Lactoferrin inhibits melanogenesis by down-regulating MITF in melanoma cells and normal melanocytes.

The aim of this study was to evaluate the effect of bovine lactoferrin (bLf) on melanin-producing cells and to elucidate its mechanism of action. We tested the anti-melanogenic effect of bLf on a 3-dimensional cultured pigmentation skin model and confirmed a 20% reduction in pigmentation, suggesting that bLf was transdermally absorbed and it suppressed melanin production. Treatment of human melanoma cells with bLf resulted in a significant, dose-dependent suppression of melanin production. Apo-bLf and holo-bLf suppressed melanogenesis to the same degree as bLf. The key feature behind this anti-melanogenic effect of bLf was the down-regulation of the microphthalmia-associated transcription factor (MITF), leading to the suppression of tyrosinase activity. Treatment with bLf resulted in both decreased expression of MITF mRNA and enhanced degradation of MITF protein. However, the primary effector was enhanced phosphorylation of extracellular signal-regulated kinase (ERK), leading to the phosphorylation and degradation of MITF. Our finding that bLf suppresses melanin production in melanocytes indicates that bLf is a possible candidate for application as a skin-whitening agent.

Antifungal Effect of Non-Woven Textiles Containing Polyhexamethylene Biguanide with Sophorolipid: A Potential Method for Tinea Pedis Prevention.

Tinea pedis is a preventable skin disease common in elderly or diabetic patients. Daily foot washing is effective for prevention, but can be difficult for many patients. Additionally, conventional methods cannot eliminate fungi within the stratum corneum, a common site for fungal invasion. This study investigates the antifungal effects, cytotoxicity, permeability, and efficacy of non-woven textiles containing polyhexamethylene biguanide (PHMB) mixed with sophorolipid. Permeability of PHMB with varying concentrations of sophorolipid was assessed via a cultured skin model. Stratum corneum PHMB concentration was quantified by polyvinylsulphuric acid potassium salt titration and cytotoxicity was assayed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Antifungal effects were evaluated via a new cultured skin/Trichophyton mentagrophytes model, with varying PHMB exposure duration. Clinically-isolated Trichophyton were applied to the feet of four healthy volunteers and then immediately treated with the following methods: washing with soap, a non-woven textile with PHMB, the textile without PHMB, or without washing. Fungal colony forming units (CFUs) were evaluated after one of these treatments were performed. Sophorolipid with various concentrations significantly facilitated PHMB permeation into the stratum corneum, which was not in a dose-dependent manner. Significant PHMB antifungal effects were achieved at 30 min, with low cytotoxicity. Textiles containing PHMB significantly reduced CFU of fungi in healthy volunteers to levels comparable to soap washing. Our results indicate the utility of this product for tinea pedis prevention in clinical settings.

Non-selective distribution of isomeric cholesterol hydroperoxides to microdomains in cell membranes and activation of matrix metalloproteinase activity in a model of dermal cells.

Cholesterol hydroperoxides (ChOOHs) are included as lipid peroxidation products in the skin exposed to ultraviolet (UV) light irradiation. They may exert physicochemical actions affecting biomembrane rigidity because cholesterol is one of the major components of cell membranes. We investigated the distribution of isomeric ChOOHs in heterogeneous cell membranes with different lipid profiles using mouse fibroblast NIH-3T3 cells as a model of the dermis. Before and after UVA irradiation in the presence of hematoporphyrin, cell membranes were partitioned to microdomains (lipid rafts and caveolae) containing a higher amount of cholesterol and non-microdomains (containing a lower amount of cholesterol) by ultracentrifugation. By a combination of diphenylpyrenylphosphine-thin-layer chromatography blotting analyses and gas chromatography-electron ionization-mass spectrometry/selected ion monitoring analyses, ChOOH isomers were determined as their trimethylsilyloxyl derivatives. Cholesterol 5α-, 7α- and 7ß-hydroperoxide were found as isomeric ChOOHs before irradiation. The amounts of the three ChOOH isomers increased significantly after photoirradiation for 2h. No difference was observed between microdomains and non-microdomains with regard to the ratio of the amounts of isomeric ChOOHs to that of cholesterol, suggesting that these ChOOH isomers were distributed equally in both parts depending on cholesterol content. When cells were treated with a purified mixture of ChOOH isomers, cell membranes incorporated ChOOHs into microdomains as well as non-microdomains evenly. Cellular matrix metalloproteinase-9 (MMP-9) activity was elevated by treatment with the purified mixture of ChOOH isomers. These results strongly suggest that ChOOHs accumulate in cell membranes irrespective of the heterogeneous microstructure and promote MMP activity if dermal cells are exposed to photodynamic actions.

Transdermal administration of lactoferrin with sophorolipid.

Lactoferrin (Lf), a multifunctional glycoprotein, is known to activate dermal fibroblasts. Enhancing percutaneous absorption without decreasing the activity of Lf is critical in making the dermal administration of Lf beneficial. Sophorolipid (SL), a glycolipid-type biosurfactant, is known to form assemblies that may elevate the efficiency of the transdermal delivery of active ingredients. Here, we investigated the role of SL in the transdermal absorption of bovine Lf (bLf) and the effect of SL on the bLf activity on dermal fibroblasts. Transdermal absorption of bLf through a model skin was enhanced by 1.3-fold to 1.7-fold when SL was added. The effects of SL on the bLf activities on dermal fibroblasts were examined by cell proliferation activities and by gene expression levels of elastic fiber components, collagen IV, and hyaluronan synthases, revealing that SL did not depress the effect of bLf to any extent. Instead, the tropoelastin gene expression was upregulated ~60-fold by bLf alone, which was further increased to ~160-fold by bLf and SL together, suggesting a significant synergism between bLf and SL. Protein levels of elastin, assessed by immunohistochemistry, correlated well with the results of gene expressions. These results indicate the feasibility of the transdermal administration of bLf with SL.