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Epigallocatechin-3-Gallate Improves Acne in Humans by Modulating Intracellular Molecular Targets and Inhibiting P. acnes

  • Author Footnotes
    4 These authors equally contributed to this work as the first authors.
    Ji Young Yoon
    Footnotes
    4 These authors equally contributed to this work as the first authors.
    Affiliations
    Acne Research Laboratory, Seoul National University Hospital, Seoul, South Korea
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  • Author Footnotes
    4 These authors equally contributed to this work as the first authors.
    Hyuck Hoon Kwon
    Footnotes
    4 These authors equally contributed to this work as the first authors.
    Affiliations
    Acne Research Laboratory, Seoul National University Hospital, Seoul, South Korea

    Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea
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  • Seong Uk Min
    Affiliations
    Acne Research Laboratory, Seoul National University Hospital, Seoul, South Korea

    Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea
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  • Diane M. Thiboutot
    Affiliations
    Department of Dermatology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA
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  • Dae Hun Suh
    Correspondence
    Department of Dermatology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, South Korea
    Affiliations
    Acne Research Laboratory, Seoul National University Hospital, Seoul, South Korea

    Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea
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  • Author Footnotes
    4 These authors equally contributed to this work as the first authors.
      Acne vulgaris is a highly prevalent skin disorder characterized by hyperseborrhea, inflammation, and Propionibacterium acnes overgrowth. Only isotretinoin and hormonal therapy reduce sebum production. To identify a new drug candidate that modulates sebum, we examined the effects of EGCG, the major polyphenol in green tea, on human SEB-1 sebocytes and in patients with acne. In SEB-1 sebocytes, we found that EGCG reduced sebum by modulating the AMPK–SREBP-1 signaling pathway. EGCG also reduces inflammation by suppressing the NF-κB and AP-1 pathways. EGCG also induces cytotoxicity of SEB-1 sebocytes via apoptosis and decreases the viability of P. acnes, thus targeting almost all the pathogenic features of acne. Finally, and most importantly, EGCG significantly improved acne in an 8-week randomized, split-face, clinical trial, and was well tolerated. Our data provide a therapeutic rationale for the use of EGCG in acne.

      Abbreviations

      AMPK
      AMP-activated protein kinase
      AP-1
      activator protein 1
      EGCG
      epigallocatechin-3-gallate
      MMP
      matrix metalloproteinase
      SREBP
      sterol regulatory element–binding protein
      TNF
      tumor necrosis factor
      VAS
      visual analogue scale

      Introduction

      Acne vulgaris is the most prevalent skin disorder, affecting more than 85% of adolescents in the United States (
      • James W.D.
      Clinical practice. Acne.
      ;
      • Thiboutot D.M.
      Overview of acne and its treatment.
      ;
      • Williams H.C.
      • Dellavalle R.P.
      • Garner S.
      Acne vulgaris.
      ). Although not life threatening, acne can persist throughout life and leave permanent scarring on the face, thereby causing significant physical and psychosocial morbidities (
      • Layton A.M.
      Optimal management of acne to prevent scarring and psychological sequelae.
      ). During the past decade, four major pathological processes have been discovered to have a critical role in the development of acne: (1) increased sebum production by the sebaceous gland, (2) altered keratinization of follicular keratinocytes, (3) activity of Propionibacterium acnes, and (4) inflammation (
      • Leyden J.J.
      Therapy for acne vulgaris.
      ;
      • Haider A.
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      Treatment of acne vulgaris.
      ;
      • Zouboulis C.C.
      • Eady A.
      • Philpott M.L.A.
      • et al.
      What is the pathogenesis of acne?.
      ). Despite this significant progress, most medical and surgical modalities developed so far to treat acne have demonstrated relatively modest efficacy, presumably because of the inherent pathological complexity of the disease. Only a few drugs target multiple pathological processes of acne and effectively improve the condition, but their use is accompanied by potentially serious side effects (
      • Sun M.
      Anti-acne drug poses dilemma for FDA.
      ;
      • Leyden J.J.
      Therapy for acne vulgaris.
      ). For example, one of the most effective treatments, isotretinoin, has serious side effects, including teratogenicity, liver enzyme abnormalities, and dyslipidemia, and its use is strictly limited in many countries (
      • James W.D.
      Clinical practice. Acne.
      ;
      • Thiboutot D.M.
      Overview of acne and its treatment.
      ). Topical retinoids, currently considered as the first-line treatment of acne, may cause burning and irritation, especially in the early stages of treatment (
      • Strauss J.S.
      • Krowchuk D.P.
      • Leyden J.J.
      • et al.
      Guidelines of care for acne vulgaris management.
      ). Therefore, new drugs that target multiple pathological processes of acne with few side effects are urgently needed.
      Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has attracted much interest in recent years because of its potent anticarcinogenic, anti-inflammatory, and antimicrobial activities (
      • Lu Y.P.
      • Lou Y.R.
      • Xie J.G.
      • et al.
      Topical applications of caffeine or (-)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice.
      ;
      • Tachibana H.
      • Koga K.
      • Fujimura Y.
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      A receptor for green tea polyphenol EGCG.
      ;
      • Yusuf N.
      • Irby C.
      • Katiyar S.
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      Photoprotective effects of green tea polyphenols.
      ). Accumulating evidence indicates that EGCG inhibits tumor progression and improves inflammatory diseases such as diabetes, allergies, and neurodegenerative disorders (
      • Dufresne C.J.
      • Farnworth E.R.
      A review of latest research findings on the health promotion properties of tea.
      ;
      • Nomura M.
      • Kaji A.
      • He Z.
      • et al.
      Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway.
      ;
      • Siddiqui I.A.
      • Malik A.
      • Adhami V.M.
      • et al.
      Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAIL-mediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis.
      ;
      • Bieschke J.
      • Russ J.
      • Friedrich R.P.
      • et al.
      EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity.
      ). In addition, a recent study showed that EGCG inhibits lipogenesis (
      • Moon H.S.
      • Chung C.S.
      • Lee H.G.
      • et al.
      Inhibitory effect of (-)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells.
      ;
      • Ku H.C.
      • Chang H.H.
      • Liu H.C.
      • et al.
      Green tea (-)-epigallocatechin gallate inhibits insulin stimulation of 3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin receptor pathway.
      ). These observations, together with the fact that acne is closely related to abnormal bacterial proliferation, inflammation, and lipogenesis, led us to hypothesize that EGCG may be beneficial in acne.
      In this study, we report that EGCG displays apoptotic, sebosuppressive, and anti-inflammatory effects on human sebocytes, and that it displays antibacterial effects on P. acnes, findings that are, to our knowledge, previously unreported. Our biochemical, genetic, and cellular studies indicate that modulation of AMPK–SREBP-1 and NF-κB/activator protein 1 (AP-1) signaling pathways mediates the sebosuppressive and anti-inflammatory effects of EGCG, respectively. Subsequently, and more importantly, we confirmed its efficacy and safety in an 8-week randomized, split-face, clinical trial. Taken together, these data demonstrate that EGCG modulates several key pathological factors of acne with very few, relatively mild side effects, suggesting the possibility that EGCG may be effective in the treatment of acne.

