IQ SYBR green Supermix (Bio-Rad, Hercules, CA) using the MiniOpticon Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in accordance to manufacturer's instructions. Briefly, PCR was performed in a 25 µl volume containing cDNA equal to 100 ng of total RNA using a temperature program as follows: 36 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 120 s. Melt curve analysis was performed to confirm the specificity of the amplified products. Signal of two housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin was chosen as reference and expression of the other genes studied was normalized to each of these reference genes. Fold changes were calculated with the comparative Ct method (ΔΔCt) according to Livak and Schmittgen [27] and are expressed as the ratio of mRNA in treated cells to that in control cells (fold 8

change from control). Total RNA from each treatment well was reverse1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

transcribed in duplicate and all PCR amplifications repeated twice from each RT reaction. The following primer sets were designed using Primer-BLAST (NCBI) and were synthesized by Eurofins MWG Operon (Ebersberg, Germany): β-Actin fwd: 5′-AAATCTGGCACCACACCTTCTAC-3′; β-Actin rev: 5′-ATAGCACAGCCTGGATAGCAAC-3′; GAPDH fwd: 5'-CAATGACCCCTTCATTGACC-3'; GAPDH rev: 5´-GACAAGCTTCCCGTTCTCAG-3´; IL-6 fwd: 5′-GTGTGAAAGCAGCAAAGAG-3′; IL-6 rev: 5′-CTCCAAAAGACCAGTGATG-3′; TNFα fwd: 5′-TCCTTCAGACACCCTCAACC-3′; TNFα rev: 5′-AGGCCCCAGTTTGAATTCTT-3′; COX2 fwd: 5′-TTCTCCTTGAAAGGACTTATGGGTAA-3′; COX2 rev: 5′-AGAACTTGCATTGATGGTGACTGTTT-3′; I L-8 fwd: 5′-GTCCTTGTTCCACTGTGCCT-3′; IL-8 rev: 5′-GCTTCCACATGTCCTCACAA-3′; IL-1β fwd: 5´-ACGCTCCGGGACTCACAGCA-3´; IL-1β rev: 5´-TGAGGCCCAAGGCCACAGGT-3´; CYP1A1 fwd: 5′-CCTGGAGACCTTCCGGCACT-3′; CYP1A1 rev: 5′-AGACACAACGCCCCTTGGGG-3′; CYP1B1 fwd: 5´-TGGTCTGTGAATCATGACCCAGTGA-3´; CYP1B1 rev: 5´-TCTTCGCCAATGCACCGCCT-3´. 2.8. ELISA assays Cells were grown to confluence in 6-well culture plates. After 18 h of treatment, the cell culture medium was collected, frozen and kept at -80°C until 9

the time of the analysis. The concentration of secreted TNF alpha, IL-6 and IL-8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

in the medium was measured by enzyme-linked immunosorbent assay with ELISA Kits (SABiosciences – Qiagen S.p.A., Milan, Italy). Samples from three independent experiments were assayed in quadruplicate for each condition. 2.9. Data analysis All measurements were done in triplicate, and data of at least three independent experiments were statistically evaluated. Statistical evaluation was carried out with Microsoft Excel 2007 software for Windows XP. Results were expressed as the mean ± S.D. To evaluate the difference between experimental groups, Student’s t-test was applied and P < 0.05 were considered to be significant.

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3. RESULTS 3.1. PPs differently interact with NHEK and have different susceptibility to UV irradiation and peroxynitrite Oxidation of PPs by peroxynitrite was followed spectrophotometrically. UV-visible absorption spectra were recorded over the range of 250-600 nm before and immediately after peroxynitrite addition (Fig. 1A-C). From the spectral changes it can be concluded that all three compounds interact with peroxynitrite, but Qr and Rv are the most oxidizable. Susceptibility

of

PPs

to

UV

irradiation

was

followed

spectrophotometrically. UV-visible absorption spectra were recorded over the range of 250-450 nm before and after 30 s irradiation (Fig. 1D-F). Rv was found to be the most susceptible to (UVA + UVB) light. 30 s of irradiation was sufficient to cause significant changes in the absorption spectrum (Fig. 1D). In contrast, the photostability of Qr and Vb was much higher. Absorption spectrum of Qr after 30 s of irradiation exhibits only small changes due to the photodegradation (Fig. 1E) and no spectral changes were observed after 30 s (Fig. 1F) and even after 10 min of Vb irradiation (data are not shown). Absorption and binding of PPs by NHEK were measured by monitoring their disappearance from the culture medium covering the cells as described in Materials and Methods. It was found that near 30 % of Qr, 10 % of Rv and less than 1 % of Vb were removed from culture medium as a result of the binding and absorption by cells for 30 min incubation (Fig. 1G).

