Effects of Oseltamivir Treatment in the Biological Behaviour and Glycosylation Pattern of Mammary Tumour Cell Lines

ANA LÍDIA ALVES DOS SANTOS EFFECTS OF OSELTAMIVIR TREATMENT IN THE BIOLOGICAL BEHAVIOUR AND GLYCOSYLATION PATTERN OF MAMMARY TUMOUR CELL LINES Dissertação de candidatura ao grau de Mestre em Oncologia - Especialização em Oncologia Molecular submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientadora - Professora Doutora Joana Tavares de Oliveira Categoria - Professora Auxiliar Afiliação - Faculdade de Medicina Veterinária da Universidade Lusófona de Humanidades e Tecnologias. Co-orientadora – Professora Doutora Maria de Fátima Moutinho Gärtner Categoria - Professora catedrática Afiliação - Instituto de Ciências Biomédicas Abel Salazar Universidade do Porto. 2 3 4 5 ACKNOWLEDGMENTS I would like to sincerely thank to: Professor Joana de Oliveira and Professor Fátima Gärtner, for having accepted me and for all the support during this past year. Professor Manuel Sobrinho-Simões, head of IPATIMUP, for hosting me for my thesis work. Professor Celso Reis, for having accepted me in the Glycobiology in Cancer group. To all the members of Glycobiology in Cancer and Differentiation in Cancer, two nice groups of which I’m grateful to be part of. Particularly to Catarina, for all the strength and support she gave me. To all of my friends, for making me know that friends are the family we choose. Finally, the most important and deepest thank. To my parents, my brother and my godmother, thank you for making of me all that I am. 6 7 8 9 ABREVIATTIONS IARC, International Agency for Research on Cancer TMC, tumores mamários de cadela TMMC, tumores mamários malignos de cadela CMT, canine mammary tumours CMMT, canine mammary malignant tumour TACA, tumour-associated carbohydrate antigens T antigen, Thomsen-Friedenreich antigen sT antigen, sialylated form of T antigen / sialylated T antigen Tn antigen, Thomsen-nouvelle antigen sTn antigen, sialylated form of Tn antigen / sialylated Tn antigen sLex antigen, sialyl-Lewis antigen x sLea antigen, sialyl-Lewis antigen a Lea antigen, Lewis antigen a Leb antigen, Lewis antigen b Lex antigen, Lewis antigen x Ley antigen, Lewis antigen y MUC1, Mucin-1 ST6Gal-I, Beta-galactoside alpha-2,6-sialyltransferase 1 Mgat5, Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A SNA, Sambucus nigra agglutinin MAL / MAL-I / MAA / MAA-1, Maackia amurensis leukoagglutinin MAH / MAL-II / MAA / MAA-2, Maackia amurensis hemagglutinin Neu1, neuraminidase/sialidase 1 Neu2, neuraminidase/sialidase 2 Neu3, neuraminidase/sialidase 3 Neu4, neuraminidase/sialidase 4 EGFR, epidermal growth factor receptor EGF, epidermal growth factor GM3, monosialoganglioside GD3, trisialoganglioside LPS, lipopolysaccharide PTNM, Pathological Tumour-Node Metastasis IDC, invasive ductal carcinomas 16 17 INTRODUCTION 18 19 Canine mammary tumours Canine mammary tumours (CMT) are the second most common type of canine neoplasia (25-50% of all diagnosed cases) (Moulton 1990). CMT are the most frequent type of female dog tumours being up to 53% of all tumours (Rutteman 2001). CMT are mainly diagnosed in elderly females, being relatively rare before 2 to 4 years of age and presenting a higher incidence between 10 and 11 years of age (Schneider 1970); (Johnston 1993). Mammary tumour initiation and progression are influenced by similar factors in both human and canine cancers, including age, nutrition, gender, reproductive status and environmental exposure (Lana 2007). In addition, several major aspects of metastasisassociated gene expression are also similar between human and canine mammary tumours (Rutteman 2001, Lindblad-Toh et al., 2005). However, the incidence of mammary tumours is 3 times higher in female dogs than in women and the time course of the disease much shorter, approximately 2 years (Sorenmo 2003). As such, it is an excellent comparative model to understand various aspects of not only breast carcinogenesis but also and perhaps most importantly metastasis in women. Also, the relatively rapid cancer progression rates, compared with those in humans, provide the opportunity to observe therapeutic effects in a shorter period of time than trials conducted in human patients (Leahy et al., 2006). In addition, a great advantage of spontaneous tumours is the crosssectional value of genetic diversity background provided, which similar to that seen in human populations as opposed to experimental animal studies (Lindblad-Toh et al., 2005). Thus, the pet dog might support the transition between mouse models and human patients (Paoloni et al., 2008). Human breast cancer Cancer is a major public health problem in the world. Breast cancer is one of the most prevalent cancers (Figure 1), and metastatic breast cancer accounts for the highest number of cancer-related deaths among women worldwide. In 2008, breast cancer incidence was higher in Western Europe, Australia/New Zealand, Northern Europe and Northern America (Ferlay et al., 2010). Currently, about 1 in 3 women in the United States develop cancer in her lifetime (Siegel et al., 2012), and it is estimated that there are nearly 3 million people with a invasive breast cancer history. Overall, 60% of these breast cancer cases are diagnosed 20 in an early stage (Howlader et al., 2011). Despite more than 1.38 million of new cases diagnosed each year and over 458.000 deaths recorded, our knowledge on systemic cancer cell dissemination is scarce (Ferlay et al., 2010). Figure1. Incidence and mortality rates of the different types of cancer affecting women worldwide, in 2008. Breast cancer presented both the higher incidence and mortality rates in women worldwide. (Data from GLOBOCAN - IARC). Glycosylation Glycosylation is one of the most important modifications in proteins and lipids, originating several glycoconjugates: glycoproteins, glycosaminoglycans, proteoglycans and glycolipids. Vital interactions between cells and the microenvironment surrounding them are mediated by the glycocalyx, a glycan layer which covers the external cell surface (Malagolini et al., 2009). In addition, cellular glycosylation has also been shown to coordinate key biological processes including cell-cell communication, signal transduction, protein folding and stability (Varki 1993); (Dwek 1995); (Wells et al., 2001). In proteins, oligosaccharide chains can be attached to the peptide backbone via two chief types of linkages: (a) aglycosidic bond of N-acetylgalactosamine (GalNAc) to the hydroxyl group of threonine or serine on the polypeptide chain, the O-linked glycans which are predominantly membrane bound or secreted on proteins; (b) a glycosidic bond of N- 21 acetylglucosamine (GlcNAc) to asparagine occurring on the consensus sequence Asn-XSer/Thr of a protein chain, the to N-linked glycan chains (Opdenakker et al., 1993). Mammalian cells have a specific enzymatic machinery that includes glycosyltransferases and glycosidases, which is required for biosynthesis of these diverse glycan structures, in the endoplasmic reticulum and Golgi apparatus. The glycan structures found are further dependent on the cell type, developmental stage and cell differentiation, and are fundamentally altered in many pathologic states, including cancer (Opdenakker et al., 1993). In cancer, glycosylation