Targeting the melanoma

Czerniak należy do najbardziej złośliwych nowotworów człowieka, a liczba zachorowań na ten typ nowotworu wzrasta na świecie w zastraszającym tempie. Komórki czerniaka charakteryzują się wyjątkową opornością na stosowane obecnie metody leczenia. Badania z ostatnich lat przyczyniły się do lepszego poznania i zrozumienia biologii nowotworu i otwarły drogę nowym terapiom celowanym. Obecnie wiele czynników znajduje się we wczesnych fazach badań klinicznych, część z nich osiągnęła już III fazę, a w sierpniu 2011 rokuFDA zaaprobowała nowy lek do walki z czerniakiem - Wemurafenib (PLX4032). Artykuł stanowi przegląd najważniejszych białek, szlaków i sieci sygnalizacyjnych zaangażowanych w rozwój i progresję czerniaka, ze szczególnym uwzględnieniem elementów będących najbardziej obiecującymi celami dla terapii antynowotworowej.Melanoma is one of the most aggressive human cancers. Its cells are inevitably resistant to conventional therapies. In recent years, better understanding of melanoma biology, has led to the development of a number of new potential therapeutic agents. Many of these compounds are being tested in early phase of clinical trials, some have already reach phase III. In August 2011 the FDA approved a new drug - Wemurafenib (PLX4032) to treatment unresectable and/or metastatic melanoma. The current article is an overview of the major proteins, signaling pathways and networks involved in the development and progression of melanoma, with particular emphasis on elements that are the most promising targets for anticancer therapy

Glycosylation of Integrins in Melanoma Progression

Each stage of melanoma development from transformed melanocytes to metastatic lesions requires the involvement of cell adhesion receptors, among which integrins are of particular importance. Strong N-glycosylation of αβ integrin heterodimers influences their processing, activation, and functions related to the modulation of cell adhesion to extracellular matrix proteins (ECM) and the basement membrane. A lack of N-glycans on integrin chains significantly reduces their interactions with the ECM. Melanoma progression is accompanied by changes in the composition of N-glycans on integrin subunits. The glycosylation profile of integrins depends on the stage of melanoma development and on the location of the metastasis. Enhanced expression of β1,6-branched complex-type oligosaccharides and altered sialylation are well-characterized changes in the N-glycosylation of integrins observed in melanoma progression. This chapter summarizes the current state of knowledge about α3β1, α5β1, and αvβ3 integrin glycosylation in melanoma and the functional consequences of changed glycosylation for the development of this cancer

Towards understanding the role of sialylation in melanoma progression

Aberrant expression of sialic acids or altered linkage types is closely associated with malignant phenotype and metastatic potential, and can have prognostic significance in human cancer. The present study was undertaken to evaluate whether expression of sialylated derivatives on melanoma cell surface is associated with tumour progression. Four cell lines (WM1552C, WM115, IGR-39 and WM266-4) were used in the study. Cell surface expression of sialic acids was evaluated by flow cytometry with the use of Maackia amurensis and Sambucus nigra lectins. Moreover, adhesion and migration potential of melanoma cells and involvement of sialic acids in these processes were analysed. We have demonstrated that WM266-4 cells have a significantly higher level of α2,3-linked sialic acid residues than other cells, whereas IGR-39 cells had lower expression of α2,6-linked sialic acids. The adhesion efficiencies of WM1552C and WM115 cells were significantly lower than that of IGR-39 and WM266-4 cells. In contrast, WM266-4 cells repaired scratch wounds at least twice as fast as other cells. Melanoma cell adhesion to fibronectin in the presence of Sambucus nigra agglutinin (SNA) was reduced only in IGR-39 and WM266-4 cells, whereas the impact of Maackia amurensis agglutinin (MAA) on this process was much more important. Migration efficiency of melanoma cells was reduced more strongly in the presence of MAA than SNA. In conclusion, our results show that melanoma progression is associated with the increased expression of α2,3-linked sialic acids on the cell surface and these residues could promote melanoma cell interaction with fibronectin

Diverse expression of N-acetylglucosaminyltransferase V and complex-type \beta1,6-1,6-branched N-glycans in uveal and cutaneous melanoma cells

