Plumbagin Suppresses α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells by Inhibiting Tyrosinase Activity

Abstract

Recent studies have shown that plumbagin has anti-inflammatory, anti-allergic, antibacterial, and anti-cancer activities; however, it has not yet been shown whether plumbagin suppresses alpha-melanocyte stimulating hormone (α-MSH)-induced melanin synthesis to prevent hyperpigmentation. In this study, we demonstrated that plumbagin significantly suppresses α-MSH-stimulated melanin synthesis in B16F10 mouse melanoma cells. To understand the inhibitory mechanism of plumbagin on melanin synthesis, we performed cellular or cell-free tyrosinase activity assays and analyzed melanogenesis-related gene expression. We demonstrated that plumbagin directly suppresses tyrosinase activity independent of the transcriptional machinery associated with melanogenesis, which includes micropthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1). We also investigated whether plumbagin was toxic to normal human keratinocytes (HaCaT) and lens epithelial cells (B3) that may be injured by using skin-care cosmetics. Surprisingly, lower plumbagin concentrations (0.5-1 μM) effectively inhibited melanin synthesis and tyrosinase activity but do not cause toxicity in keratinocytes, lens epithelial cells, and B16F10 mouse melanoma cells, suggesting that plumbagin is safe for dermal application. Taken together, these results suggest that the inhibitory effect of plumbagin to pigmentation may make it an acceptable and safe component for use in skin-care cosmetic formulations used for skin whitening.

Keywords: melanogenesis; pigmentation; plumbagin; tyrosinase.

Figure 1

Chemical structure and cytotoxicity of plumbagin. (A) Chemical structure of plumbagin; (B) toxicity of plumbagin in B16F10 mouse melanoma cells. Cells were incubated with 1, 2, 5, 10, 20 μM of plumbagin for 48 or 72 h. Values (left panel) represent mean ± SD of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01. Crystal violet staining images are shown in the right panel.

Figure 2

Effects of plumbagin on melanin production in B16F10 mouse melanoma cells. (A) Plumbagin suppressed α-MSH-induced melanin production. Cells were pre-incubated in the absence or presence of plumbagin for 1 h, following which α-MSH (0.2 mM) was added and the cells were incubated for 3 or 4 days. Color changes in the cultured medium are shown; (B) extracellular and (C) intracellular melanin content increased by α-MSH treatment alone and decreased when plumbagin treatment was also given. Cells were pre-incubated with arbutin (1 mM), kojic acid (0.2 mM), or plumbagin (0.5, 1 μM) for 1 h, and then further incubated with α-MSH (0.2 mM) for 3 or 4 days as indicated. Values represent means ± SD of three independent experiments performed in duplicate; # p < 0.05, ## p < 0.01, and ** p < 0.01.

Figure 3

Plumbagin does not affect the transcriptional machinery and signal transduction cascade associated with melanogenesis. (A) Determination of time to mRNA expression associated with melanogenesis. Cells were incubated with 0.2 mM of α-MSH for indicated time periods, following which melanogenesis-related gene-specific mRNA expression level was measured. Values represent means ± SD of two independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, and *** p < 0.001; (B) effects of plumbagin on MITF and tyrosinase protein expression levels. B16F10 cells pre-incubated with plumbagin (0.25, 0.5, 1, 2 μM) were further incubated with 0.2 mM α-MSH. MITF and tyrosinase protein expression levels were measured via immunoblotting as described in the materials and methods section; (C) effect of plumbagin on MITF, TYR, and TYRP1 mRNA expression. B16F10 cells were incubated in the absence or presence of α-MSH and plumbagin (1, 2 μM) for 4 h (MITF mRNA) or 48 h (TYR and TYRP1 mRNA). MITF, TYR, and TYRP1 mRNA expression levels were measured using quantitative RT-PCR. Values represent means ± SD of three independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, and NS (not significant); (D) regulatory effects of plumbagin on signal transduction proteins that participate in melanogenesis. Cells were pre-incubated with plumbagin for 1 h, and cells were then further incubated with α-MSH (0.2 mM) for 3 h. Indicated protein levels were measured via immunoblotting.

Figure 4

Inhibitory effects of plumbagin on (A) cellular tyrosinase activity and (B) cell-free tyrosinase activity. Tyrosinase activity was determined by measuring l-DOPA oxidation to dopachrome, and this oxidation of l-DOPA was read using an absorbance reader at 475 nm; (C) antioxidants activity of plumbagin. DPPH scavenging activity was examined at indicated concentrations using plumbagin or vitamin C as a positive control. Values represent means ± SD of three independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, *** p < 0.001 and # p < 0.05.

Figure 5

Cytotoxic effects of plumbagin in B3 and HaCaT cells. (A) Cytotoxicity of plumbagin in B3 and HaCaT cells. Cells were incubated with various concentrations (1, 2, 5, 10, 20 μM) of plumbagin for 3 days. Values (left panel) represent means ± SD of three independent experiments performed in duplicate; ** p < 0.01. Crystal violet staining images were shown (right panel); (B) plumbagin does not cause DNA damage and apoptosis in B3 and HaCaT cells. Cells were incubated with or without 2 μM of plumbagin for 3 days, and protein levels were then measured via immunoblotting.

Figure 6

Proposed regulatory mechanism of plumbagin on suppression of melanin synthesis in melanocytes. The dotted line and white arrows indicate stimulating signals. The black T bar indicates inhibitory effect. α-MSH: alpha-melanocyte stimulating hormone; MC1R: melanocortin 1 receptor; PKA: protein kinase A; CREB: cAMP response element binding protein; MITF: micropthalmia-associated transcription factor; TYR: tyrosinase; TYRP1: tyrosinase-related protein 1; l-DOPA: l-3,4-dihydroxyphenylalanine.