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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Photochem Photobiol. 2014 Oct 7;91(1):225–234. doi: 10.1111/php.12337

Fisetin inhibits UVB-induced cutaneous inflammation and activation of PI3K/AKT/NFκB signaling pathways in SKH-1 hairless mice†

Harish Chandra Pal 1, Mohammad Athar 1,2, Craig A Elmets 1,2, Farrukh Afaq 1,2,*
PMCID: PMC4294956  NIHMSID: NIHMS625193  PMID: 25169110

Abstract

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations, and alterations in signaling pathways eventually leading to skin cancer. In the present study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB exposed SKH-1 hairless mouse skin. Mice were exposed to 180 mJ/cm2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX-2, PGE2 as well as its receptors (EP1- EP4), and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL-1β and IL-6 in UVB exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB-induced expression of PI3K, phosphorylation of AKT, and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB-induced cutaneous inflammation and DNA damage.

Introduction

Skin acts as the first defense against potentially harmful physical, biological and environmental pollutants, including ultraviolet (UV) radiation (1–3). Exposure to UVB radiation has been shown to damage biological macromolecules, such as lipids, proteins and nucleic acids, resulting in a variety of cutaneous disorders, including skin cancer (4,5). Solar UVB radiation induces erythema, sunburn, hyperplasia, proliferation, inflammation, oxidative stress, DNA damage, p53 mutations, immunosuppression, and alterations in PI3K/AKT and NFκB cell survival signaling pathways eventually leading to skin cancer (3–7). UVB-induced inflammatory responses include an increase in proinflammatory cytokines (TNFα, IL-1β, and IL-6) (8,9), cyclooxygenase-2 (COX-2) activity (10), prostaglandin (PG) metabolites (11,12), infiltration of leukocytes and increased myeloperoxidase (MPO) activity (13). MPO is synthesized and secreted by infiltratory neutrophils and modulates vascular signaling and vasodilation during the processes of inflammation (14,15). UVB induced COX-2 expression plays an important role in inflammation, as well as in cell proliferation and survival (16). UVB exposure also triggers the production of PGs, which are produced from arachidonic acid via sequential pathways involving cyclooxygenases and PG synthetases. PGE2, produced abundantly by keratinocytes after UVB exposure, is the major and most effective metabolite generated by COX-2 activity and is considered to be a potent mediator of inflammatory responses (17,18). PGE2 and its receptors (EP1-EP4) have been reported to be linked with UVB-induced skin carcinogenesis (11,19–21).

The PI3K/AKT signaling pathway regulates cell proliferation and apoptosis and is an important mediator of UVB-induced cellular responses (3,22). EP4 has been reported to activate PI3K/AKT and NFκB signaling, leading to cell proliferation and survival in UVB-irradiated mouse skin (23–25). NFκB plays an important role in inflammation, cell proliferation and oncogenic responses. It has been demonstrated that NFκB is able to induce transcription of cyclin D1 and increase cell proliferation in response to variety of inflammatory stimuli (26,27). Formation of cyclobutane primidine dimers (CPDs) and pyrimidyne-(6–4)-pyrimidone photoproducts are considered to be early markers of UVB-induced DNA damage (3,28,29). Accumulation of p53 protein plays a crucial role in the cellular response to UVB-induced DNA damage. Activated p53 induces the expression of downstream effectors such as cyclin dependent kinase inhibitor protein p21, which is directly involved in DNA repair and plays an important role in cell cycle arrest and apoptosis (30–32). In UVB-induced DNA damage cells, the interaction of p21 with cyclin dependent kinases and PCNA leads to cell cycle arrest. Furthermore, over expression of p21 in UVB-exposed skin arrests cell cycle progression by inhibiting cdk2 and cdk4 kinases required for cell cycle progression (33–36).

