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Mol Med Rep
2017 Aug 01;162:1079-1086. doi: 10.3892/mmr.2017.6686.
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Inhibitory effects of Stichopus japonicus extract on melanogenesis of mouse cells via ERK phosphorylation.
Oh CT
,
Kwon TR
,
Jang YJ
,
Yoo KH
,
Kim BJ
,
Kim H
.
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Stichopus japonicus has been used as a folk medicine and as an ingredient in traditional food in East Asian countries. In recent years, the bioactive compounds found in S. japonicus have been reported to possess efficacy in wound healing and may be of potential use in the cosmeceutical, pharmaceutical and biomedical industries. Although the components and their functions require further investigation, S. japonicus extracts exhibit anti‑inflammatory properties, and may be used for cancer prevention and treatment. Although several reports have examined different aspects of S. japo-nicus, the effects of S. japonicus extract on melanogenesis in the skin has not been reported to date. Therefore the present study aimed to investigate the effects of S. japonicus extract on melanogenesis. Treatment with a mixture of S. japonicus extracts (MSCE) reduced melanin synthesis and tyrosinase (TYR) activity in mouse melanocyte cells lines, B16F10 and Melan‑A. In addition, MSCE treatment reduced the protein expression levels of TYR, tyrosinase‑related protein‑1 and tyrosinase‑related protein‑2. The reduced protein levels may be the result of decreased microphthalmia‑associated transcription factor (MITF) expression, which is an important regulator of melanogenesis. The reduced expression level of MITF was associated with delayed phosphorylation of extracellular signal‑regulated kinase (ERK) induced by MSCE treatment. A specific MEK inhibitor, PD98059, significantly blocked MSCE‑mediated inhibition of melanin synthesis. In conclusion, these results indicate that MSCE may be useful as a potential skin‑whitening compound in the skin medical industry.
Figure 1. Effects of MSCE treatment on mouse melanocyte cell viability. (A) B16F10 mouse melanoma cells and (B) Melan-A mouse melanocytes were treated with MSCE at concentrations ranging from 0–100% for 24 h in serum-free media. Cell viability was then measured by Cell Counting Kit-8 assay. Each experiment was conducted in triplicate and the data are presented as the mean ± standard deviation. MSCE, mixed Stichopus japonicus extract.
Figure 3. Effects of MSCE on melanogenesis-related proteins. B16F10 mouse melanoma cells (left panels) and Melan-A mouse normal melanocytes (right panels) were treated with MSCE (5%) for 24, 48 or 72 h. B16F10 cells were additionally induced towards melanogenesis with 1 µM α-MSH. (A) Cell lysates were analyzed by western blotting for TYR, TRP-1, TRP-2 and MITF protein expression. β-actin was used as loading control. (B) Expression of TYR was also examined by immunocytochemical staining. Representative images of (C) B16F10 cells and (D) Melan-A melanocytes from experiments performed in triplicate depict TYR expression (green), nuclear DAPI staining (blue) and merged signals (magnification, ×200). α-MSH, α-melanocyte stimulating hormone; MITF, microphthalmia-associated transcription factor; MSCE, mixed Stichopus japonicus extract; TRP, tyrosinase related protein; TYR, tyrosinase.
Figure 4. Effects of MSCE on signal transduction pathways in mouse melanocyte cells. Following 24 h of serum starvation, (A) B16F10 mouse melanoma cells and (B) Melan-A mouse normal melanocytes were treated with 5% MSCE for the indicated time periods. Cell lysates were harvested for western blot analysis using primary antibodies against p-ERK, t-ERK, p-JNK, t-JNK, p-p38 and t-p38; β-actin was used as the loading control. The results are representative of triplicate experiments. α-MSH, α-melanocyte stimulating hormone; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MSCE, mixed Stichopus japonicus extract; p, phosphorylated; p38, mitogen-activated protein kinase 14; t, total.
Figure 5. Effects of MSCE on ERK phosphorylation and melanogenesis. (A) B16F10 mouse melanoma cells were treated with 5% MSCE in the presence or absence of PD98059 (10 µM) after addition of α-MSH (1 µM) for 3 days, and melanin content was measured. Results are presented as the mean ± standard deviation and each experiment was performed in triplicate. **P<0.01. (B) B16F10 cells and Melan-A mouse normal melanocytes were treated with MSCE (5%) for 30 min in the presence or absence of PD98059 (10 µM). Cell lysates were analyzed by western blot for p-ERK and t-ERK. β-actin was used as the loading control. The results are representative of triplicate experiments. α-MSH, α-melanocyte stimulating hormone; ERK, extracellular signal-regulated kinase; MSCE, mixed Stichopus japonicus extract; p, phosphorylated; PD98059, MEK inhibitor; t, total.
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