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Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation testing plays a predominant role in the management of MM patients, because modern targeted therapies essentially consist of inhibitors of BRAF. BRAF V600 mutation must be detected using a FDA approved (USA) or CE IVD certified (Europe) test. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real time PCR rtPCR ) or next generation sequencing (NGS) assays in a real life, hospital based series of advanced MM patients.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation testing plays a predominant role in the management of MM patients, because modern targeted therapies essentially consist of inhibitors of BRAF. BRAF V600 mutation must be detected using a FDA-approved (USA) or CE IVD certified (Europe) test. The aim of this study was to compare BRAF mutational testing performed by conventiona lnucleotide sequencing approaches with either real-time PCR (rtPCR )or next-generation sequencing (NGS) assays in a real-life,hospital-based series of advanced MM patients. Consecutive patients fromSardinia, Italy, with histological diagnosis of advanced MM (AJCC stages III Cand IV) and referred a the laboratory of Biomolecular Chemistry (ICB) of the National Research Council (CNR), Sassari, Italy for molecular testing from 2012 through 2019, were enrolled into the study.

Background Identification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. Conventional methods focus on few genes per run and, therefore, are unable to screen for multiple genes simultaneously. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity. Methods We designed a customized IMI somatic gene panel for targeted sequencing of actionable melanoma mutations; this panel was tested on three different NGS platforms using 11 metastatic melanoma tissue samples in blinded manner between two EMQN quality certificated laboratory. Results The detection limit of our assay was set-up to a Variant Allele Frequency (VAF) of 10% with a coverage of at least 200x. All somatic variants detected by all NGS platforms with a VAF ≥ 10%, were also validated by an independent method. The IMI panel achieved a very good concordance among the three NGS platforms. Conclusion This study demonstrated that, using the main sequencing platforms currently available in the diagnostic setting, the IMI panel can be adopted among different centers providing comparable results.

The unique pharmacophore structure of hydroxylated biphenyls has a crucial role in the biological activity of many natural occurring compounds This structural feature allows the activation of a large and selective number of interactions with the surface of several proteins. Recently, our group has prepared an analogue of curcumin 1 (biphenol 2) We found that compound 2 was an effective breaking-chain activator in bulk lipid autoxidation , showed strong cytotoxicity against rat pheochromocytoma (PC12) cells, a slow-growing neuroendocrine tumoral cells and interacts with high affinity with alpha-synuclein . When Ph-OH groups are OMe protected, antiproliferative and proapoptotic activities against malignant melanoma and neuroblastoma were found [3a-c]. Biphenol 2 and its monomer 3 share many pharmacological features with curcumin 1 and, conversely to 1, compounds 2 and 3 are stable in organic and aqueous solutions. With the aim of searching for new compounds with enhanced biological activities, in the present study, we prepared a collection of derivatives of compound 2 and of monomeric equivalent 3. All new prepared compounds possess a common ?,?-unsaturated ketone moiety bearing a hydroxylated biphenyl or phenyl structure with Ph-OH group substituted with functionalities with different features (i.e. groups able to increase or decrease lipophilicity of the molecule) The new compounds were prepared through straightforward synthetic procedures. Different functional groups were introduced giving ether, ester, carbonate, sugar and acetal derivatives that influenced the physico-chemical properties of the molecule. The antiproliferative capacity of all the prepared compounds was preliminarly calculated.

This book represents the most current, comprehensive, and authoritative study of integrins on the market today. It provides an overview of the diverse biological functions of integrins, including: The structure and functions of integrin cytoplasmic domains, the role of b2 integrins in leukocyte adhesion, the role of platelet membrane fibrinogen receptor glycoprotein IIb-IIIa (aIIbb3) in thrombosis and hemostasis, the functions of aV integrin family, the role of integrins in signal transduction, the role of integrins in carcinoma cells, the role of integrins in internalization of microbial pathogens through the binding of Yersinia pseudotuberculosis invasin protein, the role of integrins in the skin, the integrin function in early vertebrate development using amphibian embryos.

