title: Development of Tyrosinase Promoter-Based Fluorescent Assay for Screening of Anti-melanogenic Agents.
abstract: For screening of skin-whitening ingredients that modulate inhibition of melanogenesis, tyrosinase promoter-based assay using a three-dimensional (3D) spheroid culture technique is a beneficial tool to improve the accuracy of raw material screening in cosmetics through mimicking of the in vivo microenvironment. Although the advantages of high-throughput screening (HTS) are widely known, there has been little focus on specific cell-based promoter assays for HTS in identifying skin-whitening ingredients that inhibit accumulation of melanin. The aim of this study was therefore to develop a large-scale compatible assay through pTyr-EGFP, an enhanced green fluorescent protein (EGFP)-based tyrosinase-specific promoter, to seek potential melanogenesis inhibitors for cosmetic use. Herein, a stably transfected human melanoma cell line expressing EGFP under the control of a 2.2-kb fragment derived from the tyrosinase gene was generated. Spontaneous induction of the tyrosinase promoter by 3D spheroid culture resulted in increased expression of EGFP, providing a significant correlation with the tyrosinase mRNA level, and subsequent inhibition of tyrosinase activity. Importantly, the pTyr-EGFP system provided successful tracking of the changes in the live image and real-time monitoring. Thus tyrosinase promoter-based fluorescent assay using a 3D spheroid culture can be useful as a screening system for exploring the efficiency of anti-melanogenesis ingredients.
authors.affiliation: Biospectrum Life Science Institute.
authors.forename: JaeHo
authors.initials: J
authors.lastname: Lee
authors.forename: SeungJun
authors.initials: S
authors.lastname: Lee
authors.forename: ByungMan
authors.initials: B
authors.lastname: Lee
authors.forename: KyungBaeg
authors.initials: K
authors.lastname: Roh
authors.forename: DeokHoon
authors.initials: D
authors.lastname: Park
authors.forename: EunSun
authors.initials: E
authors.lastname: Jung
mesh_terms: D001681:Biological Assay; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057166:High-Throughput Screening Assays; D006801:Humans; D008543:Melanins / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D062327:Skin Lightening Preparations / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism
chemical_list: D008543:Melanins; D062327:Skin Lightening Preparations; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014442:Monophenol Monooxygenase