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There is a growing interest in the cytotoxic effects of bioactive glycoalkaloids, such as &alpha, tomatine on tumor cells. Here, for the first time, we determine the antitumor potential of tomatine, a mixture of &alpha, tomatine and dehydrotomatine, in metastatic melanoma (MM) cell lines harboring different BRAF and MC1R variants. We performed cytotoxicity experiments and annexin-V/propidium iodide staining to assess the apoptotic/necrotic status of the cells. ER stress and autophagy markers were revealed by Western Blot, whereas antiangiogenic and vascular-disrupting effects were evaluated through a capillary tube formation assay on matrigel and by ELISA kit for VEGF release determination. Cell invasion was determined by a Boyden chamber matrigel assay. Tomatine reduced 50% of cell viability and induced a concentration-dependent increase of apoptotic cells in the range of 0.5&ndash, 1 &mu, M in terms of &alpha, tomatine. The extent of apoptosis was more than two-fold higher in V600BRAF-D184H/D184H MC1R cells than in BRAF wild-type cells and V600BRAF-MC1R wild-type cell lines. Additionally, tomatine increased the LC3I/II autophagy marker, p-eIF2&alpha, and p-Erk1/2 levels in BRAF wild-type cells. Notably, tomatine strongly reduced cell invasion and melanoma-dependent angiogenesis by reducing VEGF release and tumor-stimulating effects on capillary tube formation. Collectively, our findings support tomatine as a potential antitumor agent in MM.

Background Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported. Methods We evaluated levels and localization of eIF2α/peIF2α in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscopy analyses. Furthermore, we performed a sequence analyses and structure and dynamics studies of eIF2α protein to reveal the role of eIF2α and its correlations in different pathways involved in the invasive phase of melanoma. Results We found peIF2α both in cytoplasm and nucleus. Nuclear localization was more represented in V600BRAF melanoma cell lines. Our studies on eIF2α protein sequence indicated the presence of a predicted bipartite NLS as well as a nuclear export signal NES and an S1 domain, typical of RNA interacting proteins. Furthermore, we found high levels of transcription factor EB (TFEB), a component of the MiT/TFE family, and low β-catenin levels in V600BRAF cells. Conclusions Based on our results, we suggest that peIF2α nuclear localization can be crucial in ER stress response and in driving the metastatic spread of melanoma, through lysosomal signaling and Wnt/β-catenin pathway. In conclusion, this is the first evidence of nuclear localization of peIF2α, representing a possible target for future therapeutic approaches for metastatic melanoma. Electronic supplementary material The online version of this article (10.1186/s12967-019-1772-z) contains supplementary material, which is available to authorized users.

Summary Melanocortin-1 receptor (MC1R) plays a key role in skin pigmentation and its polymorphisms are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF-mutated MM patients treated with BRAFi. We also evaluated the effect of vemurafenib in four V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R polymorphisms and a worse outcomes in terms of both ORR (59%vs95%, p.011 univariate, p.028 multivariate analysis) and PFS shorter than 6 months (72%vs33%, p.012 univariate, p.027 multivariate analysis). No difference in OS was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase of vemurafenib IC50 in MC1R variant cell lines. This article is protected by copyright. All rights reserved.

Environmental and genetic risk factors are involved in the development of melanoma. The role of the melanocortin 1 receptor (MC1R) gene has been investigated and differences according to geographic areas have been described. To evaluate the role of some clinical and genetic risk factors in melanoma development, we performed a case-control study involving 101 melanoma patients and 103 controls coming from South-Eastern Italy (Puglia), after achieving informed consent. We confirmed the role of known clinical risk factors for melanoma. Furthermore, 42 MC1R polymorphisms were observed. Three of these variants (L26V, H232L, D294Y) were not previously reported in the literature. Their predicted impact on receptor function was evaluated using bioinformatic tools. We report an overall frequency of MC1R variants in our population higher than in Northern or Central Italy. The most common polymorphism found was V60L, that has been recently reported to spread among South Mediterranean population. This variant influenced phenotypic characteristics of our population while it did not impinge on melanoma risk. An increased risk of melanoma was associated with two or more MC1R variants, when at least one was RHC, compared to people carrying the MC1R consensus sequence or a single MC1R polymorphism. Interestingly, we observed an increased risk of melanoma in subjects with darker skin and lower nevus count, usually considered at low risk, when carrying MC1R polymorphisms.