      Results

      EGCG decreases lipogenesis in human SEB-1 sebocytes

      As excessive sebum production is considered pivotal in the pathogenesis of acne, we first examined whether EGCG can modulate lipid production in human SEB-1 sebocytes, a well-established in vitro model for the study of sebaceous gland activity (
      • Thiboutot D.
      • Jabara S.
      • McAllister J.M.
      • et al.
      Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).
      ). Lipogenesis was first analyzed by total lipid assay from SEB-1 sebocytes grown in the presence of 14C-acetate after treatment with different concentrations of EGCG and normalized by cell numbers. Then, changes in specific lipid components were further analyzed using thin-layer chromatography. EGCG significantly decreases the incorporation of 14C-acetate into total lipids, displaying 55% decreased levels of intracellular lipids in SEB-1 sebocytes treated with 40μM of EGCG (Figure 1a). This decrease mainly results from the reduction in cholesterol and triglyceride levels (Figure 1b). Thin-layer chromatography data on other sebaceous lipids are presented in Supplementary Table S1 online.
      Figure thumbnail gr1
      Figure 1Epigallocatechin-3-gallate (EGCG) decreases lipogenesis in SEB-1 sebocytes by suppressing the expression of sterol regulatory element–binding protein (SREBP)-1 through the activation of the AMP-activated protein kinase (AMPK) pathway. (a) Lipogenesis was analyzed by total lipid assay from SEB-1 sebocytes grown in the presence of 14C-acetate after treatment with EGCG for 24hours. (b) Changes in specific lipid components were further analyzed using thin-layer chromatography (C: cholesterol, FOH: fatty alcohol, OA: oleic acid, TAG: triglyceride, WE: wax ester, CO: cholesterol oleate, SQ: squalene). Units are mean±SE c.p.m/103 cells/hour. (c) Western blots for precursor and mature SREBP-1 were performed after treatment with EGCG for 24hours. (d) RNA was subjected to quantitative real-time PCR to determine the abundance of SREBP-1a, SREBP-1c, SREBP-2, fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) after treatment with EGCG for 24hours. (e) Then, SEB-1 cells were pretreated with 20μM of compound C for 30minutes, and/or were treated with EGCG. Levels of phospho-/total AMPK and phospho-ACC were measured after 3hours. Mature SREBP-1 levels were also measured after 12hours. *P<0.05, **P<0.01 compared with control (Student's t-test, a–e). (f) SEB-1 cells pretreated with 20μM of compound C for 30minutes and/or treated with EGCG for 24hours were stained with Nile Red. Bar=10μm. (g) Nile Red quantification was performed. Relative ratios compared with each control were recorded. *P<0.05 between each EGCG-treated group and control; P<0.05 between compound C–pretreated and –nontreated groups (Student's t-test, g). All experiments were repeated a minimum of five times (mean±SEM).
      To gain a mechanistic understanding of the antilipogenic effects of EGCG, we tested the hypothesis that sterol regulatory element–binding proteins (SREBPs), major transcription factors responsible for the regulation of cholesterol/fatty acid metabolism, are involved (
      • Chen G.
      • Liang G.
      • Ou J.L.
      • et al.
      Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver.
      ;
      • Engelking L.J.
      • Kuriyama H.
      • Hammer R.E.
      • et al.
      Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis.
      ;
      • Goldstein J.L.
      • DeBose-Boyd R.A.
      • Brown M.S.
      Protein sensors for membrane sterols.
      ;
      • Smith T.M.
      • Cong Z.
      • Gilliland K.L.
      • et al.
      Insulin-like growth factor-1 induces lipid production in human SEB-1 sebocytes via sterol response element-binding protein-1.
      ,
      • Smith T.M.
      • Gilliland K.
      • Clawson G.A.
      • et al.
      IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway.
      ). We found that EGCG significantly decreases both precursor and mature forms of SREBP-1 proteins (Figure 1c). In addition, EGCG also decreased mRNA levels of all SREBP isoforms, SREBP-1a, SREBP-1c, and SREBP-2, as measured by quantitative real-time PCR, suggesting that EGCG inhibits the expression of SREBPs at the transcriptional level. Consistent with this result, the expression levels of two key downstream targets of SREBPs, fatty acid synthase, and acetyl-CoA carboxylase, were significantly decreased (Figure 1d). Together, these data suggest that EGCG suppresses sebum, at least in part, through the inhibition of the SREBP signaling pathway.