changes have been often associated to a deregulation of glycosyltransferase genes expression. Glycosyltransferase genes ST6Gal-I (Nacetyllactosaminide α-2,6 sialyltransferase) and Mgat5 (Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A) are, for instance, regulated by oncogenes. However more recently, a role played by glycosidases such as sialidases in neoplasia has been increasingly recognized. Sialidases Sialidases are enzymes that exist widely in vertebrates and in a variety of microorganisms (viruses, bacteria, fungi, mycoplasma and protozoa) with evidence of great importance in various cellular functions (Schengrund et al., 1976); (Corfield et al., 1981); (Corfield et al., 1982); (Saito et al., 1995). The enzymes also are present in a large variety of cells and tissues from mammalians (Carubelli et al., 1962). Four types of mammalian sialidases have been identified and characterized to date, and designated as Neu1, Neu2, Neu3 and Neu4. The first three were classically described to localize predominantly in the lysosomes, cytosol and plasma membranes (Miyagi et al., 1999) respectively, and Neu4, the most recently identified sialidase, is often found in lysosomes, mitochondria and the endoplasmic reticulum (Roggentin et al., 1989); (Saito et al., 1996). However, their subcellular localization can vary with particular cell stimuli. Neu1, Neu2 and Neu4 show mobilization to the cell surface in certanin conditions (Saito et al., 1996). The overall amino acid identity between the four sialidases is variable. Amino acid identity between Neu1 and the other sialidases is relatively low (19-24%), while Neu2, Neu3 and Neu4 present a higher 34-40% homology between each other. Regarding comparative expression levels of sialidases in different human tissues, Neu1 usually presents the highest expression, on the opposite to Neu2, which presents extremely low expression levels (Yamaguchi et al., 2006). Sialidases have been implicated in lysosomal catabolism, and regulation of important cellular functions such as cell differentiation, cell growth and apoptosis (Thomas et al., 2001); (Miyagi et al., 2012). 22 The function of each sialidase is linked to its glycan specificity. Neu1 hydrolyzes glycoproteins and in vitro sialidase activity assays showed that Neu1 reacts with high specificity with oligosaccharides and glycopeptides (Saito et al., 1996). Moereover, it acts preferentially on oligosaccharide substrates with α2-3 sialyl linkage when comparing to those with α2-6 (Miyagi et al., 1984). Neu2 hydrolyzes native glycoproteins, oligosaccharides and also gangliosides (Miyagi et al., 1985). It acts in the cytosol preferentially on α2-8 sialyl linkages (Miyagi et al., 1985). Despite glycoconjugates being not usually present in the cytosol (Ishizuka et al., 2008), glycoproteins, oligosaccharides, gangliosides, glycosidases and lectins have been reported to occur in cytosol (Funakoshi et al., 2009). Neu3, hydrolyzes specifically gangliosides with α2-3 (GM3, monosialoganglioside) and α2-8 (GD3, trisialoganglioside) and α2-6 linkages (synthetic GM3) (Hata et al., 1998). The murine enzyme acts on oligosaccharides and glycoproteins to a certain extent (Li et al., 2001). Neu4 it is the only sialidase known toacts on mucins with high efficiency (Shiozaki et al., 2011). NEU4 was found to in vitro hydrolyze sLea and sLex antigens and to decrease their cell surface levels much more effectively than other sialidases (Shiozaki et al., 2011). Sialidases and cancer It is known that alterations in glycosylation such as increased sialylation are a common feature of malignancy (Reis et al., 2010). Sialic acids are terminal acidic monosaccharides usually found in the terminal position of the carbohydrate groups of glycoproteins and glycolipids. Their removal catalysed by sialidases may affect the conformation of glycoproteins, and therefore contribute to recognition or masking of biological sites in molecules and cells (Yogeeswaran et al., 1981); (Dennis et al., 1987); (Schauer 2000). This may influence the malignant phenotype of cancer cells, including the metastatic potential and invasiveness (Hakomori 2002). Sialidases have been found to be differentially expressed in several types of cancer, specifically in highly metastatic cells (Miyagi et al., 2008). NEU1 expression is decreased in several types of cancer. Its expression levels present an inverse relation with the metastatic cancer capacity. In different clones of mouse colon adenocarcinoma cells, lower expression of NEU1 sialidase where seen in those with higher metastatic rates when comparing to less invasive ones. There were concomitantly higher levels of sialyl Lex and GM3 in these cells (Sawada et al., 2002). Furthermore, transfection of Neu1 sialidase into mouse melanoma cells resulted in suppression of experimental pulmonary metastasis due to concomitant reduction in 23 anchorage-independent growth and increased sensitivity to apoptosis (Kato et al., 2001). Overexpression of human NEU1 with the PPCA gene resulted in similar alterations in human colonic adenocarcinoma cells with suppressed cell migration and invasion being shown. On the other hand, Neu1 knockdown resulted in the opposed effect (Galjart et al., 1988); (D'Azzo et al., 1982). NEU1-expressing cells present reduced in vivo liver metastatic potential in mice (Uemura et al., 2009). Regarding NEU2, its up-regulation also affects cancer cell behaviour. Transfection of rat gene Neu2 into highly invasive and metastatic B16-BL6 mouse melanoma cells, lead to a decrease in pulmonary metastasis, possibly related to the GM3 ganglioside decrease (Tokuyama et al., 1997). Within addition, highly metastatic mouse colon adenocarcinoma cells transfected with Neu2 showed a marked reduction in lung metastasis, invasion and cell motility, with concomitant decrease in sLex and GM3 levels. This was suggested to be related with changes in cell adhesion and/or cell motility (Sawada et al., 2002), pointing to desialylation of these and other molecules, as targets of sialidase, involvement in the suppression of metastasis. Furthermore, a human epidermoid carcinoma cell line transfected with Neu2 gene, showed reduced GM3 levels, and a concomitantly increased cell growth and tyrosine autophosphorylation of epidermal growth factor receptors (EGFR) at low EGF (epidermal growth factor)concentration levels (Meuillet et al., 1999). Moreover, human NEU2 overexpressing leukemic cells presented marked decrease in anti-apoptotic factors Bcl-XL and Bcl-2, resulting in increased sensitivity to apoptotic stimuli (Tringali et al., 2007). NEU2 overexpression in the cells reduced gene expression and activity of Bcr-Abl, together with a decrease in Bcr-Abl dependent Src and Lyn kinase activity probably through desialylation of cytosolic glycoproteins. Neu3 is up-regulated in several types of human cancer, such as colon, renal, ovarian and prostate cancers. However, Neu3 down-regulation was also observed in acute lymphoblastic leukemia and associated to disease progression (Mandal et al., 2010). NEU3 mRNA levels were found to be 3–100-fold increased in human colon cancers, compared with adjacent normal mucosa (Kakugawa et al., 2002). Furthermore, in renal cell carcinomas NEU3 mRNA levels were observed to be significantly increased (Ueno et al., 