Arch Dermatol Res DOI 10.1007/s00403-014-1529-8
Suppression of skin inflammation in keratinocytes and acute/ chronic disease models by caffeic acid phenethyl ester Kyung-Min Lim • SeungJin Bae • Jung Eun Koo Eun-Sun Kim • Ok-Nam Bae • Joo Young Lee
Received: 28 August 2014 / Revised: 17 November 2014 / Accepted: 3 December 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Skin inflammation plays a central role in the pathophysiology and symptoms of diverse chronic skin diseases including atopic dermatitis (AD). In this study, we examined if caffeic acid phenethyl ester (CAPE), a skinpermeable bioactive compound from propolis, was protective against skin inflammation using in vitro cell system and in vivo animal disease models. CAPE suppressed TNF-ainduced NF-jB activation and expression of inflammatory cytokines in human keratinocytes (HaCaT). The potency and efficacy of CAPE were superior to those of a nonphenethyl derivative, caffeic acid. Consistently, topical treatment of CAPE (0.5 %) attenuated 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced skin inflammation on mouse ear as CAPE reduced ear swelling and histologic inflammation scores. CAPE suppressed increased expression of pro-inflammatory molecules such as TNF-a, cyclooxygenase-2 and inducible NO synthase in TPAstimulated skin. TPA-induced phosphorylation of IjB and ERK was blocked by CAPE suggesting that protective effects of CAPE on skin inflammation is attributed to inhibition of NF-jB activation. Most importantly, in an oxazolone-induced chronic dermatitis model, topical application of CAPE (0.5 and 1 %) was effective in alleviating AD-like symptoms such as increases of trans-
K.-M. Lim S. Bae College of Pharmacy, Ewha Womans University, Seoul, Korea J. E. Koo J. Y. Lee (&) College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Korea e-mail: [email protected]
E.-S. Kim O.-N. Bae College of Pharmacy, Hanyang University, Ansan, Gyeonggido 426-791, Korea
epidermal water loss, skin thickening and serum IgE as well as histologic inflammation assessment. Collectively, our results propose CAPE as a promising candidate for a novel topical drug for skin inflammatory diseases. Keywords Caffeic acid phenethyl ester (CAPE) Skin inflammation Nuclear factor jB (NF-jB) Atopic dermatitis TPA-induced ear edema
Introduction Caffeic acid phenethylester (2-phenylethyl(2E)-3-(3,4dihydroxyphenyl)acrylate, CAPE), a bioactive phenolic compound found in honeybee hive propolis, has beneficial effects on inflammation and chronic diseases [12, 17]. Inhibition of nuclear factor jB (NF-jB) activation is suggested as one of the major mechanisms by which CAPE exerts anti-inflammatory effects. CAPE suppresses the activation of NF-jB induced by various inflammatory agents including lipopolysaccharide, tumor necrosis factor (TNF)-a, phorbol ester, ceramide, hydrogen peroxide, and okadaic acid in human histiocytic cell line, U937 . NFjB inhibition by CAPE is partly mediated through the suppression of IjB kinase (IKKb) complex activity . In addition, CAPE attenuates the production of pro-inflammatory cytokines like TNF-a, IL-6 and IL-8, and the expression of inducible enzymes such as cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) in RAW264.7 macrophages, ultimately accomplishing its protective effects on inflammatory symptoms . Inflammation plays a critical role in the development and progress of diverse skin diseases that include atopic dermatitis (AD), psoriasis and contact dermatitis . Especially in AD, chronic and relapsing skin inflammation
Arch Dermatol Res
constitutes the hallmark of the disease and contributes to a disturbance of epidermal barrier function, and IgE-mediated skin sensitization . To control skin inflammation, anti-inflammatory and immune-modulating drugs such as corticosteroids and calcineurin inhibitors are widely used . However, due to poor effectiveness and severe adverse effects, there is unceasing demand for new anti-inflammatory agents for skin inflammatory diseases with improved therapeutic window. While anti-inflammatory effects of CAPE are well established, its therapeutic utility in skin inflammation was known only in part. Recently, anti-inflammatory activities of caffeic acid (3,4-dihydroxycinnamic acid), a structural derivative of CAPE, against TPA-induced skin inflammation in vivo were reported . Of note, compared to CA or other natural compounds, CAPE is lipophilic and skinpermeable owing to phenethyl chain in its structure , suggesting that a topical formulation of CAPE might be more effective and efficient against diverse skin inflammation. Here, we investigated the efficacy of CAPE on skin inflammation using in vitro keratinocyte cell system and in vivo skin inflammatory disease animal models, to explore its therapeutic utility as a novel topical drug for the skin inflammatory diseases.
