Antioxidants cinnamaldehyde and Galactomyces fermentation filtrate downregulate senescence marker CDKN2A/p16INK4A via NRF2 activation in keratinocytes

Senescence is a state wherein cells are metabolically active but unable to replicate due to the increased expression of cell cycle checkpoint proteins such as cyclin-dependent kinase inhibitor 2A (CDKN2A or p16INK4A), which induce cell cycle arrest [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar,[2]Adamus J. Aho S. Meldrum H. Bosko C. Lee J.M. p16INK4A influences the aging phenotype in the living skin equivalent.J. Invest. Dermatol. 2014; 134: 1131-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. The accumulation of CDKN2A protein is a major and stable senescence marker in various cell types including keratinocytes and is induced in vitro and in vivo after successive replication and/or upon exposure to various stressors such as oxidative stress [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar,[2]Adamus J. Aho S. Meldrum H. Bosko C. Lee J.M. p16INK4A influences the aging phenotype in the living skin equivalent.J. Invest. Dermatol. 2014; 134: 1131-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. It is known that senescent keratinocytes have a 30-fold-higher intracellular concentration of reactive oxygen species (ROS) than those in the growth phase [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar]. Therefore, continuous treatment with the antioxidative enzyme catalase or the general antioxidant N-acetyl-cysteine delays the accumulation of CDKN2A protein and subsequent senescence [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar]. Senescence is generally linked to carcinogenic potential [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar]. In addition, the increase in CDKN2A + senescent keratinocytes is associated with epidermal atrophy seen in the elderly [[2]Adamus J. Aho S. Meldrum H. Bosko C. Lee J.M. p16INK4A influences the aging phenotype in the living skin equivalent.J. Invest. Dermatol. 2014; 134: 1131-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Nuclear factor-erythroid 2-related factor-2 (NRF2) is a master switch for antioxidant signaling [[3]Furue M. Uchi H. Mitoma C. Hashimoto-Hachiya A. Chiba T. Ito T. Nakahara T. Tsuji G. Antioxidants for healthy skin: the emerging role of aryl hydrocarbon receptors and nuclear factor-erythroid 2-related factor-2.Nutrients. 2017; 9 (pii: E223)Crossref PubMed Scopus (50) Google Scholar] and its activation reduces intracellular ROS levels by upregulating antioxidative enzymes such as glutathione peroxidase 2 (GPX2) and NAD(P)H quinone oxidoreductase 1 (NQO1) [[4]Takei K. Hashimoto-Hachiya A. Takahara M. Tsuji G. Nakahara T. Furue M. Cynaropicrin attenuates UVB-induced oxidative stress via the AhR-Nrf2-Nqo1 pathway.Toxicol. Lett. 2015; 234: 74-80Crossref PubMed Scopus (45) Google Scholar,[5]Walshe J. Serewko-Auret M.M. Teakle N. Cameron S. Minto K. Smith L. Burcham P.C. Russell T. Strutton G. Griffin A. Chu F.F. Esworthy S. Reeve V. Saunders N.A. Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation.Cancer Res. 2007; 67: 4751-4758Crossref PubMed Scopus (58) Google Scholar]. Various antioxidative phytochemicals and microbial products activate the NRF2 system and exert antioxidative function [[3]Furue M. Uchi H. Mitoma C. Hashimoto-Hachiya A. Chiba T. Ito T. Nakahara T. Tsuji G. Antioxidants for healthy skin: the emerging role of aryl hydrocarbon receptors and nuclear factor-erythroid 2-related factor-2.Nutrients. 2017; 9 (pii: E223)Crossref PubMed Scopus (50) Google Scholar]. Cinnamaldehyde, an active component of cinnamon, and Galactomyces fermentation filtrate (GFF) are such potent natural antioxidants acting via NRF2 activation [[6]Uchi H. Yasumatsu M. Morino-Koga S. Mitoma C. Furue M. Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes.J. Dermatol. Sci. 2017; 85: 36-43Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,[7]Takei K. Mitoma C. Hashimoto-Hachiya A. Takahara M. Tsuji G. Nakahara T. Furue M. Galactomyces fermentation filtrate prevents T helper 2-mediated reduction of filaggrin in an aryl hydrocarbon receptor-dependent manner.Clin. Exp. Dermatol. 2015; 40: 786-793Crossref PubMed Scopus (22) Google Scholar]. However, whether these natural NRF2 activators downregulate CDKN2A expression remains elusive. In this study, we stimulated normal human epidermal keratinocytes (NHEKs; Lonza, Basel, Switzerland) with cinnamaldehyde (50 μM; Sigma-Aldrich, St. Louis, MO) [[6]Uchi H. Yasumatsu M. Morino-Koga S. Mitoma C. Furue M. Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes.J. Dermatol. Sci. 2017; 85: 36-43Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar] or GFF (10%, Pitera®; P&G Innovation Godo Kaisha, Kobe, Japan) [[7]Takei K. Mitoma C. Hashimoto-Hachiya A. Takahara M. Tsuji G. Nakahara T. Furue M. Galactomyces fermentation filtrate prevents T helper 2-mediated reduction of filaggrin in an aryl hydrocarbon receptor-dependent manner.Clin. Exp. Dermatol. 