Widespread life style changes lead to increased exposure to UVB radiation. Unfortunately, wearing protective clothing and the topical application of sunscreen is not sufficient. Therefore, more effective strategies to ameliorate the adverse effects of UVB exposure are needed. Photochemoprevention is one such approach, in which pharmacologically active plant derived agents can be administered either orally or topically to prevent UVB-induced skin damage (3–5). Fisetin is a flavonoid that is abundantly present in fruits and vegetables (such as apples, grapes, strawberries, mangoes, peaches, persimmons, cucumbers, tomatoes and onions). It possesses anti-oxidative, anti-inflammatory, anti-proliferative, proapoptotic, and neuroprotective activities (37). Fisetin has been shown to inhibit COX-2, Wnt/β-catenin, PI3K/AKT and NFκB signaling pathways in various cancers (1, 38–41). Recently, we have shown that fisetin induces cell cycle arrest and apoptosis in human epidermoid carcinoma A431cells (42) and inhibits human melanoma cell invasion (43). The goal of this study was to investigate the effect of fisetin on inflammatory mediators and PI3K/AKT/NFκB cell survival signaling pathways in UVB exposed SKH-1 hairless mouse skin. We found that topical application of fisetin inhibited UVB-induced hyperplasia, infiltration of leukocytes, expression of inflammatory mediators, DNA damage, proliferation markers, and activation of PI3K/AKT/NFκB signaling pathways. In addition, fisetin treatment further enhanced UVB-mediated protein expression of p53 and p21.

Materials and Methods

Materials

Fisetin (98% pure), β-actin antibody and MPO fluorometric activity assay kit were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for F4/80, Ly-6G (Gr-1), EP1, EP2, EP3, EP4, COX-2, TNFα, IL-1β, IL-6, Cyclin D1, PCNA, p21, p53, PI3Kp110α, PI3K/p85, pAKT Ser473, NFκB p65, IKKα/β, IκBα, phospho-IκBα and lamin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidases conjugated anti-mouse, anti-goat and anti-rabbit secondary antibodies were purchased from Millipore Corporation (Billerica, MA). Anti-goat and anti-rabbit secondary antibodies labelled with either Alexa Flour 488 or 594 were purchased from Life Technologies Corporation (Grand Island, NY). PGE2 EIA kit was purchased from Cayman Chemicals Company (Ann Arbor, MI). Reagents for protein estimation were purchased from Bio-Rad Laboratories (Hercules, CA). HyBlot CL and autoradiography films were procured from Denville Scientific Inc. (Metuchen, NJ).

Treatment of animals

Six weeks old female SKH-1 hairless mice were procured from Charles River Laboratories (Wilmington, MA). Mice were housed in standard conditions where 24±2°C temperature, 50%±10% relative humidity and 12 h room light/12 h dark cycle were maintained. The phytochemical free diet AIN-76 SEMI PD (Test Diet, Richmond, IN) ad libitum was fed to the animals. The mice were divided into six groups of eight animals each. The mice in the first group were topically treated with 0.2 ml acetone and used as vehicle control. The mice in the second and third groups received topical treatment of fisetin 250 nmol/0.2 ml acetone/mouse and 500 nmol/0.2 ml acetone/mouse respectively on their dorsal skin and served as agent controls. To obtain desired concentrations, 50 mM stock solution of fisetin was prepared in DMSO and further diluted in acetone. The mice in the fourth, fifth and sixth groups were exposed to UVB (180 mJ/cm2). The mice in the fourth group received a topical application of 0.2 ml acetone after 15 min of UVB exposure designated as vehicle control. The mice in the fifth and sixth groups were treated with topical application of fisetin 250 nmol/0.2 ml acetone/mouse and 500 nmol/0.2 ml acetone/mouse respectively on to their dorsal skin after 15 min of UVB exposure. Seven treatments of UVB were given to the mice every alternate day using UV irradiation unit obtained from Daavlin Company (Bryan, OH) as described earlier (44). Studies were performed at 24 h after the last UVB exposure. To determine the effect of fisetin on UVB-induced CPD formation, mice were exposed with a single dose of 180mJ/cm2 UVB and treated with fisetin as described above. The animals were sacrificed 30 min, 24 h and 48 h post UVB irradiation and skin tissues were collected and CPD staining was performed.

Epidermal lysate and nuclear lysate preparation

As described earlier, the epidermis from the skin was separated and then epidermal lysate and nuclear lysate were prepared (44). Briefly, after homogenization of epidermis in lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, 1 mM PMSF (pH 7.4), and protease inhibitor cocktail], homogenate was centrifuged at 14 000 g for 25 min at 4°C. Supernatant was carefully transferred into a new tube and stored at −80°C. Nuclear lysate was prepared after centrifuging the epidermal homogenate in PBS at 12 000 for 10 min at 4°C and suspending pellet in buffer of 10 mM HEPES (pH 7.9), 2 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, and 0.1 mM PMSF. Suspensions were homogenized, incubated on ice for 10 min, and centrifuged at 25 000 g for 10 min. Pellets containing nuclear materials were resuspended in nuclear lysis buffer (10 mM HEPES (pH 7.9), 300 mM NaCl, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF containing 10% glycerol and protease inhibitors cocktail). After 20 min of incubation on ice, the suspension was centrifuged at 25 000 g for 10 min and the supernatants were collected as nuclear lysate and stored at −80°C.

