The role of R21 expression in differential diagnosis of melanocytic lesions.

Background: Cyclic adenosine monophosphate (cAMP) is an intracellular signal transmitter involved in the regulation of melanocyte growth, proliferation, and melanogenesis. R21 is a monoclonal antibody against the soluble adenylyl cyclase (sAC) protein. Various nuclear and cytoplasmic R21 expression patterns in melanocytic lesions have been previously reported. Pan-nuclear staining was defined as specific for melanoma and was found supportive in the assessment of surgical margins. Aims: The aim of this study is to evaluate the different expression patterns of R21 immunostain and investigate its effectiveness in the differential diagnosis of cutaneous malignant and benign melanocytic lesions. Settings and Design: Fifty invasive cutaneous melanoma and 50 benign melanocytic proliferation were included in the study. Materials and Methods: Paraffin blocks that best reflected tumor morphology were studied via immunohistochemical staining for R21. For all patterns, the cases showing staining in 25% or more tumor cells were considered as positive. Statistical Analysis used: Yates' Chi-square, Pearson Chi-square exact test, Spearman correlation were used. Results and Conclusions: Dot-like Golgi staining was characteristic for nevi (12/50) and seen only in one melanoma. Pan-nuclear staining was striking for melanoma (36/50). This pattern was observed in 2 dysplastic and 3 common melanocytic nevi too. None of the Spitz nevi expressed R21 in pan-nuclear pattern. For the diagnosis of melanoma, sensitivity and specificity of the pan-nuclear expression were 72% and 90%, respectively. Positive and negative predictive values were found as 87% and 76%. R21, a second-generation immunohistochemical marker, can be used in the differential diagnosis of benign and malignant melanocytic lesions.

Adaptability of melanocytes post ultraviolet stimulation in patients with melasma.

BACKGROUND: Dynamic in vivo changes in melanin in melasma lesions after exposure to ultraviolet (UV) irradiation have not been described. OBJECTIVES: To determine whether melasma lesions and nearby perilesions demonstrated different adaptive responses to UV irradiation and whether the tanning responses were different among different locations on face. METHODS: We collected sequential images from real-time cellular resolution full-field optical coherence tomography (CRFF-OCT) at melasma lesions and perilesions among 20 Asian patients. Quantitative and layer distribution analyses for melanin were performed using a computer-aided detection (CADe) system that utilizes spatial compounding-based denoising convolutional neural networks. RESULTS: The detected melanin (D) is melanin with a diameter >0.5 µm, among which confetti melanin (C) has a diameter of >3.3 µm and corresponds to a melanosome-rich package. The calculated C/D ratio is proportional to active melanin transportation. Before UV exposure, melasma lesions had more detected melanin (p = 0.0271), confetti melanin (p = 0.0163), and increased C/D ratio (p = 0.0152) in the basal layer compared to those of perilesions. After exposure to UV irradiation, perilesions have both increased confetti melanin (p = 0.0452) and the C/D ratio (p = 0.0369) in basal layer, and this effect was most prominent in right cheek (p = 0.030). There were however no significant differences in the detected, confetti, or granular melanin areas before and after exposure to UV irradiation in melasma lesions in all the skin layers. CONCLUSIONS: Hyperactive melanocytes with a higher baseline C/D ratio were noted in the melasma lesions. They were "fixed" on the plateau and were not responsive to UV irradiation regardless of the location on face. Perilesions retained adaptability with a dynamic response to UV irradiation, in which more confetti melanin was shed, mainly in the basal layer. Therefore, aggravating effect of UV on melasma was mainly due to UV-responsive perilesions rather than lesions.

Syringotropic Melanoma: A Diagnostic Challenge With Prognostic Implications.

ABSTRACT: The presence of neoplastic melanocytes within the eccrine apparatus into the reticular dermis and/or subcutaneous tissue is extremely rare. The staging of syringotropic melanomas and their biological behavior are still controversial. We present 6 new cases of syringotropic melanoma and their main histopathologic features; review the previous literature; and discuss about the origin, staging, and prognosis of this rare variant of melanoma.

