Inhibitory effect of 2-methyl-naphtho[1,2,3-de]quinolin-8-one on melanosome transport and skin pigmentation

Melanosomes are lysosome-related organelles with specialized capabilities of melanin synthesis and movement mediated by the Rab27a-Melanophilin-MyosinVa protein complex. In this study, we found that 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO) induced melanosome aggregation around the nucleus in melan-a melanocytes and in melan-a melanocytes/SP-1 keratinocyte co-cultures without inducing toxicity or changing the melanin content. Western blot and real-time PCR analyses showed that MNQO decreased expression of the Rab27a, Melanophilin and MyosinVa proteins and mRNAs, respectively, in melan-a melanocytes. In a reconstituted human epidermis model, treatment with 0.001% MNQO reduced skin pigmentation. Also, MNQO reduced skin pigmentation in brown guinea pigs induced by UVB irradiation. These results indicated that regulation of melanosome transport may serve as a good target for new skin depigmenting agents and MNQO itself could be a candidate.


Effect of MNQO on the perinuclear localization of melanosomes and melanin synthesis in melan-a melanocytes.
In untreated melan-a melanocytes, the melanosomes were distributed throughout the cell body ( Fig. 2A). Following treatment with MNQO at 2 μ M, 10 μ M and 20 μ M for 72 h, melanosomes were shown to cluster in the perinuclear region of the cells. The cells and dendrites did not change in shape, however, and MNQO at these concentrations for 72 h did not affect of cell viability of melan-a melanocytes (Fig. 2B).
After treatment for 72 h with MNQO, the number of cells with perinuclear melanosome aggregation was counted as described in the Methods section. The melanosome aggregation values were 6.5 ± 0.7% at 2 μ M, 48.1 ± 6.1% at 10 μ M and 74.6 ± 2.4% at 20 μ M MNQO (Fig. 2C). Treatment with MNQO at 10 μ M and 20 μ M significantly increased the proportion of cells per culture well showing perinuclear melanosome aggregation.
To test the effects of MNQO treatment on melanin synthesis in melan-a melanocytes, melanin content was measured in cells treated with MNQO at 2 μ M, 10 μ M and 20 μ M for 72 h (Fig. 2D). Phenylthiourea was used as a positive control 19 . MNQO treatment did not reduce melanin content in melan-a melanocytes. Therefore, MNQO induced perinuclear melanosome aggregation, but did not change melanin content in melan-a melanocytes.
Effect of MNQO on melanosome localization in a cell co-culture system. To assemble this model of human skin, melan-a melanocytes and SP-1 keratinocytes were seeded and cultured together. Treatments with MNQO were performed at 2 μ M and 10 μ M for 24 h and 72 h (Fig. 3A). Compared to untreated cells in co-culture, co-cultured melanocytes treated with MNQO showed significant time-dependent melanosome aggregation in the perinuclear region and significantly increased the proportion of melanosome aggregation ( Fig. 3B and Table 1). Therefore, MNQO inhibited melanosome transport in this melanocyte-keratinocyte co-culture system.

Effect of MNQO on the expression of Rab27a, Mlph and MyoVa proteins.
To identify the effect of MNQO on expression of tripartite complex proteins, we performed western blotting. Melan-a melanocytes were treated with MNQO at 2 μ M, 10 μ M and 20 μ M for 72 h. Following treatment with MNQO for 72 h, levels of Rab27a, Mlph and MyoVa proteins in melan-a melanocytes were dose-dependently reduced as compared to untreated control cells (Fig. 4A,C). In the present report, MITF regulates transcription of Rab27a as a transcription factor in melanoma cells 5 . We identified the expression of MITF protein, a known upstream regulator of Rab27a, but the level of MITF protein was not changed (Fig. 4B Effect of MNQO on melanin production in a reconstituted human skin model. Reconstituted human skin consisted of normal human-derived epidermal keratinocytes (NHEK) and melanocytes (NHEM). When we culture reconstituted human skin in vitro, NHEM undergoes melanogenesis and leads pigmentation with time. During treatment with 0.001% MNQO, pigmentation progressed more slowly and showed lower intensity after 7 days than in 1% DMSO-treated control skin (Fig. 6A). Reduced induction of pigmentation by MNQO treatment compared with DMSO control was quantified by L-values (Fig. 6B).   Melanin assays revealed a significant reduction of melanin content in MNQO treated reconstituted human skins compared with the 1% DMSO control (Fig. 6C).  Effect of MNQO on UVB-induced skin pigmentation in brown guinea pigs. To confirm the effect of MNQO on UVB-induced skin pigmentation, we used in vivo brown guinea pig models as described in method section 2.10. A reduction of pigmentation was observed in the MNQO-treated skin area compared to vehicle-treated area (Fig. 7A). A significant reduction of pigmentation was observed by 0.05% MNQO treatment  The melanin content of the lysates was measured as optical density at 490 nm after solubilizing proteins from reconstituted human skins with 1 N NaOH (n = 3). Pigmented skin equivalents were treated for 7 days with the indicated compounds. Data were analyzed using Student's unpaired t test. * p < 0.05.
at 5 weeks after UVB irradiation (Fig. 7B). Tissue biopsies were analyzed by Fontana-Masson and H&E staining (Fig. 7C,D). Accumulation of melanin was induced by UVB irradiation in the basal layer of epidermal in vehicle-treated areas (arrow head), compared with reduced melanin synthesis in 0.05% MNQO-treated areas. Any histological changes were not observed by H&E stained skin section.