Western blot analysis

Western blot analysis of lysates were performed by resolving 25–40 μg of protein over SDS–PAGE gels composed of 10–12% Tris–glysine. Resolved proteins were transferred onto PVDF membranes and incubated with 5% nonfat dry milk in 20mM TBS (pH 7.6) containing 0.1% Tween-20 for 1 h at room temperature. Membranes were then incubated overnight at at 4°C with primary antibody following 1h incubation with anti-mouse or anti-rabbit or anti-goat secondary antibody conjugated with horseradish peroxidase. Membranes were then washed with TBST (20mM TBS (pH 7.6) containing 0.5% Tween-20). Membranes were developed by chemiluminescence using Pierce ECL Western Blotting Substrate reagents (Thermo Scientific, Rockford, IL) and autoradiography using HyBlot CL Autoradiography Film obtained from Densville Scientific Inc., (Metuchen, NJ). ImajeJ scientific software program (http://rsbweb.nih.gov/ij/index.html) was used to obtain densitometric measurements of the bands.

Immunohistochemical and immunofluorescence staining

After euthanizing the mice skin was harvested and fixed in 10% neutralized formalin and embedded in paraffin. Five micrometer sections were deparaffinized in xylenes, rehydrated in ethanol, and H&E was performed to analyze hyperplasia and infiltration of inflammatory cells in the dermis. For immunohistochemical and immunofluorescence staining, 5μm thick sections were deparaffinized in xylenes, rehydrated in ethanol and subjected to antigen retrieval by heating at 95°C for 30 min in citrate buffer (pH 6.0). For CPD staining nuclear DNA was denatured by incubating the slides in 70mM NaOH in 70% ethanol for 2 min. They were then neutralized with 100 mM Tris-HCl (pH 7.5) for 1 min. Sections were washed with phosphate-buffered saline, blocked with blocking buffer at room temperature for 30 min, and incubated overnight at 4°C with appropriated primary antibodies. Peroxidase blocking with 3% H2O2 for 20 min was performed and sections were incubated with a specific horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Sections were developed by incubating 3–5 min with diaminobenzidene peroxidase substrate solution (DAB) and counterstained with Mayer’s Hematoxylin solution. For immunofluorescence staining after antigen retrieval, sections were incubated overnight at 4°C with primary antibodies and then incubated with secondary antibody conjugated with Alexafluor-488 or -594 for 1 h at room temperature. Vectashield mounting media containing DAPI was used to mount the slides and then analyzed under fluorescence microscope.

Determination of PGE2 levels

PGE2 levels were determined using PGE2 EIA kit from Cayman Chemicals Company (Ann Arbor, MI). This assay is based on the competition between PGE2 and a PGE2-acetylcholinesterase conjugate (PGE2 Tracer) for a limited amount of PGE2 monoclonal antibody. Briefly, skin samples were homogenized in 0.1M phosphate buffer (pH 7.4) containing 1 mM EDTA and 10 μM indomethacin. The supernatants were collected for analysis of PGE2 levels and assay was performed according to the instruction provided in the manufacture’s manual.

Determination of MPO activity

MPO activity was determined using MPO fluorometric activity assay kit purchased from Sigma-Aldrich (St. Louis, MO). In this assay, MPO catalyzes the formation of hypochlorous acid which reacts with the substrate, aminophenyl fluorescein, to generate fluorescein (λex= 485/λem=525nm). One unit of MPO is the amount of enzyme that will oxidize the MPO substrate to yield 1.0 umole of fluorescein per min at room temperature. Assay was performed according to the instruction provided in the manufacture’s manual.

Statistical analysis

The results are expressed as mean ± SEM. Levels of significance of differences among the groups were calculated using Students t-test and a p<0.05 is considered significant.

