title: Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells.
abstract: There are two established depigmenting agent assays currently in use. However, these methods are unreliable and time-consuming. Therefore, it will be valuable to establish a better assay system for depigmenting agent analysis. In this study, we established a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the microphthalmia-associated transcription factor (MITF), tyrosinase (Tyr) and dopachrome tautomerase (Dct) genes in MeWo human melanoma cells. We used several melanogenesis regulators, including theophylline, hesperetin, arbutin and rottlerin, to confirm the function of this assay system. The established MeWo/pMITF-EGFP, MeWo/pTyr-EGFP and MeWo/pDct-EGFP stable cells integrated the pMITF-EGFP, pTyr-EGFP and pDct-EGFP plasmids into their genomic DNA. These stably transfected cells were used to examine alterations in the expression of the MITF, Tyr and Dct genes. All of the tested compounds, including theophylline, hesperetin, arbutin and rottlerin, could be analyzed in the stable cells, producing reliable results. Therefore, we believe that this melanogenesis regulation assay system can be used as a rapid and reliable assay system to analyze the regulation of melanogenesis by many known or unknown compounds.
authors.affiliation: Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC. Electronic address: chchlin@pu.edu.tw.
authors.forename: Chih-Chien
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC.
authors.forename: Chao-Hsun
authors.initials: CH
authors.lastname: Yang
authors.affiliation: Department of Medical Research, China Medical University Hospital, 2 Yuh-Der Road, Taichung 40447, Taiwan, ROC; School of Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan, ROC.
authors.forename: Ying-Ju
authors.initials: YJ
authors.lastname: Lin
authors.affiliation: Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC.
authors.forename: Ya-Wen
authors.initials: YW
authors.lastname: Chiu
authors.affiliation: Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC; Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan, ROC.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Chen
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D001104:Arbutin / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004790:Enzyme Induction / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D005813:Genes, Synthetic; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006569:Hesperidin / Q000494:pharmacology; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D008543:Melanins / Q000032:analysis / Q000096:biosynthesis*; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D008546:Melanoma, Experimental / Q000473:pathology; D051739:Microphthalmia-Associated Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D014442:Monophenol Monooxygenase / Q000096:biosynthesis / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D062327:Skin Lightening Preparations / Q000494:pharmacology*; D013806:Theophylline / Q000494:pharmacology; D014162:Transfection
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; C497234:MITF protein, human; D008543:Melanins; D051739:Microphthalmia-Associated Transcription Factor; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D062327:Skin Lightening Preparations; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013806:Theophylline; D001104:Arbutin; C085746:rottlerin; D006569:Hesperidin; D014442:Monophenol Monooxygenase; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase; C013015:hesperetin
keywords: Dopachrome tautomerase (Dct); Fluorescent protein; Melanogenesis; Microphthalmia-associated transcription factor (MITF); Reporter gene system; Tyrosinase (Tyr)