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. 2012 Sep 20;17(4):616–632. doi: 10.2478/s11658-012-0033-4

Evaluation of melanogenesis in A-375 melanoma cells treated with 5,7-dimethoxycoumarin and valproic acid

Ewa Chodurek 1,✉, Arkadiusz Orchel 1, Joanna Orchel 2, Sławomir Kurkiewicz 3, Natalia Gawlik 4, Zofia Dzierżewicz 1, Krystyna Stępień 3
PMCID: PMC6275710  PMID: 23001511

Abstract

Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10–150 μM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.

Key words: A-375 cell line; Malignant melanoma; Valproic acid; Tyrosinase; Gene expression; 5,7-dimethoxycoumarin; Melanin; Pyrolysis-gas chromatography/mass spectrometry

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Abbreviations used

BCL2

B cell lymphoma 2

BCL-X

B cell lymphoma-X

DMC

5,7-dimethoxycoumarin

DMSO

dimethyl sulfoxide

DOPA

3,4-dihydroxyphenylalanine

HDACIs

histone deacetylase inhibitors

PCR

polymerase chain reaction

Py-GC/MS

pyrolysis-gas chromatography/mass spectrometry

ROS

reactive oxygen species

SDS

sodium dodecyl sulfate

TNF

tumor necrosis factor

VPA

valproic acid

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