Results

Fisetin treatment inhibits UVB-induced hyperplasia and infiltration of inflammatory cells

UVB exposure is known to induce hyperplasia and infiltration of inflammatory cells in the skin (45). Therefore, we first assessed the effect of topical application of fisetin on hyperplasia and infiltration of inflammatory cells in the skin of SKH-1 hairless mice after UVB exposure. UVB exposure to the mouse skin resulted in increased epidermal thickness and infiltration of inflammatory cells as compared to control mice (Figure 1A). Topical application of fisetin (250 and 500 nmol on the dorsal skin) after UVB exposure resulted in inhibition in the induction of epidermal hyperplasia and infiltration of inflammatory cells when compared with the skin of UVB exposed mice (Figure 1A). Moreover, fisetin alone did not induce epidermal hyperplasia in the mouse skin when compared to control (Figure 1A). UVB exposure resulted in a marked increase in activated macrophages and neutrophils, demonstrated by increased immunostaining of F4/80 and Gr-1 respectively when compared with the skin of control mice (Figure 1B,C). However, topical application of fisetin in UVB exposed mice skin resulted in a marked decrease in the infiltration of macrophages (F4/80 positive cells) and neutrophils (Gr-1 positive cells) compared to the skin of UVB exposed mice (Figure 1B,C).

Figure 1. Effect of fisetin on UVB-induced cutaneous hyperplasia and infiltration of leukocytes.

Figure 1

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The groups of mice were either unexposed or exposed to UVB (180mJ/cm2) and treated with fisetin (250 nmol and 500 nmol) 15 min after UVB irradiation. The details of mice treatment are described in Materials and Methods. The animals were sacrificed 24 h after the last UVB exposure and skin tissues were collected. (A) Hematoxylin and eosin staining. Representative photomicrographs from each treatment group are shown. Bar represents 25 μm. (B & C) Immunofluorescence staining for macrophages (F4/80) and neutrophils (Gr-1) was performed on 5μm thick skin sections as described in Materials and Methods. F4/80 staining is shown in green, Gr-1 in red and DAPI in blue. Representative photomicrographs from each treatment group are shown. Bar represents 25 μm.

Fisetin treatment inhibits UVB-induced MPO activity

MPO is synthesized and secreted by activated leukocytes and is characterized by potent prooxidative and proinflammatory properties (46,47). MPO catalyzes the formation of hypochlorous acid and tyrosyl radicals during the respiratory burst process (15,16). Since fisetin treatment inhibited the infiltration of inflammatory cells such as neutrophils and macrophages, MPO activity was determined in the skin of UVB exposed mice treated with fisetin. Consistent with the inhibition of infiltration of neutrophils and macrophages in the skin, fisetin treatment also significantly inhibited MPO activity in UVB exposed mouse skin as compared to the group only exposed to UVB (Figure 2D).

Figure 2. Effect of fisetin on UVB-induced inflammatory mediators.

Figure 2

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The details of mice treatment are described in Materials and Methods. The animals were sacrificed 24 h after the last UVB exposure and skin tissues were collected. (A & B) Western blot analysis and relative density were performed to determine the protein expression of COX-2 and EP receptors. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The western blots shown here from a representative experiment repeated three times with similar results. Difference is shown as fold change ± SEM. Significant differences were measured as **P<0.01 vs control. #P<0.05 and ##P<0.01 vs UVB. (C & D) PGE2 level and MPO activity in skin tissue was performed using commercially available kits. Level of significance was determined as **P<0.01 vs control. #P<0.05 and ##P<0.01 vs UVB.

Fisetin treatment inhibits UVB-induced expression of COX-2, production of PGE2 and expression of PGE2 receptors (EPs)

COX-2 can be readily induced in response to inflammatory stimuli leading to production of PGs, especially PGE2 (48). In addition, PGE2 regulates COX-2 expression via a positive autoregulatory feedback loop (49). Therefore, the effect of fisetin treatment on COX-2 expression was determined in the skin of UVB exposed mice. After UVB exposure COX-2 expression was significantly increased as compared to control mice. However, fisetin treatment significantly inhibited UVB-mediated induction of COX-2 expression (Figure 2A,B). As a consequence of increased COX-2 expression, significantly elevated levels of PGE2 were detected in the UVB exposed mice as compared to control mice. Fisetin treatment also significantly reduced the enhanced level of PGE2 in UVB exposed mice (Figure 2C). Moreover, PGE2 is known to exert its biological effects through four membranes bound G-protein coupled E prostanoid (EP) receptors: EP1, EP2, EP3 and EP4. Therefore, expression of these receptors was determined in fisetin treated UVB exposed mice. Fisetin significantly reduced the expression of EP receptors (EP1-EP4) in the skin of UVB exposed mice as compared to UVB control mice (Figure 2A,B).

