Melanin synthesis occurs in melanocytes and is an essential physiological process that determines the color of human skin and protects its DNA from UV damage. It is closely related with the occurrence of pigmentary disorders: the imbalanced regulation of melanin synthesis results in many pigmentary skin diseases that commonly affect men and women of all ethnic groups, including hyperpigmentation disorders, such as melanocytic nevus, seborrheic keratosis, and melanoma, and hypopigmentation disorders, such as piebaldism, pityriasis, and vitiligo.
Melanin is synthesized in melanosomes, which are transferred to surrounding keratinocytes where they protect cells against DNA damage. Melanogenesis is critically regulated by the expression of various melanogenesis-related enzymes, such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). Tyrosinase, the rate-limiting enzyme for controlling melanin synthesis, is involved primarily in the production of L-dopaquinone. TRP-1 and TRP-2 catalyze specific steps in melanogenesis and stabilize tyrosinase activity. As tyrosinase is a key enzyme that catalyzes a rate-limiting step of melanin synthesis, numerous inhibitors that target tyrosinase have been investigated for their ability to inhibit this process. These include well-known tyrosinase inhibitors, such as hydroquinone, arbutin, kojic acid, and salicylic acid. However, due to the side effects of these inhibitors and the increasing demand for safe and effective cosmetics, many continuing efforts are being made to identify or produce new skin-whitening agents. Some researchers have screened natural products and found that chalcones, resveratrol, and coumarins exhibit inhibitory activity against mushroom tyrosinase. Other groups have sought to develop bioactive materials. Particular attention has been paid to various bioactive peptides, including short-sequence oligopeptides, kojic acid-tripeptide compounds, dipeptides and tripeptides derived from natural products, and cysteine-containing dipeptides, all of which have been used as cosmetic peptides. In the context of topical application, peptides have strong advantages due to their stability, easy synthesis and modification, and diverse availability. For example, the tetrapeptide, PKEK, inhibits the UVB-induced upregulation of the genes encoding IL-1α, IL-6, IL-8, and TNF-α and the protein levels of POMC and tyrosinase, and it may be suitable as a skin tone-modulating agent in cosmetic products. At present, bioactive peptides are widely used by cosmeceutical companies as cosmetics.
We recently reported that D-tyrosine, the enantiomer of L-tyrosine, suppresses melanogenesis induced by α-MSH treatment or UV irradiation, two key inducers of melanogenesis, in melanocytes by inhibiting the enzymatic activity of tyrosinase. Based on this, we hypothesized that the presence of D-tyrosine could enable a cosmetic peptide to negatively regulate melanin synthesis. Here, we investigated whether D-tyrosine-containing cosmetic peptides can regulate melanin synthesis in melanoma cells and melanocytes.
Our observation that D-tyrosine negatively regulates melanin synthesis prompted us to further investigate whether a D-tyrosine-containing peptide could have a similar anti-melanogenic effect. We selected Leuphasyl (pentapeptide-18; YdAGFL), a commercialized cosmetic peptide that acts as a neurotransmitter inhibitor to reduce fine lines and wrinkles. We synthesized pentapeptide-18 peptides in which we either replaced L-tyrosine with D-tyrosine at the N-terminus or added a L- or D-tyrosine at the C-terminus and treated human melanoma MNT-1 cells with various doses of each peptide. Expectedly, 500 μM of D-tyrosine reduced the melanin content of MNT-1 cells by about 50%. Notably, 500 μM of peptapeptide-18 containing D-tyrosine at the N-terminus (N-D) or C-terminus (C-D) decreased the melanin contents and tyrosinase activities of MNT-1 cells by 18% and 25%, respectively. Consistently, 500 μM of N-D or C-D decreased the expression of tyrosinase and microphthalmia-associated transcription factor (MITF), which plays a critical role in melanogenesis. L-DOPA staining also showed that the level of intracellular tyrosinase activity was decreased in MNT-1 cells treated with 500 μM of N-D or C-D, and tyrosinase activity was reduced by C-D treatment in in vitro. However, MTT assays showed that 500 μM of N-D or C-D did not affect the proliferation of MNT-1 cells. Together, these data suggest that pentapeptide-18 containing terminal D-tyrosine inhibits melanin synthesis in human melanoma cells.
As α-MSH is known to be a key regulator of melanogenesis in melanocytes, we investigated whether the D-tyrosine-containing peptides could suppress α-MSH-induced melanin synthesis. Indeed, 500 μM of N-D and C-D suppressed the α-MSH-induced increases of tyrosinase in MNT-1 cells. Similarly, N-D and C-D downregulated the α-MSH-induced increases of melanin synthesis and intracellular tyrosinase activity, as assessed by L-DOPA staining. Since ultraviolet (UV) irradiation is also known to stimulate melanogenesis in the skin, we examined whether N-D and C-D could suppress UV-induced melanin synthesis. 500 μM of N-D and C-D suppressed the UV-induced increases in tyrosinase protein expression and the mRNA levels of tyrosinase and MITF. Similarly, N-D and C-D down-regulated the UV-induced increases in melanin synthesis and intracellular tyrosinase activity. In addition, 250 μM of C-D suppressed the α-MSH (100 nM)-induced increases of tyrosinase and MITF mRNA levels and melanin synthesis in MNT-1 cells, confirming that terminal D-Tyrosine-containing pentapeptide-18 can regulate the melanogenesis triggered by α-MSH and UV irradiation, which are the most common environmental factors that cause melanin synthesis.
