alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Pancreatic-Neoplasms

Topics: Amylases; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biogenic Polyamines; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; GPI-Linked Proteins; Humans; Isoenzymes; Membrane Glycoproteins; Mesothelin; Oligosaccharides; Pancreatic Elastase; Pancreatic Neoplasms; Sialyl Lewis X Antigen

Late diagnosis of pancreatic ductal adenocarcinoma (PDA) is one of the reasons of its low 5-year survival rate and it is due to its unspecific symptoms during the first stages of the disease and the lack of reliable serological markers. Since PDA shows an altered glycan expression, here we have focused on finding novel potential biomarkers, namely glycoproteins that express the tumor associated carbohydrate structure sialyl-Lewis x (sLe

Topics: Adenocarcinoma; Biomarkers, Tumor; Carrier Proteins; Extracellular Matrix Proteins; Glycoproteins; Humans; Microfibrils; Pancreatic Neoplasms; Sialyl Lewis X Antigen

Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumour-associated carbohydrate antigens sialyl-Lewis x (SLe

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Epitopes; Female; Glycosylation; Humans; Male; Middle Aged; Mucin 5AC; Mucin-1; Neoplasm Staging; Oligosaccharides; Pancreatic Neoplasms; Sialyl Lewis X Antigen

Pancreatic adenocarcinoma (PDAC) usually shows an enhanced expression of sialyl-Lewis X (sLe(x)) and related epitopes. PDAC may secrete some of the proteins carrying such increased sLe(x) determinant into serum, so they could be used as PDAC markers. Previously, we identified acute-phase proteins with increased sLe(x) in both PDAC and in chronic pancreatitis patients. In this study, depleted sera from the main acute-phase proteins has been analysed for the search of proteins with increased sLe(x) levels in PDAC. Sera from healthy controls, chronic pancreatitis and PDAC patients were depleted, electrophoresed and subjected to sLe(x) immunodetection. Proteins that differentially expressed sLe(x) in PDAC were trypsin digested and identified by LC-ESI-QTOF mass spectrometry. Five protein bands that differentially expressed sLe(x) in PDAC were identified and corresponded to seven different acute-phase proteins. Among them, ceruloplasmin (CP) was selected for further analysis. N-glycan sequencing of CP confirmed the increase of sLe(x) levels in CP in PDAC patients. Healthy controls, chronic pancreatitis and PDAC patients' sera were immunoprecipitated with anti-CP antibodies, and their sLe(x) and CP levels were analysed by western blot. The sLe(x)/CP ratio tended to be higher for the PDAC group, which altogether suggests that the sLe(x)/CP ratio could be a useful biomarker for PDAC.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Cell Line, Tumor; Ceruloplasmin; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Male; Middle Aged; Oligosaccharides; Pancreatic Neoplasms; Pancreatitis, Chronic; Polysaccharides; Sialyl Lewis X Antigen

The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19-9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19-9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer. Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19-9, is elevated in the plasma of patients who are low in CA19-9, potentially enabling more comprehensive detection and classification of pancreatic cancers.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; CA-19-9 Antigen; Carbohydrate Sequence; Carcinoma, Pancreatic Ductal; Gene Expression; Humans; Immunoassay; Molecular Sequence Data; Oligosaccharides; Pancreatic Neoplasms; Polysaccharides; Sensitivity and Specificity; Sialyl Lewis X Antigen

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewis(x) (SLe(x)) through the regulation of specific glycosyltransferase genes.. Two human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1β, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens' biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens.. IL-1β stimuli increased SLe(x) and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLe(x) biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLe(x) and Le(y) antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens' biosynthesis, were increased. Most PDAC tissues stained for SLe(x) and SLe(a) and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells.. The inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLe(x), which contributes to pancreatic tumor malignancy.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cytokines; Disease Progression; Epitopes; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycosyltransferases; Humans; Immunohistochemistry; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Lewis X Antigen; Oligosaccharides; Pancreatic Neoplasms; Polysaccharides; Sialic Acids; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha

Tumor cells modulate their extracellular matrix (ECM) adhesion and migration to become more metastatic. Moreover, they show an increase in sialic acid, which could have an effect on their ECM adhesion and migration. This work describes the influence of pancreatic adenocarcinoma cell surface α2,3- and α2,6-sialic acid determinants on the aforementioned processes.. We have characterized the cell surface α2,3- and α2,6-sialic acids, and sialyl-Lewis x levels and the integrin levels of 2 pancreatic adenocarcinoma cell lines, Capan-1 and MDAPanc-28, grown at different cell densities, and also of the ST3Gal III overexpressing Capan-1 cells, C31. We have measured their adhesion to several ECM proteins and their migration through collagen with and without blocking their sialic acid determinants.. Adhesion to ECM proteins of Capan-1 and MDAPanc-28 grown at different cell densities, and of C31, depended on their cell surface sialic acid determinants repertoire, correlating the higher α2,6-sialic acid levels with their increased ECM adhesion. Cell migration also depended on their sialic acid determinants expression; and in this case, higher α2,3-sialic acid levels correlated with a more migratory phenotype.. This study shows how the intrinsic heterogeneity of cell membrane sialylation regulates the adhesive and migratory potential of pancreatic adenocarcinoma cells.