Discussion
In transport of melanosomes from the perinuclear region of a cell to the dendrite tips, a "melanosome transport complex, " consisting primarily of Rab27a, Mlph and MyoVa proteins, is involved. Rab27a, serving as a transporter protein, binds to the melanosome surface and engages in actin-dependent melanosome movement through direct interaction with the effector molecule Mlph and indirect interaction with exon F-containing isoforms of MyoVa 20 . All three proteins are essential for normal melanosome transport to the periphery of the cell. A defect in any one of them perturbs melanosome transport and ultimately affects skin pigmentation, without any change in melanin synthesis. As a result, skin coloring may be patchy or pale.
To identify compounds that inhibit melanosome transport, we tested a variety of plant derivatives, and MNQO appeared to induce melanosome aggregation within melan-a melanocytes. We observed that MNQO inhibited melanosome transport in monolayer cultures of melan-a melanocytes and in co-cultures consisting of SP-1 keratinocytes and melan-a melanocytes. Significant clustering of melanosomes in perinuclear areas was shown in cells treated with 10 μ M and 20 μ M MNQO as compared to control melan-a melanocytes. It was reported that close associations exist between melanocytes and their neighboring keratinocytes, and these two cell types together compose the epidermal melanin unit 21 . Keratinocytes receive melanin from the melanocytes via endo/exocytosis, and skin coloring develops through this interaction 22 . Melanosome clustering was also observed in a co-culture system composed of SP-1 keratinocytes and melan-a melanocytes treated with 10 μ M MNQO. Melanosomes transfers to SP-1 cells could be reduced by MNQO in co-culture systems by clustered melanosome in melan-a cells. To test the effect of MNQO on melanin production, we treated melan-a melanocytes with MNQO at 2 μ M, 10 μ M and 20 μ M for 72 h; however, none of these treatments affected melanin synthesis detectably. Using Western blot analysis, we found that Rab27a, Mlph and MyoVa protein levels declined significantly with  common regulatory factor of three genes has not been reported yet. Therefore, it will be helpful for understanding the regulatory mechanism of melanosome transport and vesicle transport if we can identify the upstream regulator of these genes.
Manassantin B and Hesperidin have been reported as an inhibitor of melanosome transport 24,25 . Manassantin B disrupts Mlph-MyoVa interaction and inhibits melanosome transport. Hesperidin blocks the Rab27a-Mlph interaction in silico without inhibiting melanin synthesis or directly inhibiting tyrosinase. Although these two compounds could inhibit melanosome transport by reducing the interaction of protein-protein binding, but MNQO inhibited melanosome transport by decreasing the Rab27a, Mlph and MyoVa gene expression and it does not affect melanin synthesis in melanocytes.
Finally, we investigated the effect of MNQO in reconstituted human skin maintained in vitro and in brown guinea pigs in vivo. Treatment of MNQO reduced skin pigmentation in reconstituted human skin and brown guinea pigs, without any side effects such as toxicities.
Many compounds have been developed for skin whitening or treating pigmentation disorders 26 . Most of these substances decreased melanin production by inhibition of tyrosinase, but they have not been used in clinic for efficacy or safety issues. Recently, Rhododendrol, a tyrosinase inhibitor was developed for skin whitening cosmetics 27 . However, rhododendrol was identified to trigger a depigmentary disorder, leukoderma and products containing rhododendrol were withdrawn in the market. Therefore, it is necessary to develop differentiated compounds for skin whitening or treating pigmentation disorders.
Controlling melanosome transport, such as Mlph, could be more efficient and safe way to modulate skin pigmentation. The capacity of MNQO to reduce skin pigmentation at a very low concentration suggested that controlling melanosome transport could be a good target for regulation of skin pigmentation and this compound could be used as a potential skin whitening agent for treating hyper-pigmentation disorders.

Materials and Methods
Materials. MNQO  Cytotoxicity assay. Melan-a melanocytes were seeded in 96-well plates in RPMI-1640 medium and incubated at 37 °C for 24 h. Each well was then rinsed with DPBS, and DMSO or MNQO was added. After 24 h, 48 h and 72 h, wells were rinsed with DPBS and refilled with medium containing 10% EZ-Cytox (Daeil Lab Service, Korea). After further incubation at 37 °C for 1 h, the optical density of medium in each well was measured at 450 nm using a microplate reader (TECAN, Switzerland).

