Development of an in vitro Primary Screen for Skin Depigmentation and Antimelanoma Agents

Abstract

An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC₅₀) at < 100 µg/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphe-nol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.

Fluvastatin increases tyrosinase synthesis induced by UVB irradiation of B16F10 melanoma cells.

“…When cells reached 40% of confluence, the medium was changed to DMEM supplemented with either 10% FCS alone or 10% FCS with fluvastatin at concentrations: 0.1 μM; 1 μM; 10 μM. Cell viability was determined in triplicates, using crystal violet assay [10,11]. After incubating B16F10 cells with test substances for up to 72 hours, culture medium was removed and the cells were stained with 0.1% crystal violet in 10% ethanol for 5 min at 37°C and rinsed four times.…”

Occupational Leukoderma in Workers Engaged in 4‐(p‐Hydroxyphenyl)‐2‐Butanone Manufacturing

“…Some studies supported this hypothesis"). Recently, advances in research on melanogenesis, cell culture assays and development of antimelanoma agents have identified potential pigmentation-reducing agents [16][17][18]. These studies suggested that depigmentation agents have a specific cytotoxic action on melanocytes except for a few agents which inhibit synthesis of melanine without cytotoxic action16.…”

Extract of Enzyme-Hydrolyzed Green Tea Seed as Potent Melanin Synthesis Inhibitor

“…Recently, the application of naturally occurring products as melanin synthesis inhibitors in cosmetics has attracted much interest. 3 For example, plant polyphenols have been the target of several studies, 4 resulting in repeated reviews of their classification, occurrence, structural aspects, reactivity, biochemistry, and biogenesis. Extensive literature is available on the screening of tyrosinase inhibitors, among phenolics of plant origin, and polyphenols are currently the target of numerous studies.…”