Fisetin treatment inhibits UVB-induced proinflammatory cytokines

Skin inflammation due to UVB exposure has been characterized by infiltration of leukocytes and production of proinflammatory cytokines such as TNFα, IL-1β and IL-6 (8,9). TNFα facilitates local inflammatory reactions within the epidermis and plays an important role in photodamage (50). IL-6 also plays a key role in the acute phase response and governs leukocyte infiltration during acute inflammation (51). Production of IL-6 increases in the skin after UVB exposure. IL-1β is involved in hemostatic function in normal skin, but is implicated in various pathophysiological conditions including inflammation when over produced. IL-1β can be released from infiltrated macrophages, mast cells, and keratinocytes after UVB exposure (8,9). Expression of these proinflammatory cytokines was evaluated in the skin of UVB exposed mice treated with fisetin. In UVB exposed mice the protein expression of TNFα, IL-1β and IL-6 was significantly higher as compared to the control mice. However, fisetin treatment significantly reduced the protein expression of TNFα, IL-1β and IL-6 cytokines in the skin of UVB exposed mice as compared to mice exposed to UVB alone (Figure 3).

Figure 3. Effect of fisetin on UVB-induced inflammatory cytokines.

Figure 3

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The details of mice treatment are described in Materials and Methods. The animals were sacrificed 24 h after the last UVB exposure and skin tissues were collected. (A & B) Western blot analysis and relative density were performed to determine the protein expression of TNFα, IL-1β and IL-6. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The western blots shown here from a representative experiment repeated three times with similar results. Difference is as fold change ± SEM. Significant differences were measured as *P<0.05 and **P<0.01 vs control. #P<0.05 and ##P<0.01 vs UVB. (C) Immunostaining analysis for TNFα and IL-1β was performed on 5μm thick sections as described in Materials and Methods. Representative photomicrographs from each treatment group are shown. Bar represents 25 μm.

Fisetin treatment inhibits UVB-induced markers of cell proliferation

UVB-induces keratinocyte survival and proliferation by inducing COX-2 expression and production of PGE2. PGE2 acts through EP receptors leading to cell survival (18–20). Therefore, we determined the effect of fisetin on the proliferative potential of epidermal cells after UVB exposure. Exposure of mice to UVB resulted in increased expression of PCNA and cyclin D1 as compared to control mice. Furthermore, fisetin treatment significantly inhibited this increased expression of PCNA and cyclin D1 in the skin of UVB exposed mice (Figure 4).

Figure 4. Effect of fisetin on UVB-induced cell proliferation markers.

Figure 4

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The details of mice treatment are described in Materials and Methods. The animals were sacrificed 24 h after the last UVB exposure and skin tissues were collected. (A & B) Western blot analysis and relative density were performed to determine protein expression of cyclin D1 and PCNA. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The western blots shown here from a representative experiment repeated three times with similar results. Difference is shown as fold change ± SEM. Significant differences were measured as **P<0.01 vs control. ##P<0.01 and vs UVB.

Fisetin treatment accelerates the removal of UVB-induced CPDs

CPD formation occurs immediately after UVB irradiation and is considered to be an early biomarker of UVB-induced DNA damage (28,29). Due to the abundance and slow repair process of CPDs, they are considered to be an important type of pre-mutagenic DNA damage (3). Therefore, we evaluated the effect of fisetin treatment on CPD formation in the skin of UVB exposed (180mJ/cm2) SKH-1 hairless mice after a single exposure. Skin samples were collected 30 min, 24 h and 48 h after UVB exposure. The number of CPD+ cells was significantly higher in UVB exposed groups at all these time points when compared to control (Figure 5A,B). In the skin biopsies collected 30 min after UVB exposure the number of CPD+ cells was almost similar in UVB exposed mice and UVB exposed-fisetin treated groups (Figure 5A,B). However, the number of CPD+ cells was significantly reduced in UVB exposed mice treated with fisetin when compared to UVB alone groups at 24 and 48 h (Figure 5A,B). Therefore, the reduction in the number of CPD+ cells at these time points may be due to enhanced DNA repair by fisetin.