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3.2. Effects of pre- and post-exposure treatment with plant polyphenols on inflammatory response and overexpression of detoxication enzymes in NHEK exposed to solar UV irradiation The impact of plant polyphenols on the NHEK responses to UV was investigated at 6 h post-irradiation ( UVA 1.0 J/cm2 + UVB 0.1 J/cm2) using realtime RT-PCR (Fig. 2) and ELISA (Fig. 3) techniques. We demonstrated previously that this dose and post-irradiation time are optimal for testing antiinflammatory agents [28]. Two different experimental designs were applied: pretreatment (protocol 1) and post-treatment (protocol 2) of NHEK with PPs. Effects of post-exposure treatment. Under these experimental conditions all tested polyphenols significantly affected UV-activated inflammatory and metabolic signaling pathways (Fig. 2B). Namely, RT-PCR data indicate that all compounds completely abolished overproduction of IL-1 beta mRNA that occurred in UV irradiated NHEK. Both Qr and Rv, but not Vb statistically significantly reduced the level of mRNA for IL-6 and COX-2. At the same time Vb statistically significantly abolished overexpression of IL-8 and TNF alpha. Quantitative cytokine analysis at the protein level (Fig. 3) is generally consistent with RT-PCR results. In particular, the level of IL-6 in culture medium after 18 h of treatment was significantly decreased by Qr and Rv, but not Vb. Qr and Vb significantly reduced the level of IL-8, and Vb statistically significantly lowered the level of TNF alpha in the medium after 18 h of treatment. Solar-simulated irradiation affected xenobiotic signaling pathways in NHEK, resulting in increased levels of Cyp1a1 and Cyp1b1 mRNAs. Post-exposure treatment with PPs not only completely prevented the UV-induced overexpression of the detoxication enzymes, but Qr and Rv significantly downregulated Cyp1a1 and Cyp1b1 gene expression in UV irradiated NHEK compared to sham-irradiated cells (Fig. 2B). 12

Effects of pre-exposure treatment. In contrast to the above results the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

effects of PPs on inflammatory signaling were less marked (Fig. 2A). Namely only Qr significantly reduced the level of IL-1 beta, Qr and Vb reduced the level of IL-6, Vb reduced the level of IL-8. Cellular response to UV irradiation via Cyp1a1 and Cyp1b1 gene expression was also less affected in the case of pretreatment compared to post-exposure treatment of NHEK with Rv and Vb. However, pretreatment with Qr completely abolished UV-induced overexpression of Cyp1a1 and Cyp1b1 genes (Fig. 2A). 3.3. Effects of pre- and post-exposure treatment with plant polyphenols on oxidation of fluorescent probes in NHEK exposed to UV irradiation Intracellular NO production was measured using the cell-permeable dye DAF-2DA that is hydrolyzed to oxidation DAF-2 by intracellular esterases. Upon reaction with N2O3, the immediate product of NO oxidation, DAF-2 DA forms the highly green fluorescent DAF-2T [24]. Formation of intracellular peroxides such as hydrogen peroxide and peroxynitrite was detected using DHR, which is specifically oxidized to fluorescent rhodamine by peroxides. Intracellular levels of both NO and peroxides (Fig. 4) were quantified in arbitrary units and plotted as percentage of sham-irradiated cells. As shown in Fig. 4A, irradiation of NHEK with solar-simulated UV light (UVA 2.5 J/cm2 + UVB 0.5 J/cm2) resulted in a strong increase of intracellular NO level. Treatment of cells with PPs, before (protocol 1) and after the exposure (protocol 2) has poor effect on UV-induced NO production. Only treatment with Qr slightly, but statistically significant abolished the effect of UV. Exposure of NHEK to solar-simulated UV light also caused a remarkable increase of intracellular peroxide level (Fig. 4B). The level of peroxides in the cells pretreated with Rv or Qr was slightly lower than that in 13

untreated cells, but the difference was not statistically significant. At the same 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

time treatment of cells with PPs after UV-irradiation not only completely abolished the effect of UV, but also reduced the level of intracellular peroxides below the basal level.