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer.

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.

Clear Cell Tumor With Melanocytic Differentiation and ACTIN-MITF Translocation: Report of 7 Cases of a Novel Entity.

Clear cell morphology is an uncommon finding in tumors. A subset of clear cell neoplasms also shows melanocytic differentiation, including clear cell sarcoma, PEComa, and some subtypes of renal cell carcinoma. A hallmark of these tumor types is the activation of a member of the MIT/TFE family of transcription factors, which includes MITF, TFE3, TFEB, and TFEC. Microphthalmia transcription factor (MITF is the master regulator of melanin synthesis, while TFEB plays a critical role in lysosome biogenesis. Cytogenetic translocations involving TFE3 and TFEB are now well described in multiple tumor types, but there has been little evidence to suggest similar regulation of MITF. Here we describe a series of 7 clear cell cutaneous neoplasms with melanocytic differentiation that are characterized by ACTIN-MITF gene fusions, either ACTB-MITF or ACTG1-MITF. The chromosomal breakpoints preserve MITF's dimerization and transcriptional activation domains, suggesting that these fusion proteins likely result in hyperactive MITF function, analogously to the previously reported TFE3 and TFEB fusions. Our findings indicate that MITF gene rearrangements may be key drivers of tumor pathogenesis and expand the spectrum of neoplasia associated with the MIT/TFE family.

Digital Quantification of Melanocytic Density in Resection Margins of Lentigo Maligna Using SOX10 Versus Hematoxylin-Eosin Staining.

ABSTRACT: Lentigo maligna (LM) represents an overgrowth of atypical melanocytes at the dermal-epidermal junction of chronically sun-damaged skin. The presence of LM on sun-damaged skin poses a diagnostic challenge because the solar-induced melanocytic hyperplasia makes it difficult to assess the LM margins. Melanocytic density can be used to discriminate sun-damaged skin from LM. The aim of this study was to quantify the melanocytic density at the surgical margins of scanned whole-slide images of LM comparing sections stained with H&E and SOX10. Twenty-six surgically excised LM diagnosed at the Department of Pathology at Sahlgrenska University Hospital were collected. The slides that contained the closest surgical margin or harbored the highest density of melanocytes at the margin were selected for serial sectioning using H&E and SOX10. Whole-slide imaging at ×40 magnification was used, and a circular field with a diameter of 0.5 mm at the surgical margin was superimposed on the image. Five blinded pathologists reviewed the slides in a randomized order. In the majority of the cases (24/26), the pathologists identified more melanocytes on the SOX10 slides than those on the H&E slides. On average, 2.5 times more melanocytes were counted using SOX10 compared with H&E (P < 0.05). Furthermore, the average group SD on the H&E slides was 4.12 compared with 2.83 on the SOX10 slides (P = 0.004). Thus, the use of SOX10 staining leads to higher melanocytic density counts compared with H&E staining when assessing the surgical margins of LM. The use of SOX10 staining also significantly decreased the interobserver variability between pathologists.

Defining the Histopathological Term Atypical Intraepidermal Melanocytic Proliferation: A Retrospective Cross-Sectional Study.

BACKGROUND: Atypical intraepidermal melanocytic proliferation (AIMP) is a general term assigned to melanocytic proliferations of uncertain biological potential when a definitive histopathological diagnosis cannot be achieved. There are few data available describing the possibility of malignancy of AIMP, or ways to further define diagnosis. OBJECTIVE: To determine the rate of diagnostic change of AIMP to melanoma or melanoma in situ (MIS) after conventional excision. In addition, to determine the role of immunohistochemistry (IHC) in defining AIMP biopsies. METHODS: Retrospective cross-sectional, single-center review of biopsies with a diagnosis of AIMP with a follow-up conventional excision from 2012-2016 was performed. In a separate analysis, a search was performed for AIMP biopsied lesions in which IHC was subsequently performed. RESULTS: The rate of diagnostic change of AIMP to MIS was 4.8% (8/167) after excision. Punch biopsy was a risk factor for diagnostic change to MIS (odds ratio 12.94, confidence interval 2.56-65.38, P = 0.008). The rate of diagnostic change of AIMP biopsies after examining with IHC was 21.3% (34/160) to MIS and 4.4% (7/160) to melanoma. CONCLUSION: The possibility of malignancy of AIMP lesions must be taken into consideration when counseling patients and when planning treatment options. IHC is a useful tool and should be used in the evaluation of AIMP specimens.