Materials and methods Reagents 12-O-tetradecanoylphorbol-13-acetate (TPA), oxazolone, CAPE and CA were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TNF-a was purchased from R&D Systems (Minneapolis, MN, USA). Cell culture Human keratinocyte cell line (HaCaT) was obtained from Cell Lines Service (Heidelberg, Germany). HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10 % heat-inactivated fetal bovine serum (Thermo Scientific Hyclone, Logan, UT, USA), 100 units/mL penicillin, and 100 lg/mL streptomycin (Welgene, Daegu, Korea). Cells were maintained in 95 % air, 5 % CO2 at 37 °C. Experiments were performed with HaCaT cells at 90 % confluency. RT-quantitative real-time PCR HaCaT cells were treated with TNF-a (20 ng/mL) in the presence or absence of CAPE (1, 10 lM) or CA (10 lM) for 6 h, and total cellular RNA was isolated using Trizol reagent (Life Technologies, Carlsbad, CA, USA). cDNA
was synthesized from total RNA using ReverTra AceÒ qPCR RT Kit (Toyobo, Osaka, Japan). Quantification of gene copies was carried on CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), using iQTM SYBR Green supermix (Bio-Rad). PCR cycles consisted of an initial step at 95 °C for 3 min followed by 45 cycles at 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 10 s. Relative mRNA expressions were calculated by the comparative CT method (2-DDCt), normalized to the endogenous 18S control. Primers for TNF-a, IL-1b, and IL-6 were obtained from GENOTECH (Daejeon, Korea) and the sequences were as follows: hTnf-a: 50 -AGC CCA TGT TGT AGC AAA CC-30 (for), 50 -GGC ACC ACC AAC TGG TTA TC-30 (rev); hIl-1b, 50 -AGG CCT CTC TCA CCT CTC CT-30 (for), 50 -AGA ATG TGG GAG CGA ATG AC-30 (rev); hIl-6, 50 -AAA GAG GCA CTG GCA GAA AA-30 (for); 50 -TTT CAC CAG GCA AGT CTC CT30 (rev); and h18S, 50 -GTA ACC CGT TGA ACC CCA TT30 (for), 50 -CCA TCC AAT CGG TAG TAG CG-30 (rev). Nuclear translocation of NF-jB Nuclear translocation of NF-jB p65 was examined by immunoblotting using Tris–HCl SDS-PAGE. HaCaT cells (2 9 105) were seeded in six-well plates and incubated at 37 °C for 48 h. After cells were treated with TNF-a (20 ng/mL) in the presence and absence of CAPE (10 lM) for 1 h, cells were harvested and washed twice with cold PBS. Cytoplasmic and nuclear extracts were prepared by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. Protein concentrations in each fraction were measured by PierceTM BCA Protein Assay Kit (Thermo Scientific). After electrophoresis, the proteins were transferred to PVDF membrane (Bio-Rad), and probed with primary antibody against NF-jB p65, Lamin A/C (Cell Signaling Technology, Danvers, MA, USA) or GAPDH (Merck Millipore, Darmstadt, Germany) overnight at 4 °C, followed by incubation with horseradish peroxidase-linked secondary antibody for 1 h, The levels of proteins were analyzed using SuperSignalÒ Chemiluminescent Substrate (Thermo Scientific). Bands were quantified by NIH Image J program, and normalized by the corresponding loading controls. Animals Animal care and the study protocols were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of The Catholic University of Korea (Permission No. 2013-020). Mice were purchased from Orient bio (Seoul, Korea) and acclimated under specific pathogen-free conditions in an animal
Arch Dermatol Res
facility for at least a week before use. The mice were housed in a temperature (23 ± 3 °C) and relative humidity (40–60 %)-controlled room. Lighting was adjusted automatically at a cycle of 12 h light and 12 h dark. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model Efficacy of CAPE and mechanism of action on skin inflammation were examined in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model. Skin inflammation was induced by topical application of TPA (2.5 lg/20 ll in acetone) to ear of male BALB/c mice. CAPE (0.5 %) or indomethacin (Indo; 2.5 %) was topically treated on the ear twice per day for 2 days before TPA treatment and 30 min and 6 h after TPA treatment. Twentyfour hours after TPA application, the weight of 6 mm biopsy of ears was measured and pathological scoring was determined by a blinded histologist. The mRNA levels of TNF-a, COX-2, and iNOS were determined by RT-quantitative real-time PCR. The sequence of primers were as follows: Tnf-a, 50 -AAAATTCGAGTGACAAGCCTGT AG-30 (for), 50 -CCCTTGAAGAGAACCTGGGAGTAG30 (rev); Cox-2, 50 -CATATTTGATTGACAGTCCACC30 (for), 50 -TCCTTATTTCCCTTCACACC-30 (rev); iNos 50 GATGTTGAACTATGTCCTATCTCC-30 (for), 50 -AACA CCACTTTCACCAAGAC-30 (rev); b-actin, 50 -TCATGA AGTGTGACGTTGACATCCGT-30 (for), 50 -TTGCGGTG CACGATGGAGGGGCCGGA-30 (rev). Immunoblotting was performed using primary antibody for COX-2 (Cayman, Ann Arbor, MI, USA), phosphoIjBa, GAPDH, phospho-ERK, and ERK (Cell Signaling Technology, Boston, MA, USA). Oxazolone-induced AD model in hairless mice Therapeutic utility of CAPE against AD was investigated in a chronic hapten (oxazolone)-induced AD model in hairless mice developed by Man et al. . AD was induced by repeated hapten (oxazolone) treatment for 16 days. CAPE (0.5, 1 % in acetone) or betamethasone (BM) (0.01 %) was applied twice daily from day 7 to day 16. Twenty-four hours after the final treatment, trans-epidermal water loss (TEWL), skin thickness of the treated skin area, and serum IgE levels, were determined. IgE level was determined by a commercial IgE assay ELISA kit, Opt EIA Mouse set (BD Bioscience, Mississauga, Canada). Histology evaluation Ear and dorsal skin biopsies of mice were isolated and fixed in 10 % neutral phosphate-buffered formalin, embedded in paraffin, sectioned at 5 lm thickness and
stained with hematoxylin and eosin (H&E). All sections stained simultaneously were analyzed under light microscope (1009 magnification for ear and 2009 for skin biopsy, Olympus Bx-41 microscopy, Olympus, Japan) and photomicrographs were taken with a Leica DC 300F (Leica, Switzerland). Scale bar (100 lm) was added if necessary. A representative area was selected for qualitative light microscopic analysis. Statistical analysis Data are expressed as mean ± standard error of mean (S.E.M.). The statistical analyses were performed by oneway ANOVA followed by Dunnett’s post hoc test. A p value \0.05 was considered statistically significant.
Results CAPE suppresses the expression of inflammatory mediators in TNF-a-stimulated human keratinocyte cell line Keratinocytes constitute a major cell population in the outermost layer of skin. They are sensitive to pro-inflammatory stimuli and are responsible for the initiation of inflammatory responses of skin. Therefore, first we investigated if CAPE inhibited the expression of inflammatory cytokines in human keratinocyte cell line (HaCaT). Since caffeic acid (3,4-dihydroxycinnamic acid, CA), one of the most abundant bioactive polyphenol compounds in plants, is structurally similar to CAPE, we intended to compare the potency of CAPE and CA in the regulation of inflammatory signals in keratinocytes. CAPE significantly blocked TNFa-induced expression of inflammatory cytokines, IL-1b, IL-6 and TNF-a, in HaCaT cells (Fig. 1). However, CA failed to inhibit expression of the cytokines in HaCaT cells demonstrating that CAPE was more effective to reduce inflammatory signals than CA, non-phenethyl derivative, in keratinocytes. CAPE suppresses NF-jB activation induced by TNF-a in human keratinocyte cell line To delineate the underlying mechanism, we examined whether CAPE modulated NF-jB activation in keratinocytes. CAPE suppressed nuclear translocation of NF-jB p65 induced by TNF-a in HaCaT cells (Fig. 2). These suggest that CAPE is effective to alleviate inflammatory responses in keratinocytes stimulated by proinflammatory signals, at least partly mediated by inhibition of NF-jB activation.