Co-culture.
Melan-a melanocytes were seeded in six-well plates containing RPMI-1640 medium. Two days later, the plates were rinsed twice with DPBS. The SP-1 keratinocytes were seeded at 1.5 × 10 5 cells per well to each well containing melanocytes, The initial seeding ratio of keratinocytes to melanocytes was 4:1 and co-cultures of melanocytes and keratinocytes were maintained in EMEM medium (with 0.05 mM Ca 2+ ). At 2, 3 and 5 days later, the cells were rinsed with DPBS and resuspended in EMEM (with 0.05 mM Ca 2+ ). One day after that, the cultures were photographed.
Detection and quantification of melanosome aggregation. Melan-a melanocytes were seeded in 24-well plates and maintained for 24 h. The cells were then rinsed in DPBS and treated with samples in RPMI-1640 containing 2% FBS for 3 days. The cells were observed in bright field using an Olympus CKX41 culture microscope (Olympus, Japan), and images were photographed using a DMC camera (INS Industry, Korea) and DMC advanced software adapted to the microscope. Evaluation of melanosome aggregation was performed by counting cells with perinuclear melanosome aggregates in three random microscopic fields per well at x200 magnification. Values are presented as the mean ± SD from three wells (n = 3).

Maintenance of reconstituted 3-dimensional human skin tissue in culture.
Neoderm-ME was purchased from Tego Science, Seoul, Korea, and maintained as instructed by the manufacturer. Neoderm-ME is a viable, reconstituted, 3-dimensional human epidermis containing melanocytes and keratinocytes. In brief, Neoderm-ME was removed from the medium-containing agar and transferred onto 6-well plates for equilibration at 37 °C in 5% CO 2 for 1 day. The Neoderm-ME was then treated with 1% DMSO and 0.001% MNQO for 7 days. Each compound was dissolved in vehicle and applied directly to the cultured skin surface three times each week, and the tissue samples were kept in an incubator at 37 °C with 5% CO 2 throughout the course of the experiments. The degree of pigmentation was assessed on photographic images from the brightness value (L value) as determined using editing software (Adobe Photoshop CS; Adobe Systems, USA).

Measurement of melanin contents.
Melan-a melanocytes were seeded in 24-well plates and maintained for 24 h. The cells were then washed in DPBS and treated with samples in RPMI-1640 containing 10% FBS for 3 days. After washing in DPBS, the cells were dissolved in 1 N NaOH at 55 °C for 30 min. The resulting cell lysates were transferred to 96-well plates for optical density (OD) measurements at 490 nm using a microplate reader. For melanin assay using reconstituted 3-dimensional human tissue, Neoderm-ME was dissolved in 1N NaOH and sonicated. The debris was clarified by centrifugation at 13,000 rpm for 5 min, and supernatant ODs were measured at 490 nm using a microplate reader. Protein concentrations of cell lysates were measured using a BCA Protein Assay Reagent Kit (Pierce Biotechnology, Inc, Rockland, IL, USA) with bovine serum albumin as the standard.

UVB-induced pigmentation in brown guinea pigs.
Eight-week-old female brown guinea pigs were supplied by Central Laboratory Animal Inc. (Seoul, Korea). Animal experimental protocol was approved by the ethical committee of Kyung Hee University (no: KHU-12-04) and was carried out in accordance with the approved guidelines. Brown guinea pigs were kept in an air-conditioned room under a temperature 24 °C ± 1 and light/dark cycle at 12 h. After acclimation one week, brown guinea pigs were housed in individual cages. Guinea pigs were anesthetized using ketamine and rompun (4:1 ratio, 1 ml/kg) and five separate areas (1.5 cm × 1.5 cm) on the back of each animal were exposed to UVB radiation (six UVB lamps: TL 20W/12RS, Phillips). The total energy dose of UVB irradiation was 380 mJ/cm 2 per exposure. Five skin sites from three animals were exposed to the UVB irradiation three times a week for three consecutive weeks. MNQO was then applied topically to the UVB-induced areas once a day for five consecutive weeks. The vehicle (propylene glycol:ethanol:water = 5:3:2) was used as a control. Colorimeter (CR-300, Minolta, Japan) was applied once a week for five consecutive weeks to determine the L value (lightness). The difference of L value was measured: ∆ = − L Lvalue (on the measuring day) L value (on the first day of the test, before treated with sample) Immunohistochemical staining. Skin biopsy specimens were taken from the MNQO-treated and vehicle-treated area of guinea pigs after 5 weeks. Biopsied sections were stained with Fontana-Masson method and Hematoxylin and eosin (H&E) method for identified melanin content and skin histological property.