Metastatic Melanoma Negative for 5 Melanocytic Markers, Complete Regressed Primary Cutaneous Melanoma, and Melanoma-Associated Leukoderma in the Same Patient.

Melanomas with complete histological regression have been seen very infrequently. On the other hand, the diagnosis of metastatic melanoma is based on the histopathology and positivity of markers such as S100, Melan-A, and HMB-45 whose sensitivity is 99%, 82%, and 76%, respectively. It is very rare that metastatic melanomas and even more primary melanoma are negative for all of these markers. In these rare cases, there is usually a known primary. We present the case of a 82-year-old woman with a erythematous mass in the left groin and a 1-cm black-bluish irregular nodule on the skin of the ipsilateral foot. This lesion was clinical and dermoscopically compatible with primary melanoma. In the histological evaluation of the skin, a dermis full of melanophages and hemosiderophages were found in a background of fibrosis, scarce lymphocytic infiltrate, and neovascularization. Any cells expressing melanocytic markers were observed. It was diagnosed as tumoral melanosis. Lymph nodes showed a proliferation of atypical epithelioid cells with eosinophilic cytoplasm. Mitosis was conspicuous. Tumoral cells were vimentin and CD99 positive, and S100, CD34, HMB-45, Melan-A, SOX 10, tyrosinase, C-KIT, CD45, and CKAE1/AE3 negative, and BRAF-V600 mutated was detected. During follow-up, atypical vitiligo-like lesions were discovered, suggesting the diagnosis of metastatic melanoma totally regressed in our patient.

Syntenin regulates melanogenesis via the p38 MAPK pathway.

Melanogenesis is the synthesis of the skin pigment melanin, which serves a critical role in the study of pigmentary skin diseases. Syntenin has been identified as a melanosome protein, but its role in melanogenesis is not completely understood. The present study aimed to investigate the effects and mechanisms underlying syntenin on melanogenesis in immortalized human melanocytes. Depletion of syntenin expression increased both tyrosinase (Tyr) activity and melanin content. Syntenin silencing also increased the protein expression levels of Tyr, pre­melanosomal protein and microphthalmia­associated transcription factor. In addition, the results indicated that syntenin regulated melanogenesis by upregulating the phosphorylation of p38 mitogen­activated protein kinase (p38 MAPK). Taken together, these findings suggested that the regulation of melanogenesis by syntenin may be mediated by the activation of p38 MAPK and that syntenin might provide new insights into the pathogenesis of pigmented diseases.

l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus Trichoderma erinaceum F1-1.

The effects of a single-amino-acid culture strategy on secondary metabolite production in the marine-derived fungus Trichoderma erinaceum F1-1 were investigated by culturing the fungus in GPY medium supplemented or not supplemented with l-phenylalanine. A suite of secondary metabolites, including seven terpenoids (1-7) and one polyketide (8), among which are four new compounds, harziandione A (1), cyclonerodiols A and B (3, 4), and trichodermaerin A (6), were isolated from the GPY medium without l-phenylanine, whereas 18 aromatic compounds (9-26), including six new compounds, trichoderolides A-F (9, 10, and 14-17), were isolated from the culture grown in the GPY medium with l-phenylalanine. The structures of the new compounds were determined by high-resolution mass spectrometry, NMR spectroscopic analysis, optical rotation calculations, chemical methods, and X-ray crystallography. Compounds 10, 12, 13, and 26 exhibited cytotoxic activities against MDA-MB-435 human melanocyte cancer cells. Compound 26 was cytotoxic to A549 adenocarcinomic human alveolar basal epithelial cells.