Arch Dermatol Res
Fig. 1 Caffeic acid phenethyl ester (CAPE) suppresses the expression of pro-inflammatory cytokines in TNF-a-stimulated human keratinocytes. HaCaT cells were stimulated with TNF-a (20 ng/mL) in the presence or absence with CAPE (1 and 10 lM) or caffeic acid (CA, 10 lM) for 6 h. a Chemical structure of CAPE or CA was
shown. b The mRNA levels of pro-inflammatory cytokines (IL-1b, IL-6, and TNF-a) were analyzed by quantitative real-time PCR. Values are mean ± SEM (n = 3). Asterisk significantly different from TNF-a alone, p \ 0.05
Topical application of CAPE reduces skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate in mice
COX-2 protein expression induced by TPA was blocked by CAPE (Fig. 4b). Intracellular signaling pathways activated by TPA including phosphorylation of IjBa and ERK were also suppressed by CAPE (Fig. 4b), suggesting that reduction of pro-inflammatory protein expression by CAPE in skin is mediated through the blockade of NF-jB activation. The results show that topical treatment with CAPE alleviates acute inflammatory symptoms of skin stimulated with TPA in mice. Consistently with results in keratinocytes, CAPE inhibited activation of NF-jB in TPA-stimulated skin tissue.
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model is widely used for the study of anti-inflammatory agents in the skin . Treatment with TPA on mouse ear induced edema formation and inflammatory responses as determined by increased ear weights and pathological scores that collectively evaluate the neutrophil influx, epidermal hyperproliferation and edema with 0–4 scores (Fig. 3a). Meanwhile, topical application of CAPE (0.5 %) almost completely blocked TPA-induced ear swelling and pathological scores (Fig. 3a). Consistently, histological observation revealed the protective effect of CAPE on TPA-induced skin inflammation (Fig. 3b). CAPE reduces the expression of pro-inflammatory mediators and the activation of NF-jB and ERK in 12O-tetradecanoylphorbol-13-acetate-treated mouse skin To investigate anti-inflammatory mechanism of CAPE in the skin inflammation, we measured the expression of proinflammatory cytokine and enzymes in mouse skin. mRNA levels of TNF-a, COX-2, and iNOS that were significantly increased by TPA treatment were decreased by CAPE (Fig. 4a). Furthermore, immunoblot results showed that
CAPE blocks inflammatory symptoms in a hapteninduced atopic dermatitis animal model Atopic dermatitis (AD) is one of the commonest allergic and chronic inflammatory skin diseases. Hapten-induced AD model well recapitulates the chronic inflammatory and allergic symptoms of AD . Repeated topical application of oxazolone-induced robust AD symptoms in female hairless mice as determined by skin barrier damage [increased trans-epidermal water loss (TEWL)], epidermal thickening from inflammation and increased serum immunoglobulin E (IgE) levels (Fig. 5a–c). In contrast, topical treatment of CAPE attenuated skin barrier damage and epidermal thickening associated with AD. Most importantly, CAPE significantly alleviated the increase in
Arch Dermatol Res
Fig. 2 Caffeic acid phenethyl ester (CAPE) inhibits TNF-a-induced nuclear translocation of NF-jB in human keratinocytes. HaCaT cells were stimulated with TNF-a (20 ng/mL) in the presence or absence with CAPE (10 lM) for 1 h. a Immunoblotting of cytosolic and nuclear fractions was performed to determine nuclear translocation of NF-jB p65. GAPDH and Lamin A/C were used as a specific marker for cytosolic and nuclear fractions, respectively. b Band densities of immunoblots were measured by ImageJ program. Values are mean ± SEM (n = 3). Asterisk significantly different from nontreated group, p \ 0.05. Hash significantly different from TNF-a alone, p \ 0.05
serum IgE, which plays a critical role in the mediation of hyper-allergic reactions in AD. The protective effects of CAPE against hapten-induced AD were almost as potent as those of betamethasone, one of the widely used, moderately potent topical corticosteroids. Histological examination also showed an improvement of dermal inflammation and edema when the mice were treated with CAPE (Fig. 5d). The results suggest that topical application of CAPE is effective to treat chronic skin inflammatory diseases including AD (Fig. 6). Collectively, our results demonstrate that CAPE would be beneficial for blocking detrimental events occurring in acute and chronic skin inflammation, possibly mediated through the suppression of intracellular signaling pathways such as NF-jB in keratinocytes.
