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Kinetin Improves Barrier Function of the Skin by Modulating Keratinocyte Differentiation Markers

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Kinetin Improves Barrier Function of the Skin by Modulating Keratinocyte Differentiation Markers S An, et al pISSN 1013-9087ㆍeISSN 2005-3894 Ann Dermatol Vol. 29, No. 1, 2017 https://doi.org/10.5021/ad.2017.29.1.6 ORIGINAL ARTICLE Kinetin Improves Barrier Function of the Skin by Modulating Keratinocyte Differentiation Markers Sungkwan An, Hwa Jun Cha, Jung-Min Ko, Hyunjoo Han, Su Young Kim, Kyung-Suk Kim, Song Jeong Lee, In-Sook An1, Sangwon Kim2, Hae Jeong Youn3, Kyu Joong Ahn3,*, Soo-Yeon Kim* Korea Institute for Skin and Clinical Sciences, Konkuk University, Seoul, 1KISCS Incorporated, Cheongju, Korea, 2Orangewood Christian School, Maitland, FL, USA, 3Department of Dermatology, Konkuk University School of Medicine, Seoul, Korea Background: Kinetin is a plant hormone that regulates texture. Conclusion: Kinetin induced the expression of kera- growth and differentiation. Keratinocytes, the basic building tinocyte differentiation markers, suggesting that it may affect blocks of the epidermis, function in maintaining the skin differentiation to improve skin moisture content, TEWL, and barrier. Objective: We examined whether kinetin induces other signs of skin aging. Therefore, kinetin is a potential new skin barrier functions in vitro and in vivo. Methods: To eval- component for use in cosmetics as an anti-aging agent that uate the efficacy of kinetin at the cellular level, expression of improves the barrier function of skin. (Ann Dermatol 29(1) 6∼ keratinocyte differentiation markers was assessed. Moreover, 12, 2017) we examined the clinical efficacy of kinetin by evaluating skin moisture, transepidermal water loss (TEWL), and skin -Keywords- surface roughness in patients who used kinetin-containing Cell culture techniques, Differentiation, Keratinocytes, Kine- cream. We performed quantitative real-time polymerase tin, Skin barrier chain reaction to measure the expression of keratinocyte dif- ferentiation markers in HaCaT cells following treatment. A clinical trial was performed to assess skin moisture, TEWL, INTRODUCTION and evenness of skin texture in subjects who used ki- netin-containing cream for 4 weeks. Results: Kinetin in- The effects of human aging are mostly visible in the skin in creased involucrin, and keratin 1 mRNA in HaCaT cells. such forms as increased wrinkling and sagging, as well as Moreover, use of a kinetin-containing cream improved skin decreased elasticity1. Aging is also associated with physical moisture and TEWL while decreasing roughness of skin disorders of the skin because its barrier function is dis- rupted, leading to a dry appearance and enhanced risk of skin disorders2,3. Understanding the mechanisms of skin ag- Received November 12, 2015, Revised April 8, 2016, Accepted for publication May 9, 2016 ing is important for developing skin care products that de- *These authors contributed equally to this study. lay aging and reduce damage4,5. Skin aging is induced by Corresponding author: Kyu Joong Ahn, Department of Dermatology, Konkuk both intrinsic and extrinsic factors4,6 that lead to a reduction University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul of structural integrity and loss of physiological function6. 05029, Korea. Tel: 82-2-2030-5181, Fax: 82-2-2030-5179, E-mail: kjahn@ kuh.ac.kr Kinetin, a cytokine isolated in 1955, is an essential plant Soo-Yeon Kim, Korea Institute for Skin and Clinical Sciences, Konkuk growth hormone that regulates cell growth and differ- University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Tel: 82- entiation7,8. Kinetin has also been reported to be present 2-450-4054, Fax: 82-2-3436-3778, E-mail: [email protected] in human cell extracts9 and urine10, and has been identi- This is an Open Access article distributed under the terms of the Creative fied as a naturally occurring base modification agent of Commons Attribution Non-Commercial License (http://creativecommons. 11 org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, DNA . Kinetin has been reported to have multiple func- distribution, and reproduction in any medium, provided the original work tions, including anti-aging effects in cultured cells12,13 and is properly cited. fruit flies14, antioxidant properties15,16, antithrombotic ac- Copyright © The Korean Dermatological Association and The Korean tivity17,18, and cell differentiation effects19-21. Therefore, in Society for Investigative Dermatology 6 Ann Dermatol Kinetin Inproves Barrier Function of the Skin this study, we demonstrate that kinetin may induce an- lative expression levels of each gene were calculated us- −ΔΔ 22 ti-aging effects in skin by improving its barrier function. ing the 2 Ct method . Subjects for clinical evaluation MATERIALS AND METHODS The study protocols were approved by the Institutional Cell culture Review Board of KISCS Incorporated (Cheongju, Korea) HaCaT human keratinocytes (American Type Culture (IRB no. 1-70005239-A-N-01-2013-KISCS-ACA001-KSH). Collection, Boulecard Manassas, VA, USA) were cultured All subjects were informed