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.

CYSLTR2-mutant Cutaneous Melanocytic Neoplasms Frequently Simulate "Pigmented Epithelioid Melanocytoma," Expanding the Morphologic Spectrum of Blue Tumors: A Clinicopathologic Study of 7 Cases.

Recurrent activating Gαq mutations in the spectrum of blue nevi have been well studied. However, the clinicopathologic characteristics of the recently described CYSLTR2-mutant and PLCB4-mutant blue nevi remain limited, owing to their rarity. Herein, we present 7 CYSLTR2-mutant melanocytic neoplasms, including 1 cellular blue nevus, 4 atypical cellular blue nevi, and 2 blue nevus-like melanomas. They occurred on the scalp, breast, flank, forearm, thigh, leg, and ankle of 3 male patients and 4 female patients, with a median age of 43 (25 to 81) years at diagnosis. Five exhibited an exophytic growth, and 6 were heavily pigmented. A fascicular arrangement of medium to large spindle melanocytes was seen in 6 cases, but epithelioid cytology was present in only 2 cases, one of them being focal. A junctional component was present in 3 cases. Immunoreactivity for HMB45 was diffusely present, except in 1 cellular blue nevus. BAP1 nuclear immunoexpression was lost in 1 melanoma case. A canonical CYSLTR2 L129Q hotspot mutation was present in all cases. Altogether, these histopathologic findings suggest that CYSLTR2-mutant melanocytic blue neoplasms frequently exhibit a heavily pigmented exophytic tumor with a silhouette resembling "pigmented epithelioid melanocytoma" rather than usual cellular blue nevus. Moreover, most of these tumors were not clinically recognized as blue nevi and not located in the classic topography of cellular blue nevus aside from the scalp. However, a fascicular arrangement of medium to large-sized spindled melanocytes, as well as a lack of epithelioid or nevoid melanocytes, could be potential diagnostic clues to morphologically distinguish CYSLTR2-mutant tumors from "pigmented epithelioid melanocytoma."

Semi-automated standardisation of melanin bleaching procedures of heavily pigmented melanocytic lesions for immunohistochemical analysis on an automated platform.

Background: The diagnosis of heavily pigmented melanocytic lesions is problematic. This is often compounded by lack of visibility of nuclear detail of tumour cells due to physical masking by melanin pigment. Similarly, there can be colour merging of chromogenic final reaction products with melanin, making an evidence of antigenic localisation problematic. There are a number of melanin bleaching techniques available for immunohistochemical assessments.Material and methods: All methods to date have involved the bleaching of melanin as a manually performed primary step before loading subsequently bleached slides onto automated immunohistochemical platforms. Here we define a semi-automated bleaching procedure that allows full integration on one of the most widely employed automated IHC staining platforms (Roche Ventana BenchMark Ultra). The bleaching protocol was defined on the BenchMark Ultra and involved the assessment of 24 histological cases of heavily pigmented malignant melanoma lesions (13 cutaneous and 11 metastatic) routinely fixed processed and paraffin wax embedded.Results: Completion of the bleaching was assessed on H&E preparations performed following the semi-automated bleaching step and employing the Roche Ventana BenchMark Ultra machine for 60 min at 42°C. Complete immunohistochemical staining was achieved on the automated platform within 5-6 h including the bleaching step. Results were consistent across all tissue evaluated.Discussion: This data provides evidence that the hydrogen peroxide bleaching procedure can be adapted for integration on one of the most widely employed automated IHC staining platforms and as a result, improve the efficiency and reproducibility of the technique.

[Primitive myxoid melanoma: An unusual histological aspect]. / Mélanome myxoïde primitif : une forme histologique inhabituelle.