Figure 5. Effect of fisetin on UVB-induced DNA damage, p53 and p21 protein expression.

Figure 5

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The groups of mice were either unexposed or exposed to UVB (180mJ/cm2) and treated with fisetin (250 nmol and 500 nmol) 15 min after UVB irradiation. After 30 min, 24 and 48 h post UVB irradiation, the animals were sacrificed and skin tissues were collected. (A) Five micrometer thick skin sections were cut and processed for CPD staining. Representative photomicrographs from each treatment group are shown. (B) The number of CPD positive cells after CPD staining were counted in at least three different areas of the sections under a microscope. The numbers of CPD positive cells are represented as percent of CPD positive cells ± SEM. Significant differences were measured as *P<0.05 and **P<0.01 vs control. #P<0.05 vs UVB (24 h). $P<0.05 vs UVB (48 h). Bar represents 10 μm. (C & D) The groups of mice were either unexposed or exposed to UVB (180mJ/cm2) and treated with fisetin (250 nmol and 500 nmol) 15 min after UVB irradiation. The details of mice treatment are described in Materials and Methods. The animals were sacrificed 24 h after the last UVB exposure and skin tissues were collected. Western blot analysis and relative density were performed to determine protein expression of p53 and p21. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The western blots shown here from a representative experiment repeated three times with similar results. Difference is shown as fold change ± SEM. Significant differences were measured as *P<0.05 and **P<0.01 vs control. #P<0.05 and ##P<0.01 vs UVB.

Fisetin treatment augments UVB-induced protein expression of p53 and p21

Mitigation of UVB-induced skin damage depends on the ability of cells to detect and repair DNA damage, regulates the cell cycle, and if necessary induces apoptosis. In UVB exposed skin, DNA damage induces expression of p53 and its downstream target protein p21. During DNA damage, expression of these proteins inhibits progression of the cell cycle at G1–S phase, which allows for either correction of DNA lesions or, if the cell is severely damaged, induction of apoptosis. The discomposure of this regulation leads to neoplastic transformation of DNA damaged cells resulting in UVB-induced skin carcinogenesis (31,30). Therefore, protein expression of p53 and p21 was determined in the skin of UVB exposed mice after fisetin treatment. As shown in figure 5C,D, UVB exposure increased the protein expression of both p53 and p21 as compared to control. In addition, fisetin treatment of UVB exposed mice further enhanced the expression of p53 and p21 (Figure 5C,D).

Fisetin treatment inhibits UVB-induced expression of PI3K and phosphorylation of AKT

Activation of the PI3K/AKT signaling pathway is another key mediator of UVB-induced skin malignancies. PI3K/AKT signaling regulates various cellular processes including apoptosis, proliferation and growth (3,22). EP4 has also been shown to activate PI3K, leading to AKT phosphorylation (21). In the present study, we found that UVB exposure significantly increased the protein expression of PI3K/p110α, PI3K/p85 and phosphorylation of AKT as compared to control. However, fisetin treatment significantly inhibited the protein expression of PI3K/p110α, PI3K/p85 and phosphorylation of AKT when compared with UVB exposed mice (Figure 6A,B).

Figure 6. Effect of fisetin on UVB-induced activation of PI3K/AKT and NFκB signaling.

Figure 6

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The details of mice treatment are described in Materials and Methods. After 24 h of last UVB exposure and fisetin treatment, animals were sacrificed and skin tissues were collected. Western blot analysis and relative density were performed to determine the protein expression of (A & B) PI3Kp110α, PI3K/p85 and phosphorylation of AKT, and (C & D) IKKα/β, IκBα, and phosphorylation of IκBα. The NFκBp65 was determined in the nuclear lysate. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin or lamin. The western blots shown here from a representative experiment repeated three times with similar results. Difference is shown as fold change ± SEM. Significant differences were measured as **P<0.01 vs control. #P<0.05 and ##P<0.01 vs UVB.

