Małgorzata Przybyło

Increased levels of bone remodeling biomarkers (osteoprotegerin and osteopontin) in hypertensive individuals

OBJECTIVES Osteoprotegerin (OPG) and osteopontin (OPN) are bone metabolism biomarkers which are involved in the regulation of vascular calcification processes and prediction of future adverse cardiac events. DESIGN AND METHODS OPG, OPN levels and classic risk factors were determined in 130 asymptomatic and hypertensive subjects. Receiver operator characteristic (ROC) analysis was performed and the area under the curve (AUC) was calculated. RESULTS The hypertensive subjects had elevated OPG, OPN, fibrinogen, CRP and fasting glucose levels in comparison to the normotensive ones. There were significant correlations between age, CRP and OPG. Multiple regression analysis showed that as well as inflammation (CRP), age and hypertension were predictors of increased OPG levels. OPN increase was correlated with CRP and glucose levels. The AUCs were similar for OPG and OPG biomarkers. CONCLUSIONS Plasma OPG and OPN levels were significantly associated with inflammation and arterial hypertension. They might be useful as additional biomarkers for monitoring endothelial dysfunction and prognosis of cardiovascular diseases.

Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma.

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

BackgroundThe 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.ResultsHuman 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.ConclusionsThese results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis.

The new face of nucleolin in human melanoma

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin.

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors - integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.

Does glycosylation of lysosomal proteins show age-related changes in rat liver?

We studied the pattern of lectins binding by liver lysosomal proteins from rats between 18 days of gestation and 72 weeks of age. An analysis of the carbohydrate structure was carried out after an electrophoresis and blotting, followed by a very sensitive detection system with highly specific digoxigenin-labelled lectins. The only age-related differences were observed in the reaction with sialic acid--(MAA; Macckia amurensis, SNA; Sambucus nigra) and fucose--(AAA; Aleuria aurantia) specific lectins. Sialylation increased and fucosylation decreased with age. We also observed a specific reaction with Galanthus nivalis (GNA), Phaseolus vulgaris (PHA-L) and peanut agglutinin (PNA), without any significant changes with age.

Is oestrogen an important player in melanoma progression

The oestrogen-dependent regulation of cell behaviour is realised by stimulation of specific oestrogen receptors. The classical oestrogen receptors ERα and ERβ are transcription factors, and they modulate expression of hormonally regulated genes, while the third one, GPER, is thought to be responsible for the observed rapid, non-genomic cellular response. Oestrogen dependency is attributed to a number of cancers, including breast, ovarian and endometrial cancer; however, there is still growing evidence that melanoma should also be cited as a hormonally dependent tumour. This comes from the observations of gender-related differences in melanoma progression and reports concerning the history of the malignant course of melanomas during pregnancy. Although, the observations of oestrogen regulation of melanoma progression are controversial, the effect of oestrogen should not be neglected, as the skin possesses its own hormonal microenvironment. This aspect of melanoma progression should be taken under careful consideration as it may offer new therapeutic possibilities.

Deciphering the role of ectosomes in cancer development and progression: focus on the proteome

Ectosomes are small heterogeneous membrane vesicles generated by budding from the plasma membrane in a variety of cell types and, more frequently, in tumor cells. They are shed into the extracellular space and are proposed as a novel form of intracellular communication in which information is transmitted from the originating cell to recipient cells without direct cell-to-cell contact. This review focuses on a single population of extracellular vesicles—ectosomes. We summarize recent studies of tumor-derived ectosomes which examine their biogenesis and protein cargo, and their influence on different aspects of cancer progression. We discuss possible clinical implications involving ectosomes as potential biomarkers, diagnostic tools and treatment targets in oncology. The unique composition of the molecules (cargo) that ectosomes carry, and their functional role, depends largely on the state of their originating cell. Through horizontal transfer of a variety of biologically active molecules (including proteins, lipids and nucleic acids) between donor and recipient cells, tumor-derived ectosomes may play functional roles in oncogenic transformation, tumor progression, invasion, metastasis, angiogenesis promotion, escape from immune surveillance, and drug resistance, thereby facilitating disease progression. The presence of tumor-derived ectosomes in body fluids such as the blood and urine of cancer patients makes them potentially useful prognostic and predictive biomarkers. Tumor-derived ectosomes also offer possible targets for multiple therapeutic strategies.

Glycans in melanoma screening. Part 1. The role of β1,6-branched N-linked oligosaccharides in melanoma.

Melanoma, which is one of the most aggressive human tumours, originates from melanin-producing melanocytes. As no effective systemic therapy exists for advanced-stage melanoma, the best chance of recovery remains surgical removal of thin early-stage melanoma. Aberrant glycosylation is a hallmark of malignancy and a well-studied class of β1,6-branched oligosaccharides is associated with malignant transformation of rodent and human cells, and poor prognosis in cancer patients. It is evident that increased β1,6 branching significantly contributes to the phenotype of melanoma cells, influencing the adhesion to extracellular matrix components and motility as well as invasive and metastatic potential. Despite the considerable success in establishing the role of β1,6-branched N-linked oligosaccharides in melanoma biology, there is virtually no progress in using these glycans as a screening tool for the early diagnosis of the disease, or a target-specific therapeutic agent.