2006). In the case of prostate cancer, Neu3 is also up-regulated and it’s correlated with malignancy (Kawamura et al., 2012). Prostate cancer cells revealed a significant decrease in invasion and migration capacity after NEU3 knockdown in vitro.NEU3 activates molecules such as EGFR, FAK, ILK, Shc and integrin β4, frequently up-regulated in carcinogenesis, being possibly the reason for the development of a malignant phenotype. In addition, this sialidase was found to increase azoxymethaneinduced aberrant crypt foci formation in colon mucosa by suppression of apoptosis 24 possibly due to the activation of EGF signalling. NEU3 is thus involved in the regulation of transmembrane signalling at the cell surface through both modulation of gangliosides as the result of enzyme reactions and by interaction with other signal molecules, including caveolin-1, Rac-1, integrin β4, Grb-2 and EGFR (Miyagi et al., 2008). In what regardsNEU4 sialidase, its levels are markedly decreased in human colon cancer (Yamanami et al., 2007). In addition, human colon adenocarcinoma cell lines transfected with NEU4 showed increased apoptosis and decreased invasiveness capacity and cellular motility (Shiozaki et al., 2011). To elucidate the significance of NEU4 downregulation in colon cancer, sialyl-Lewis antigens, sLea and sLex, were investigated, and Neu4 was found to hydrolyze these antigens in vitro and decrease their cell surface levels farly more than the other sialidases (Shiozaki et al., 2011). These multiple features suggest that each type of sialidase may play a unique role according to its properties. The role of sialidases in cancer is probably closely related to the available substrate which poses several therapeutic implications. Sialylation and cancer A general increase in sialylation is often found in cell surface glycoproteins of malignant cells (Neufeld et al., 2001). Altered sialylation of glycolipids is also observed to be a ubiquitous phenotype (Achyuthan et al., 2001). In fact, tumour-associated antigens are often sialylated. Tn antigen is the precursor of the T antigen, also known as Core 1. The Tn antigen does not occur in abundance in normal cells and tissues of adult animals, however it can be detected in almost all kinds of carcinomas (Julien et al., 2001). Almost 90% of breast cancers express Tn antigen while it is scarcely detected in normal mammary tissue (Springer 1997). This antigen was found to be associated to with the Pathological Tumour-Node Metastasis (PTNM staging) tumour stage in Invasive Ductal Carcinoma of the breast (Wang et al., 1997). Tn antigen can be sialylated at the C6 position of GalNAc residue, resulting in the dissacharide Neu5Acα2-6GalNAc-R, sTn antigen (sialylated Tn antigen). sTn is suggested to have an important role in carcinogenesis. Its expression is rarely observed in normal tissues but highly expressed in most several carcinomas such as gastric (David et al., 1992); (Victorzon et al., 1996); (Baldus et al., 2000), colorectal (Itzkowitz et al., 1990), ovarian (Kobayashi et al., 1992), breast (Leivonen et al., 2001); (Yonezawa et al., 1992), and pancreatic (Kim et al., 2002). Thus, sTn has been associated with carcinoma aggressiveness and poor prognosis. 25 T antigen is an oncofetal glycan antigen. It is originated by a non-elongated form of Core 1. In normal epithelium, T antigen is masked by sialic acids, sulphates or by addition of other sugar chains to form branched and complex O-glycans (Springer 1984); (Hanisch et al., 1997). T antigen is a pancarcinoma antigen expressed in several types of cancer including breast (Kumar et al., 2005), colon (Baldus et al., 2000), bladder (Coon et al., 1982), prostate (Janssen et al., 1996), liver (Cao et al., 1996), ovary (Ghazizadeh et al., 1990) and stomach (Baldus et al., 2001), suggesting that changes in O-glycosylation provide some advantage to tumour development (Cazet et al., 2010). In vitro and in vivo studies show that T antigen facilitates metastases by binding galectin-3 which clusters MUC1 mucin at the cell surface thereby facilitating not only heterotypic adhesion between tumour and endothelial cells (Yu et al., 2007), but also homotypic adhesion between tumour cells essential for their survival in the blood stream (Zhao et al., 2010). Inhibition of galectin-3 with T antigen interaction was shown to reduce metastatic capacity (Glinskii et al., 2012). Core 1 was found to be mostly sialylated (sT antigen) in CMT, however its nonsialylated form (T antigen) is expressed in tumour emboli (de Oliveira et al., 2011). The histoblood group Lewis antigens are found in most human epithelial tissues, where they are expressed at the terminal part of glycolipid and glycoprotein carbohydrate chains (Ravn et al., 2000). These antigens derive from the substitution of type 1 (Galβ13GlcNAc) or type 2 (Galβ1-4GlcNAc) dissacharide sequences by fucose and sialic acid residues. Lea, Leb and sLea antigens derive from type 1 sequences, and Lex Ley and sLex antigens derive from type 2 (Cazet et al., 2010). In a normal context, sLex antigen expression is restricted to the immune system cells contributing to leukocyte function in the inflammatory response event via interaction with E-selectin expressed on endothelial cells (Cazet et al., 2010). On the other hand, sialyl-Lewis antigens are usually altered in cancer cells and show a good correlation with the metastatic risk in breast cancer patients. sLea and sLex antigens expression are found to be increased in breast cancer tissues, including primary breast carcinoma lesions (Renkonen et al., 1997). Levels of expression of sLex antigen are higher in breast cancer patients who had distant metastasis, when compared with patients presenting non-metastatic lesions (Matsuura et al., 1997); (Jeschke et al., 2005). Sialidase inhibition Oseltamivir phosphate (Tamiflu®, Roche) is an effective sialidase inhibitor, extensively used as an anti-influenza virus drug. It is a sialic acid analogue which interacts with the active sites of the influenza sialidase enzymes. Once in the liver, after oral 32 Cell viability assay Cell viability was determined using a commercial available kit CellTiter 96® AQueous One Solution reagent (Promega Corporation, U. S. A.), and performed according to manufacturer’s instructions. Briefly, cells were plated in triplicate in a 96 wells plate (Orange Scientific, Belgium), with a density of 5x103 cells per well. After cell adherence to the wells, oseltamivir was added in 125 ng/ml, 1.25 µg/ml, 12.5 µg/ml and 125 µg/ml oseltamivir concentrations, and PBS was used as control. Cell viability was measured by adding MTS tetrazolium reagent and absorbance was recorded at 490nm 2 hours after reagent addition. The assays were performed in triplicate or both CMA07 and CMT-U27 cell lines, during 48 hours, with time-points at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hours. An additional control measurement was performed at time-point 0, in a culture well without cells. Wound-healing The wound-healing assay was performed in a time-lapse microscope, and wound image acquisition was done with 5 minutes intervals during 48 hours, using the program Axio Vision Release 4.8.2. and converted in video. Briefly, 20x104 cells were plated into a 24 wells culture plate (Falcon by Becton Dickinson Labware, U. S. A.) and after reaching high confluence an artificial "wound" was made with a pipette tip. Culture medium was replaced with the different oseltamivir doses: 125 ng/ml, 1.25 µg/mL and 12.5 µg/ml oseltamivir and PBS as control. The migration was evaluated considering the “healing capacity” of cells during 48 hours. Matrigel Invasion Assay A matrigel invasion assay was performed to evaluate the invasive capacity of CMT-U27 cells. Briefly, inserts were re-hydrated with RPMI 1640 and maintained for 1 hour at 37º in a humidified 5% CO2 incubator (Thermo Scientific, U.S.A.) to complete the hydration process. After insert rehydration, 1x105 cells were seeded on Matrigel-coated chambers in the presence of different oseltamivir doses (125 ng/ml, 1.25 µg/ml, 12.5 µg/ml oseltamivir), and PBS as control, and cultured during 6 hours. After that, the content of each insert was removed and washed twice with PBS. Then, invasive cells were fixed with cold methanol for 20 minutes. After fixation, inserts were transferred to slides (Industrial Quality, Germany) and mounted with Vectashield mounting medium with DAPI 33 (Vector Laboratories, U.S.A.). Cell invading capacity was measured by counting the number of cells that passed through the Matrigel-coated filter. Fluorescence Cells were cultured in glass coverslips in 24 wells culture plates, and the culture medium was supplemented with 125 ng/ml, 1.25 µg/ml and 12.5 µg/ml oseltamivir and PBS as control, during 24 hours. After 24 hours of treatment, cells were washed with PBS and fixed with cold methanol for 20 minutes. Following methanol fixation, cells were rehydrated with PBS and then blocked with 10% BSA for 20 min. BSA was replaced with plant lectins SNA (Biotinylated Ederberry bark lectin, B-1305, Vector Laboratories, U.S.A.), MAL I (Biotinylated Maackia amurensis lectin I, B-1315, Vector Laboratories, U.S.A.), and MAL II (Biotinylated Maackia amurensis lectin II, B-1265, Vector Laboratories, U.S.A.) 1:300 diluted in PBS, incubated for 1 hour, at room temperature. For galectin-3-ligands fluorescence, BSA was replaced with biotinylated galectin-3 1:100 diluted in PBS, and slides were incubated for 1 hour, at room temperature. After two washes with PBS, Streptavidin FITC-conjugated was incubated for 1 hour at room temperature in a 1:1000 dilution in PBS, protected from the light. For galectin-3 fluorescence, BSA was replaced with anti-gal-3 antibody, 1:100 diluted in PBS, and slides were incubated overnight, at 4ºC. After two washes with PBS, slides were incubated with FITC-conjugated anti-rat secondary antibody diluted 1:200 in 5% BSA for 1 hour, protected from light, at room temperature. After two washes with PBS, slides were incubated for 10 minutes with DAPI (Sigma-Aldrich, U.S.A.) in PBS and slides were mounted in Vectashield mounting medium for fluorescence (Vector Laboratories, U.S.A.). Slides were analysed and images were taken in a Carl Zeiss fluorescent microscope (Carl Zeiss Microscopy, Germany). Western Blot analysis Cells from CMA07 and CMT-U27 cell lines were grown to confluence in 6 wellplates. Then, oseltamivir was added (125 ng/ml, 1.25 µg/ml and 12.5 µg/ml oseltamivir), and cells were incubated for 24 hours. Then, cells were washed three times with PBS, and 200µl of a pre-prepared mixture of 1000µl of RIPA lysis buffer (50 mM Tris HCl, pH 8; 150 mM NaCl; 1% NP-40; 0,5% sodium desoxicolate; 0,1% SDS) containing 40µl of complete protease inhibitor cocktail (Roche, Switzerland), 10µl of PMSF (phenylmethyl sulfonyl fluoride), and 10µl Na3VO4 (sodium orthovanadate) were added to each well. 34 After 10 minutes incubation, cells were scrapped, collected to tubes and centrifuged (Eppendorf AG, Germany) at 13000 rpm, for 10 minutes, at 4°C. After centrifugation, supernatants were collected and protein amount was quantified using the biocinchoninic acid method, Pierce™ BCA Protein Assay Kit (Pierce/ Thermo Scientific, U.S.A.), according to the manufacturer’s instructions. The total extracts were boiled for 5 min at 95°C in Laemmli sampling buffer, and runned in 10% SDS-PAGE. After electrophoresis, gels were transferred to a nitrocellulose membrane (Amersham Biosciences/GE Healthcare Life Sciences, U.K.) and incubated with lectins: Biotinylated Maackia amurensis lectin I (B-1315, Vector Laboratories, U.S.A.), Biotinylated Maackia amurensis lectin II (B-1265, Vector Laboratories, U.S.A.) and Biotinylated Ederberry bark lectin, SNA (B-1305, Vector Laboratories, U.S.A.) diluted 1:500 in 5% BSA (Sigma-Aldrich, U.S.A.) in 1x PBS with 0.05% Tween-20 (Sigma-Aldrich, U.S.A.). After lectins incubation, three washes with PBS 0,05% Tween-20 were performed and membranes were incubated with avidin-biotin complex kit (Vectastain ABC kit Standard, Vector Laboratories, U.S.A.) for 1 hour at room temperature. For galectin-3ligand analysis, membranes were incubated with galectin-3 biotinylated 1:200 diluted. After three washes with PBS 0,05% Tween-20 were performed, membranes were incubated with avidin-biotin complex kit for 1 hour at room temperature. For galectin-3 analysis membranes were incubated with anti-galectin-3 antibody 1:200 diluted (eBioscience, San Diego, CA) followed by HRP-conjugated anti-rat secondary antibody for 1 hour incubation. Analysis were done by chemiluminescence using the ECL Western blotting detection reagent and films (both from GE Healthcare, U.K.). Western blot for actin diluted 1:4000 (Santa Cruz Biotechnology) was used as loading control. Statistical analysis Whenever adequate, the results were presented as mean ± standard deviation. Statistical analysis was performed using One Way ANOVA (Analysis of variance) test and for multiple comparisons Dunnett and Tukey’s tests with p<0,05 as the level of significance, in GraphPad Prism 5.02 version. 35 RESULTS 36 37 CMT cell morphology assessment in the absence and presence of oseltamivir Cell morphology assay was performed in CMA07 and CMT-U27 cell lines during 7 days in order to determine if oseltamivir leads to morphologic alterations of cells. Different concentrations of oseltamivir were tested, 125 ng/mL, 1.25 µg/mL, 12.5 µg/mL, and PBS was used as control. Cell morphology was observed in an inverted microscope in days 1, 3 and 7, and photographs were taken. No differences in cell clustering were observed in both CMA 07 and CMTU27 cells in the different doses of oseltamivir, when compared with control cells. CMA07 and CMT-U27 cell lines showed no differences in cell morphology for both cell lines with the different oseltamivir doses, when compared to non-treated cells (Figure 1). Figure 1. Evaluation of CMA07 and CMT-U27 cell morphology upon oseltamivir treatment. Morphology of cells was evaluated after oseltamivir addition. Compared with control cells (A, I respectively, x4 magnification), no differences in cell clustering were observed in both CMA 07 (B – D, x4 magnification) and CMTU27 cells (J – L, x4 magnification) in the different doses of oseltamivir. Compared with non-treated cells (E, M x20 magnification), no differences were observed in both CMA07 (F-H, x20 magnification) and CMTU27 cells (N-P, x20 magnification) in the different doses of oseltamivir. 