7,3',4'-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses α-Melanocyte-Stimulating Hormone-Induced Melanogenesis by Targeting Melanocortin 1 Receptor

Abstract

7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is a metabolite of daidzein which is a representative isoflavone found in soybean. Recent studies suggested that 7,3',4'-THIF exerts a hypopigmentary effect in B16F10 cells, however, its underlying molecular mechanisms and specific target protein remain unknown. Here, we found that 7,3',4'-THIF, but not daidzein, inhibited α-melanocyte-stimulating hormone (MSH)-induced intracellular and extracellular melanin production in B16F10 cells by directly targeting melanocortin 1 receptor (MC1R). Western blot data showed that 7,3',4'-THIF inhibited α-MSH-induced tyrosinase, tyrosinase-related protein-1 (TYRP-1), and tyrosinase-related protein-2 (TYRP-2) expressions through the inhibition of Microphthalmia-associated transcription factor (MITF) expression and cAMP response element-binding (CREB) phosphorylation. 7,3',4'-THIF also inhibited α-MSH-induced dephosphorylation of AKT and phosphorylation of p38 and cAMP-dependent protein kinase (PKA). cAMP and Pull-down assays indicated that 7,3',4'-THIF strongly inhibited forskolin-induced intracellular cAMP production and bound MC1R directly by competing with α-MSH. Moreover, 7,3',4'-THIF inhibited α-MSH-induced intracellular melanin production in human epidermal melanocytes (HEMs). Collectively, these results demonstrate that 7,3',4'-THIF targets MC1R, resulting in the suppression of melanin production, suggesting a protective role for 7,3',4'-THIF against melanogenesis.

Keywords: 3′; 4′-Trihydroxyisoflavone; 7; depigmentation; melanocortin 1 receptor; melanogenesis; α-MSH.

FIGURE 1

Effect of 7,3′,4′-THIF or daidzein on the α-MSH-induced melanin content in B16F10 melanoma cells. (A,B) The chemical structures of daidzein (A) and 7,3′,4′-THIF (B). (C,D) B16F10 cells were treated as described in “Materials and Methods,” and the melanin contents were measured 3 days later. (C) Effects of 7,3′,4′-THIF or daidzein on α-MSH-induced intracellular melanin in untreated control cells (a) and in cells treated with α-MSH (b), α-MSH and 100 μM arbutin (c), α-MSH and 10 μM daidzein (d), α-MSH and 20 μM daidzein (e), α-MSH and 40 μM daidzein (f), α-MSH and 10 μM 7,3′,4′-THIF (g), α-MSH and 20 μM 7,3′,4′-THIF (h), and α-MSH and 40 μM 7,3′,4′-THIF (i). (D) Effects of 7,3′,4′-THIF or daidzein on the α-MSH-induced extracellular melanin level in the culture media of B16F10 melanoma cells. The cells were pretreated with samples at the indicated concentrations (10, 20, or 40 μM) for 1 h before being exposed to α-MSH (100 nM) for 3 days. The secreted melanin levels were determined as described in “Materials and Methods.” Asterisks indicate a significant difference (*p < 0.05; **p < 0.01; ***p < 0.001) compared with α-MSH treated groups.

FIGURE 2

Effect of 7,3′,4′-THIF on melanogenic protein expression in B16F10 melanoma cells. (A) 7,3′,4′-THIF suppressed tyrosinase and TYRP-1 but not TYRP-2 expression in B16F10 melanoma cells. To determine the expression of melanogenic enzymes, the cells were pretreated with 7,3′,4′-THIF at the indicated concentrations (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH for 3 days. (B) 7,3′,4′-THIF suppressed MITF transcription factor expression and the phosphorylation of CREB in B16F10 melanoma cells. To examine transcription factor expression, the cells were pretreated with 7,3′,4′-THIF at the indicated concentrations (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH for 3 days or 30 min. The levels of indicated proteins were determined by Western blot analysis, as described in “Materials and Methods,” using specific antibodies. The data are representative of more than two independent experiments that gave similar results. Asterisks indicate a significant difference (*p < 0.05; **p < 0.01; ***p < 0.001) compared with α-MSH treated group.

FIGURE 3

Effect of 7,3′,4′-THIF on the α-MSH-induced dephosphorylation of AKT signaling or phosphorylation of p38 and PKA signaling in B16F10 melanoma cells. (A) 7,3′,4′-THIF activated the α-MSH-induced dephosphorylation of AKT. Cells were treated with 7,3′,4′-THIF (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH and harvested 1 h later. (B) 7,3′,4′-THIF inhibited the α-MSH-induced phosphorylation of p38. Cells were treated with 7,3′,4′-THIF (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH and harvested 15 min later. (C) 7,3′,4′-THIF inhibited the α-MSH-induced phosphorylation of PKA. Cells were treated with 7,3′,4′-THIF (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH and harvested 15 min later. The cells were disrupted and the levels of phosphorylated and total proteins were determined by Western blot analysis, as described in section “Materials and Methods,” using specific antibodies against the respective phosphorylated and total proteins. The data are representative of three independent experiments that gave similar results. The levels of indicated proteins were determined by Western blot analysis, as described in section “Materials and Methods,” using specific antibodies. The data are representative of more than two independent experiments that gave similar results. Asterisks indicate a significant difference (*p < 0.05; **p < 0.01; ***p < 0.001) compared with α-MSH treated group.

