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

Chih-Chien Lin; Chao-Hsun Yang; Ying-Ju Lin; Ya-Wen Chiu; Cheng-Yu Chen

doi:10.1016/j.enzmictec.2014.09.008

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

Enzyme Microb Technol. 2015 Jan:68:1-9. doi: 10.1016/j.enzmictec.2014.09.008. Epub 2014 Sep 28.

Authors

Chih-Chien Lin¹, Chao-Hsun Yang², Ying-Ju Lin³, Ya-Wen Chiu², Cheng-Yu Chen⁴

Affiliations

¹ Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC. Electronic address: chchlin@pu.edu.tw.
² Department of Cosmetic Science, Providence University, 200, Section 7, Taiwan Boulevard, Shalu District, Taichung 43301, Taiwan, ROC.
³ 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.
⁴ 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.

PMID: 25435499
DOI: 10.1016/j.enzmictec.2014.09.008

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.

Keywords: Dopachrome tautomerase (Dct); Fluorescent protein; Melanogenesis; Microphthalmia-associated transcription factor (MITF); Reporter gene system; Tyrosinase (Tyr).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acetophenones / pharmacology
Arbutin / pharmacology
Benzopyrans / pharmacology
Cell Line, Tumor
Drug Evaluation, Preclinical / methods
Enzyme Induction / drug effects
Gene Expression Regulation, Neoplastic / drug effects
Genes, Reporter
Genes, Synthetic
Green Fluorescent Proteins / analysis*
Green Fluorescent Proteins / genetics
Hesperidin / pharmacology
Humans
Intramolecular Oxidoreductases / biosynthesis
Intramolecular Oxidoreductases / genetics
Melanins / analysis
Melanins / biosynthesis*
Melanoma / genetics
Melanoma / pathology*
Melanoma, Experimental / pathology
Microphthalmia-Associated Transcription Factor / biosynthesis
Microphthalmia-Associated Transcription Factor / genetics
Microscopy, Fluorescence
Monophenol Monooxygenase / biosynthesis
Monophenol Monooxygenase / genetics
Neoplasm Proteins / biosynthesis
Neoplasm Proteins / genetics
Promoter Regions, Genetic / genetics*
RNA, Messenger / biosynthesis
Recombinant Fusion Proteins / analysis
Skin Lightening Preparations / pharmacology*
Theophylline / pharmacology
Transfection

Substances

Acetophenones
Benzopyrans
MITF protein, human
Melanins
Microphthalmia-Associated Transcription Factor
Neoplasm Proteins
RNA, Messenger
Recombinant Fusion Proteins
Skin Lightening Preparations
enhanced green fluorescent protein
Green Fluorescent Proteins
Theophylline
Arbutin
rottlerin
Hesperidin
Monophenol Monooxygenase
Intramolecular Oxidoreductases
dopachrome isomerase
hesperetin