Immacolata Maida

ULTRASTRUCTURAL AND FUNCTIONAL STUDY OF THE LIVER PIGMENT CELLS FROM RANA ESCULENTA L.

SummaryA 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.

Aurora kinase B inhibition reduces the proliferation of metastatic melanoma cells and enhances the response to chemotherapy

BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThese findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

MORPHO-FUNCTIONAL CHARACTERIZATION OF CULTURED PIGMENT CELLS FROM RANA ESCULENTA L. LIVER

SummaryA simple method to isolate and culture liver pigment cells fromRana 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 fromRana 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.

Melanogenesis in visceral tissues of Salmo salar. A link between immunity and pigment production

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 Peyers 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.

Sporadic melanoma in South-Eastern Italy: the impact of melanocortin 1 receptor (MC1R) polymorphism analysis in low-risk people and report of three novel variants.

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.

Molecular cloning and biochemical characterization of the skin tyrosinase from Rana esculenta L.

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.

Effects of copper on the tyrosinase of liver pigment cells from Rana esculenta L.

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. The effects of addition and/or removal of Cu on the DOPA-oxidase activity of the system were studied. 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.

The mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines: correlations with BRAF mutational status

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.

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants 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 (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four V600BRAF melanoma cell lines with/without MC1R variants.

New insight into the role of metabolic reprogramming in melanoma cells harboring BRAF mutations.

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.