FIGURE 4

Binding of 7,3′,4′-THIF to MC1R. (A) 7,3′,4′-THIF reduced the forskolin-induced cAMP level in B16F10 melanoma cells. The intracellular cAMP level was determined by a cAMP immunoassay as described in section “Materials and Methods.” The results are expressed as the cAMP level relative to the forskolin-treated control. All data are presented as the mean ± SD of three independent determinations. Asterisks (*) indicate a significant difference (p < 0.05) compared with the forskolin-treated group. (B) 7,3′,4′-THIF binds MC1R directly in vitro and ex vivo. MC1R-7,3′,4′-THIF binding was confirmed by immunoblotting using antibodies against human MC1R (upper panel) or mouse MC1R (lower panel). Lane 1 (input control), human MC1R protein standard or whole B16F10 cell lysates; lane 2 (negative control), Sepharose 4B was used to pull-down MC1R, as described in section “Materials and Methods,” or a B16F10 cell lysate precipitated with Sepharose 4B beads; and lane 3, 7,3′,4′-THIF-Sepharose 4B affinity beads were used to pull-down MC1R or whole B16F10 cell lysates precipitated with 7,3′,4′-THIF-Sepharose 4B affinity beads. (C) Daidzein did not bind with MC1R in vitro and ex vivo. MC1R-daidzein binding was confirmed by immunoblotting using antibodies against human MC1R (upper panel) or mouse MC1R (lower panel). Lane 1 (input control), human MC1R protein standard or whole B16F10 cell lysates; lane 2 (control), Sepharose 4B was used to pull-down MC1R, as described in section “Materials and Methods,” or a B16F10 cell lysate precipitated with Sepharose 4B beads; and lane 3, daidzein-Sepharose 4B affinity beads were used to pull-down MC1R or whole B16F10 cell lysates precipitated with daidzein-Sepharose 4B affinity beads. (D) 7,3′,4′-THIF binds to MC1R competitively with α-MSH. B16F10 cellular supernatant fraction (1,000 μg) was incubated with α-MSH at the concentrations indicated (0, 0.1, 1, 10, or 100 μM) and 100 μL of 7,3′,4′-THIF-Sepharose 4B or Sepharose 4B (as a negative control) in a reaction buffer to a final volume of 500 μL. The pulled-down proteins were detected by western blot analysis as described in “Materials and Methods”: lane 1 (input control), whole B16F10 cell lysates; lane 2 (negative control), indicating that neither MC1R binds with Sepharose 4B and lane 3 is the positive control, which indicates that MC1R binds with 7,3′,4′-THIF-Sepharose 4B. Each experiment was performed three times; representative blots are shown. Asterisks indicate a significant difference.

FIGURE 5

Modeling study of MC1R binding to 7,3′,4′-THIF and daidzein. (A–D) 7,3′,4′-THIF binding with MC1R. 7,3′,4′-THIF binding (shown as stick) with MC1R formed hydrogen bonds (yellow dash line) at Glu94 and Leu106 (A–C). Ligand interaction diagram of 7,3′,4′-THIF binding with MC1R (D). The hydroxyl group at the 3’ position of 7,3′,4′-THIF binding with MC1R, formed hydrogen bonds (yellow dash line) with the backbone oxygen of Gly239 and the backbone nitrogen of Lys243 in the MC1R pocket. (E,F) Modeling study of MC1R binding to daidzein. Daidzein binding (shown as stick) with MC1R, formed hydrogen bonds (yellow dash line) at Leu106 (E) Ligand interaction diagram of daidzein binding with MC1R (F).

FIGURE 6

Effect of 7,3′,4′-THIF and daidzein on the α-MSH-induced melanogenesis of HEMs. (A) Effects of 7,3′,4′-THIF on α-MSH-induced intracellular melanin in untreated control cells (a) and in cells treated with α-MSH (b), α-MSH and 100 μM arbutin (c), α-MSH and 10 μM daidzein (d), α-MSH and 20 μM daidzein (e), α-MSH and 40 μM daidzein (f), α-MSH and 10 μM 7,3′,4′-THIF (g), α-MSH and 20 μM 7,3′,4′-THIF (h), and α-MSH and 40 μM 7,3′,4′-THIF (i). (B) 7,3′,4′-THIF suppressed tyrosinase in HEMs. To determine the expression level, the cells were pretreated with 7,3′,4′-THIF at the indicated concentrations (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH for 3 days. (C) Effect of 7,3′,4′-THIF on the α-MSH-regulated phosphorylation of AKT, p38 and PKA. Cells were treated with 7,3′,4′-THIF (10, 20, or 40 μM) for 1 h before being exposed to 100 nM α-MSH and harvested 0.5 h later. The cells were disrupted and the levels of phosphorylated and total proteins were determined by Western blot analysis, as described in section “Materials and Methods,” using specific antibodies against the respective phosphorylated and total proteins. The data are representative of more than two independent experiments that gave similar results. Asterisks indicate a significant difference (*p < 0.05; **p < 0.01; ***p < 0.001) compared with α-MSH treated group.

FIGURE 7

Hypothetical mechanism of the depigmenting activity of 7,3′,4′-THIF.