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. 2019 Sep 26;20(19):4785.
doi: 10.3390/ijms20194785.

The Organogermanium Compound THGP Suppresses Melanin Synthesis via Complex Formation with L-DOPA on Mushroom Tyrosinase and in B16 4A5 Melanoma Cells

Junya Azumi  1 , Tomoya Takeda  2   3 , Yasuhiro Shimada  4 , Hisashi Aso  5 , Takashi Nakamura  6
Affiliations

The Organogermanium Compound THGP Suppresses Melanin Synthesis via Complex Formation with L-DOPA on Mushroom Tyrosinase and in B16 4A5 Melanoma Cells

Junya Azumi et al. Int J Mol Sci. .

Abstract

The organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) has various biological activities. We previously reported that THGP forms a complex with cis-diol structures. L-3,4-Dihydroxyphenylalanine (L-DOPA), a precursor of melanin, contains a cis-diol structure in its catechol skeleton, and excessive melanin production causes skin darkening and staining. Thus, the cosmetic field is investigating substances that suppress melanin production. In this study, we investigated whether THGP inhibits melanin synthesis via the formation of a complex with L-DOPA using mushroom tyrosinase and B16 4A5 melanoma cells. The ability of THGP to interact with L-DOPA was analyzed by 1H-NMR, and the influence of THGP and/or kojic acid on melanin synthesis was investigated. We also examined the effect of THGP on cytotoxicity, tyrosinase activity, and gene expression and found that THGP interacted with L-DOPA, a precursor of melanin with a cis-diol structure. The results also showed that THGP inhibited melanin synthesis, exerted a synergistic effect with kojic acid, and did not affect tyrosinase activity or gene expression. These results suggest that THGP is a useful substrate that functions as an inhibitor of melanogenesis and that its effect is enhanced by combination with kojic acid.

Keywords: THGP; melanogenesis; organogermanium.

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Conflict of interest statement

The authors declare no conflict of interest. The Asai Germanium Research Institute Co., Ltd. had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Formation of a complex of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3-(trihydroxygermyl)propanoic acid (THGP). (A) Structures of THGP, L-DOPA, and 3-trimethylgermylpropanoic (TMGP). (B,C) The formation of a complex between THGP or TMGP and L-DOPA was analyzed by 1H-NMR (300 MHz). (D) Reaction for the formation of the THGP and-L-DOPA complex.
Figure 2
Figure 2
Inhibitory effect of THGP, TMGP, and kojic acid on the synthesis of melanin via a reaction mediated by mushroom tyrosinase. (A–C) THGP, TMGP, or kojic acid was mixed with L-DOPA and mushroom-derived tyrosinase and allowed to react at 37 °C for 20 min to synthesize melanin. (D) The suppressive effect of the combination of THGP and kojic acid on melanin synthesis was examined. The results were statistically analyzed using Dunnett’s method, and 0 µM THGP was used as the control. Circles: 0 µM THGP; filled circles: 1000 µM THGP; triangles: 2500 µM THGP; squares: 5000 µM THGP. The data are presented as the means ± SDs from six independent experiments. ** p < 0.01 vs. the control.
Figure 3
Figure 3
Cell viability after treatment with THGP or kojic acid. B16 4A5 melanoma cells were treated with THGP or kojic acid for 72 h, and the resulting cell viability was assessed by the MTS assay. The results were statistically analyzed by Dunnett’s method, and 0 μM THGP was used as the control. The results are presented as the means ± SDs from eight independent experiments. ** p < 0.01 vs. the control.
Figure 4
Figure 4
Inhibitory effect of THGP and kojic acid on melanogenesis in B16 4A5 melanoma cells. B16 4A5 cells were treated with THGP or kojic acid for 72 h, and the (A) extracellular and (B) intracellular melanin amounts were then evaluated. (C) Optical microscopy images of cells treated with THGP (6000 μM). The arrow means melanin accumulated in large amount in B16 4A5 and the circle means the enlarged location in the figure below. (D) The suppressive effect of the combination of THGP and kojic acid on melanin synthesis was evaluated. The results were statistically analyzed using Dunnett’s method, and 0 μM THGP served as the control. The data are presented as the means ± SDs from six independent experiments. * p < 0.05 and ** p < 0.01 vs. the control.
Figure 5
Figure 5
Effect of THGP on tyrosinase activity and the production of melanin production-related genes in B16 4A5 melanoma cells. (A) Tyrosinase activity was measured after treatment with THGP for 24 h. (B) The expression of genes after treatment with THGP for 24 h was assessed by real-time RT-PCR. (C) The expression of genes after treatment with THGP for 24 h with 100 μM L-DOPA was assessed by real-time RT-PCR. NC is nontreated L-DOPA and THGP. The values shown are relative to the expression of β-actin (Actb), a housekeeping gene. The results were statistically analyzed by Dunnett’s method, and 0 μM THGP with 100 μM L-DOPA served as the control. The data are presented as the means ± SDs from six independent experiments. ** p < 0.01 vs. the control.
Figure 5
Figure 5
Effect of THGP on tyrosinase activity and the production of melanin production-related genes in B16 4A5 melanoma cells. (A) Tyrosinase activity was measured after treatment with THGP for 24 h. (B) The expression of genes after treatment with THGP for 24 h was assessed by real-time RT-PCR. (C) The expression of genes after treatment with THGP for 24 h with 100 μM L-DOPA was assessed by real-time RT-PCR. NC is nontreated L-DOPA and THGP. The values shown are relative to the expression of β-actin (Actb), a housekeeping gene. The results were statistically analyzed by Dunnett’s method, and 0 μM THGP with 100 μM L-DOPA served as the control. The data are presented as the means ± SDs from six independent experiments. ** p < 0.01 vs. the control.
Figure 6
Figure 6
Inhibitory effect of THGP on melanin production after stimulation with α-MSH or L-DOPA. (A) The extracellular melanin levels in B16 4A5 melanoma cells under α-MSH activation were measured after treatment with THGP or kojic acid for 72 h. (B) The amount of extracellular melanin in B16 4A5 melanoma cells stimulated with L-DOPA was evaluated after treatment with THGP or kojic acid for 48 h. The results were statistically analyzed by Dunnett’s method, and 0 µM THGP with α-MSH or L-DOPA stimulation served as the control. “−” means untreated group and “+” means treated group about THGP, α-MSH or L-DOPA. The data are reported as the means ± SDs from six independent experiments. ** p < 0.01 vs. the control.
Figure 7
Figure 7
Mechanism underlying the inhibitory effect of the combination of THGP and kojic acid on melanin synthesis. Compared with the control condition, THGP administration inhibits the amount of extracellular melanin but slightly increases the amount of intracellular melanin. In addition, THGP does not affect tyrosinase activity or the expression of melanin production-related genes. Moreover, THGP shows a synergetic effect with kojic acid. “up arrow” means up-regulated, “down arrow” means down-regulated, “grey arrow” means reaction path about melanin syhthesis and “T arrow” means inhibitory effects.
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