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3. DISCUSSION Cellular response after exposure to solar UV may be divided at least into two phases. The first phase includes photophysical and photochemical events following light absorption by cellular photosensitizers and occurring during and immediately after irradiation. These events trigger a number of biochemical and cellular alterations at the second phase. Keratinocytes, that play a crucial role in the regulation of skin inflammation, produce during the second phase of cellular response to solar UV numerous inflammatory mediators including prostaglandins, chemokines, cytokines and surface adhesion molecules [3,5-7]. Excessive production of inflammatory mediators leads to chronic inflammation which in turn could raise the risk of carcinogenesis and tumor progression in a variety of cancers [13,14]. It has been suggested that plants polyphenols may provide an effective strategy for skin photoprotection and the reduction of incidence of skin cancer [17,29]. Anti-inflammatory effect of PPs associated with modulation of signal transduction has been demonstrated in cellular studies [18-23]. However, a particular effect of PPs on a particular phase of cellular response to solar UV remains unclear. The current study was designed to clarify this issue and to obtain data on the effect of PPs on particular phases of the cellular response to solar UV. Two protocols for the treatment of NHEK with PPs were utilized in order to distinguish the effects of pre- and post-exposure administration and the results indicate substantial differences between them. It was found that inflammatory (overexpression of IL-6, IL-1 beta and COX2), metabolic (overexpression of Cyp1a1 and Cyp1b1) and oxidative (formation of intracellular ROS) responses of UV-irradiated NHEK are very sensitive to post-treatment with Qr or Rv. In other words, these PPs affect molecular and cellular events at the second phase of 15

cellular responses. In the case of Qr the effects of pre- and post-treatment on 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

inflammatory and metabolic responses are very similar, however, it have to be mentioned that substantial amount of Qr added to culture medium before UVirradiation was bound and absorbed by the cells (Fig. 1G). Probably, so many of Qr could be sufficient to affect inflammatory and xenobiotic signaling pathways at the second phase of cellular responses to UV-irradiation. Rv can also be bound and absorbed by the cells, however Rv was found to be very susceptible to UVA+UVB light (Fig. 1D) and the time of exposure of cells to irradiation was sufficient to almost completely destroy absorbed substance. As a result pretreatment with Rv not only did not reduce, but even further enhanced UVinduced inflammatory and metabolic responses. One explanation of this finding may be related to the proinflammatory effect of products of Rv photodestruction. Effects of treatment with Vb on the NHEK responses to UV-irradiation were similar with that for Qr and Rv except that pretreatment, but not post-treatment with Vb significantly abolished overexpression of IL-6 mRNA. Besides, pretreatment of NHEK with Vb moderately but statistically significantly attenuated the overexpression of TNF alpha and IL-8 mRNAs. Several speculations may explain these findings. Firstly, Vb, which was bound and absorbed by the cells during preincubation may affect the photophysical and photochemical events occurring during and immediately after irradiation. However, there is doubt that the amount of bound Vb may be sufficient to affect the first phase of the cellular response to UV-irradiation. The other explanation may be that Vb during preincubation affected inflammatory signaling pathways in such a way that this may reduce inflammatory response NHEK to solar UV.

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It have be mentioned that pretreatment with PPs is often used to examine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

their photoprotective effect. For example, Vicentini et al. [20] demonstrated that 16 h pretreatment of NHEK with 50 µmol/L Qr resulted in strong suppression of UV-induced overexpression of inflammatory cytokines IL-1 beta, IL-6 and IL-8. However; here we show that a similar effect could be achieved if the keratinocytes were treated with 10 µmol/L Qr after UV-irradiation. Cellular oxidative response was assessed with fluorescent probes. In agreement with published data [2,30] we found significant elevation of intracellular levels of NO and peroxides in UV-irradiated NHEK. PPs exhibited poor impact on UV-induced changes in intracellular level of NO, but at the same time post-treatment with all PPs completely abolished the effect of UV on rhodamine

fluorescence.