      AMPK pathway is mainly responsible for the suppression of lipogenesis by EGCG treatment in SEB-1 sebocytes

      To identify upstream regulators responsible for the EGCG-mediated suppression of the SREBP pathway, we have focused on the AMP-activated protein kinase (AMPK) pathway for the following reasons: (1) previous studies showed that EGCG modulates the activity of AMPK in hepatocytes and adipocytes (
      • Hwang J.T.
      • Park I.J.
      • Shin J.I.
      • et al.
      Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase.
      ;
      • Collins Q.F.
      • Liu H.Y.
      • Pi J.
      • et al.
      Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5′-AMP-activated protein kinase.
      ); (2) AMPK is well known to act as a master regulator of lipid metabolism in various cell lines (
      • Zhou G.
      • Myers R.
      • Li Y.
      • et al.
      Role of AMP-activated protein kinase in mechanism of metformin action.
      ;
      • Canto C.
      • Gerhart-Hines Z.
      • Feige Z.N.
      • et al.
      AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.
      ;
      • Chau M.D.
      • Gao J.
      • Yang Q.
      • et al.
      Fibroblast growth factor 21 regulates energy metabolism by activating the AMPK-SIRT1-PGC-1alpha pathway.
      ); and (3) recent studies described that AMPK directly phosphorylates and inhibits SREBP activity and also regulates the transcription of SREBPs in hepatocytes (
      • Li Y.
      • Xu S.
      • Mihaylova M.M.
      • et al.
      AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.
      ).
      We found that EGCG increases phosphorylation (Thr172) of AMPK and acetyl-CoA carboxylase (Ser79), a well-characterized target of AMPK, suggesting that EGCG activates the AMPK pathway in SEB-1 sebocytes, as shown in other cell lines (
      • Hwang J.T.
      • Park I.J.
      • Shin J.I.
      • et al.
      Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase.
      ;
      • Collins Q.F.
      • Liu H.Y.
      • Pi J.
      • et al.
      Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5′-AMP-activated protein kinase.
      ) (Figure 1e). To test whether AMPK activation is required for the EGCG-mediated decrease of SREBP-1, we used a selective chemical inhibitor of AMPK, compound C (
      • McCullough L.D.
      • Zeng Z.
      • Li H.
      • et al.
      Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke.
      ). We confirmed that compound C efficiently inhibits the EGCG-induced activation of AMPK. More importantly, compound C blocked the reduction of SREBP-1 expression induced by EGCG, suggesting that AMPK is a critical mediator of EGCG's inhibition of SREBP-1 expression (Figure 1e). Subsequently, we found that inhibition of AMPK also reverses the sebosuppressive effects of EGCG. SEB-1 sebocytes treated with EGCG showed a significant decrease in intracellular lipid levels, as measured by Nile Red staining (Figure 1f and g), consistent with the total lipid assay data. However, when SEB-1 sebocytes were pretreated with compound C, EGCG failed to decrease intracellular lipid levels (Figure 1f and g). These data suggest that EGCG suppresses lipogenesis in SEB-1 sebocytes via modulation of the AMPK-SREBP-1 pathways.

      EGCG decreases inflammation induced by heat-inactivated P. acnes through the inhibition of NF-κB and AP-1 pathways