38 In day 3, CMA07 cells treated with 1.25 and 12.5 μg/mL oseltamivir presented less cell clustering, compared with non-treated and 125 ng/mL oseltamivir treated cells. Concerning CMT-U27 cells treated with 12.5 ng/mL oseltamivir, less cell clustering was observed, compared with non-treated, 125 ng/mL oseltamivir, and 1.25 ng/mL oseltamivir treated cells. CMA07 cells showed no alterations concerning cell morphology in the different oseltamivir doses, when compared to non-treated cells. However, CMT-U27 cells treated with 12.5 µg/mL oseltamivir present larger and irregular cytoplasm and protrusions, when compared to non-treated cells (Figure 2). Figure 2. Evaluation of CMA07 and CMT-U27 cell morphology three days after oseltamivir treatment. After three days of oseltamivir treatment, cell morphology was evaluated. Compared with non-treated and 125 ng/mL oseltamivir treated cells (A, B, x4 magnification),CMA07 cells treated with 1.25 and 12.5 μg/mL oseltamivir (C, D, x4 magnification) presented less cell clustering. Compared with non-treated, 125 ng/mL oseltamivir, and 1.25 ng/mL oseltamivir treated cells (I - K, x4 magnification), CMT-U27 cells treated with 12.5 ng/mL oseltamivir (L, x4 magnification) showed less cell clustering. Compared with non-treated cells (E, x20 magnification), CMA07 cells treated with oseltamivir (F - H, x20 magnification) showed larger and irregular cytoplasm, and protrusions. Compared with non-treated cells (M, x20 magnification), CMT-U27 cells treated with 12.5 μg/mL oseltamivir (P, x20 magnification) showed larger and irregular cytoplasm, and protrusions. 39 In day 7, no differences in cell clustering were observed in both CMA 07 and CMTU27 cells in the different doses of oseltamivir, when compared with control cells. Differences in CMA07 cell morphology were observed in both 125 ng/mL and 12.5 µg/mL oseltamivir doses, presenting more round and shiny cells, when compared to non-treated cells. Concerning CMT-U27 cell line, no differences were observed in cell morphology in the different oseltamivir doses, when compared with non-treated cells (Figure 3). Figure 3. Evaluation of CMA07 and CMT-U27 cell morphology seven days after oseltamivir treatment. Compared with control cells (A and I respectively, x4 magnification), no differences in cell clustering were observed in both CMA 07 (B – D, x4 magnification) and CMTU27 cells (J – L, x4 magnification) in the different doses of oseltamivir. After seven days of oseltamivir treatment, compared to non-treated cells (E, x20 magnification), differences in CMA07 cell morphology where observed in both 125 ng/mL (F, x20 magnification) and 12.5 μg/mL oseltamivir treatment (H, x20 magnification), presenting more round and shiny cells. Compared with non-treated cells (M, x20 magnification), CMT-U27 cells treated with treated with oseltamivir (N-P, x20 magnification) showed no differences in cell morphology. 40 Cell proliferation assay in CMT cell lines in the absence and presence of oseltamivir In this study, cell proliferation assay was performed both with CMA07 and CMTU27 cell lines, using oseltamivir in order to determine if cell proliferation is affected by its presence. Oseltamivir was tested in different doses (125 ng/mL, 1.25 µg/mL, 12.5 µg/ml and 125 µg/mL oseltamivir) and PBS was added to the non-treated cells group. Cell growth was monitored during 7 days by counting cells every day (Figure 4). Proliferation rates of CMA07 and CMT-U27 cell lines were not significantly affected by treatment with different oseltamivir doses, when compared to the proliferation curve of non-treated cells. However, an exception was observed for the highest dose tested, 125 µg/mL of oseltamivir, which seemed to impair CMA07 cell growth, though with no statistical significance was found when compared to non-treated cells. Figure 4. In vitro proliferation analysis of CMA07 and CMT-U27 cell lines upon oseltamivir treatment. Cells were cultured in 24-well dishes in the presence of different oseltamivir doses, and cell growth was monitored during 7 days by counting cells every single day. CMA07 and CMT-U27 cell lines in the presence of 41 different doses of oseltamivir did not present significantly different proliferation rates when compared with nontreated cells. The highest dose administered, 125 μg/mL oseltamivir, impairs CMA07 cell growth (although with no statistical significant difference, ns p = 0.3492). Cell viability assay of CMT cell lines in the absence and presence of oseltamivir Cell viability assay performed with the CellTiter 96® AQueous One Solution reagent (Promega Corporation, U. S. A.) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The MTS tetrazolium compound in the reagent is bioreduced by cells into a colored formazan product and analysed after recording the absorbance at 490nm. The quantity of formazan product as measured by the absorbance at 490nm is directly proportional to the number of living cells in culture. With the purpose of evaluate the effect of oseltamivir treatment in both CMA07 and CMT-U27 cells’ viability, CellTiter 96® AQueous One Solution Cell Proliferation Assay was performed in both CMA 07 and CMTU27 cell lines, during 48 hours, with time-points at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hours (Figure 5). For CMA07 and CMT-U27 cell lines treated with 125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir doses, no significant differences (ns p>0.05) were observed in cells’ viability, when compared to non-treated cells. However, the highest tested dose, 125 µg/ml oseltamivir, severely impaired cell viability both in CMA07, ***p=0.0005, and CMTU27, ****p<0.0001, cell lines, since the number of viable cells decreased over time. For this reason, this dose was excluded from the further studies. 48 Regarding Siaα2-3Galβ1-4GlcNAc terminal structures, their expression in CMA07 and CMT-U27 cell lines was assessed by MAL I lectin labelling (Figure 10). CMA07 cells treated with 12.5 μg/mL oseltamivir showed an increased in Siaα2-3Galβ1-4GlcNAc structures, when compared to non-treated cells. CMT-U27 cells treated with oseltamivir in different doses, showed an increased in Siaα2-3Galβ1-4GlcNAc structures when compared to non-treated cells. Figure 10. Expression of Siaα2-3Galβ1-4GlcNAc terminal structures in CMA07 and CMT-U27 cell lines upon oseltamivir treatment, assessed by MAL I lectin labelling. Compared to non-treated cells (A, x20 magnification), CMA07 cells treated with 12.5 μg/mL oseltamivir (D, x20 magnification) show an increased in Siaα2-3Galβ1-4GlcNAc structures. Compared to non-treated cells (E, x20 magnification), CMT-U27 cells treated with oseltamivir in different doses (F-H, x20 magnification), show an increased in Siaα2-3Galβ14GlcNAc structures. Concerning Siaα2-3Galβ1-3GlcNAc terminal structures, expression in CMA07 and CMT-U27 cell lines was accessed by MAL II lectin labelling (Figure 11). CMA07 cells treated with different doses of oseltamivir did not present alteration in Siaα2-3Galβ13GlcNAc expression, when compared to non-treated cells. CMT-U27 cells treated with 1.25 μg/mL oseltamivir showed increased expression of Siaα2-3Galβ1-3GlcNAc, when compared to non-treated cells. 