Since

peroxynitrite-mediated

oxidation

of

dihydrorhodamine 123 and formation of fluorescent rhodamine was clearly established [25] it is reasonable to assume that in UV-irradiated NHEK, DHR can be specifically oxidized to rhodamine by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. This conclusion is supported by the result of Aitken et al. [2] that found enhanced levels of superoxide and NO for a rather long period after UV irradiation of keratinocytes. Since all polyphenols studied easily react with peroxynitrite they can act on peroxynitrite-mediated dihydrorhodamine oxidation as competitive inhibitors. The concept that ROS, such as superoxide anion, H2O2, peroxynitrite can contribute to the control of signal transduction and gene expression has become widespread in recent years [14]. From this point of view it is possible to consider that peroxides, particularly peroxynitrite can mediate inflammatory and metabolic responses to solar UV-irradiation and peroxynitrite scavenging by PPs may partially diminish effects of solar UV. Indeed, we observed that the diminution of 17

inflammatory and metabolic responses by Rv and Vb is generally consistent with 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

their ability to scavenge peroxides. At the same time, we found that pretreatment NHEK with Qr strongly diminished response of pro-inflammatory cytokines and completely prevented the overexpression of Cyp1a1 and Cyp1b1 mRNAs, but had a weak impact on dihydrorhodamine oxidation by peroxides. Apparently, quercetin can affect inflammatory and metabolic responses through another mechanism rather than peroxynitrite scavenging. In conclusion, our data strongly suggest that PPs predominantly affect delayed molecular and cellular events initiated in keratinocytes by solar UV rather than primary photochemical reactions. Therefore, post-exposure treatment of NHEK with PPs are effective against inflammatory response and metabolic disorders. Among the cellular events that are sensitive to impact of PPs is extended production of peroxides, particularly peroxynitrite. Finally, PPs may be important component in cosmetic formulations for post-sun skin care.

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ACKNOWLEDGEMENTS The work was partly financed by the grant from Italian Ministry for Health (RC. IDI IRCCS2012).

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17. Nichols JA, Katiyar SK. Skin photoprotection by natural polyphenols: anti-inflammatory, antioxidant and DNA repair mechanisms. Arch Dermatol Res. 2010;302:71-83. 18. Liu ML, Yu LC. Potential protection of green tea polyphenols against ultraviolet irradiation-induced injury on rat cortical neurons. Neurosci Lett. 2008;444:236-9. 19. Wu LY, Zheng XQ, Lu JL, Liang YR. Protective effect of green tea polyphenols against ultraviolet B-induced damage to HaCaT cells. Hum Cell. 2009;22:18-24. 20. Vicentini FTMC, He T, Shao Y, Fonseca MJV, Verri WA, Fisher GJ, Xu Y. Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kB pathway. J Dermatol Sci. 2011;61:162-8. 21. Pastore S, Potapovich A, Lulli D, Fidanza P, Kostyuk V, De Luca C, Mikhal'chik E, Korkina L. Plant Polyphenols Regulate Chemokine Expression and Tissue Repair in Human Keratinocytes Through Interaction with Cytoplasmic and Nuclear Components of Epidermal Growth Factor Receptor (EGFR) System. Antioxid Redox Signal. 2012;16:314-28. 22. Pastore S, Lulli D, Potapovich AI, Fidanza P, Kostyuk VA, Dellambra E., de Luca C, Maurelli R, Korkina LG. Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells. J Dermatol Sci. 2011;63:104-14. 23. Adhami VM, Afaq F, Ahmad N. Suppression of ultraviolet B exposure-mediated activation of NFkappaB in normal human keratinocytes by resveratrol. Neoplasia. 2003;5:74-82. 24. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 1998;70:2446-53. 25. Kooy NW, Royall JA, Ischiropoulos H, Beckman JS. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med. 1994;16:149-56. 26. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA. 1990;87:1620-4. 27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402-8. 28. Kostyuk VA, Potapovich AI, Lulli D, Stancato A, De Luca C, Pastore S, Korkina L. Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols as a Basis for Chemoprevention of Non-Melanoma Skin Cancers. Curr Med Chem. 2013;20 (in press). 29. Afaq F, Katiyar SK. Polyphenols: skin photoprotection and inhibition of photocarcinogenesis. Mini Rev Med Chem. 2011;11:1200-15. 30. Wu S, Wang L, Jacoby AM, Jasinski K, Kubant R, Malinski T. Ultraviolet B lightinduced nitric oxide/peroxynitrite imbalance in keratinocytes--implications for apoptosis and necrosis. Photochem Photobiol. 2010;86:389-96.