      P. acnes colonization of the follicule is another critical pathological factor for acne, which increases the inflammatory response within the pilosebaceous unit (
      • Graham G.M.
      • Farrar M.D.
      • Cruse-Sawyer J.E.
      • et al.
      Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.
      ;
      • Kang S.
      • Cho S.
      • Chung J.H.
      • et al.
      Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo.
      ;
      • Nagy I.
      • Pivarcsi A.
      • Kis K.
      • et al.
      Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes.
      ). Heat-inactivated P. acnes induced several molecular markers of inflammation, including IL-1α, IL-1β, tumor necrosis factor (TNF)-α, and IL-8, in SEB-1 sebocytes (Figure 2a). ELISA confirmed the increased expression of TNF-α and IL-8, and western blot analysis also demonstrated increased levels of the gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9 (Figure 2b and c). To further investigate the underlying molecular mechanisms, we checked the activation of several transcription factors responsible for regulation of the inflammatory response. Among them, phosphorylation of c-Jun and phospho-IκB, a molecular marker for the activation of AP-1 and NF-κB pathway, respectively, is significantly increased (Figure 2c). Nuclear translocation of NF-κB p65 is also increased as expected (Figure 2d). In addition, IL-1α, a critical cytokine involved in follicular hyperkeratosis, was induced in HaCaT keratinocytes stimulated with heat-inactivated P. acnes, confirming the role of skin parenchymal cells in the pathological inflammatory response associated with acne (Figure 2e and f).
      Figure thumbnail gr2
      Figure 2Epigallocatechin-3-gallate (EGCG) decreases inflammation induced by SEB-1 sebocytes stimulated by heat-inactivated Propionibacterium acnes through the inhibition of NF-κB and activator protein 1 (AP-1) pathways. (ac) SEB-1 cells stimulated with heat-inactivated P. acnes were treated with EGCG for 24hours. (a) RNA was isolated and subjected to quantitative real-time PCR (qRT–PCR) to determine the abundance of cytokines (IL-1α, IL-1β, tumor necrosis factor (TNF)-α, and IL-8). (b) ELISA for TNF-α and IL-8 proteins in the supernatant of cultured cells was performed before and after treatments by EGCG for 24hours. (c) Western blots of phospho-c-Jun, phospho IκBα, matrix metalloproteinase (MMP)-2, and MMP-9 in SEB-1 cells stimulated with heat-inactivated P. acnes pretreated with 20μM of compound C for 30minutes and/or treated with EGCG were performed. (d) SEB-1 cells stimulated with heat-inactivated P. acnes were incubated with EGCG for 30minutes and 24 hours, and then nuclear and cytosolic fractions were further isolated and assayed for NF-κB p65 by western blotting, respectively. (e) Western blotting of phospho-IκBα, NF-κB p65, and IL-1α were performed in HaCaT keratinocytes stimulated with heat-inactivated P. acnes before and after treatments with EGCG for 24hours. (f) Then, qRT–PCR was performed to determine the abundance of IL-1α mRNA before and after EGCG treatments. All experiments in ae were repeated a minimum of five times. Data in (a, b, f) are mean±SEM. Student’s t-test was performed for statistical comparisons of data presented in this figure: *P<0.05 between control and each P. acnes–stimulated group. P<0.05 between P. acnes–stimulated, EGCG-nontreated group, and each EGCG-treated group.
      Supporting its potential anti-inflammatory effects, EGCG significantly decreased mRNA levels of IL-1α, IL-1β, IL-8, and TNF-α in SEB-1 sebocytes stimulated with heat-inactivated P. acnes (Figure 2a). Consistent with these results, protein expression of those targets was similarly affected. Specifically, EGCG significantly decreased IL-8, TNF-α, MMP-2, MMP-9, phospho-c-Jun, and phospho-IκB (Figure 2b and c). EGCG also inhibited the nuclear translocation of NF-κB p65 in SEB-1 cells, confirming that EGCG inhibits heat-inactivated P. acnes–triggered upstream signaling of NF-κB activation (Figure 2d and Supplementary Figure S1 online). EGCG significantly suppressed increased levels of IL-1α, NF-κB, and phospho-IκB in HaCaT keratinocytes (Figure 2e and f and Supplementary Figure S1 online). Furthermore, when SEB-1 sebocytes stimulated with heat-inactivated P. acnes were pretreated with 20μM of compound C, the effects of EGCG on the levels of phospho-c-JUN, phospho-IκB, and gelatinases were reversed, suggesting that the AMPK pathway also has a role in the anti-inflammatory effects of EGCG (Figure 2c). Our findings suggest that EGCG can decrease the inflammatory response in SEB-1 sebocytes and HaCaT keratinocytes associated with P. acnes, by regulating AP-1 and NF-κB signaling pathways.

      EGCG induces cytotoxicity of SEB-1 sebocytes via apoptosis

      To investigate the temporal effects of EGCG on the viability of SEB-1 sebocytes, MTT assays were performed. When treated with EGCG, a significant reduction of cellular growth was observed (Figure 3a). We observed a similar reduction in the viability of HaCaT cells, yet to a lesser extent, indicating that SEB-1 sebocytes were more sensitive to growth inhibition by EGCG. DAPI (4’,6-diamidino-2-phenylindole) staining revealed that nuclei of SEB-1 sebocytes became condensed and fragmented after EGCG treatments, resembling apoptotic nuclei (Figure 3b). To determine whether EGCG induces apoptosis, SEB-1 sebocytes were treated with increasing concentrations of EGCG, and then TUNEL staining was performed. After 48hours of EGCG treatment, there was an increase in TUNEL-positive nuclei, suggesting increased levels of apoptosis (Figure 3c and d). To precisely characterize the molecular mechanisms involved in EGCG-induced apoptosis, the expression levels of proteins related to the apoptotic pathway were examined. A statistically significant increase in the ratio between Bax and Bcl-2, a critical marker of the intrinsic apoptotic pathway, was observed in SEB-1 sebocytes treated with EGCG. Consistent with these, EGCG also increases cleaved (active) caspase-3, confirming increased levels of apoptosis. In addition, cyclin D1, an important regulator of G1-/S-phase progression (
      • Alao J.P.
      The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention.
      ), is also decreased significantly, suggesting that EGCG can also halt cell cycle progression (Figure 3e). These findings indicate that EGCG decreases the viability of SEB-1 sebocytes by increasing apoptosis and arresting cell cycle progression, possibly leading to the suppression of hyperproliferation of sebocytes in acne lesions.
      Figure thumbnail gr3
      Figure 3Epigallocatechin-3-gallate (EGCG) induces cytotoxicity of SEB-1 sebocytes via apoptosis and inhibits the growth of Propionibacterium acnes. (a) Cell survival of both SEB-1 sebocytes and HaCaT keratinocyte was measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after treatment with EGCG for 24 and 48hours. (b) DAPI (4’,6-diamidino-2-phenylindole) staining was performed after 48hours of incubation (scale bar, 10μm; Inset is shown at × 400 original magnification). (c) TUNEL staining was performed after 48hours of incubation (Bar, 20μm). Nuclei of each sample were stained by methyl green. (d) Quantification of the percentage of TUNEL-positive stained cells after EGCG treatment at 48hours. (e) Expression levels of Bcl-2, Bax, capase-3, and cyclin D1 were measured after 12, 24, and 48hours of EGCG treatments, respectively. The ratio of Bax/Bcl-2 proteins was calculated to compare the relative intensity. (f) Then, P. acnes was incubated under vehicle control and increasing concentrations of EGCG. (g) Percentile of colony numbers of P. acnes according to increasing EGCG concentrations compared with control was described. All experiments (ag) were repeated a minimum of five times (mean ± SEM). Student's t-test was performed for statistical comparisons, and P-values of <0.05 and <0.01 in comparison with the control were denoted with * and **, respectively.