Novel Aspect Secretome analysis is a feasible and efficient method to find, identify and characterize clinical biomarkers Introduction Secretome (proteins secreted by cells into the extracellular environment) can reflect a large variety of pathological conditions and can be a useful source of biomarkers. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. During the last years the incidence and mortality of melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated to cancer progression with poor prognosis and survival. Deregulation of dynamic interactions between tumor melanocytes and neighbouring stromal cells leads to the acquisition of cell proliferation capabilities and invasiveness. Proteomic profiling of secretomes of human metastatic cell lines derived from the same patient was performed by a shotgun LC-MS/MS-based approach. Methods Cell lines PES 41-43-47 were isolated from different metastases of the same patient (PES 41 and 47 from sub cutaneous metastases and PES 43 from lung metastases). For secretome analysis, cells were seeded in 30 cm plates in 10% FCS DMEM. After 24 h, culture media were replaced with 0.5% FCS DMEM and after 24h culture media were replaced with serum-free media. Media were collected after 24 h, lyophilized and resuspended in MilliQ water. Proteins were precipitated with TCA, resuspended in AMBIC buffer, reduced, alkylated and digested with trypsin. Aliquots of the supernatant were analyzed by LC-MS/MS. For Western Blot analyses proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and incubated with SPARC, ECM1 and OPN antibodies. Preliminary Data Results provide a list of candidates associated to the metastatic potential of PES human melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e. SPARC, osteopontin, galectins). In addition, the extracellular matrix protein 1 that stimulates the proliferation and angiogenesis of endothelial cells and fibronectin that is involved in cell adhesion and motility were also identified. The secretion of selected proteins (i.e. SPARC, ECM1 and OPN) was further validated by Western Blot analysis. Equal amounts of SPARC and ECM1 were detected in the three cell lines analyzed, whereas OPN secretion was found to be less pronounced in PES 47 cells secretome. Bioinformatic approaches can support the experimental work by providing algorithms for pathway analysis that aims at connecting proteins identified in "omics" surveys. To better understand the mutual interactions of identified proteins, network maps were constructed using the Ingenuity Pathway Analysis software. The graphical view of the resulting network revealed that several secreted proteins were clustered on the basis of their reciprocal interactions. The two pathways obtained for PES secretomes show that several proteins differentially expressed were found to converge on the JNK, ERK, and NF-?? molecular complexes. In addition, a second network was found to converge on Myc and FOS signaling pathways. Overall, proteins identified in PES secretomes converge on pathways involved in the activation of cancer signaling cascades. Although network data need to be further investigated, information derived from the analysis of these pathways will provide useful suggestions for future experimental works.

Background: In malignant melanoma (MM), overexpression of alfavbeta3 integrin is linked to a more metastatic phenotype. Development of anti-alfavbeta3 agents able to counteract melanoma progression would be helpful for disease treatment. A new selective ligand of alfavbeta3, RGDechi-hCit, has anti-angiogenic properties against endothelial cells in angiogenesis animal models. Aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. Material and Methods: Cytofluorimetric analysis characterized cell surface expression of alfavbeta3 integrin on seven MM cell lines. Cell proliferation, adhesion, and migration assays were carried out using the alfavbeta3-antagonist RGDechi-hCit. Results: Proliferation was not significantly inhibited by RGDechi-hCit, though striking morphological changes were detected in MM cell lines highly expressing alfavbeta3. Conversely, assays on fibronectin-coated plates showed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion and migration. Conclusions: Data demonstrate anti-adhesion and anti-migration, but not anti-proliferative, activities of RGDechi-hCit against MM cells.