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FIGURE LEGENDS

Fig. 1. Photo- and

oxidative alteration (chromophore degradation) of plant polyphenols (PPs) and their loss from culture media as a results of cellular binding and uptake. (A-C) UV-visible absorption spectra of 100 µmol/L resveratrol (Rv), quercetin (Qr), verbascoside (Vb) in 0.1 mol/L potassium phosphate buffer (pH 7.4) recorded prior to and immediately after addition of peroxynitrite in the final concentration 500 µmol/L; (D-F) UV-visible absorption spectra of 10 µmol/L Rv, Qr, Vb in 0.1 mol/L potassium phosphate buffer (pH 7.4) recorded prior to and immediately after solar-simulated irradiation (UVA 1.0 J/cm2 + UVB 0.1 J/cm2); (G) cellular binding and expressed as percentage of baseline values.

uptake of PPs by NHEK for 30 min incubation,

Fig. 2. Effects of plant polyphenols (PPs) on inflammatory and metabolic responses of NHEK exposed to ultraviolet radiation. (A) Cells were pretreated with 10 µmol/L resveratrol (Rv), 10 µmol/L quercetin (Qr), 50 µmol/L verbascoside (Vb) for 30 min and then were exposed to solar-simulated irradiation (UVA 1.0 J/cm2 + UVB 0.1 J/cm2). Before irradiation the medium was replaced by PBS, immediately after UV irradiation PBS was replaced with fresh medium without PPs; (B) cells were irradiated in PBS without pretreatment with PPs, immediately after UV irradiation PBS was replaced by medium with PPs. Cells were harvested 6 h later, and mRNA levels were measured by real-time RT-PCR. Results were normalized to the housekeeping genes (GAPDH and beta-actin) using the 2−ΔΔCt method. Data (mean ± S.D., n=6) are expressed as fold change relative to levels in sham-irradiated controls. ‡p < 0.001 vs. sham-irradiated controls; §p < 0.05, #p < 0.01 and ұp < 0.001 vs. cells irradiated with UV. Fig. 3. Effects of plant polyphenols on release of inflammatory cytokines by UV-irradiated keratinocytes. Cells were exposed to solar-simulated irradiation (UVA 1.0 J/cm2 + UVB 0.1 J/cm2). Before irradiation the medium was replaced by PBS, immediately after UV irradiation PBS was replaced by medium with 10 µmol/L resveratrol (Rv), 10 µmol/L quercetin (Qr), 50 µmol/L verbascoside (Vb). Conditioned medium was collected 18 h later, and inflammatory cytokines were analyzed by ELISA. Data (mean ± S.D.) of 3 experiments are shown. *p < 0.05, †p < 0.01 and ‡ p < 0.001 vs. sham-irradiated controls; §p < 0.05 and ұp < 0.001 vs. cells irradiated with UV. Fig. 4. Effects of plant polyphenols (PPs) on oxidative response of NHEK exposed to UV irradiation (UVA 2.5 J/cm2 + UVB 0.5 J/cm2). A (PPs; pretreatment): cells were pretreated with 10 µmol/L resveratrol (Rv), 10 µmol/L quercetin (Qr), 50 µmol/L verbascoside (Vb) for 30 min, then the medium was replaced by PBS and immediately after UV irradiation PBS was replaced by fresh medium with DAF-2DA (fluorescent probe for NO); A (PPs; post-treatment): cells were irradiated in PBS without pretreatment with PPs, immediately after UV irradiation, PBS was replaced with fresh medium containing PPs and DAF-2DA. B: protocols were the same as in A, except that DHR (fluorescent probe for peroxides) was used instead of DAF-2DA. Data (mean ± S.D., n=12) are presented as percentage of sham-irradiated controls. ‡p < 0.001 vs. sham-irradiated controls; §p < 0.05, #p < 0.01 and ұp < 0.001 vs. cells irradiated with UV.

