The investigation of the changes in the surface glycoconjugates using two different spheroid models of breast cancer cells and availability assessment of these spheroid models for rapid diagnosis

The importance of early cancer diagnosis has led to development of many different diagnostic methods. In this context, the studies investigating the presence and amount of sugar residues to use as indicators in the identification of cancer cell type have become prominent. In the present study, sialic acids found on the membrane surfaces of ER (+) MCF-7 and ER (-) MDA-MB-231 breast cancer cell lines were labeled using three-dimensional (3D) cell culture (Spheroid) model as the closest method to the patient sample, thus its natural environment, among the cell culture methods. These sugar units that play a role in regulation of important immune characteristics such as recognition, binding and metastasis were made visualizable by applying fluorescent-labeled lectins such as FITC-(Wheat Germ Agglutinin) specifically binding to sialic acid units (GlcNAc, Neu5Ac) including particularly ß-GlcNAc and FITC-(Maackia Amurensis-Lectin-1) specifically binding to Galß4GlcNAc type sialic acids. These glycan units were specifically labeled with FITC-(Maackia Amurensis-Lectin-1) and FITC- (Wheat Germ Agglutinin) and radiation intensities were analyzed relatively. The two different breast cancer cell cultures were compared with respect to change in the amounts of sialic acid residues containing α-2,3- and α-2,6 bonds using fluorescent-labeled lectins. In the present study, we have performed a precise, accurate and rapid determination of the sugar content in the different breast cancer cell surface lines by means of fluorescent-labeled lectins and carried out a relative comparison between the micrographs.

Increase of MAL-II Binding Alpha2,3-Sialylated Glycan Is Associated with 5-FU Resistance and Short Survival of Cholangiocarcinoma Patients

Background and objectives: Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA). Materials and Methods: The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays. Results: MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA. Conclusions: Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Distribution of Sialoglycoconjugates in the Rat Cerebellum and Its Change with Aging

We examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, Maackia amurensis lectin (MAL) for Siaα2-3Gal and Sambucus sieboldiana agglutinin (SSA) for Siaα2-6Gal. Each lectin showed characteristic staining patterns. In young adult rats, MAL stained a strongly granular layer, a weakly molecular layer, and the medullary lamina, while SSA more strongly stained the medullary lamina than the molecular and granular layers. After aging, different staining patterns were obtained. Intense SSA reactivity was observed in the granular layer and intense MAL reactivity was observed in the medullary lamina of the aged groups. The reactivity of Purkinje cells with MAL was downregulated in the aged rats. These results indicated that Siaα2-3Gal and Siaα2-6Gal were expressed in distinct regions of the rat cerebellum and that their expression patterns changed in the aged brain.

Location of Sialoglycoconjugates Containing the Siaα2–3Gal and Siaα2–6Gal Groups in the Rat Hippocampus and the Effect of Aging on Their Expression

The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Siaα2–3Gal, and Sambucus sieboldiana agglutinin, specific for Siaα2–6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Siaα2–3Gal and Siaα2–6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum moleculare. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Siaα2–3Gal and Siaα2–6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain.