J. Cosmet. Sci., 62, 515–523 (September/October 2011) 515 Use of non-melanocytic HEK293 cells stably expressing human tyrosinase for the screening of anti-melanogenic agents MIJIN KIM, SANG MI AN, JAE-SOOK KOH, DONG-IL JANG, and YONG CHOOL BOO, Department of Molecular Medicine and Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine, Daegu (M.K., Y.C.B.), Dermapro Skin Research Center, Dermapro Ltd., Seoul (S.M.A., J.-S.K.), and Cotde Inc., Cheonan (D.-I.J.), Republic of Korea. Accepted for publication June 7, 2011. Synopsis Tyrosinase (TYR) from mushrooms has been inappropriately used in the screening assay for hypopigmenting agents even though its biochemical properties are different from those of human TYR. Cell-free extracts of human epidermal melanocyes (HEMs) could be another choice for the assay, but HEMs grow too slowly to get a suffi cient amount of cell-free extracts. In the present study, human embryonic kidney (HEK) 293 cells were transfected with a human TYR construct to establish a cell line that grows rapidly and expresses human TYR constitutively. Cell-free extracts of the established cell line, HEK293-TYR, were tentatively used in the screening assays for 11 phenylpropanoids that have chemical structures similar to that of L-tyrosine, the substrate of TYR. Of the 11 compounds, the strongest inhibition of TYR activity was shown by p-coumaric acid (IC50, 3 μM), followed by 3-(4-hydroxyphenyl)propionic acid (50 μM) and 3-(4-hydroxyphenyl)lactic acid (70 μM). The results indicate that p-coumaric acid has an optimal chemical structure for the inhibition of TYR. The effects of these phenylpropanoids on melanin synthesis in HEMs correlated well with their ef- fects on TYR activity in vitro. This study demonstrated that HEK293-TYR cells can be a good source of the human TYR enzymes needed in the screening assay of anti-melanogenic agents. INTRODUCTION Control of skin hyperpigmentation represents a major challenge in dermatology and cos- metics. Although both ablative and non-ablative laser-based techniques have been used to remove unwanted pigmentation including melasma, the results are not always consis- tent. In addition, no safe and effective pharmacological methods are currently available to combat hyperpigmentation. The synthesis of melanin, a major pigment responsible for the coloration of the skin, hair, and eye, occurs principally in specialized organelles called melanosomes. Thus melanin Address all correspondence to Yong Chool Boo at ycboo@knu.ac.kr.

JOURNAL OF COSMETIC SCIENCE 516 synthesis is restricted to melanocytic cells that contain melanosomes. A number of en- zymes including tyrosinase (TYR) are involved in the oxidative polymerization of the amino acid L-tyrosine to eumelanin or pheomelanin. TYR (EC 1.14.18.1) is a copper- containing enzyme that catalyzes two distinct reactions, i.e., the hydroxylation of L- tyrosine to dihydroxyphenylalanine (DOPA) and the subsequent two-electron oxidation to DOPA quinone (1,2). These reactions represent the rate-limiting steps of overall melanin synthesis, and therefore TYR inhibitors have been sought as potential hypopigmenting agents (3). Mushroom TYR is often used as a substitute for human TYR in order to screen TYR inhibitors, probably because the latter is hardly available (4,5). However, the use of mush- room TYR for this purpose can be problematic because it is quite different from human TYR in terms of the amino acid sequence and substrate specifi city (6–8). Indeed, many compounds have had inconsistent effects on the activity of TYR of mushroom, murine, or human origin (9–11). These observations have underscored the importance of using human TYR enzyme for the screening of hypopigmenting agents. Under a situation where human TYR is not commercially available, cell-free extracts of human epidermal melanocytes (HEMs) have been used instead in the initial screening of TYR inhibitors (11). However, it has been diffi cult to obtain suffi cient amounts of HEMs for this purpose because the cells grow very slowly. In addition, the culturing of HEMs requires a specialized medium that is very expensive. Highly proliferating melanin-pro- ducing melanoma cells may be a good alternative to HEMs, but they are not readily avail- able either. Therefore, this study attempted to prepare a cell line that proliferates rapidly and expresses human TYR constitutively. Human embryonic kidney (HEK) 293 cells were chosen for this purpose because these cells are commercially available, relatively easy to culture in vitro, and widely used in the production of viruses or proteins. In the present study, HEK293 cells were stably transfected with a human TYR construct, and the cell- free extracts of the established cell line, HEK293-TYR, were tentatively used in the screening of potential TYR inhibitors. MATERIALS AND METHODS CHEMICALS L-tyrosine and DOPA were purchased from Sigma-Aldrich (St. Louis, MO). The phenyl- propanoids, including p-coumaric acid, o-coumaric acid, m-coumaric acid, cinnamic acid, p-methoxycinnamic acid, caffeic acid, ferulic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)lactic acid, 3-(4-hydroxyphenyl)pyruvic acid, and 3-phenyllactic acid, were purchased from Sigma-Aldrich. CELL CULTURE Human embryonic kidney (HEK) 293 cells were purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in the growth medium Dulbecco’s minimum Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U ml-1 penicillin, 0.1 mg ml-1 streptomycin, and 0.25 μg ml-1 amphotericin B. The cells were cultured at 37°C in a humidifi ed atmosphere containing 5% CO2 and 95% air. HEMs,