49 Figure 11. Expression of Siaα2-3Galβ1-3GlcNAc terminal structures in CMA07 and CMT-U27 cell lines upon oseltamivir treatment, assessed by MAL II lectin labelling. Compared to non-treated cells (A, x20 magnification), CMA07 cells treated with different doses of oseltamivir (B-D, x20 magnification) do not present alteration in Siaα2-3Galβ1-3GlcNAc expression. Compared to non-treated cells (E, x20 magnification), CMTU27 cells treated with 1.25 μg/mL oseltamivir (F, x20 magnification) show increased expression of Siaα23Galβ1-3GlcNAc. Evaluation of galectin-3-ligands expression in CMT cell lines in the absence and presence of oseltamivir Fluorescence assay was performed in order to evaluate galectin-3-ligands expression in CMA07 and CMT-U27 cell lines treated with oseltamivir, when compared to non-treated cells (Figure 12). CMA07 cells treated with oseltamivir showed no differences in galectins-3-ligands expression, when compared with control cells. Treatment of CMT-U27 cells with 1.25 μg/mL and 12.5 μg/mL oseltamivir resulted in a decreased expression of galectin-3-ligands when compared to non-treated cells. 50 Figure 12. Evaluation of galectin-3 ligands expression in both CMA07 and CMT-U27 cell lines upon oseltamivir treatment. Compared to non-treated cells (A, x20 magnification), treatments of CMA07 cells (BD, x20 magnification) showed no differences in galectin-3-ligands expression. Compared to non-treated cells (E, x20 magnification), treatment of CMT-U27 cells with 1.25 μg/mL and 12.5 μg/mL oseltamivir (G,H, x20 magnification), show a decreased in expression of galectin-3 ligands. Evaluation of galectin-3 expression in CMT cell lines in the absence and presence of oseltamivir Immunofluorescence assay was performed in order to evaluate galectin-3 expression in CMA07 and CMT-U27 cell lines upon oseltamivir treatment, when compared to non-treated cells (Figure 13). Results showed that galectin-3 is increased in both CMA07 and CMT-U27 cells upon treatment with 1.25 μg/mL and 12.5 μg/mL oseltamivir, when compared to both nontreated cells and to cells treated with 125 ng/mL oseltamivir. Moreover, differences in galectin-3 expression were also observed between the two cell lines, with more intense galectin-3 expression pattern in CMT-U27, when compared with CMA07. 51 Figure 13. Expression of galectin-3 in CMA07 and CMT-U27 cell lines upon oseltamivir treatment. Compared to both non-treated cells (A,E, x20 magnification) and to cells treated with 125ng/mL of oseltamivir (E,F, x20 magnification), there is increased expression of galectin-3 in both CMA07 (C-D, x20 magnification) and CMT-U27 cells (G-H, x20 magnification) upon treatment with 1.25 μg/mL and 12.5 μg/mL oseltamivir. Furthermore, galectin-3 expression pattern in CMT-U27 is more intense when compared with CMA07. Evaluation of terminal α2-6 and 2-3 sialic acid structures expression in proteins from total cell lysates of CMT cell lines in the absence and presence of oseltamivir Expression of terminal α2-6 and α2-3 sialylated structures were evaluated by lectin blot analysis with SNA, MAL I and MAL II lectins. The results demonstrated an increased expression of sialylated proteins upon oseltamivir treatment (Figure 14). Both CMA07 and CMT-U27 cells presented differences in the expression of terminal α2-6 and α2-3 sialic acid structures in some proteins upon oseltamivir treatment. These results demonstrated the effect of oseltamivir in sialidase inhibition, and consequent inhibition of sialic acid cleavage, since it was observed an increased α2-6 and α2-3 sialic acid terminal structures in cells treated with oseltamivir. The results from SNA lectin blot demonstrated an increase in the expression of terminal α2,6 sialic acid structures in proteins from CMT-U27 cells treated with oseltamivir (125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir) when compared to proteins from non-treated cells (PBS), especially in a molecular weight around 120 kDa. Regarding MAL I lectin blot, no differences were observed in α2,3 sialic acid structures in proteins from both CMA07 and CMT-U27 cells treated with oseltamivir (125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir), when compared to proteins from non-treated cells (PBS). The 52 results from MAL II lectin blot demonstrated an increase in terminal α2,3 sialic acid structures expression in proteins from lysates of cells treated with 125 ng/mL, 1.25 µg/mL oseltamivir, in a molecular weight around 120 kDa, when compared to proteins from nontreated cells (PBS) and cells treated with 12.5 µg/mL oseltamivir. 53 Figure 14. Western analysis of SNA, MAL I and MAL II lectins in proteins from total cell lysates of both CMA07 and CMT-U27 cell lines upon oseltamivir treatment. Expression of terminal sialylated structures were evaluated by western blot analysis with SNA (A), MAL I (B) and MAL II (C) plant lectins. The results from SNA lectin blot (A) demonstrated an increase in the expression of terminal α2,6 sialic acid structures in proteins from CMT-U27 cells treated with oseltamivir (125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir) when compared to proteins from non-treated cells (PBS), especially in a molecular weight around 120 kDa. Regarding MAL I lectin blot (B), no differences were observed in α2,3 sialic acid structures in proteins from both CMA07 and CMT-U27 cells treated with oseltamivir (125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir), when compared to proteins from non-treated cells (PBS). The results from MAL II lectin blot (C) CMT-U27 cells demonstrated an increase in terminal α2,3 sialic acid structures expression in proteins from lysates of cells treated with 125 ng/mL, 1.25 µg/mL oseltamivir, in a molecular weight of about 120 kDa, when compared to proteins from lysate correspondent to non-treated cells (PBS) and cells treated with 12.5 µg/mL oseltamivir. 54 Evaluation of galectin-3-ligands expression in proteins from total cell lysates of CMT cell lines in the absence and presence of oseltamivir Proteins from CMA07 and CMT-U27 cell lines treated with oseltamivir were labelled with biotinylated galectin-3 in order to evaluate galectin-3-ligands expression in proteins from total cell lysates of both cell lines (Figure 15). Regarding galectin-3-ligands expression in proteins from CMA07 cell line treated with oseltamivir, no differences were observed for when compared to proteins from nontreated cells (PBS). Concerning galectin-3-ligands expression in proteins from CMT-U27 cell line treated with oseltamivir, we observed a decrease in galectin-3 ligands in proteins when compared to proteins from non-treated cells, especially at the molecular weight around 120 kDa. Figure 15. Western blot analysis of galectin-3-ligands in proteins from total cell lysates of both CMA07 and CMT-U27, upon oseltamivir treatment. Expression of galectin-3-ligands was evaluated by western blot analysis with a biotinylated galectin-3. Regarding galectin-3-ligands expression in proteins from CMA07 cell line treated with oseltamivir, no differences were observed for when compared to proteins from non-treated cells (PBS). Concerning galectin-3-ligands expression in proteins from CMT-U27 cell line treated with oseltamivir, we observed a decrease in galectin-3 ligands in proteins when compared to proteins from nontreated cells, principally at the molecular weight around 120 kDa. 55 Evaluation of galectin-3 expression in proteins from total cell lysates of CMT cell lines in the absence and presence of oseltamivir CMA07 cells treated with oseltamivir showed no differences in galectin-3 expression, when compared to non-treated cells (Figure 16). CMT-U27 cells treated with 1.25 µg/mL and 12.5 µg/mL oseltamivir treatments, showed increased galectin-3 expression, when compared to non-treated cells. These results are in accordance with those observed for galectin-3 immunofluorescence. Figure 16. Western blot analysis of galectin-3 in proteins from total cell lysates of both CMA07 and CMT-U27, upon oseltamivir treatment. CMA07 cells treated with oseltamivir, showed no differences in galectin-3 expression, when compared to non-treated cells. CMT-U27 cells treated with 1.25 µg/mL and 12.5 µg/mL oseltamivir treatments, showed increased galectin-3 expression, when compared to non-treated cells. 