Discussion It has been well known that CAPE exerts various biological activities for infection , inflammation, cancer, diabetes, ischemic injury , hepatotoxicity  and neurodegeneration . Recently, anti-melanoma  and immunomodulatory  activities of CAPE have been reported,
drawing attention to its therapeutic utility as a novel botanical drug for skin diseases. Our results showed that topical application of CAPE alleviated the skin inflammatory symptoms in both acute TPA-induced ear edema model and chronic hapten-induced AD model. The inhibitory effect of topical application of CAPE on TPA-mediated skin tumor promotion processes has been well reported . In addition, it was reported that CAPE blocked TPA-induced oxidative processes in skin, including polymorphonuclear leukocytes infiltration, hydrogen peroxide production, and oxidized DNA formation in addition to the decrease of ear edema . The beneficial role of CAPE in inhibiting TPA-induced oxidative stress in skin was suggested to be mediated by the blockade of ROS production by CAPE . In addition, our results presented the suppression of NF-jB activation as another mechanism by which CAPE blocked TPA-mediated inflammatory responses in skin. NF-jB activation was suggested to play an important role in skin inflammation and immune reactions [13, 27]. TNF-a-induced NF-jB activation triggers transcription of pro-inflammatory cytokines and enzymes like IL-6, IL-8, iNOS and COX-2 , that are important for the progression of skin inflammation. Epidermal knockdown of RelA resulted in restricted contact allergy responses. Epidermis-specific deletion of IKK2, which results in NF-jB activation, leads to severe inflammatory skin diseases that are mediated by TNF-a . IjBa degradation was observed in epidermis stimulated by oxazolone indicating the role of NF-jB activation in hapteninduced AD-like symptoms . Our results show the blockade of NF-jB activation by CAPE both in vitro keratinocyte cell system and in vivo TPA-induced edema animal models (Figs. 2, 3). Therefore, these suggest that suppression of NF-jB activation may be the action mechanism by which CAPE exerts protective effects on skin tissue inflammation. In addition to NF-jB activation, our results also show that CAPE suppressed phosphorylation of ERK1/2 suggesting that there may be multiple targets for CAPE to regulate skin inflammatory responses (Fig. 4). It is reported that CAPE has other anti-inflammatory targets such as TLR4/MD2 complex and Nrf2 [12, 15] and 5-lipoxygenase . In addition, CAPE can modulate a variety of targets for anti-cancer effects, including tumor suppressor protein p53, p38 MAPK, activator protein-1 (AP-1), PI-3K/Akt, and mTOR. It will be revealed whether these molecules are involved in the regulation of skin tissue inflammation by CAPE in a future study. CAPE has an extra advantage for topical application to skin diseases as compared with other polyphenols. CAPE has a high lipophilicity [computed log P (water/octanol partition coefficient) = 4.2] and high cell permeability that may facilitate skin penetration, thereby resulting in higher
Arch Dermatol Res
Fig. 3 Topical application of caffeic acid phenethyl ester (CAPE) reduces skin inflammation induced by 12-O-tetradecanoylphorbol-13acetate (TPA) in mice. Skin inflammation was induced by topical application of TPA (2.5 lg/20 ll in acetone) to ear of male BALB/c mice. CAPE (0.5 %) or indomethacin (Indo; 2.5 %) was topically treated on the ear twice per day for 2 days before TPA treatment and 30 min and 6 h after TPA treatment. a Twenty-four hours after TPA
Fig. 4 Topical application of caffeic acid phenethyl ester (CAPE) reduces TPA-induced inflammatory gene expression and signal activation in skin. Skin samples were obtained as described in the legend of Fig. 3. a The mRNA levels of TNF-a, COX-2, and iNOS were determined and presented in fold induction as compared with vehicle alone. The value from individual mouse was expressed as a dot in addition to mean ± SEM of the groups (n = 4–5). Asterisk p \ 0.05. b Immunoblotting was performed to detect COX-2, phospho-IjBa, GAPDH, phospho-ERK, and ERK. Hash represents the individual mouse skin
application, the weight of 6 mm biopsy of ears was measured and pathological scoring was determined by a blinded histologist. Values are mean ± SEM (n = 8-12). Asterisk significantly different from TPA alone, p \ 0.05. b After weighing, biopsy tissues were processed and stained with hematoxylin and eosin for histological examination (9100 magnification). Representative pictures are presented