Topics: Adenocarcinoma; Analysis of Variance; beta-Galactoside alpha-2,3-Sialyltransferase; Cell Adhesion; Cell Count; Cell Line, Tumor; Cell Membrane; Cell Movement; Collagen Type I; Extracellular Matrix; Humans; Integrins; Oligosaccharides; Pancreatic Neoplasms; Sialic Acids; Sialyl Lewis X Antigen; Sialyltransferases

Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed in pancreatic adenocarcinoma-sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)-during the progression of pancreatic cancer from early stages to metastatic disease.. Immunohistochemical analyses of mucin and associated glycan expression on primary tumor and liver metastatic tumor samples were conducted with matched sets of tissues from 40 autopsy patients diagnosed with pancreatic adenocarcinoma, 14 surgically resected tissue samples, and 8 normal pancreata.. There were significant changes in mucin expression patterns throughout disease progression. MUC1 and MUC4 were differentially glycosylated as the disease progressed from early pancreatic intraepithelial neoplasias to metastatic disease. De novo expression of several mucins correlated with increased metastasis indicating a potentially more invasive phenotype, and we show the expression of MUC6 in acinar cells undergoing acinar to ductal metaplasia. A "cancer field-effect" that included changes in mucin protein expression and glycosylation in the adjacent normal pancreas was also seen.. There are significant alterations in mucin expression and posttranslational processing during progression of pancreatic cancer from early lesions to metastasis. The results are presented in the context of how mucins influence the biology of tumor cells and their microenvironment during progression of pancreatic cancer.

Topics: Acinar Cells; Adenocarcinoma; Aged; Aged, 80 and over; Antigens, Viral, Tumor; Autopsy; CA-19-9 Antigen; Disease Progression; Female; Glycosylation; Humans; Immunohistochemistry; Male; Middle Aged; Mucin-1; Mucin-4; Mucin-6; Mucins; Oligosaccharides; Pancreatic Neoplasms; Polysaccharides; Protein Isoforms; Sialyl Lewis X Antigen

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Topics: Adenocarcinoma; Aged; Animals; beta-Galactoside alpha-2,3-Sialyltransferase; Cell Membrane; Cell Movement; E-Selectin; Female; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Mice; Mice, Nude; Middle Aged; N-Acetylneuraminic Acid; Neoplasm Metastasis; Oligosaccharides; Pancreatic Neoplasms; Protein Binding; RNA, Messenger; Sialyl Lewis X Antigen; Sialyltransferases

Topics: Biomarkers, Tumor; Female; Humans; Lewis X Antigen; Lung Neoplasms; Male; Ovarian Neoplasms; Pancreatic Neoplasms; Sialyl Lewis X Antigen

Ductal adenocarcinoma of the pancreas has recently been suggested to arise from histologically identifiable ductal lesions known as pancreatic intraepithelial neoplasia (PanINs). Altered levels and patterns of mucin gene expression have been reported to occur in epithelial cancers.. To examine the pattern of expression of membrane-associated mucins, MUC3 and MUC4, and a mucin-associated carbohydrate tumor antigen, sialyl Le(x), in these precursor lesions and ductal adenocarcinoma of the pancreas.. A total of 144 PanIN lesions and 85 cases of ductal adenocarcinoma of the pancreas were examined by using immunohistochemistry and in situ hybridization methods.. MUC3 showed a progressive increase in expression in PanINs of increasing dysplasia and was also highly expressed in ductal adenocarcinoma. In contrast, neoexpression of MUC4 and sialyl Le(x) antigen was observed, mainly in PanIN-3 and ductal adenocarcinoma. In addition, a decrease in the expression of MUC3 and MUC4 was correlated with the degree of de-differentiation of the tumor.. Aberrant expression of membrane mucins MUC3 and MUC4 and of a mucin-associated carbohydrate tumor antigen Sialyl Le(x) in PanINs and adenocarcinoma further supports the progression model for pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Differentiation; Disease Progression; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Mucin-3; Mucin-4; Mucins; Oligosaccharides; Pancreas; Pancreatic Neoplasms; RNA, Messenger; Sialyl Lewis X Antigen

The clinicopathological factors and expression of sialyl Lewis antigens which are the cell adhesion molecules to endothelial cells were compared in relation to the extent of the postoperative hepatic metastasis in 23 consecutive patients with pancreatic ductal adenocarcinoma whose clinical courses were carefully monitored and documented. The overall survival of cases with massive hepatic metastasis (MHM) was significantly poorer than that of those with local or no hepatic metastasis (p = 0.0453). Postoperative MHM was significantly correlated with the presence of duodenal invasion (p = 0.0418), the presence of portal vein invasion (p = 0.0435), the presence of extratumoral venous invasion (p = 0.0052) and high expression of sialyl Lewis x antigen (p = 0.0022). Multivariate analysis confined significant correlation between the high expression of sialyl Lewis x antigen and the development of MHM (p = 0.0402). Kaplan-Meier analysis revealed that the overall survival of patients with a high expression of sialyl Lewis x antigen was significantly poorer than that of patients with a low expression of the antigen (p = 0.0216). These results indicate that the overexpression of sialyl Lewis x antigen plays an important role in the development of MHM, and also predicts a poorer overall survival of these patients. Further studies with more cases are warranted to confirm these results.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Oligosaccharides; Pancreatic Neoplasms; Postoperative Period; Prognosis; Sialyl Lewis X Antigen; Survival Rate; Veins

