KHG26792 Inhibits Melanin Synthesis in Mel-Ab Cells and a Skin Equivalent Model

Abstract

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.

Keywords: ERK; KHG26792; Melanogenesis; Skin equivalent; Tyrosinase.

Fig. 1

The structure of KHG26792 [3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride]. Pr, propyl.

Fig. 2

Effects of KHG26792 on melanogenesis. (A) After serum starvation for 24 h, Mel-Ab cells were treated with KHG26792 (0.1~50 µM) in serum-free media for 24 h. Cell viability was measured by crystal violet assay. (B) Mel-Ab cells were treated with KHG26792 (0.1~20 µM) for 3 days. Phase contrast photographs were obtained using a digital video camera. Melanin content (C) and tyrosinase activity in a cell-free system (D) were measured as described in Methods. Each assessment was performed in triplicate, and all data are shown as mean±S.D. ^*p<0.05, ^**p<0.01 compared to the untreated control.

Fig. 3

Effects of KHG26792 on melanogenic proteins and related signaling pathways. (A) Mel-Ab cells were cultured with KHG26792 (10 µM) for 24, 48, and 72 h. Western blot analysis was conducted with antibodies specific to MITF and tyrosinase. (B) After 24 h of serum-starvation, Mel-Ab cells were treated with 10 µM KHG26792 for 0~360 min. Cell lysates were assayed by Western blot analysis using antibodies against phospho-Akt, phospho-GSK3β, and phospho-ERK. Equal protein loading was confirmed using an anti-actin antibody

Fig. 4

Effects of PD98059 on KHG26792-induced hypopigmentation. Mel-Ab cells were exposed to 10 µM KHG26792 in the presence of 10 µM PD98059 for 3 days. (A) Phase contrast photographs were obtained using a digital video camera. (B) Melanin content was measured as described in Methods. Each determination was performed in triplicate, and the resulting data are shown as mean±S.D. ^**p<0.01 compared with the KHG26792-treated group.

Fig. 5

Effects of KHG26792 on pigmented skin equivalents (SEs). Pigmented SEs were reconstructed and treated with 10 µM and 50 µM KHG26792 as described in Methods. (A) Photographs of SEs in 24-mm trans-well culture plates after 13 days of SE reconstruction. (B) H&E staining. (C) Melanin in the pigmented SEs detected by Fontana-Masson staining.