Soyasaponin-I Attenuates Melanogenesis through Activation of ERK and Suppression of PKA/CREB Signaling Pathways

Abstract

Soyasaponin-I (SS-I), a sialyltransferase inhibitor naturally found in soybeans, has antioxidant, anticarcinogenic, and hepatoprotective properties. In this study, we explored the possibility to use SS-I as an antimelanogenic agent for the treatment of skin hyperpigmentation disorders. When melanoma cell lines, MNT-1 and B16F10, were treated with SS-I, significant suppression of both α-2,3 and α-2,6 sialylations was observed by using lectin fluorescence staining with sialic acid-binding lectins-Sambucus nigra agglutinin (SNA) and Maackia amurensis lectin-II (MAL-II). SS-I significantly attenuated the α-MSH-induced melanogenesis of MNT-1 and B16F10 cells without a cytotoxic effect. SS-I could activate ERK and suppress the PKA/CREB signaling pathways of melanoma cells. Moreover, SS-I treatment caused significant downregulation of the expression of melanosome-related proteins; tyrosinase-related protein 1 (TRP1), TRP2, and premelanosome protein (PMEL) and the melanogenic-related transcription factor microphthalmia-associated transcription factor (MITF). Consequently, the expression of tyrosinase-the key enzyme regulating melanin production-was significantly suppressed after SS-I treatment. These results suggest the role of sialylation in melanogenesis and the possibility of using SS-I as an alternative antimelanogenic agent. In conclusion, we have demonstrated the antimelanogenic effect of SS-I, an active compound produced in soybeans. SS-I can be an antimelanogenic agent in cosmetic products for the treatment of hyperpigmentation disorders.

Figure 1

Effect of SS-I on sialylation of MNT-1 and B16F10 melanoma cell lines. The expression of the sialylated glycans was examined in (A) MNT-1 and (B) B16F10 cells using lectin fluorescence staining with MAL-II and SNA after treatment with SS-I for 72 h. Sialylated glycans were represented by Alexa 488 (green), and the nuclei were stained with Hoechst 33342 (blue). Scale bar: 50 μm.

Figure 2

Effect of SS-I on melanin production and cell viability. (A) Melanin was dissolved using an alkaline–DMSO solution after SS-I and α-MSH treatments, and the absorbance was measured at 450 nm. The melanin content was calculated as % of the control. (B) The MTT assay was conducted to determine the cell viability 72 h after SS-I and kojic acid treatment. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 3

Effect of SS-I on tyrosinase activity. Effect of SS-I on the tyrosinase activity was examined using (A) mushroom tyrosinase and (B) human tyrosinase (extracted from MNT-1 melanoma cells). The enzymes were mixed with 0, 10, 20, 50, and 100 μM SS-I or kojic acid, using ethanol as a solvent control. The DOPA chrome was measured at 450 nm. The experiment was conducted in triplicate and repeated at least twice. Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 4

Effects of SS-I on the expression of melanogenesis-related proteins and cell signaling pathways. MNT-1 cells were treated with SS-I, compared with PBS, for 72 h. Western blot analysis was used to determine (A) the expression of TYR, TRP1, TRP2, PMEL, and MITF and (B) the melanogenesis-related signaling pathways: PKA/CREB, Akt, and ERK. Phosphorylated forms of each protein: pPKA (T197), pCREB (S133), pAKT (S473), and pERK (T202/Y204) were normalized by their total forms. β-Actin was used as an internal control. The graph was generated from an average value from three independent experiments. The blot images were from a representative experiment. Asterisks represented significant difference (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).