Malignant melanoma (MM) is one of the most aggressive cancer and chemotherapeutic agents currently in use are still unsatisfactory. Prevention and early diagnosis are the only tools against this cancer whose incidence and mortality rates have been highly increased during the last decades in western countries. On the way of developing new therapeutic drugs against cancer, we aimed to characterize different compounds of natural origin with cytotoxic and/or antiproliferative activity in MM. Eugenol and curcumin are main components of several spices such as clove oil and turmeric spice, respectively, and both have been described as antiossidant, anti-inflammatory and antitumoral agents. We have tested several eugenol related biphenyls for their antiproliferative activity against MM cell lines showing that the enantiomeric form (aS) of the dibromo-deidrodieugenol (S7-S)was the most active with IC50 ranging around 20-30 ?M and without affecting cultured human fibroblasts (M.Pisano et al., 2007). Pro-apoptotic activity? Curcumin itself has been shown to suppress cellular transformation, proliferation, invasion and angiogenesis and we confirmed its potent antiproliferative activity in our MM cell lines (IC50

Cytogenetic and molecular studies suggest that chromosome 1p might contain oncosuppressor genes involved in the pathogenesis of neuroblastoma and other adult tumors. The isolation of these genes by the 'positional cloning' approach will be facilitated by the characterization of cell lines with well defined chromosomal aberrations. In the present report we provide molecular data on the NGP neuroblastoma cell line which contains a reciprocal t(1;15) translocation. Two regions, possibly hosting oncosuppressor genes, have been identified: one is distal to the ENO1 locus, the other one is comprised between PND and A12M2 and corresponds to that of a constitutional t(1;17) translocation described in a neuroblastoma patient. Genetic data also suggest that the NGP cell line, despite the presence of two chromosomes 1, might be hemizygous for the subtelomeric 1p region.

Differentiation of human neuroblastoma (NB) cells is a very interesting biologic event, providing useful insights in both basic neurobiology and clinical management of this malignancy. Investigation of in vitro NB differentiation exploits several NB cell lines that can be induced to differentiate by an array of natural or synthetic chemicals, as well as biological factors such as some cytokines. The hallmarks of neuronal differentiation are represented by a partial or complete block of cell proliferation, morphological alterations and acquisition of biological features typical of mature neurons (for example, induced synthesis and storage of monoamines and neuropeptides, expression of peculiar cytoskeletal proteins and membrane antigens). The possibility to transfer the differentiative approach to the treatment of NB patients opens exciting therapeutic perspectives.

The integrin alpha 6 beta 4 is a heterodimer predominantly expressed by epithelia. While no definite receptor function has yet been assigned to it, this integrin may mediate adhesive and/or migratory functions of epithelial cells. We have determined the complete primary structure of both the alpha 6 and beta 4 subunits from cDNA clones isolated from pancreatic carcinoma cell line libraries. The deduced amino acid sequence of alpha 6 is homologous to other integrin alpha chains (18-26% identity). Antibodies to an alpha 6 carboxy terminus peptide immunoprecipitated alpha 6 beta 4 complexes from carcinoma cells and alpha 6 beta 1 complexes from platelets, providing further evidence for the association of alpha 6 with more than one beta subunit. The deduced amino acid sequence of beta 4 predicts an extracellular portion homologous to other integrin beta chains, and a unique cytoplasmic domain comprised of greater than 1,000 residues. This agrees with the structures of the beta 4 cDNAs from normal epithelial cells (Suzuki, S., and Y. Naitoh. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:757-763; Hogervost, F., I. Kuikman, A. E. G. Kr. von dem Borne, and A. Sonnenberg. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:765-770). Compared to these structures, however, the beta 4 cDNAs that we have cloned from carcinoma cells contain extra sequences. One of these is located in the 5'-untranslated region, and may encode regulatory sequences. Another specifies a segment of 70 amino acids in the cytoplasmic tail. Amplification by reverse transcription-polymerase chain reaction of mRNA indicated that multiple forms of beta 4 may exist, possibly due to cell-type specific alternative splicing. The unique structure of beta 4 suggests its involvement in novel cytoskeletal interactions. Consistent with this possibility, alpha 6 beta 4 is mostly concentrated on the basal surface of epithelial cells, but does not colocalize with components of adhesion plaques.