      EGCG inhibits the growth of P. acnes

      The previous finding that EGCG exerts antimicrobial activity against a diverse range of microorganisms (
      • Bruggemann H.
      • Henne A.
      • Hoster F.
      • et al.
      The complete genome sequence of Propionibacterium acnes, a commensal of human skin.
      ;
      • Yoda Y.
      • Hu Z.Q.
      • Zhao W.H.
      • et al.
      Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatechin gallate.
      ;
      • Hauber I.
      • Hohenberg H.
      • Holstermann B.
      • et al.
      The main green tea polyphenol epigallocatechin-3-gallate counteracts semen-mediated enhancement of HIV infection.
      ), together with the pathogenic role of P. acnes in the development of acne, led us to hypothesize that EGCG might show an antibacterial effect against P. acnes. To test this hypothesis, mean colony-forming units of P. acnes cultured in different concentrations of EGCG was compared with vehicle control (Figure 3f). EGCG significantly decreases mean colony-forming units in a dose-dependent manner, suggesting that EGCG suppresses the growth of P. acnes (Figure 3g). These results suggest that EGCG could target the abnormal colonization of P. acnes in the development of acne.

      EGCG improves acne and is well tolerated in a clinical trial setting

      To investigate the clinical efficacy and tolerability of EGCG in the treatment of facial acne, an 8-week randomized, double-blinded, split-face clinical trial was conducted. Of the 37 subjects enrolled, 35 (17 men and 18 women, mean age: 22.1) completed the 8-week study. Beginning at the baseline visit, the affected areas on a randomly allocated half side of the face of the subjects were treated with 1 or 5% EGCG solution twice a day, whereas those of the affected the areas on the opposite side were treated with vehicle control only (3% ethanol). Clinical visits were scheduled at baseline, and at 1, 2, 4, 6, and 8 weeks (Figure 4a). The revised Leeds score that reflects both inflammatory (papules, pustules, nodules, cysts) and noninflammatory (comedones) acne lesions was used as a global assessment of acne severity. The mean revised Leeds acne grading at baseline was 5.1±0.4 (mean±SD, range 2–7;
      • O’Brien S.C.
      • Lewis J.B.
      • unliffe W.J.
      Leeds revised acne grading system.
      ). Treatment with 1 and 5% EGCG significantly decreased the mean revised Leeds score to 1.2±0.4 and 1.7±0.6, respectively, at week 8 (Figure 4b). Specifically, the mean noninflammatory and inflammatory lesion counts significantly decreased from the mean baseline value of 53.8±19.8 and 10.0±3.1 to 15.6±6.2 and 1.1±0.5, respectively, after 8 weeks of 1% EGCG application, corresponding to 79 and 89% reduction compared with the baseline (Figure 4c and d). Treatments with 5% EGCG showed a parallel improvement in acne lesion counts. Patients’ subjective assessments were analyzed using a visual analogue scale (VAS) scale ranging from 0 to 10, and was set to 10 in all subjects at the baseline visit. Two weeks after treatment, both 1 and 5% EGCG treatments decreased VAS scores significantly. At the 8-week visit, VAS scores were reduced to 3.5 and 4.9 in the 1 and 5% EGCG-treated side, respectively (Figure 4e). The average global assessment, lesion counts, and VAS data are presented in Supplementary Table S2 online.
      Figure thumbnail gr4
      Figure 4Topical application of 1 and 5% epigallocatechin-3-gallate (EGCG) significantly improves acne lesions in an 8-week human clinical study. (a) A total of 35 patients were instructed to apply EGCG solution only to one side of the face, and vehicle containing 3% ethanol to the other side. The applied side was randomly selected for each patient. Seventeen subjects were designated to use 1% EGCG and 18 subjects were designated to use 5% EGCG to evaluate a dose–response relationship. (b) Revised Leeds grading and acne lesion counts were performed. Changes in both (c) noninflammatory lesions (whiteheads and blackheads, comedones) and (d) inflammatory lesions (papules, pustules, and nodules) during EGCG application period were calculated. (e) VAS was also measured, to assess the subjective assessment of patients with time. Statistical significance was determined by Wilcoxon signed rank test: *P<0.05 between each EGCG-treated group and baseline. P<0.05 between each EGCG-treated group and control (Wilcoxon signed rank test). VAS, visual analogue scale.
      Furthermore, we examined the expression of several proteins associated with acne in vivo. Skin specimens were acquired from active acne lesions. Consistent with in vitro results, EGCG significantly decreased the expression of IL-8, SREBP-1, phospho-c-Jun, and NF-κB, as measured by immunohistochemistry. TUNEL-positive nuclei were increased after application of EGCG, suggesting that EGCG treatment induces apoptosis, thereby suppressing hyperproliferation of sebocytes in acne lesions (Figure 5). These immunohistological results indicate that EGCG treatment decreases the inflammatory response and increases apoptosis in acne lesions in vivo.
      Figure thumbnail gr5
      Figure 5Histopathological changes after applying epigallocatechin-3-gallate (EGCG) for 8 weeks. IL-8, phospho-c-JUN, SREBP-1, and NF-κB p65 were identified by staining with specific antibodies at baseline and final visits. TUNEL assay was also performed as listed in the Materials and Methods section. All are at the same magnification (bar=100μm), and all experiments were repeated a minimum of five times (mean±SEM). Quantitative analysis was performed for each sample. Wilcoxon signed rank test was performed, and *P-value<0.05 was considered statistically significant compared with baseline.
      EGCG treatment was generally well tolerated, and no severe adverse effects were observed during the time window of our study. Subtle adverse effects, such as erythema and irritation, were observed in only four patients receiving 5% EGCG (4 out of 18), which subsided spontaneously within a few hours. No side effects were observed in patients receiving 1% EGCG. In conclusion, our clinical results strongly suggest that EGCG is effective in both inflammatory and noninflammatory acne lesions, with few, mild side effects. A schematic of our overall results is illustrated in Figure 6.
      Figure thumbnail gr6
      Figure 6Possible therapeutic mechanisms based on our in vitro results. These mechanisms could elucidate the efficacy of epigallocatechin-3-gallate (EGCG) in our 8-week, randomized, double-blinded, split-face human clinical trial.