56 57 DISCUSSION 64 kDa. Regarding the results of western blot analysis with MAL I and MAL II lectins, differences in the terminal α2,3 sialic acid structures were observed in few proteins from MAL II blot in CMT-U27 cells treated with oseltamivir. Apparently, MAL II showed increased protein recognition in the same molecular weight of proteins that had increased terminal α2,6 sialic acid structures in SNA blot. In addition, and regarding galectin-3ligands expression in proteins from CMT-U27 cells upon oseltamivir treatment, it was observed a decrease in galectin-3-ligands in proteins in a molecular weight around 120 kDa. This observation strengthened the result of the western blot analysis with SNA lectin that demonstrated an increase in terminal α2,6 sialic acid structures expression in proteins from CMT-U27 cells treated with oseltamivir (125 ng/mL, 1.25 µg/mL and 12.5 µg/mL oseltamivir), more specifically, in a molecular weight a around 120 kDa. We conclude that, when we have increase in terminal α 2,6 sialic acid structures, galectin-3-ligands decrease, suggesting a masking of these ligands by α2,6 sialylated structures. This observation was reinforced by our fluorescence results where we observed a decrease in galectin-3-ligands in CMT-U27 cells treated with 1.25 μg/mL and 12.5 μg/mL oseltamivir with a concomitant increase expression of terminal α2,6 sialic acid structures. Regarding galectin-3 expression in proteins from total cell lysates of CMT-U27 cells, upon treatment with oseltamivir we observed an increase in galectin-3 expression. This observation was strengthened by the immunofluorescence galectin-3 expression in CMT-U27 cell line showed an increase in galectin-3 when cells were treated with 1.25 μg/mL and 12.5 μg/mL oseltamivir. In summary, these results lead us to highlight the role of sialylation during CMT progression, since upon oseltamivir treatment we observed an increase in sialylated structures accompanied by an increase in invasion capacity and migration of cells, which are common features of enhanced malignancy. Sialidase inhibition is possibly a bottom line for further studies and anti-cancer therapy. 65 CONCLUSIONS 66 67 The present study showed that oseltamivir plays an important role in the biological behaviour and in glycosylation pattern of CMT (canine mammary tumour) cell lines. Sialidases inhibition by treatment with oseltamivir demonstrated that they act on glycoproteins during mammary tumour progression since we found an increase in invasion capacity and also in migration of cells. Moreover, in what regards glycosylation pattern, highly malignant CMT-U27 cells treated with oseltamivir, showed an increase in sialylated structures, with concomitant decrease in galectin-3-ligands. These observations point to an important dynamic expression of sialidases in CMMT (canine mammary malignant tumour). Thus, the results of this study are of the utmost importance suggesting in what concerns about differential/dynamic expression and activity of sialidases in invading cells in CMMT, possibly involved in sialic acid capping and uncapping of glycans during tumour progression and invasion. These results indicate that the discovery of a powerful inhibitor capable of inhibit aberrant sialylation could potentially be used for anti-cancer therapy. 68 69 REFERENCES 70 71 Achyuthan K. E. and Achyuthan A. M. (2001). Comparative enzymology, biochemistry and pathophysiology of human exo-alpha-sialidases (neuraminidases). Comp Biochem Physiol B Biochem Mol Biol. 129. (1): 29-64. Almaraz R. T., Tian Y., Bhattarcharya R., Tan E., Chen S. H., Dallas M. R., . . . Yarema K. J. (2012). Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis. Mol Cell Proteomics. 11. (7): M112 017558. Amith S. R., Jayanth P., Franchuk S., Finlay T., Seyrantepe V., Beyaert R., . . . Szewczuk M. R. (2010). Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling. Cell Signal. 22. (2): 314-324. Baldus S. E., Engelmann K. and Hanisch F. G. (2004). MUC1 and the MUCs: a family of human mucins with impact in cancer biology. Crit Rev Clin Lab Sci. 41. (2): 189-231. Baldus S. E., Zirbes T. K., Glossmann J., Fromm S., Hanisch F. G., Monig S. P., . . . Dienes H. P. (2001). Immunoreactivity of monoclonal antibody BW835 represents a marker of progression and prognosis in early gastric cancer. Oncology. 61. (2): 147-155. Baldus S. E., Zirbes T. K., Hanisch F. G., Kunze D., Shafizadeh S. T., Nolden S., . . . Dienes H. P. (2000). Thomsen-Friedenreich antigen presents as a prognostic factor in colorectal carcinoma: A clinicopathologic study of 264 patients. Cancer. 88. (7): 1536-1543. Bresalier R. S., Niv Y., Byrd J. C., Duh Q. Y., Toribara N. W., Rockwell R. W., . . . Kim Y. S. (1991). Mucin production by human colonic carcinoma cells correlates with their metastatic potential in animal models of colon cancer metastasis. J Clin Invest. 87. (3): 1037-1045. Bull C., Boltje T. J., Wassink M., de Graaf A. M., van Delft F. L., den Brok M. H. and Adema G. J. (2013). Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth. Mol Cancer Ther. Cao Y., Linden P., Shima D., Browne F. and Folkman J. (1996). In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. J Clin Invest. 98. (11): 2507-2511. Carubelli R., Trucco R. E. and Caputto R. (1962). Neuraminidase activity in mammalian organs. Biochim Biophys Acta. 60. 196-197. Castronovo V., Van Den Brule F. A., Jackers P., Clausse N., Liu F. T., Gillet C. and Sobel M. E. (1996). Decreased expression of galectin-3 is associated with progression of human breast cancer. J Pathol. 179. (1): 43-48. 72 Cazet A., Julien S., Bobowski M., Burchell J. and Delannoy P. (2010). Tumour-associated carbohydrate antigens in breast cancer. Breast Cancer Res. 12. (3): 204. Chambers A. F., Groom A. C. and MacDonald I. C. (2002). Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2. (8): 563-572. Chavas L. M., Kato R., Suzuki N., von Itzstein M., Mann M. C., Thomson R. J., . . . Wakatsuki S. (2010). Complexity in influenza virus targeted drug design: interaction with human sialidases. J Med Chem. 53. (7): 2998-3002. Chavas L. M., Tringali C., Fusi P., Venerando B., Tettamanti G., Kato R., . . . Wakatsuki S. (2005). Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J Biol Chem. 280. (1): 469-475. Choufani G., Nagy N., Saussez S., Marchant H., Bisschop P., Burchert M., . . . Hassid S. (1999). The levels of expression of galectin-1, galectin-3, and the Thomsen-Friedenreich antigen and their binding sites decrease as clinical aggressiveness increases in head and neck cancers. Cancer. 