Fisetin treatment inhibits UVB-induced activation of IKKα/β and NFκB, and phosphorylation and degradation of IκBα

The activation of NFκB by extracellular stimuli such as UVB depends on the phosphorylation and subsequent degradation of IκB proteins through a family of serine/threonine kinases known as IKK. UVB is a known activator of NFκB (44, 52–54), and activation of NFκB leads to the production of the proinflammatory cytokines IL-1β, IL-6 and TNFα (55). UVB exposed skin of SKH-1 hairless mice showed increased expression of IKKα/β, resulting in increased phosphorylation and degradation of IκBα. In addition, fisetin treatment of the UVB exposed mice significantly restored the IκBα protein level by reducing the UVB-induced expression of IKKα/β and phosphorylation of the IκBα protein (Figure 6C,D) as compared to UVB exposed mice not treated with fisetin. Furthermore, fisetin treatment also inhibited UVB-induced activation and nuclear translocation of NFκBp65 as compared to the skin of UVB exposed mice not treated with fisetin (Figure 6C,D).

Discussion

Skin cancer is the most common cancer in the USA, accounting for nearly half of all the human cancers. Solar UVB radiation is a ubiquitous environmental carcinogen, which causes a variety of cutaneous disorders including melanoma and non-melanoma skin cancers (NMSCs). In the USA, more than 2 million people are diagnosed annually with NMSCs (56). Plant-derived products possess potential anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties and are gaining considerable attention for the prevention of UVB-induced skin damage (3,5). In this study, fisetin, a flavonoid abundantly present in fruits and vegetables, was topically applied in UVB exposed SKH-1 hairless mice to investigate its effects on inflammatory mediator markers and PI3K/AKT/NFκB signaling pathways. We demonstrated that topical application of fisetin significantly inhibited UVB-induced hyperplasia and infiltration of inflammatory leukocytes. Activated leukocytes synthesize and secrete MPO, a prooxidative and proinflammatory enzyme, which is known to induce inflammation by modulating vascular signaling and vasodilation in UVB exposed skin (46,47). Consistent with inhibition of hyperplasia, and infiltration of neutrophils and macrophages in the skin of UVB exposed mice, fisetin treatment also significantly inhibited MPO activity.

There is an accumulating body of evidence demonstrating exposure of skin to UVB exposure to the skin leads to induction of COX-2 expression and production of PGE2. Both expression of COX-2 and PGE2 production promote keratinocyte proliferation and survival. In addition, COX-2 and PGE2 play a pivotal role in induction of UVB-induced skin hyperplasia and inflammation (57–59). The expression of COX-2 was increased after UVB exposure, and fisetin treatment significantly inhibited COX-2 expression in the skin of UVB exposed mice. As a consequence of inhibition of COX-2 expression by fisetin treatment, a significant reduction in the level of PGE2 was also observed. PGE2 manifests its biological response through EP receptors (EP1–EP4) and expression of these receptors has been linked with inflammation, angiogenesis, decreased host immunity, enhanced invasion, and metastasis (19,20). Fisetin treatment significantly inhibited the inflammatory response by reducing the expression of EP receptors in the skin of UVB exposed mice. In addition to increased expression of these inflammatory mediators, proinflammatory cytokines such as TNFα, IL-1β and IL-6 also play an important role in UVB-induced photodamage (8,9). Increased expression of proinflammatory cytokines further promotes hyperproliferation of keratinocytes and infiltration of inflammatory cells (50,51). Fisetin treatment significantly inhibited the expression of these proinflammatory cytokines in the skin of UVB exposed mice.

DNA damage caused by UVB radiation leads to stabilization and accumulation of p53 protein through transcriptional activation which play a crucial role in cell cycle arrest at the G1 phase. Once activated, p53 protein translocates into the nucleus and activates transcription of several DNA-binding proteins and downstream effectors involved cell cycle arrest, DNA repair and/or induction of apoptosis (31,32). In UVB exposed skin, activation of p53 leads to activation of p21, which in turn inhibits the cyclin E/cdk2 and cyclin A/cdk2 kinases resulting in cell cycle arrest. Inhibition of the cell cycle permits repair of damaged DNA through nucleotide excision repair mechanism (31,34). Western blot analysis in the present study clearly demonstrated increased p53 and p21 expression in the skin of UVB exposed mice. Fisetin treatment further augmented protein expression of p53 and p21 in UVB exposed mice. Furthermore, generation of DNA photoproducts (mainly CPD formation) is a direct response of UVB-induced DNA damage (3). Expression and accumulation of p53 protein leads to repair of photoproducts including CPDs. Consistent with these findings, quantification of immunohistochemical analysis revealed that CPD repair was significantly enhanced in fisetin treated, UVB exposed mice.