      Discussion

      Despite the advances in our understanding of the pathophysiological mechanisms underlying acne, the development of drugs that target multiple pathological processes has been limited. Our in vitro results demonstrated that EGCG can directly target three out of four pathological processes in acne, including excessive sebum production, inflammation, and P. acnes overgrowth, and possibly indirectly target altered keratinization (see below). Furthermore, our clinical trial demonstrates that EGCG is effective in both inflammatory and noninflammatory acne lesions, with few, mild side effects. Together, these data suggest that EGCG may represent a new therapeutic opportunity in acne vulgaris.
      For many years, excessive sebum production has been known to be one of the key factors in acne (
      • Zouboulis C.C.
      • Seltmann H.
      • Hiroi N.
      • et al.
      Corticotropin-releasing hormone: an autocrine hormone that promotes lipogenesis in human sebocytes.
      ;
      • Harper J.C.
      • Thiboutot D.M.
      Pathogenesis of acne: recent research advances.
      ), yet until recently there has been no effective medication to reduce sebum production except isotretinoin, whose detailed mechanism of action is not fully known (
      • Nelson A.M.
      • Gilliland K.L.
      • Cong Z.
      • et al.
      13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.
      ,
      • Nelson A.M.
      • Zhao W.
      • Gilliland K.L.
      • et al.
      Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells.
      ). In our current study, we demonstrate that EGCG has sebosuppressive effects mainly through the regulation of AMPK-SREBP-1 signaling pathways. EGCG activates AMPK in SEB-1 sebocytes, which in turn decreases the expression of SREBPs and lipogenesis. Whether the EGCG-induced downregulation of SREBPs results solely from the direct action of AMPK on SREBPs remains to be determined. It should be noted that EGCG additionally inhibits the IGF-1/PI3K/Akt pathway (Supplementary Figure S2 online) in an AMPK-dependent manner. These findings, together with the fact that IGF-1/PI3K/Akt pathway regulates lipogenesis through SREBPs (
      • Smith T.M.
      • Cong Z.
      • Gilliland K.L.
      • et al.
      Insulin-like growth factor-1 induces lipid production in human SEB-1 sebocytes via sterol response element-binding protein-1.
      ,
      • Smith T.M.
      • Gilliland K.
      • Clawson G.A.
      • et al.
      IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway.
      ), suggest the possibility that AMPK may regulate SREBPs through the IGF-1/PI3K/Akt pathway. Taken together, our data indicate that AMPK has a critical role in the EGCG-induced sebosuppression in SEB-1 sebocytes, offering therapeutic strategies to target lipogenesis in acne.
      Many human diseases are often associated with a chronic inflammatory response, which also has a critical role in the development of acne (
      • Webster G.F.
      Inflammation in acne vulgaris.
      ;
      • Harper J.C.
      • Thiboutot D.M.
      Pathogenesis of acne: recent research advances.
      ;
      • Yamasaki K.
      • Di Nardo A.
      • Bardan A.
      • et al.
      Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.
      ). We found that EGCG suppresses the inflammatory response induced by heat-inactivated P. acnes in SEB-1 sebocytes, a well-established in vitro model of inflammatory acne, through the inhibition of NF-κB and AP-1 pathways. Although the mechanism by which EGCG regulates NF-κB and AP-1 pathways remains elusive, we hypothesize that it acts upstream of IκB degradation, based on our results showing that EGCG decreases phosphorylation of IκB. It is noteworthy that inhibition of AMPK rescued the EGCG-induced suppression of NF-κB and AP-1 pathways, suggesting that EGCG-activated AMPK suppresses the activity of the NF-κB and AP-1 pathways. Consistent with these observations, an increasing body of evidence demonstrates that AMPK inhibits the NF-κB pathway as well as the inflammatory response in the different types of cells (
      • Navarro-Peran E.
      • Cabezas-Herrera J.
      • Sanchez-Del-Campo L.
      • et al.
      The anti-inflammatory and anti-cancer properties of epigallocatechin-3-gallate are mediated by folate cycle disruption, adenosine release and NF-kappaB suppression.
      ;
      • Green C.J.
      • Macrae K.
      • Fogarty S.
      • et al.
      Counter-modulation of fatty acid-induced pro-inflammatory nuclear factor kappaB signalling in rat skeletal muscle cells by AMP-activated protein kinase.
      ;
      • Morizane Y.
      • Thanos A.
      • Takeuchi K.
      • et al.
      AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts.
      ). It is also remarkable that EGCG decreases IL-1α in HaCaT keratinocytes, based on the fact that IL-1α induces hypercornification of the infundibulum in a manner similar to that seen in comedones (
      • Guy R.
      • Green M.R.
      • Kealey T.
      Modeling acne in vitro.
      ). These results suggest that EGCG might reverse the altered keratinization of follicular keratinocytes through the regulation of IL-1α. Together, these data provide insight to the molecular basis of the therapeutic effects of EGCG on the inflammatory acne lesions in our clinical trial.