2015; 40: 786-793Crossref PubMed Scopus (22) Google Scholar] for 24 h. Western blot analysis revealed that both cinnamaldehyde (Fig. 1A, D, and E) and GFF (Fig. 1B, G, and H) upregulated the protein expression of GPX2 (detected by rabbit polyclonal GPX2 antibody, ab137431; Abcam, Cambridge, UK) and NQO1 (mouse monoclonal NQO1 antibody, ab28947; Abcam), suggesting their NRF2-activating capacity. In parallel with this, both antioxidative agents appeared to inhibit the protein expression of CDKN2A (rabbit monoclonal, p16INK4A, #80772; Cell Signaling Technology, Danvers, MA, USA) (Fig. 1A–C and F). β-Actin (mouse monoclonal β-actin antibody, #3700; Cell Signaling Technology) protein level served as an internal housekeeping protein control. Output TIFF images were analyzed using ImageJ (http://imagej.nih.gov/ij) to quantify antibody expression. To examine the dependence of these behaviors on NRF2, we performed a similar experiment using NHEKs transfected with NRF2 small interfering (si)RNA (s9492; Ambion, Austin, TX, USA) or control siRNA (Ambion). Transfection of NRF2 siRNA reduced the protein level of NRF2 (82.5 ± 4.6% reduction). The basal expression and cinnamaldehyde-induced upregulation of GPX2 and NQO1 proteins were downregulated in NRF2-silenced keratinocytes compared with the levels in those transfected with control siRNA (Fig. 2A, D, and E). The basal expression and GFF-induced upregulation of GPX2 and NQO1 proteins were also downregulated in NRF2-silenced keratinocytes compared with the levels in those transfected with control siRNA (Fig. 2B, G, H). Both cinnamaldehyde and GFF again inhibited the CDKN2A protein expression (Fig. 2A-C, F). Notably, NRF2 silencing enhanced the CDKN2A protein expression in untreated, cinnamaldehyde-treated and GFF-treated keratinocytes (Fig. 2A-C, F). These results indicate that the natural antioxidants cinnamaldehyde and GFF are capable of inhibiting protein expression of the senescence marker CDKN2A in a NRF2-dependent manner. The skin is continuously exposed to various oxidative stressors including ultraviolet rays and environmental pollutants [1Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar, 2Adamus J. Aho S. Meldrum H. Bosko C. Lee J.M. p16INK4A influences the aging phenotype in the living skin equivalent.J. Invest. Dermatol. 2014; 134: 1131-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 3Furue M. Uchi H. Mitoma C. Hashimoto-Hachiya A. Chiba T. Ito T. Nakahara T. Tsuji G. Antioxidants for healthy skin: the emerging role of aryl hydrocarbon receptors and nuclear factor-erythroid 2-related factor-2.Nutrients. 2017; 9 (pii: E223)Crossref PubMed Scopus (50) Google Scholar]. As such, the accumulation of CDKN2A protein in keratinocytes is observed in normal aged skin as well as neoplastic and inflammatory skin disorders [[1]Abbadie C. Pluquet O. Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?.Cell. Mol. Life Sci. 2017; 74: 4471-4509Crossref PubMed Scopus (32) Google Scholar,[2]Adamus J. Aho S. Meldrum H. Bosko C. Lee J.M. p16INK4A influences the aging phenotype in the living skin equivalent.J. Invest. Dermatol. 2014; 134: 1131-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,[8]Uryu M. Kido-Nakahara M. Nakahara T. Chiba T. Furue M. Epidermal p16(INK)(4a) expression is more frequently and intensely upregulated in lichen planus than in eczema, psoriasis, drug eruption and graft-versus-host disease.J. Dermatol. 2017; 44: 343-344Google Scholar]. Numerous natural antioxidative agents activate the cellular NRF2 system and protect the body from oxidative stress [[3]Furue M. Uchi H. Mitoma C. Hashimoto-Hachiya A. Chiba T. Ito T. Nakahara T. Tsuji G. Antioxidants for healthy skin: the emerging role of aryl hydrocarbon receptors and nuclear factor-erythroid 2-related factor-2.Nutrients. 2017; 9 (pii: E223)Crossref PubMed Scopus (50) Google Scholar]. Very recently, it was reported that endogenous hormones such as 1,25-dihydroxyvitamin D and oxytocin alleviate cellular senescence via NRF2 activation [[9]Chen L. Yang R. Qiao W. Zhang W. Chen J. Mao L. Goltzman D. Miao D. 1,25-Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2-antioxidant signaling and inactivation of p16/p53-senescence signaling.Aging Cell. 2019; 18e12951Google Scholar,[10]Cho S.Y. Kim A.Y. Kim J. Choi D.H. Son E.D. Shin D.W. Oxytocin alleviates cellular senescence through oxytocin receptor-mediated ERK/Nrf2 signalling.Br. J. Dermatol. 2019; (Feb 22. in press)Google Scholar]. The present study proved that the natural NRF2 activators cinnamaldehyde and GFF downregulate the expression of CDKN2A protein in keratinocytes. These results support the notion that the topical or systemic administration of these natural NRF2 activators may reduce the process of senescence of human epidermis. This work was partly supported by grants from The Ministry of Health, Labour, and Welfare of Japan (H30-Shokuhin-Shitei-005) and by The Leading Advanced Projects for Medical Innovation in Japan (LEAP) .