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Authors' Response to Reviewers' Comments Click here to download Authors' Response to Reviewers' Comments: A LIST OF CHANGES AND A REBUTTA1.doc

A LIST OF CHANGES AND A REBUTTAL Reviewer #1: Interesting manuscript,w hich describes a useful addition to the literature concerning the effects of PP with respect to UVR. This reviewer has a number of comments: Abstract Q. Methods. It would be more useful to describe the endpoints studied, rather than the approaches used. Ansv. Appropriate changes were made in the revised version Introduction Q. P3, l17. Define NO. "...were also..." should this be "...have also..."? Ansv. Appropriate changes were made in the revised version Q. l25. There are more DNA nucleobase products than simply 8-hydroxylation of guanine. Ansv. Instead of “and increase mutagenesis via DNA single-strand breaks, the 8-hydroxylation of guanine and cross-linking of DNA“ written “and promote mutagenesis via DNA single-strand breaks, oxidative DNA adducts and DNA-protein cross-links “ Materials and Methods Q. P6, l61. Here and elsewhere 'L' not 'l' for litre. Ansv. Appropriate changes were made in the revised version Q. P7, l19. Why are the two UV filter and dose protocols? How do these two relate and why? Ansv. In this study we used different experimental approaches to identify the effects pre- and posttreatment with plant polyphenols on oxidative and inflammatory responses. To induce inflammatory response we exposed cells to (UVA 1.0 J/cm2 + UVB 0.1 J/cm2) with emission spectrum from 300 nm, emission peak at 375 nm. As we previously reported (Kostyuk VA, Potapovich AI, Lulli D, Stancato A, De Luca C, Pastore S, Korkina L. Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As

exposure of cells to such dose had no impact on NHEK viability and did not induce apoptosis, but resulted in the remarkable overexpression of inflammatory mediators. At the same time the exposure of cells to such dose resulted in a weak oxidative stress and this protocol of cell exposure was hardly used to analyze effects of polyphenols on oxidative response. To compare the effects pre- and posttreatment with plant polyphenols on oxidative response we must induce more severe oxidative stress exposing cells to UV irradiation at the total dose of 3.0 J/cm2 (emission spectrum from 280 nm, emission peak at 375 nm). a Basis for Chemoprevention of Non-Melanoma Skin Cancers. Curr Med Chem. 2013 Mar 1;20(7):869-79.)

Q. P9. RT PCR. Why were these genes chosen? What's their significance in keratinocyte responses to UVR? Ansv. TNFα, IL-6, IL-1β, L-8, COX2 are usually discussed as the main pro-inflammatory mediators. Expression of CYP1A1 and CYP1B1 genes have long been used as a biomarker for aryl hydrocarbon receptor (AhR) activation. As shown recently, the physiological role of AhR is not limited to the control of xenobiotic metabolism, but it extends to numerous cell functions such as breakdown of endogenous metabolites, proliferation, cell-to-cell contacts, immune and inflammatory responses, melanogenesis, and circadian rhythm. Effect of UVA 1.0 J/cm2 + UVB 0.1 J/cm2 on expression of MCP1, ICAM1, NOX-1, MMP1 and 9, IL-1α, and some other inflammatory related mediators was studied (Kostyuk VA, Potapovich AI, Lulli D, Stancato A, De Luca C, Pastore S, Korkina L. Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As a Basis for Chemoprevention of Non-Melanoma Skin Cancers. Curr Med Chem. 2013 Mar 1;20(7):869-79., Korkina L, Kostyuk V, De Luca C, Pastore S. Plant Phenylpropanoids as Emerging AntiInflammatory Agents. Mini Rev Med Chem. 2011, 11: 823-835,

