Ethyl linoleate inhibits α-MSH-induced melanogenesis through Akt/GSK3β/β-catenin signal pathway

Abstract

Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in α-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) and reduced the level of β-catenin, suggesting that ethyl linoleate inhibits melanogenesis through Akt/GSK3β/β-catenin signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.

Keywords: Akt; Ethyl linoleate; GSK3β; Melanogenesis; β-catenin.

Fig. 1. Effects of cell cytotoxicity and melanin production of ethyl linoleate.

The cells were treated for 48 h with indicated concentration of ethyl linoleate. Cell viabilities were determined by MTT assay on B16F10 murine melanoma (A) and human dermal fibroblast cells (B). (C) The B16F10 cells were exposed to α-melanocyte-stimulating hormone (α-MSH, 500 nM) in the presence of ethyl linoleate (EL) for 48 h. Melanin content in B16F10 cells was visualized and determined at the indicated concentrations of EL or positive whitening agents (AR, arbutin 2 mM; KA, kojic acid 400 µM; RSV, resveratrol 20 µM). The data are expressed as the means±SD. ^*p<0.01, compared with the α-MSH control; ^#p<0.01, compared with the vehicle control.

Fig. 2. Effects of ethyl linoleate on tyrosinase activity in B16F10 cells.

The B16F10 cells were exposed to α-melanocyte-stimulating hormone (α-MSH, 500 nM) in the presence of ethyl linoleate (EL) for 48 h. (A) In situ intracellular tyrosinase activity determined by L-DOPA staining. Resveratrol (RSV, 20 µM) was used as a positive control. Images were captured under identical conditions using bright field microscopy. Bar=20 µm. (B) Intracellular tyrosinase activity was determined using lysates obtained from B16F10 cells treated with EL or RSV. (C) The direct effect of ethyl linoleate on tyrosinase activity was measured with mushroom tyrosinase. Kojic acid (KA, 400 µM) was used as a positive control. The data are expressed as the means±SD. ^*p<0.01, compared with the α-MSH control; ^#p<0.01, compared with the vehicle control.

Fig. 3. Effects of ethyl linoleate on expression of melanogenic enzyme proteins in B16F10 cells.

The B16F10 cells were exposed to α-melanocyte-stimulating hormone (α-MSH, 500 nM) in the presence of ethyl linoleate (EL) for 48 h. (A) The levels of tyrosinase, TRP1, and β-actin from ethyl linoleate treated B16F10 cells were detected using western blot. Band intensity of tyrosinase (B) and TRP1 (C) compared to the α-MSH control was determined by ImageJ software. Resveratrol (RSV, 20 µM) was used as a positive control. The data are expressed as the means±SD. ^*p<0.05, compared with the α-MSH control; ^#p<0.05, compared with the vehicle control.

Fig. 4. Effects of ethyl linoleate on expression of MITF through Akt/GSK3β/β-catenin signal in B16F10 cells.

The B16F10 cells were exposed to α-melanocyte-stimulating hormone (α-MSH, 500 nM) in the presence of ethyl linoleate (EL) or resveratrol (RSV, 20 µM) for 4 h. The expression levels of protein including Akt, p-Akt, GSK3β, p-GSK3β, β-catenin, and β-actin were detected by using western blot. Band intensity compared to the α-MSH control was determined by ImageJ software. (A) MITF, (B) Akt and p-Akt, (C) GSK3β and p-GSK3β, and (D) β-catenin. The data are expressed as the means±SD. ^*p<0.05, compared with the α-MSH control; ^#p<0.05, compared with the vehicle control.