Arch Dermatol Res
Fig. 5 Caffeic acid phenethyl ester (CAPE) reduces inflammatory symptoms in a hapten-induced atopic dermatitis animal model. AD was induced by repeated hapten (oxazolone) treatment for 16 days. CAPE (0.5 %, 1 % in acetone) or betamethasone (BM) (0.01 %) was applied twice daily from day 7 to day 16. Twenty-four hours after the final treatment, a trans-epidermal water loss (TEWL), b skin
thickness of the treated skin area, and C. serum IgE levels, were determined. Values are mean ± SEM (n = 6–8). Asterisk significantly different from hapten alone, p \ 0.05. d Skin tissues were stained with hematoxylin and eosin for histological examination (9200 magnification). Representative pictures are presented
efficacy in skin diseases. In contrast, CA, a structural derivative lacking phenethyl motif has limited skin permeability , that may stem from high hydrophilicity (computed log P = 1.2) and free carboxylic group. Indeed, our results demonstrate that CAPE is superior to CA in suppressing expression of pro-inflammatory cytokines, IL-1b, IL-6 and TNF-a in keratinocytes (Fig. 1). Since CA is one of the most predominant phenolic acids in plants, there have been numerous studies on the bioactivity of CA, and some of them often focused on the comparison of biological benefits between CA and CA-related hydroxylcinnamic acid compounds including CAPE. Both CA and CAPE show similar biological activities such as anti-oxidant, enzyme-inhibiting, anti-tumor activities , the relative potencies of CA and CAPE are not consistent between studies. For example, even in anti-oxidant capacity, CA showed higher activity in inhibition of lipid oxidation than CAPE, while CAPE showed higher DPPH scavenging activity than CA , suggesting that CA and CAPE may have different mode of action. In our study, CAPE significantly inhibited upregulation of inflammatory cytokines induced by TNF-a, while CA failed to reduce expression of these cytokines at the same concentration tested (Fig. 1). Moreover, CAPE was
effective in vivo at the concentrations as low as 0.5 %. Recent report showed that the application of 6 % CA suppressed multiple TPA challenge-induced ear edema only by *24 %  while in our study, CAPE almost completely blocked TPA-induced ear edema at 0.5 % (Fig. 3). The effective dosages of CA in TPA-induced chronic inflammation model were 2, 4, and 6 %  whereas our results showed that CAPE was effective at 0.5 and 1 % in hapteninduced chronic dermatitis model (Fig. 5). Furthermore, Frenkel et al.  compared the potency of CAPE and CA for TPA-induced ear edema and epidermal ornithine decarboxylase in CD-1 mice showing that CAPE was almost twofold more potent than CA. These results support that CAPE is more potent than CA for treating and preventing skin inflammation. Of note, the efficacy of CAPE was almost equivalent to that of a topical steroid, betamethasone, supporting that the topical formulation of CAPE can be used for treatment of skin diseases. Collectively, our results demonstrate that CAPE is effective in alleviating in vitro and in vivo skin inflammation as well as in vivo hapten-induced AD-like symptoms. The inhibitory effects of CAPE were mediated through the suppression of pro-inflammatory gene
Arch Dermatol Res
Fig. 6 Schematic illustration for the role of caffeic acid phenethyl ester (CAPE) in skin inflammation. CAPE blocks the activation of NF-jB induced by inflammatory stimuli and oxidative stress resulting in the decreased expression of pro-inflammatory cytokines and enzymes in keratinocytes
expression associated with acute and chronic inflammation in the skin. CAPE inhibited phosphorylation of IjBa and ERK in the skin, which could ultimately block the activation of NF-jB and its nuclear translocation. The potency of CAPE was superior to CA to inhibit the expression of pro-inflammatory cytokines and the activation of NF-jB in keratinocytes. Most notably, topical treatment of CAPE manifested potent anti-inflammatory activities in AD-like symptoms that were comparable to those of a topical corticosteroid, betamethasone, suggesting that CAPE might be used in treatment of diverse skin diseases. Acknowledgments KM-Lim designed and JE-Koo, ES-Kim, ONBae performed experiments. SJ-Bae analyzed the data. KM-Lim and JY-Lee wrote the manuscript. Authors thank Chae-Wook Lee for technical support for experiments. This study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF-2012R1A1A3004541), and the Research Fund, 2013 of the Catholic University of Korea. Conflict of interest
The authors declare no conflict of interest.