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon ( Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyer’s patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.

Melanomas of the oral cavity are extremely rare. Their rarity and their independence on exposure to UV radiation make them particularly interesting. The authors analyzed an oral multiphasic melanoma composed by a nodular nonpigmented ulcerated central region, a nodular ulcerated pigmented area, a pigmented nonulcerated region, and an area similar to a dysplastic nevus. They determined the expression of some genes involved in the differentiation and cellular transformation in morphologically different regions of melanoma. All these areas were also analyzed by electron microscopy. The various regions composing the melanoma expressed genes involved in melanogenesis and melanoma progression in a different manner. Electron microscopy observation of ultrathin sections of each region evidenced ultrastructural differences, being the cellular architecture more compromised in the most aggressive parts of the neoplasm. This pilot study identified morphological, molecular, and ultrastructural differences that characterize each region of the multiphasic melanoma.

Amphibian tyrosinases display unique and poorly understood properties such as seasonal activity variations, different activities in dorsal and ventral skin and the occurrence as inactive forms requiring proteolytic activation. For the first time we have sequenced and characterized Rana esculenta L. tyrosinase by functional expression of the cloned cDNA, and compared it with frog skin extracts. R. esculenta tyrosinase ORF is well conserved compared with tyrosinases of various sources. The amino acid similarities between the tyrosinases from R. esculenta and other amphibia range from 85% to 98%. Homology remains high with mammalian tyrosinases (65% identity with Homo sapiens, and 63% with Mus musculus) and with bird orthologues (66% identity with Gallus gallus). Tyrosinase was expressed in HEK293T cells as an active enzyme. Activity staining on non reducing SDS-PAGE revealed two bands around 63 and 68 kDa. R. esculenta skin extracts were mildly active and reached maximal activity upon protease treatment, revealing a high molecular weight dopa-positive band in the 200 kDa range and one of higher MW, after nagarse treatment, in activity stainings. The different behaviour of recombinant tyrosinase compared to skin extracts suggests formation in vivo of a multimeric complex.

Various enzymes are known to be involved in melanin biosynthesis, but the key role appertains to tyrosinase. In amphibians this enzyme displays peculiar characteristics: i) it requires an activation process; ii) its level of enzymatic activity in the animal skin changes depending on the season. In this work, by using chymotrypsin, subtilisin and SDS as putative activators, we studied the activation process of the skin pro-tyrosinase of Rana esculenta L. in different seasons over a period of two years. We found that chymotrypsin and subtilisin were able to yield an active enzyme, but not SDS. The maximum levels of tyrosinase activity were recorded in winter and the minimum in summer. We detected tyrosinase activity in the melanosomal fraction, where the enzyme form was least sensitive to proteolytic activation, probably corresponding to a “ mature ” tyrosinase. The enzyme forms found in the microsomal and soluble fractions were more sensitive to proteolytic activation, probably corresponding to “ immature ” tyrosinase. On SDS-PAGE, the tyrosinase activity assays showed a dopa-positive band at 200 kDa and a second aggregated band with a still higher molecular mass. The significance of these results in frog melanogenesis regulation is discussed.

The liver of Amphibia and Reptilia shows a dark-brown pigmentation due to the presence of particular melanin-containing cells that are different from melanocytes and derive from cells of macrophage lineage (Kupffer Cells), which have been shown to have an autonomous capacity to synthesize melanins. To date, as far as we know, there are no reports in the literature about the genetic system of tyrosinase as regards these melanin-synthesizing cells; we carried out the present study to analyze how the tyrosinase gene may function. We showed that the Kupffer cells of Rana esculenta L. do indeed have a transcriptionally active tyrosinase gene. Evidence of this was obtained by reverse transcription polymerase chain reaction analysis carried out on both the liver tissue and the Kupffer cells in culture. Moreover, analysis of the cells in culture enabled us to observe that, by increasing the culture time from 0 to 72 hr, an appreciable increase occurred in the amplification products of the tyrosinase gene, as well as in the level of dopa oxidase activity and in the quantity of melanin in the cells. The results of the present study demonstrate that frog Kupffer cells possess an active tyrosinase gene and that the increase of the tyrosinase mRNA accumulation closely correlates with phenotypic differentiation, in terms of increased dopa oxidase activity and melanosome content. This provides further strong support of the hypothesis that amphibian Kupffer cells possess an endogenous ability to synthesize melanin and suggests the involvement of the transcriptional level of control in the modulation of their melanogenic activity.