about the objective of the in Dulbecco’s modified Eagle medium (Gibco/Life Tech- study and provided informed consent and agreed to use nologies, Carlsbad, CA, USA) supplemented with 10% fetal products for skin care during the study. Forty women bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% greater than 40 years of age were enrolled in a random- penicillin/streptomycin (Gibco/Life Technologies) at 37oC ized, double-blind clinical trial (control group: 46.80± in an atmosphere of 5% CO2. Kinetin was purchased from 4.83 years, experiment group: 46.70±0.83 years). The Sigma-Aldrich and dissolved in dimethyl sulfoxide. subjects were selected based on age, the absence of skin conditions other than age-related conditions, and were not Cell viability assay pregnant or nursing. All subjects were informed about the HaCaT cells were seeded at a density of 3×103 cells in objective of the study, provided signed informed consent, 96-well plates and incubated for 24 h. The cells were then and agreed to use only products for skin care during the incubated with kinetin (0∼600 μM) for 24 h. HaCaT cell study. Reasons for dropping out were itching, erythema, toxicity due to kinetin was evaluated using a water-soluble or excessive drinking or smoking. Subjects were divided tetrazolium salt (WST)-1 assay (EZ-Cytox Cell Viability into control and experimental groups consisting of 20 sub- Assay Kit; Itsbio, Seoul, Korea). WST-1 solution was added jects each. All conditions were identical, other than the to cultured cells at a volume equal to 10% that of the cul- exposure of the experimental group to the test material. ture medium, and then the cells were incubated at 37oC The study lasted four weeks, except no drop out. Clinical for 1 h. Cell viability was evaluated by measuring the ab- parameters were evaluated three times, namely, before ap- sorbance at 450 nm using an iMark microplate reader plication, and after 2 and 4 weeks of use. The investigator (Bio-Rad, Hercules, CA, USA). asked subjects about the condition of their skin and per- formed a visual examination of their skin condition, such Isolation of total RNA and quantitative real-time poly- as erythema, itching, scale, edema, tingling, and burning merase chain reaction sensation, at every visit. The cream was prepared by in- Total RNA was isolated using TRIzol reagent (Invitrogen corporating the ingredients in the three phases (A, B, C). Life Technologies, Carlsbad, CA, USA) according to the Ingredients in the A phase (distilled water, glycerin, manufacturer's protocol. The purity and concentration of 1,3-butylene glycol) were combined and heated until all the RNA were evaluated using a MaestroNanoⓇ, a micro- the components were melted, and ingredients in the B volume spectrophotometer (Maestrogen, Las Vegas, NV, phase (distilled water, dipotassium phosphate, sodium hy- USA). The recommended parameters of RNA quality for droxylate, kinetin) were combined and heated to the same cDNA synthesis were OD 260/230 ＞1.8 and an OD temperature, to ensure homogeneity. The A and B phases 260/280 ratio within the range of 1.8∼2.0. cDNAs were were combined and emulsified using a homo mixer synthesized using the miScript II RT Kit (Qiagen, Hilden, (Tokushu Kika Kogyo Co., Ltd., Osaka, Japan) at 5,000 Germany) according to the manufacturer's protocol. To rpm for 10 min. The mixture was cooled to 60oC and evaluate the expression of INV (forward primer: 5'- blended with the homogenized phase C (emulium delta, GGGTGGTTATTTATGTTTGGGTGG-3', reverse primer: sepipuls 400) at 5,000 rpm for 10 min. By then, the tem- 5'-GCCAGGTCCAAGACATTCAAC-3') and KRT1 (forward perature of the mixture had dropped to 45oC. The mixture primer: 5'-ATTTCTGAGCTGAATCGTGTGATC-3', reverse was combined and homogenized, while maintaining the primer: 5'-CTTGGCATCCTTGAGGGCATT-3'), quantitative pH at 6.2. The cream provided to the experimental group real-time polymerase chain reaction was performed using contained 2% (wt%) kinetin and the cream provided to EvaGreen dye (Solis BioDyne, Tartu, Estonia) with Line-Gene the control group was prepared using the same volume of K software (Bioer Technology Co., Ltd., Hangzhou, China). water in place of kinetin. The C value for each gene was normalized to β-actin T Experimental procedures (forward primer: 5'-GGATTCCTATGTGGGCGACGA-3', reverse primer: 5'-CGCTCGGTGAGGATCTTCATG-3'). Re- To investigate the improvement in skin barrier function, Vol. 29, No. 1, 2017 7 S An, et al subjects were instructed to apply 2 g of test material to the Statistical and mathematical analysis face every morning and night for 4 weeks. Subjects and investigators were blinded to test and control treatments. In cellular efficacy tests, all results are presented as the Moisture and transepidermal water loss (TEWL) were mean percentage±standard deviation of three indepen- measured on the right cheek and skin texture was eval- dent experiments. Differences with a p-value of less than uated on the left side of the forehead. At every visit, before 0.05 or 0.001, as determined by Student’s t-test, were con- measurements were taken, all subjects washed with the sidered statistically significant.
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