      As apoptosis and cell cycle arrest can lead to drastic decreases in sebaceous glands’ size and lipid contents, we examined whether EGCG has an antiproliferative effect. Indeed, we found that EGCG increases the levels of apoptosis and cell cycle arrest. The molecular mechanisms by which EGCG induces apoptosis and cell cycle arrest remain elusive, yet it is plausible to assume that inhibition of either NF-κB or Akt, or both, by EGCG causes apoptosis and cell cycle arrest, based on the fact that the NF-κB and Akt pathways are well recognized as prosurvival signals (
      • Beg A.A.
      • Baltimore D.
      An essential role for NF-kappaB in preventing TNF-alpha-induced cell death.
      ;
      • Chen W.
      • Borchers A.H.
      • Dong Z.
      • et al.
      UVB irradiation-induced activator protein-1 activation correlates with increased c-fos gene expression in a human keratinocyte cell line.
      ). In addition, it is possible that EGCG-activated AMPK may induce apoptosis or cell cycle arrest, as described in recent studies (
      • Motoshima H.
      • Goldstein B.J.
      • Igata M.
      • et al.
      AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer.
      ;
      • Hwang J.T.
      • Ha J.
      • Park I.J.
      • et al.
      Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway.
      ). Further studies are needed to address this question. Our findings suggest that EGCG may promote apoptosis and cell cycle arrest in sebocytes, contributing to decreased sebaceous glands, and therefore mitigated acne lesions, similar to isotretinoin.
      P. acnes, a Gram-positive anaerobic bacterium, is a commensal of human skin, and its overgrowth is closely implicated in the progression of inflammation in acne (
      • Kim J.
      Review of the innate immune response in acne vulgaris: activation of Toll-like receptor 2 in acne triggers inflammatory cytokine responses.
      ;
      • Michalak-Stoma A.
      • Tabarkiewicz J.
      • Olender A.
      The effect of Propionibacterium acnes on maturation of dendritic cells derived from acne patients’ peripherial blood mononuclear cells.
      ). Until recently, various antibiotics have been used to control the overgrowth of P. acnes, yet increasing antibiotic resistance (
      • Strauss J.S.
      • Krowchuk D.P.
      • Leyden J.J.
      • et al.
      Guidelines of care for acne vulgaris management.
      ;
      • Thiboutot D.M.
      Overview of acne and its treatment.
      ) and biofilm formation (
      • Burkhart C.G.
      • Burkhart C.N.
      Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug.
      ) lead to a poor outcome. An accumulating body of evidence suggests that EGCG shows antimicrobial activity against a diverse range of microorganisms, including bacteria, viruses, and fungi (
      • Alexis A.F.
      • Jones V.A.
      • Stiller M.J.
      Potential therapeutic applications of tea in dermatology.
      ;
      • Dufresne C.J.
      • Farnworth E.R.
      A review of latest research findings on the health promotion properties of tea.
      ;
      • Yoda Y.
      • Hu Z.Q.
      • Zhao W.H.
      • et al.
      Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatechin gallate.
      ). Indeed, we found that EGCG significantly inhibits the growth of P. acnes. As a possible mechanism, sophisticated molecular interactions between triphenols of EGCG and a peptide structure of bacterial proteins, including peptidoglycan, was suggested, because neither amino acid, alone or in combination, showed any effect on the antibiotic effect of EGCG (
      • Yoda Y.
      • Hu Z.Q.
      • Zhao W.H.
      • et al.
      Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatechin gallate.
      ). This antibiotic effect of EGCG may provide an advantage as a therapeutic strategy for the treatment of acne, especially considering increasing concerns about antibiotic-resistant bacteria.
      Following the promising results of our in vitro studies, a clinical trial in acne patients was conducted to examine the therapeutic potential of EGCG on acne. Our clinical trial revealed that 1 and 5% EGCG treatment significantly improves both inflammatory and noninflammatory acne lesions. Remarkably, the efficacies of EGCG in this small study were better than those of a new compound product reported in recent large-scale clinical trials (
      • Thiboutot D.M.
      • Weiss J.
      • Bucko A.
      • et al.
      Adapalene-benzoyl peroxide, a fixed-dose combination for the treatment of acne vulgaris: results of a multicenter, randomized double-blind, controlled study.
      ;
      • Gollnick H.P.
      • Draelos Z.
      • Glenn M.J.
      Adapalene-benzoyl peroxide, a unique fixed-dose combination topical gel for the treatment of acne vulgaris: a transatlantic, randomized, double-blind, controlled study in 1670 patients.
      ). Furthermore, the rate of overall side effects observed in our clinical trial is less than that of the aforementioned studies. As the follicular pathway, in contrast to the conventional transdermal pathway, is especially favorable for the delivery of small molecules (
      • Mitragotri S.
      Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways.
      ), topically administered EGCG might be especially effective on acne lesions connected to hair follicles.
      In summary, our data demonstrate that EGCG can modulate several key pathological factors of acne, including hyperseborrhea, inflammation, and P. acnes overgrowth, with few, mild side effects, suggesting that EGCG may be used for the effective and rapid treatment of both inflammatory and noninflammatory acne.