and unpublished data). However, effect of UV on the expression mRNA for these genes was rather weak. Q. P10. L5. Samples were assayed in quadruplicate, but how many BIOLOGICAL replicates? Ansv. Data of three independent experiments were statistically evaluated. Appropriate changes were made in the revised version Discussion. Q. P15, l54. Wasn't nitrosative as well as oxidative stress intended to be considered? Ansv. Here we considered that treatment of NHEK with PPs after UV-irradiation completely abolished the effect of UV on of intracellular peroxides but did not affect NO production. Q. P16. Is qPCR sufficiently robust to act as a proxy for protein levels. Shouldn't Western blotting have been performed (instead or at a least) to confirm some of the qPCR data? Ansv. On the protein level, we studied the release of TNFα, IL-6 and IL-8 in the medium using ELISA. Q. P17. reactive oxygen species and hydrogen peroxide already defined. Ansv. Appropriate changes were made in the revised version Figure legends Q. n=? how many experiments? Ansv. Appropriate changes in the legends for figure 2-4 were made in the revised version Q. How were mRNA expression levels corrected? Ansv. Appropriate changes were made in the revised version: Results were to normalized to the housekeeping genes (GAPDH and beta-actin) using the 2−ΔΔCt method.

Reviewer #2 Q. Figure 1 title does not represent the figure. The change in spectra do not demonstrate degradation and further analysis is required to demonstrate this. Similarly, the final part fo teh figure does not show binding to cells and other analyses are required to confirm uptake by cells. In such a case, the effect of pre-irradiation on uptake should be clarified. Ansv. We agree with the reviewer and have modified the title to: Photo- and oxidative alteration (chromophore degradation) of plant polyphenols (PPs) and their loss from culture media as a results of cellular binding and uptake Q. What is the effect on cell viability of treatment. Where is the evidence that failure to elicit and inflammatory response is not due to cell death. Ansv. To induce inflammatory response we exposed cells to (UVA 1.0 J/cm2 + UVB 0.1 J/cm2). As we previously reported (Kostyuk VA, Potapovich AI, Lulli D, Stancato A, De Luca C, Pastore S, Korkina L. Modulation of Human Keratinocyte Responses to Solar UV by Plant Polyphenols As a Basis for Chemoprevention of Non-Melanoma Skin Cancers. Curr Med Chem. 2013 Mar 1;20(7):869-79.) exposure of cells

to such dose is optimal for testing anti-inflammatory agents and had no impact on NHEK viability and did not induce apoptosis. In the text there is an appropriate reference P.12, l.15 Q. What is the impact of chromophores anf fluorescence of probes? Ansv. 1. DHR is commonly used to evaluate the influence of PPs on cellular oxidative stress (see: Bestwick and Milne. Quercetin modifes reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells. BBA 1528 (2001) 49-59; Borra M.T. et al. Mechanism of Human SIRT1 Activation by Resveratrol. The Journal of Biological Chemistry, 2005, 280, 17187-17195.).

It also has been found that quercetin in the concentration up to 20 µM had no destructive impact on rhodamine chromophore (See Fig. 2 in Annaert PP, Brouwer KL. Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwichcultured rat hepatocytes. Drug Metab Dispos. 2005 Mar;33(3):388-94.)

2. Post treatment PPs has no effect on fluorescence of NO probe (DAF-2T). Since we used fluorescence parameters of (excitation 485 nm, emission 535 nm) are similar for both probes we may conclude that treatment with PPs had no screen effect on the emission of peroxide probe (rhodamine) Q. the statistical analysis is incorrect. T tests are not appropriate and reanalysis must be undertaken. Ansv. As a result of experiments described in this paper we received several different groups of data, that were evaluated using t test. Presumably, the reviewer suggests to use for data analysis the methods for multiple comparisons. However, all conclusions and assumptions in this paper were made from the Comparison only two data set columns e.g. “control data” column and “UV data” column; “UV data” column and “UV+RV data” column, etc. We did not compare simultaneously three or more data columns. From this point of view, we suppose that using Student's t test for data evaluation is corrected.

Figure 1 Click here to download high resolution image