BACKGROUND: Myxoid melanoma is a rare variant of melanoma that must be recognised. Herein we describe a new metastatic case. PATIENTS AND METHODS: A 78-year-old woman consulted for a firm, pinkish nodule measuring 25mm and present for six months on her left leg. Analysis of the biopsy revealed achromic fusiform tumour cells separated by large myxoid plaques. Labeling of SOX10, HMB45 and PS100 was diffuse and of moderate to strong intensity. A diagnosis of myxoid melanoma was considered, with Breslow thickness of 9mm. Surgery was carried out with a 2-cm margin and confirmed the diagnosis. Dermatological follow-up at one year revealed metastatic spread to the ganglia, pleura, liver and bone. DISCUSSION: Few cases of primary myxoid melanoma have been described, and the condition is probably underdiagnosed. The classic clinical presentation of this condition consists of a solitary achromic nodule found chiefly on the limbs. The microscopic appearance is relatively non-specific. Immunohistochemical analysis may indicate melanocytic involvement: cells exhibit expression of SOX10, diffuse expression of protein S100, and less consistent and more variable expression of HMB45. The increasingly common use of anti-SOX10 is of value since it is expressed in the nucleus of melanocytes. Mastocytes and TGF-ß secretion appear to be involved in myxoid stroma production. In the absence of specific codification, management of myxoid melanoma is comparable to that of other types of melanoma. There is uncertainty about the prognosis, with the involvement of TGF-ß possibly indicating the aggressive potential of this type of tumour.

"Pigmented Extramammary Paget Disease"-A Potential Mimicker of Malignant Melanoma and a Pitfall in Diagnosis: A Case Report and Review of the Literature.

Extramammary Paget disease (EMPD) is a rare intraepithelial carcinoma and an uncommon variant of Paget disease affecting areas of the apocrine-rich skin of the perineum, vulva, and less commonly, axilla. Women in their sixth to eighth decades are commonly affected. It is exceedingly rare for EMPD to present on the face, chest, abdomen, or other nonapocrine sites and even more unusual for EMPD to present as a pigmented lesion. The relationship between Paget cells in pigmented extramammary Paget disease (PEMPD) and reactive proliferation and colonization by melanocytes has been poorly explored. The relevance of this rare entity resides in its potential to be misdiagnosed clinically and histopathologically as malignant melanoma in situ. Therefore, application of a panel of immunostains and careful analysis and interpretation of these findings are essential to arrive at the correct diagnosis. We report a new case of PEMPD on a nonapocrine site. The specimen was examined by routine microscopy including hematoxylin and eosin stain as well as immunostains. Histologic examination revealed characteristic features of PEMPD confirmed with immunohistochemical stains.

SMAD7 is a novel independent predictor of survival in patients with cutaneous melanoma.

Overexpression of SMAD7-a hallmark inhibitor of transforming growth factor ß (TGFß) signaling-has been documented and related with adverse prognosis in a number of epithelial malignancies, suggesting that it may be responsible for resistance to TGFß-induced growth arrest of cancer cells. The involvement of SMAD7 in development and progression of malignant melanoma is unclear, and its expression has not been characterized so far at the protein level in clinical melanoma tissue samples. We evaluated SMAD7 expression in 205 skin melanoma primary tumors by immunohistochemistry and correlated the findings with clinicopathological profiles of patients. Melanocytic SMAD7 was evidenced in 204 cases, and the expression pattern was predominantly nuclear. High expression of SMAD7 was positively associated with several features of tumor aggressiveness, for example, presence of ulceration (P < 0.001), higher tumor thickness (P < 0.001), and mitotic rate (P < 0.001), but not presence of regional or distant metastases. Moreover, high SMAD7 expression independently predicted unfavorable outcome: melanoma-specific survival (hazard ratio = 3.16, P < 0.001) and recurrence-free survival (hazard ratio = 2.88, P < 0.001). Taken together, our results underline the importance of TGFß signaling in cancer and define SMAD7 as a marker of aggressive tumor behavior and adverse clinical outcomes in melanoma patients.