1. Albukhari AA, Gashlan HM, El-Beshbishy HA, Nagy AA, Abdel-Naim AB (2009) Caffeic acid phenethyl ester protects against tamoxifen-induced hepatotoxicity in rats. Food Chem Toxicol 47(7):1689–1695. doi:10.1016/j.fct.2009.04.021 2. Alomar A, Berth-Jones J, Bos JD, Giannetti A, Reitamo S, Ruzicka T, Stalder JF, Thestrup-Pedersen K, European Working Group on Atopic D (2004) The role of topical calcineurin inhibitors in atopic dermatitis. Br J Dermatol 151(Suppl 70):3–27. doi:10.1111/j.1365-2133.2004.06269.x 3. Bieber T (2008) Atopic dermatitis. N Engl J Med 358(14): 1483–1494. doi:10.1056/NEJMra074081 4. Chen JH, Ho C-T (1997) Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J Agric Food Chem 45(7):2374–2378. doi:10.1021/jf970055t 5. Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, Kim SH, Kim JG, Kim CH (2004) Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEB J 18(14):1670–1681. doi:10.1096/fj.042126com 6. da Cunha FM, Duma D, Assreuy J, Buzzi FC, Niero R, Campos MM, Calixto JB (2004) Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties. Free Radic Res 38(11): 1241–1253. doi:10.1080/10715760400016139 7. Frenkel K, Wei H, Bhimani R, Ye J, Zadunaisky JA, Huang MT, Ferraro T, Conney AH, Grunberger D (1993) Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester. Cancer Res 53(6):1255–1261 8. Huang MT, Ma W, Yen P, Xie JG, Han J, Frenkel K, Grunberger D, Conney AH (1996) Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells. Carcinogenesis 17(4):761–765 9. Jung WK, Choi I, Lee DY, Yea SS, Choi YH, Kim MM, Park SG, Seo SK, Lee SW, Lee CM, Park YM, Choi IW (2008) Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways. Int J Biochem Cell Biol 40(11):2572–2582 10. Kart A, Cigremis Y, Ozen H, Dogan O (2009) Caffeic acid phenethyl ester prevents ovary ischemia/reperfusion injury in rabbits. Food Chem Toxicol 47(8):1980–1984. doi:10.1016/j.fct. 2009.05.012 11. Khan AQ, Khan R, Qamar W, Lateef A, Ali F, Tahir M, Muneeb UR, Sultana S (2012) Caffeic acid attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced NF-kappaB and COX-2 expression in mouse skin: abrogation of oxidative stress, inflammatory responses and proinflammatory cytokine production. Food Chem Toxicol 50(2):175–183. doi:10.1016/j.fct.2011. 10.043 12. Kim SY, Koo JE, Seo YJ, Tyagi N, Jeong E, Choi J, Lim KM, Park ZY, Lee JY (2013) Suppression of Toll-like receptor 4 activation by caffeic acid phenethyl ester is mediated by interference of LPS binding to MD2. Br J Pharmacol 168(8): 1933–1945. doi:10.1111/bph.12091 13. Kumari S, Herzberg B, Pofahl R, Krieg T, Haase I (2014) Epidermal RelA Specifically Restricts Contact Allergen-Induced Inflammation and Apoptosis in Skin. J Invest Dermatol. doi:10. 1038/jid.2014.193 14. Lee JH, Jung KM, Bae IH, Cho S, Seo DB, Lee SJ, Park YH, Lim KM (2009) Anti-inflammatory and barrier protecting effect of Lithospermum erythrorhizon extracts in chronic oxazolone-
Arch Dermatol Res