A simple method to isolate and culture liver pigment cells from Rana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells from Rana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance.

The present study reports the results of a morpho-functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin-synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosine-hydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p-phenylenediamine-pyrocatechol (PPD-PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenol-oxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue-specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.

Background Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works [1,2] provided new insights concerning molecular mechanisms involved in mitochondrial biogenesis of melanoma. This work aims to find possible correlations between pathways involved in the onset and progression of the disease in order to provide supporting information in this field. In particular we studied the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines and its relations with BRAF mutational status.

A study of the liver pigment cells of Rana esculenta L. has been performed on both liver in toto and cells in culture. Ultrastructural and cytochemical analyses showed a close relationship between this visceral pigment cell system and the cells of hepatic macrophage lineage. Like the latter, the liver pigment cells present phagocytic activity, in the sinusoids and in vitro, and give a positive response to tests for peroxidase and lipase. The liver pigment cells are isolated, together with the Kupffer cells, from the sinusoidal cell fraction of the liver. In culture, they maintain their melanogenetic ability, demonstrated by the presence of dopaoxidase activity in the soluble, membranous, and melanosome fractions. Analysis of the cultures showed that as culture time increased, so did melanosome dopaoxidase activity, the number of pigmented fields, and the level of pigmentation of the cells. The values of dopaoxidase activity of the pigment cells in culture show the same seasonal oscillations as the system in toto, indicating that the cells maintain an internal clock, at least in the first 72 h of culture. There is evidence that the pigment cells are macrophages which can express a melanogenetic function. Our results and other experimental data provide a basis for hypothesizing that the pigment cells in Rana esculenta L. liver may derive from, or have a common origin with, the Kupffer cells.

1. 1. The liver pigment cells of R. esculenta L. constitute a peculiar pigment cell system of histiocytic nature and contain a tyrosinase-like activity localized in the protein component of melanosomes. 2. 2. The effects of addition and/or removal of Cu on the DOPA-oxidase activity of the system were studied. 3. 3. It was concluded that: (a) this tyrosinase behaves as a Cu-enzyme; (b) Cu could be involved in the regulation of the enzyme activity; and (c) mixtures of apoenzyme and active enzyme coexist in the melanosomes.

This work demonstrates the presence of a tyrosinase-like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.

Background Metastatic melanoma represents the most deadly form of skin cancer. The poor response to chemotherapy and the brief response to the anti-BRAF vemurafenib in selected population of patients, make the identification of new therapeutic approaches an urgent need. Our goal is the evaluation of the efficacy of barasertib, an aurora B kinase inhibitor impairing cytokinesis, in both mutated and non-mutated melanoma cell lines. Materials and methods Panel of melanoma cells: BRAFV600E mutated cells (MBA72 and Hmel1), the same cell in which the resistance to vemurafenib was induced by chronic exposure to it (MBA72R and Hmel1R) and BRAF wt (HBL and LND1). Cells were characterized for vemurafenib and barasertib effectiveness on cell growth by MTT assay after 3 and 6 days of continuous exposure. Cell cycle was determined by flowcytometry and migration was evaluated by wound-healing assay. Results Cells with BRAFV600E mutation are sensitive to vemurafenib conversely, those with BRAF wt and the resistant ones showed an IC50 of at least 10 folds higher. 3 days-barasertib exposure strongly reduced cell growth (30-60% at 30 and 300nM, respectively) in all cell lines; when the drug was given together with vemurafenib, no gain in effectiveness was evident. Prolonged exposure to barasertib (6 days) showed a progressive increase of effectiveness particularly in cells BRAF wt. The analysis of cell death mechanisms involved in determining the effectiveness of barasertib and vemurafenib showed that the first drug induced both apoptosis and necrosis conversely, the latter mainly apoptosis. Cell cycle analysis demonstrated that barasertib induced an increase in cell size and in polyploidia, suggesting also the mitotic catastrophe as a further cell death mechanism. Moreover, the anti-metastatic behaviour of this agent has been evaluated in function of drug concentration and time exposure. Preliminary results showed a strong reduction of cell migration after drug exposure. Conclusions

Background The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. Methods The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. Results The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. Conclusions These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0385-4) contains supplementary material, which is available to authorized users.