      Materials and Methods

      Cell culture

      The SEB-1-immortalized human sebocyte cell line was generated by transfection of secondary sebocytes with SV40 large T antigen, as previously described (
      • Thiboutot D.
      • Jabara S.
      • McAllister J.M.
      • et al.
      Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).
      ). SEB-1 cells were cultured and maintained in standard culture medium containing DMEM (Invitrogen, Carlsbad, CA), 5.5mM glucose/Ham's F-12 3:1 (Invitrogen), fetal bovine serum 2.5% (HyClone, Logan, UT), adenine 1.8 × 10−4M (Sigma, St Louis, MO), hydrocortisone 0.4μgml−1 (Sigma), insulin 10ngml−1 (Sigma), epidermal growth factor 3ngml−1 (Austral Biologicals, San Ramon, CA), and cholera toxin 1.2 × 10−10M (Sigma) at 37°C in a 5% CO2 incubator. The human keratinocyte cell line HaCaT was maintained in DMEM (Invitrogen) supplemented with 5% fetal bovine serum, 20mM L-glutamine, 1mM sodium pyruvate, and antibiotic/antimycotic solution (10Uml−1 penicillin, 10μgml−1 streptomycin, and 0.25μgml−1 amphoterin; Invitrogen) at 37°C in a 5% CO2 incubator.

      Western blot analysis

      Protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA). Protein contents in lysates were determined using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were run on 10% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane. The blots were primarily probed with cleaved caspase-3 (Asp175) (5A1) rabbit antibody, cyclin D1 (DCS6) mouse antibody, Bax rabbit antibody, BCL-2 mouse antibody, Akt rabbit antibody, Phospho-Akt (Thr308) rabbit antibody, Phospho-PI3K p85(Tyr 458)/p55 (Tyr199) rabbit antibody, Phospho-IRS-1 (Ser307) rabbit antibody, Phospho-ACC (Ser79) rabbit antibody, Phospho-AMPKα (Thr172) rabbit antibody, AMPKα rabbit antibody, Phospho-c-Jun (Ser63) rabbit antibody, Phospho-IκB (Ser32/36) mouse antibody (Cell Signaling Technology), β-actin mouse antibody, Lamin A, NF-κB p65 mouse antibody, SREBP-1 rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA), Phospho-IGF 1 receptor rabbit antibody (Abcam, Cambridge, UK), MMP-2 mouse antibody, MMP-9 mouse antibody, and IL-1α mouse antibody (R&D Systems, Minneapolis, MN). Secondary anti-rabbit IgG and anti-mouse IgG antibody (Cell Signaling Technology) were used to detect primary antibodies. Blots were developed with WESTSAVE Up (LabFrontier, Seoul, South Korea) and exposed to the film. Films of blots were analyzed and quantified using a densitometric program (TINA; Raytest Isotopenmebgerate, Straubenhardt, Germany).

      Thin-layer chromatography

      SEB-1 cells were grown to 80% confluence in 35-mm dishes and treated with control or different concentrations of EGCG for 24hours. In all experiments, cells were counted to normalize the data. The remaining cells were suspended in a DMEM solution containing 1μCi 14C-acetate and incubated for 2hours at 37°C with agitation, and extracted twice with ethyl ether and nonradioactive carrier lipids. Samples were dissolved in a small volume of ethyl acetate and spotted on 20-cm silica gel thin-layer chromatography plates (Merck, Darmstadt, Germany), which were run until the solvent front reached 19.5cm in hexane, followed by 19.5cm in benzene, and finally to 11cm in hexane:ethyl ether:glacial acetic acid (69.5:30:1.5). Lipid spots were visualized, excised, and radioactivity in each spot was quantified in a liquid scintillation counter. Each experiment was conducted at least five separate times.
      This clinical study was performed in accordance with the Declaration of Helsinki (2000) and was approved by the Institutional Review Board of Seoul National University Hospital. Written informed consent was obtained from each participant before study enrollment. The study was registered with ClinicalTrials.gov under registration ID# NCT01687556; the CONSORT participant flowchart is provided as Supplementary Figure S3 online.

      ACKNOWLEDGMENTS

      This work was supported by grant (04-2005-0430) from the SNUH Research Fund and by grant (A080258) from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea. JYY and HHK developed the conceptual framework of the study, designed the experiments, conducted studies, analyzed the data, and wrote the paper. SUM conducted studies and analyzed the data. DMT provided cell lines and adviced on in vitro aspects of the study. DHS conceived of and supervised the whole project and wrote the manuscript.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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