induced murine atopic dermatitis. J Dermatol Sci 56(1):64–66. doi:10.1016/j.jdermsci.2009.07.001 Lee Y, Shin DH, Kim JH, Hong S, Choi D, Kim YJ, Kwak MK, Jung Y (2010) Caffeic acid phenethyl ester-mediated Nrf2 activation and IjB kinase inhibition are involved in NFjB inhibitory effect: structural analysis for NFjB inhibition. Eur J Pharmacol 643(1):21–28. doi:10.1016/j.ejphar.2010.06.016 Man MQ, Hatano Y, Lee SH, Man M, Chang S, Feingold KR, Leung DY, Holleran W, Uchida Y, Elias PM (2008) Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges. J Invest Dermatol 128(1):79–86. doi:10.1038/sj.jid.5701011 Michaluart P, Masferrer JL, Carothers AM, Subbaramaiah K, Zweifel BS, Koboldt C, Mestre JR, Grunberger D, Sacks PG, Tanabe T, Dannenberg AJ (1999) Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Res 59(10):2347–2352 Murtaza G, Karim S, Akram MR, Khan SA, Azhar S, Mumtaz A, Bin Asad MH (2014) Caffeic acid phenethyl ester and therapeutic potentials. Biomed Res Int 2014:145342. doi:10.1155/2014/ 145342 Natarajan K, Singh S, Burke TR Jr, Grunberger D, Aggarwal BB (1996) Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc Natl Acad Sci USA 93(17):9090–9095 Nestle FO, Di Meglio P, Qin JZ, Nickoloff BJ (2009) Skin immune sentinels in health and disease. Nat Rev Immunol 9(10):679–691. doi:10.1038/nri2622 Pasparakis M, Courtois G, Hafner M, Schmidt-Supprian M, Nenci A, Toksoy A, Krampert M, Goebeler M, Gillitzer R, Israel A, Krieg T, Rajewsky K, Haase I (2002) TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417(6891):861–866. doi:10.1038/nature00820
22. Pramanik KC, Kudugunti SK, Fofaria NM, Moridani MY, Srivastava SK (2013) Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway. Carcinogenesis 34(9):2061–2070. doi:10.1093/carcin/bgt154 23. Rao TS, Currie JL, Shaffer AF, Isakson PC (1993) Comparative evaluation of arachidonic acid (AA)- and tetradecanoylphorbol acetate (TPA)-induced dermal inflammation. Inflammation 17(6):723–741 24. Sud’ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD (1993) Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 329(1–2):21–24 25. Tian B, Nowak DE, Jamaluddin M, Wang S, Brasier AR (2005) Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling. J Biol Chem 280(17):17435–17448. doi:10.1074/jbc.M500437200 26. Velazquez C, Navarro M, Acosta A, Angulo A, Dominguez Z, Robles R, Robles-Zepeda R, Lugo E, Goycoolea FM, Velazquez EF, Astiazaran H, Hernandez J (2007) Antibacterial and freeradical scavenging activities of Sonoran propolis. J Appl Microbiol 103(5):1747–1756. doi:10.1111/j.1365-2672.2007. 03409.x 27. Wullaert A, Bonnet MC, Pasparakis M (2011) NF-kappaB in the regulation of epithelial homeostasis and inflammation. Cell Res 21(1):146–158. doi:10.1038/cr.2010.175 28. Zhang M, Zhou J, Wang L, Li B, Guo J, Guan X, Han Q, Zhang H (2014) Caffeic acid reduces cutaneous tumor necrosis factor alpha (TNF-a), IL-6 and IL-1b levels and ameliorates skin edema in acute and chronic model of cutaneous inflammation in mice. Biol Pharm Bull 37(3):347–354 29. Zilius M, Ramanauskiene K, Briedis V (2013) Release of propolis phenolic acids from semisolid formulations and their penetration into the human skin in vitro. Evid Based Complement Alternat Med 2013:958717. doi:10.1155/2013/958717