Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

Is the lectin binding pattern of human breast and colon cancer cells influenced by modulators of sialic acid metabolism?

Sialic acid residues are the most abundant terminal carbohydrate residues of mammalian cells. Modification of the sialic acid residues by exposure of cells in culture to sialic acid precursor analogues resulted in a modifed susceptibility to polyoma viruses. In the present study, human breast and colon cancer cell lines were exposed for 65 h to these acid precursor analogues at 5 mM and their lectin binding pattern was analysed. Use of a panel of several different lectins indicated that the pretreatment of these cell lines with the sialic acid analogues did not change their lectin binding profile. The incorporation of these precursors into membrane glycoproteins was assessed by reversed phase high-performance liquid chromatography, which clearly demonstrated that the precursors were incorporated. The results therefore indicate that these analogues are highly specific for sialic acid and do not interfere with other biosynthetic pathways of membrane glycoconjugates.     

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3⁺ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3⁺ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3⁺ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Accessibility of glycolipid and oligosaccharide epitopes on rabbit villus and follicle-associated epithelium

The initial step in many mucosal infections is pathogen attachment to glycoconjugates on the apical surfaces of intestinal epithelial cells. We examined the ability of virus-sized (120-nm) and bacterium-sized (1-microm) particles to adhere to specific glycolipids and protein-linked oligosaccharides on the apical surfaces of rabbit Peyer's patch villus enterocytes, follicle-associated enterocytes, and M cells. Particles coated with the B subunit of cholera toxin, which binds the ubiquitous glycolipid GM1, were unable to adhere to enterocytes or M cells. This confirms that both the filamentous brush border glycocalyx on enterocytes and the thin glycoprotein coat on M cells can function as size-selective barriers. Oligosaccharides containing terminal beta(1,4)-linked galactose were accessible to soluble lectin Ricinus communis type I on all epithelial cells but were not accessible to lectin immobilized on beads. Oligosaccharides containing alpha(2, 3)-linked sialic acid were recognized on all epithelial cells by soluble Maackia amurensis lectin II (Mal II). Mal II coated 120-nm (but not 1-microm) particles adhered to follicle-associated enterocytes and M cells but not to villus enterocytes. The differences in receptor availability observed may explain in part the selective attachment of viruses and bacteria to specific cell types in the intestinal mucosa.     

Assay of lectin binding sites in colon cancer-associated mucins: comparison of fluorescence microscopy with a quantitative chromatographic technique

We have developed a technique for quantitation of binding of fluorescent lectins to glycoconjugates in specimens of tumors derived from cultured human colorectal cancer cells. Tumor cells were injected subcutaneously into nude mice, giving rise to xenografts that resemble primary human colorectal cancers. The tumors were extracted with saline and were subjected to dialysis and lyophilization. Standardized amounts of the tumor extract were then incubated with fluorescent lectins and subjected to gel permeation liquid chromatography to separate lectin bound to high molecular weight glycoproteins from free (unbound) lectin, and were quantitated using a spectrofluorometer. This assay permitted quantitative measurement of the lectin bound to high molecular weight glycoconjugates such as mucin. The results of this assay were compared with the standard histochemical assessment of tissue labeling by fluorescent lectins. A close correlation between the two techniques was found, especially when little or no labeling was present. Greater variations were observed at higher levels of labeling. The quantitative assay confirms that lectins bind to high molecular weight mucin-type glycoconjugates on fixed sections of tumors, and supports the use of semi-quantitative histochemical assessments of tissue labeling.     

Selective Expression of PNA-Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential

Alterations of cell-surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III-V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.     

Plant lectin,ATF1011, on the tumor cell surface augments tumor-specific immunity through activation of T cells specific for the lectin

Summary The possibility that a plant lectin as a carrier protein would specifically activate T cells, resulting in the augmentation of antitumor immunity was investigated. ATF1011, a nonmitogenic lectin for T cells purified from Aloe arborescens Mill, bound equally to normal and tumor cells. ATF1011 binding on the MM102 tumor cell surfaces augmented anti-trinitrophenyl (TNP) antibody production of murine splenocytes when the mice were primarily immunized with TNP-conjugated MM102 tumor cells. The alloreactive cytotoxic T cell response was also augmented by allostimulator cells binding ATF1011 on the cell surfaces. These augmented responses may be assumed to be mediated by the activation of helper T cells recognizing ATF1011 as a carrier protein. Killer T cells were induced against ATF1011 antigen in the H-2 restricted manner using syngeneic stimulator cells bearing ATF1011 on the cell surfaces. When this lectin was administered intralesionally into the tumors, induction of cytotoxic effector cells was demonstrated. These results suggest that intralesionally administered ATF1011 binds to the tumor cell membrane and activates T cells specific for this carrier lectin in situ, which results in the augmented induction of systemic antitumor immunity.     

Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.     

Sialofucosylated podocalyxin is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells

Selectin-mediated interactions in the vasculature promote metastatic spread by facilitating circulating tumor cell binding to selectin-expressing host cells. Therefore, identifying the selectin ligand(s) on tumor cells is critical to the prevention of blood-borne metastasis. A current challenge is to distinguish between structures expressed by circulating tumor cells that can bind selectins in vitro from the functional ligands whose depletion suppresses selectin-dependent binding under flow in vivo. Interestingly, podocalyxin (PODXL), which can bind E- and L-selectin, is upregulated in a number of cancers, including those of the breast, colon, and pancreas. In this work, we show that metastatic pancreatic cancer cells overexpress PODXL compared with nonmalignant pancreatic epithelial cells. We further demonstrate via tissue microarray that 69% of pancreatic ductal adenocarcinomas stain positive for PODXL. In cases of focal expression, positive staining is restricted to the invasive front of primary tumors. By combining immunoblot, immunodepletion, short-hairpin RNA-mediated gene silencing, and flow-based adhesion assays, we evaluated the functional role of sialofucosylated PODXL in selectin-mediated adhesion under flow. Our data indicate that sialofucosylated PODXL is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells, as specific depletion of this molecule from the cell surface significantly interferes with selectin-dependent interactions. Cumulatively, these data support a correlation between sialofucosylated PODXL expression and enhanced binding to selectins by metastatic pancreatic cancer cells and offer additional perspective on the upregulation of PODXL in aggressive cancers.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.     

Influence of temperature on the interaction of Concanavalin A with chick fibroblasts during embryo development

The interaction between Concanavalin A and chick embryo fibroblasts was studied. Cells from younger and older embryos had the same number of lectin receptor sites per cell at 4°, 21° and 37°C but the affinity constants increased with increasing temperature. Analysis of the binding data according to Scatchard showed that the apparent changes in binding as a function of temperature might be related to thermodynamic properties. The lectin binding sites on the cell surface proved homogeneous with regard to binding properties.The age-related differences noted in the affinities of the cells to bind Concanavalin A could be related to differences in the degree of rearrangement of the cell surface components and/or to a change in the structure of cell surface glycoconjugates, and may serve to explain the differences in the effect of Concanavalin A on cell growth.     

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.     

Two Chitotriose-Specific Lectins Show Anti-Angiogenesis,Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle

The antiproliferative activity of two chito- specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI₅₀ values were approximately 8.4 μg ml^-1 (0.247 μM) and 142 μg ml^-1(14.8 μM) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G₀/G₁ arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.