Tyrosinase and nestin immunohistochemical expression in melanocytic nevi as a histopathologic pattern to trace melanocyte differentiation and nevogenesis.

While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.

Resolution of conjunctival melanoma with topical interferon alpha 2b in a patient with mitomycin C intolerance. / Resolución de un melanoma conjuntival con interferón tópico alfa 2b en un paciente con intolerancia a la mitomicina C.

OBJECTIVE: To describe the clinical and histological resolution of a case of an inexcisable conjunctival melanoma using topical interferon alpha 2b (INFα2b) in a patient with mitomycin C (MMC) intolerance. CASE REPORT: Conjunctival melanoma is a rare, but potentially sight- and life-threatening, tumour. In cases of multiple lesions, or when surgical excision is not possible, topical combination chemotherapy with MMC and INFα2b has been described as first line therapy. The case is presented of a 77 year-old woman with a multifocal conjunctival in situ melanoma, who was intolerant to initial treatment with MMC and was switched to long-term INFα2b therapy, with a good outcome. CONCLUSIONS: When topical MMC is given as chemotherapy treatment for primary acquired melanosis with atypia or in situ melanoma is not well tolerated, switching to INFα2b seems to be a good option. This approach could replace surgical management of pigmented tumours, especially the larger ones, with potential benefits that include less dependence on surgical margins. This report prompts a need for prospective studies designed to examine the role of INFα2b as primary treatment for heavily pigmented conjunctival tumours avoiding the ocular surface toxicity caused by MMC.

Mechanism underlying inhibitory effect of six dicaffeoylquinic acid isomers on melanogenesis and the computational molecular modeling studies.

Dicaffeoylquinic acid (DCQA), which contain 2 caffeic acids and a quinic acid, is 6 isomeric compounds (1,3-, 1,4-, 1,5-, 3,4-, 3,5-, and 4,5-DCQA). In this study, the mechanism underlying the inhibitory effect of DCQA isomers on melanogenesis in B16F1 murine melanoma cells stimulated by melanocyte stimulating hormone (α-MSH) was evaluated. DCQA isomers showed inhibitory effects on melanogenesis in α-MSH-stimulated B16F1 cells. Furthermore, the anti-melanogenesis activities of 1,5-DCQA and 4,5-DCQA were 61% and 84%, respectively, which were greater than that of arbutin (35%). For cell-free tyrosinase, 3,4-DCQA and 4,5-DCQA indicated high inhibitory effects, similar to the activity to arbutin (35%) at 25 µM. DCQA isomers inhibited the melanogenic enzymes including tyrosinase and dopachrome tautomerase (DCT) on α-MSH-stimulated B16F1 cells. Interestingly, 4,5-DCQA, the most potent inhibitor of melanogenesis among the six DCQA isomers, significantly downregulated the expression of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TRP1) containing tyrosinase, and DCT. In particular, the inhibitory mechanism of 4,5-DCQA on MITF expression was elucidated, revealing that 4,5-DCQA inhibits the phosphorylation of cAMP response element-binding protein (CREB) by attenuating cAMP generation during melanogenesis. A molecular docking study was conducted to elucidate the inhibitory mechanism of 4,5-DCQA on cAMP production. DCQA isomers dock to the residues of adenylyl cyclase with a distance of <3 Å, except for 1,3-DCQA. Especially, 4,5-DCQA showed Full Fitness of -1304.68 kcal/mol and △G of -8.33 kcal/mol, as well as H-bonding with adenylyl cyclase at ILE953 and LYS930 residues. In conclusion, DCQA isomers have different effects on melanogenesis depending on their structure. Especially, 4,5-DCQA has depigmentation activity through the inhibitory effect on cellular tyrosinase directly and binding effect on adenylyl cyclase, resulting in the downregulation of MITF protein, thereby reducing the expression of melanogenic enzymes.

UVA-induced carbon-centred radicals in lightly pigmented cells detected using ESR spectroscopy.

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.