Although both uveal (UM) and cutaneous (CM) melanoma cells derive from the transformed melanocytes, their biology varies significantly in several aspects. Malignant transformation is frequently associated with alternations in cell glycosylation, in particular those concerning branched complex-type N-glycans. These changes occur principally in β1,4-N-acetylglucosaminyltransferase III (GnT-III) that catalyzes the synthesis of glycans with bisected N-acetylglucosamine (GlcNAc) and β1,6-N-acetylglucosaminyltransferase V (GnT-V) that is involved in forming β1,6-branched antenna in complex-type glycans. We searched for the reasons of a different behavior of CM and UM cells in the expression of GnT-III and GnT-V and their oligosaccharide products. Our study showed that UM cells have more β1,6-branched glycans than CM cells, what results from a higher expression of MGAT5 gene encoding GnT-V. The higher β1,6-branching of glycans in UM may contribute to their higher potential to migrate on fibronectin and weaker binding to main extracellular matrix proteins, observed in our previous studies

Adhesion properties of human bladder cell lines with extracellular matrix components : the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the α2, a5 and β1 integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the β1 integrin subunit. Antibodies to α3 integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with colagen. It seems that α3 subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy

Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines

BACKGROUND: The aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins. RESULTS: Human non-malignant epithelial cell of ureter HCV29, v-raf transfected HCV29 line (BC3726) and transitional cell cancers of urine bladder Hu456 and T24 were grown in cell culture. Equal amounts of protein from each cell extracts were separated by SDS-PAGE electrophoresis and were blotted on an Immobilon P membrane. Cadherins were immunodetected using anti-pan cadherin mAb and lectin blotting assays were performed, in parallel. N-oligosaccharides were analysed by specific reaction with Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Datura stramonium agglutinin (DSA), Aleuria aurantia agglutinin (AAA), Phaseolus vulgaris agglutinin (PHA-L) and wheat germ agglutinin (WGA). The cadherin from HCV29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans, some of which were α2,6-sialylated. The cadherin from BC3726 cell line exhibited exclusively high mannose type glycans. Cadherins from Hu456 and T24 cell lines expressed high mannose type glycans as well as β1,6-branched oligosaccharides with poly-N-acetyllactosamine structures and α2,3-linked sialic acid residues. Additionally, the presence of fucose and α2,6-sialic acid residues on the cadherin from T24 cell line was detected. CONCLUSIONS: These results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis

The lectin-binding pattern of nucleolin and its interaction with endogenous galectin-3

Unlike nuclear nucleolin, surface-expressed and cytoplasmic nucleolin exhibit Tn antigen. Here, we show localization-dependent differences in the glycosylation and proteolysis patterns of nucleolin. Our results provide evidence for different paths of nucleolin proteolysis in the nucleus, in the cytoplasm, and on the cell surface. We found that full-length nucleolin and some proteolytic fragments coexist within live cells and are not solely the result of the preparation procedure. Extranuclear nucleolin undergoes N- and O-glycosylation, and unlike cytoplasmic nucleolin, membrane-associated nucleolin is not fucosylated. Here, we show for the first time that nucleolin and endogenous galectin-3 exist in the same complexes in the nucleolus, the cytoplasm, and on the cell surface of melanoma cells. Assessments of the interaction of nucleolin with galectin-3 revealed nucleolar co-localization in interphase, suggesting that galectin-3 may be involved in DNA organization and ribosome biogenesis

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with α2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with α-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with α2-3 sialic acid (positive reaction with Maackia amurensis agglutinin - MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells

An analysis of fungal propagules transported to the Henryk Arctowski Antarctic Station

During three austral summer seasons, dust and soil from clothes, boots and equipment of members of scientific expeditions and tourists visiting the Polish Antarctic Station Henryk Arctowski were collected and analysed for the presence of fungal propagules. Of a total of 60 samples, 554 colonies of fungi belonging to 19 genera were identified. Colonies of the genus Cladosporium, Penicillium and non−sporulating fungus (Mycelia sterilia) dominated in the examined samples. The microbiological assessment of air for the presence of fungi was also conducted at two points in the station building and two others outside the station. A total of 175 fungal colonies belonging to six genera were isolated. Colonies of the genus Penicillium were the commonest in the air samples. The potential epidemiological consequences for indigenous species as a result of unintentional transport of fungal propagules to the Antarctic biome are discussed in the light of rapid climate change in some parts of the Antarctic and adaptation of fungi to extreme conditions