86. (11): 2353-2363. Coon J. S., Weinstein R. S. and Summers J. L. (1982). Blood group precursor T-antigen expression in human urinary bladder carcinoma. Am J Clin Pathol. 77. (6): 692-699. Corfield A. P., Veh R. W., Wember M., Michalski J. C. and Schauer R. (1981). The release of Nacetyland N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases. Biochem J. 197. (2): 293-299. Corfield A. P., Wember M., Schauer R. and Rott R. (1982). The specificity of viral sialidases. The use of oligosaccharide substrates to probe enzymic characteristics and strain-specific differences. Eur J Biochem. 124. (3): 521-525. Crain S. M. and Shen K. F. (2004). Neuraminidase inhibitor, oseltamivir blocks GM1 gangliosideregulated excitatory opioid receptor-mediated hyperalgesia, enhances opioid analgesia and attenuates tolerance in mice. Brain Res. 995. (2): 260-266. Croce M. V., Isla-Larrain M., Rabassa M. E., Demichelis S., Colussi A. G., Crespo M., . . . SegalEiras A. (2007). Lewis x is highly expressed in normal tissues: a comparative immunohistochemical study and literature revision. Pathol Oncol Res. 13. (2): 130-138. 73 D'Azzo A., Hoogeveen A., Reuser A. J., Robinson D. and Galjaard H. (1982). Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man. Proc Natl Acad Sci U S A. 79. (15): 4535-4539. David L., Nesland J. M., Clausen H., Carneiro F. and Sobrinho-Simoes M. (1992). Simple mucintype carbohydrate antigens (Tn, sialosyl-Tn and T) in gastric mucosa, carcinomas and metastases. APMIS Suppl. 27. 162-172. de Oliveira J. T., de Matos A. J., Santos A. L., Pinto R., Gomes J., Hespanhol V., . . . Gartner F. (2011). Sialylation regulates galectin-3/ligand interplay during mammary tumour progression--a case of targeted uncloaking. Int J Dev Biol. 55. (7-9): 823-834. Dennis J. W. and Laferte S. (1987). Tumor cell surface carbohydrate and the metastatic phenotype. Cancer Metastasis Rev. 5. (3): 185-204. Dwek R. A. (1995). Glycobiology: "towards understanding the function of sugars". Biochem Soc Trans. 23. (1): 1-25. Ellerhorst J., Troncoso P., Xu X. C., Lee J. and Lotan R. (1999). Galectin-1 and galectin-3 expression in human prostate tissue and prostate cancer. Urol Res. 27. (5): 362-367. Ferlay J., Shin H. R., Bray F., Forman D., Mathers C. and Parkin D. M. (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 127. (12): 2893-2917. Funakoshi Y. and Suzuki T. (2009). Glycobiology in the cytosol: the bitter side of a sweet world. Biochim Biophys Acta. 1790. (2): 81-94. Galjart N. J., Gillemans N., Harris A., van der Horst G. T., Verheijen F. W., Galjaard H. and d'Azzo A. (1988). Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases. Cell. 54. (6): 755-764. Ghazizadeh M., Oguro T., Sasaki Y., Aihara K., Araki T. and Springer G. F. (1990). Immunohistochemical and ultrastructural localization of T antigen in ovarian tumors. Am J Clin Pathol. 93. (3): 315-321. Glinskii O. V., Sud S., Mossine V. V., Mawhinney T. P., Anthony D. C., Glinsky G. V., . . . Glinsky V. V. (2012). Inhibition of prostate cancer bone metastasis by synthetic TF antigen mimic/galectin-3 inhibitor lactulose-L-leucine. Neoplasia. 14. (1): 65-73. 80 Springer G. F. (1997). Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J Mol Med (Berl). 75. (8): 594-602. Tokuyama S., Moriya S., Taniguchi S., Yasui A., Miyazaki J., Orikasa S. and Miyagi T. (1997). Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA. Int J Cancer. 73. (3): 410-415. Tringali C., Lupo B., Anastasia L., Papini N., Monti E., Bresciani R., . . . Venerando B. (2007). Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling. J Biol Chem. 282. (19): 14364-14372. Uemura T., Shiozaki K., Yamaguchi K., Miyazaki S., Satomi S., Kato K., . . . Miyagi T. (2009). Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin beta4. Oncogene. 28. (9): 1218-1229. Ueno S., Saito S., Wada T., Yamaguchi K., Satoh M., Arai Y. and Miyagi T. (2006). Plasma membrane-associated sialidase is up-regulated in renal cell carcinoma and promotes interleukin-6induced apoptosis suppression and cell motility. J Biol Chem. 281. (12): 7756-7764. van den Brule F. A., Berchuck A., Bast R. C., Liu F. T., Gillet C., Sobel M. E. and Castronovo V. (1994). Differential expression of the 67-kD laminin receptor and 31-kD human laminin-binding protein in human ovarian carcinomas. Eur J Cancer. 30A. (8): 1096-1099. Varghese J. N., Smith P. W., Sollis S. L., Blick T. J., Sahasrabudhe A., McKimm-Breschkin J. L. and Colman P. M. (1998). Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. Structure. 6. (6): 735-746. Varki A. (1993). Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 3. (2): 97-130. Vereecken P., Zouaoui Boudjeltia K., Debray C., Awada A., Legssyer I., Sales F., . . . Heenen M. (2006). High serum galectin-3 in advanced melanoma: preliminary results. Clin Exp Dermatol. 31. (1): 105-109. Victorzon M., Nordling S., Nilsson O., Roberts P. J. and Haglund C. (1996). Sialyl Tn antigen is an independent predictor of outcome in patients with gastric cancer. Int J Cancer. 65. (3): 295-300. Wang B. L., Springer G. F. and Carlstedt S. C. (1997). Quantitative computerized image analysis of Tn and T (Thomsen-Friedenreich) epitopes in prognostication of human breast carcinoma. J Histochem Cytochem. 45. (10): 1393-1400. 81 Wells L., Vosseller K. and Hart G. W. (2001). Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science. 291. (5512): 2376-2378. Wilson J. C. and von Itzstein M. (2003). Recent strategies in the search for new anti-influenza therapies. Curr Drug Targets. 4. (5): 389-408. Woronowicz A., Amith S. R., Davis V. W., Jayanth P., De Vusser K., Laroy W., . . . Szewczuk M. R. (2007). Trypanosome trans-sialidase mediates neuroprotection against oxidative stress, serum/glucose deprivation, and hypoxia-induced neurite retraction in Trk-expressing PC12 cells. Glycobiology. 17. (7): 725-734. Yamaguchi K., Hata K., Wada T., Moriya S. and Miyagi T. (2006). Epidermal growth factor-induced mobilization of a ganglioside-specific sialidase (NEU3) to membrane ruffles. Biochem Biophys Res Commun. 346. (2): 484-490. Yamanami H., Shiozaki K., Wada T., Yamaguchi K., Uemura T., Kakugawa Y., . . . Miyagi T. (2007). Down-regulation of sialidase NEU4 may contribute to invasive properties of human colon cancers. Cancer Sci. 98. (3): 299-307. Yogeeswaran G. and Salk P. L. (1981). Metastatic potential is positively correlated with cell surface sialylation of cultured murine tumor cell lines. Science. 212. (4502): 1514-1516. Yonezawa S., Tachikawa T., Shin S. and Sato E. (1992). Sialosyl-Tn antigen. Its distribution in normal human tissues and expression in adenocarcinomas. Am J Clin Pathol. 98. (2): 167-174. Yu L. G., Andrews N., Zhao Q., McKean D., Williams J. F., Connor L. J., . . . Rhodes J. M. (2007). Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion. J Biol Chem. 282. (1): 773-781. Zaccai N. R., Maenaka K., Maenaka T., Crocker P. R., Brossmer R., Kelm S. and Jones E. Y. (2003). Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin. Structure. 11. (5): 557-567. Zhao Q., Barclay M., Hilkens J., Guo X., Barrow H., Rhodes J. M. and Yu L. G. (2010). Interaction between circulating galectin-3 and cancer-associated MUC1 enhances tumour cell homotypic aggregation and prevents anoikis. Mol Cancer. 9. 154. 82