FGIN-1-27 Inhibits Melanogenesis by Regulating Protein Kinase A/cAMP-Responsive Element-Binding, Protein Kinase C-β, and Mitogen-Activated Protein Kinase Pathways

Abstract

FGIN-1-27 is a synthetic mitochondrial diazepam binding inhibitor receptor (MDR) agonist that has demonstrated pro-apoptotic, anti-anxiety, and steroidogenic activity in various studies. Here we report, for the first time, the anti-melanogenic efficacy of FGIN-1-27 in vitro and in vivo. FGIN-1-27 significantly inhibited basal and α-melanocyte-stimulating hormone (α-MSH)-, 1-Oleoyl-2-acetyl-sn-glycerol (OAG)- and Endothelin-1 (ET-1)-induced melanogenesis without cellular toxicity. Mushroom tyrosinase activity assay showed that FGIN-1-27 did not directly inhibit tyrosinase activity, which suggested that FGIN-1-27 was not a direct inhibitor of tyrosinase. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, FGIN-1-27 downregulated the expression levels of key proteins that function in melanogenesis. FGIN-1-27 played these functions mainly by suppressing the PKA/CREB, PKC-β, and MAPK pathways. Once inactivated, it decreased the expression of MITF, tyrosinase, TRP-1, TRP-2, and inhibited the tyrosinase activity, finally inhibiting melanogenesis. During in vivo experiments, FGIN-1-27 inhibited the body pigmentation of zebrafish and reduced UVB-induced hyperpigmentation in guinea pig skin, but not a reduction of numbers of melanocytes. Our findings indicated that FGIN-1-27 exhibited no cytotoxicity and inhibited melanogenesis in both in vitro and in vivo models. It may prove quite useful as a safer skin-whitening agent.

Keywords: FGIN-1-27; SK-MEL-2 cells; human epidermis melanocytes; melanogenesis; mitogen-activated protein kinase; protein kinase A/cAMP-responsive element-binding; protein kinase C-β.

FIGURE 1

Effect of FGIN-1-27 (FGIN) on melanogenesis in SK-MEL-2 cells. (A) The chemical structure of FGIN-1-27, (B) after incubation of with various concentrations (1–16 μM) of FGIN-1-27 for 48 h, cell viability was determined using MTT assay, (C) SK-MEL-2 cells were treated with FGIN-1-27 in the presence or absence of α-MSH (50 nM) for 48 h. Melanin contents were measured as described in methods. (D) SK-MEL-2 cells were treated with FGIN-1-27 (4 μM) for 48 h and were stained with Masson–Fontana ammoniacal silver stain. Bar = 20 μm (E,F) SK-MEL-2 cells were treated with 200 μM OAG or 10 nM ET-1 in the presence or absence of FGIN-1-27. Melanin contents were measured as described in methods. Data are expressed as the mean ± SD (n = 3). *p < 0.05, ***p < 0.001 vs. non-treated cells. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. α-MSH-, OAG-, or ET-1-treated cells.

FIGURE 2

Effect of FGIN-1-27 (FGIN) on the expression of tyrosinase and the activity of tyrosinase. (A) SK-MEL-2 cells were treated with FGIN-1-27 (0, 1, 2, 4 μM) in the presence or absence of α-MSH (50 nM) for 48 h and western blot was then applied to detect the tyrosinase, TRP-1 and TRP-2 levels. (B) SK-MEL-2 cells were treated with FGIN-1-27 (0, 1, 2, 4 μM) in the presence or absence of ET-1 (10 nM) for 48 h and western blot was then applied to detect the tyrosinase, TRP-1 and TRP-2 levels. SK-MEL-2 cells were treated with FGIN-1-27 (4 μM) in the presence or absence of OAG (200 μM) for 12 h. (C) Cellular tyrosinase activity was determined by L-DOPA oxization as described in methods, and (D) western blot was applied to detect the tyrosinase levels. (E) Mushroom tyrosinase activity was determined as described in methods. Data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. α-MSH, ET-1 or OAG-treated cells.

FIGURE 3

Effect of FGIN-1-27 (FGIN) on the expression of MITF in SK-MEL-2 cells. (A) SK-MEL-2 cells were treated with FGIN-1-27 for 4 h and RT-qPCR was then applied to detect MITF gene expression. (B and C) SK-MEL-2 cells were treated with α-MSH (50 nM) or ET-1 (10 nM) in the presence or absence of FGIN-1-27 for different times and western blot was then applied to detect MITF protein levels. Data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. α-MSH or ET-1-treated time-matched cells.

FIGURE 4

Effect of FGIN-1-27 on the activity of PKC-β, PKA/CREB and MAPK signaling pathways in SK-MEL-2 cells. SK-MEL-2 cells were treated with FGIN-1-27 (4 μM) for the indicated time period (0–120 min), and the expression of PKC-β, p-PKA cat, PKA cat, p-CREB, CREB, p-p38, p38, p-JNK, JNK, p-ERK and ERK were measured by western blot. Data are expressed as the mean ± SD (n = 3). **p < 0.01, ***p < 0.001 vs. non-treated cells.

FIGURE 5

Effect of FGIN-1-27 (FGIN) on melanogenesis in human melanocytes. (A) Human melanocytes were treated with FGIN-1-27 for 48 h and melanin contents were measured as described in methods. (B) Human melanocytes were treated with FGIN-1-27 for 48 h and the expression of tyrosinase, TRP-1 and TRP-2 were measured using western-blot as described in methods. (C) Human melanocytes were treated with α-MSH (50 nM) in the presence or absence of FGIN-1-27 for different times and western blot was then applied to detect MITF protein levels. (D) Human melanocytes were treated with FGIN-1-27 for the indicated time period (0–120 min), and the expression of PKC-β, p-PKA cat, PKA cat, p-CREB, CREB, p-p38, p38, p-ERK and ERK were measured by western blot. Data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells.

FIGURE 6

Effect of FGIN-1-27 (FGIN) on pigmentation in zebrafish. Representative photographs of zebrafish. Zebrafish embryos were treated with PTU or FGIN-1-27 from 35 to 60 h. The effects on the pigmentation of zebrafish were observed under the stereomicroscope.

FIGURE 7

Effect of FGIN-1-27 on pigmentation in guinea-pig skin. (A) Representative photographs of dorsal skin of guinea pigs. (B) The degree of depigmentation was determined by a chromameter (CR-300; Minolta, Osaka, Japan) once a week for 4 weeks. The ΔL value was calculated using the L value (brightness index) measured with the chromameter follows: ΔL = L (at each week measured) − L (at day 0). Negative ΔL values indicate an UV-induced darkening of the skin. An increase in the ΔL value indicates a decrease in hyperpigmentation induced by UV. (C) Masson–Fontana ammoniacal silver staining of skin biopsies. (D) Immunohistochemical staining of skin biopsies for the detection of S-100 as a melanocyte marker protein (E) Number of melanocytes per microscopic field in skin sections. Bar = 50 μm *p < 0.05 vs. vehicle-treated groups.

FIGURE 8

Schematic description of changes in pigmentation upon FGIN-1-27(FGIN) treatment.