In addition, p53 and p21 proteins are involved in the regulation of PCNA and cyclin D1. UVB exposure has been shown to induce keratinocytes proliferation and increase expression of PCNA (60,61). In UVB exposed cells, p21 inhibits DNA replication by interacting with PCNA. PCNA promotes degradation of the replication initiation factor complex induced by DNA damage, and increased expression of PCNA in UVB-induced DNA damaged cells is a result of binding of p53 to PCNA promoter (33–36). In the present study, fisetin treatment significantly inhibited the protein expression of PCNA in the skin of UVB exposed mice. Cyclin D1 is another important proliferation marker that regulates G1 to S phase progression. Overexpression of cyclin D1 has been linked to the development and progression of many cancers, including UVB-induced skin carcinogenesis (27). In the present study, UVB exposure induced a significant increase in the protein expression of cyclin D1; however, fisetin treatment significantly inhibited its expression in the skin of UVB exposed mice.

UVB radiation is known to activate several signal transduction pathways, including the PI3K/AKT pathway in cultured cells as well as in both mouse and human skin (22–24,62). In UVB exposed skin PI3K/AKT signaling increases the survival response of keratinocytes and facilitates tumorigenesis. Furthermore, in UVB exposed skin, PI3K/AKT signaling promotes transcriptional activation of COX-2. Thus the PI3K/AKT signaling pathway is a potential target for suppressing UVB mediated COX-2 expression and the resulting PGE2-medited inflammation (18,20,21). In the present study, protein expression of PI3K (catalytic subunit p110 and a regulatory subunit p85) and phosphorylation of AKT was significantly elevated in the skin of UVB exposed mice as compared to control. However, increased expression of PI3K protein and phosphorylation of AKT was significantly inhibited in the skin of UVB exposed, fisetin treated mice as compared to the skin of the UVB exposed mice alone. As a consequence of PI3K/AKT signaling inhibition by fisetin, reduced expression of COX-2 with decreased levels of PGE2 was observed in UVB exposed mice and correlated with reduced inflammation.

NFκB regulates production of inflammatory mediators and induces transcription of pro-inflammatory genes. NFκB also contributes to the regulation of cell proliferation and survival. Studies have shown that UVB radiation activates NFκB signaling in keratinocytes and mouse skin (44, 52–54). NFκB mediated secretion of cytokines (such as TNFα, IL-1β, IL-6), expression of COX-2 and production of PGE2 in UVB exposed skin (63,64). In addition, p53 and NFκB signaling have many mutually antagonistic functions; specifically p53 can inhibit NFκB signaling (65–67). Furthermore, induction of the cyclin-dependent kinase inhibitor p21 inhibits NFκB-mediated keratinocyte proliferation (68–70). It has been demonstrated that fisetin inhibits cell cycle progression at G0/G1 phase and induces apoptosis by increasing p53 and p21 expression, and down regulating NFκB signaling in cancer cells (71,72). In the present study, we found that fisetin treatment significantly inhibited UVB-induced activation of IKKα/β, phosphorylation and degradation of IκBα proteins, and nuclear translocation of NFκBp65. Our results suggest that fisetin inhibits NFκB signaling in UVB exposed mice that leads to induction of p53 and p21 protein expression, reduced expression of proinflammatory cytokines (TNFα, IL-1β, IL-6) and inhibition of COX-2, resulting in decreased infiltration of inflammatory cells and reduced inflammation.

Collectively, our data suggests that topical application of fisetin to SKH-1 mice after UVB exposure results in a significant decrease in leukocyte infiltration, inflammatory mediators (MPO, COX-2 and PGE2), inflammatory cytokines (TNFα, IL-1β, IL-6) and proliferation markers (PCNA and cyclin D1). In addition, fisetin inhibited PI3K/AKT/NFκB signaling, which is involved in UVB-induced inflammation, cell survival and proliferation. Fisetin treatment also augmented UVB-mediated protein expression of p53 and p21, and reduced the number of CPD+ cells caused by UVB exposure. Importantly, fisetin treatment is not damaging to mouse skin. Overall, these findings suggest that fisetin could be developed as a novel photochemopreventive agent for the management of UVB-induced skin malignancies.

Acknowledgments

This work was supported by NIH Grant 1R21CA173043-01A1, and UAB Skin Disease Research Center Pilot and Feasibility Grant (P30AR050948).

Footnotes

†

This paper is part of the Special Issue commemorating the 65th birthday of Dr. Craig A. Elmets

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