The carbohydrate antigen sialyl-Lewis(a) is important to pancreatic tumour biology because the circulating antigen is used in serological tests for malignancy and because cell surface antigen is involved in tumour cell binding to the endothelial adhesion molecule, E-selectin, in extravasation. In this study, we examined the effects of the adenylyl cyclase activator, forskolin, and the diacylglycerol analogue, phorbol 12-myristate 13-acetate (PMA), on the expression and release of sialyl-Lewis(a) in human pancreatic cancer cells. Increases in the release of sialyl-Lewis(a) from SW1990 cells produced by forskolin and PMA were associated with increases in the activities of protein kinases A and C, respectively, and could be blocked by inhibitors specific for these enzymes. Immunoprecipitation experiments showed that sialyl-Lewis(a) was associated with MUC1 mucin. Forskolin also increased the cellular content of antigen and MUC1 mRNA. Actinomycin D and a protein kinase A inhibitor, H8, blocked these effects. In contrast, PMA reduced cellular antigen and MUC1 mRNA levels, although it produced a temporary increase in release of the antigen. The effects of PMA were blocked by the protein kinase C inhibitor, H7. PMA also reduced cell binding to the adhesion molecule E-selectin. In summary, PKA and PKC alter cell MUC1-associated sialyl-Lewis(a) in opposite directions. These changes may have clinical utility in the diagnosis of pancreatic cancer and the prevention of metastases.

Topics: Blotting, Western; Colforsin; Cyclic AMP-Dependent Protein Kinases; Dactinomycin; Humans; Mucin-1; Oligosaccharides; Pancreatic Neoplasms; Phorbol Esters; Protein Kinase C; RNA, Messenger; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Previous studies have shown that sialyl Lewis a (SLea) and sialyl Lewis x (SLex) correlated to hematogenous metastasis of human cancers. Although SLea/SLex and E-selectin act as a set of adhesion molecules in vitro, it is not clear whether the in vivo correlation is exclusively mediated by the adhesion function. To address this issue, we investigated whether or not the role of SLea/SLex antigens on hematogenous metastasis to the liver in SCID mice was exclusively mediated by adhesion by using antibodies for these antigens and SLea/SLEx-negative, human pancreas adenocarcinoma cell line PCI-6. The absence of SLea/SLex expression was supported by the absent flow cytometric detection of the antigens as well as by the absent attachment augmentation to activated endothelial cells. PCI-6 cells are xenotransplantable to nude and SCID mice and produce vascular endothelial cell growth factor (VEGF) in a significant amount. PCI-6 cells, 1 x 10(6), were injected into the spleens of SCID mice, and resultant liver metastases were evaluated six weeks later. We observed an inhibitory effect on the establishment and growth of metastatic colonies when anti-SLea or anti-SLex antibody was administered. This indicates that SLea/x antigens have an important in vivo role, even in the metastasis of SLea/SLex-negative tumor cells. This implies that there may be an in vivo function of SLea/x antigens other than that of the attachment between tumor and endothelial cells.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Line; Endothelial Growth Factors; Endothelium, Vascular; Flow Cytometry; Humans; Interleukin-1; Lewis Blood Group Antigens; Lymphokines; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Oligosaccharides; Pancreatic Neoplasms; Sialyl Lewis X Antigen; Specific Pathogen-Free Organisms; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Several alpha(1,3/1,4) fucosyltransferases expressed in human pancreatic cancer cells can participate in the biosynthesis of cell surface sialyl-Lewis a and sialyl-Lewis x antigens that contribute to hematogenous metastatis. Previously, we observed a significant increase of the alpha(1,4) fucosyltransferase activity in tumoral pancreatic cell lines, suggesting that FUT3 could be involved in the sialyl-Lewis antigen expression. Therefore, we invalidated the expression of FUT3 by expressing FUT3 antisense sequence in the human pancreatic tumor BxPC-3 cell line, which expresses the alpha(1,4) fucosyltransferase activity and harbors the cell surface sialyl-Lewis antigens. The decrease of FUT3 transcript after transfection of antisense cDNA of FUT3 in these cells results in a substantial reduction of sialyl-Lewis antigen expression on cell surface. This decreased antigen expression was associated with an inhibition of adhesive properties to E-selectin and a decrease of metastatic power of FUT3 antisense-transfected BxPC-3 cells as tested in nude mice. Our study provides evidence that the expression level of FUT3 may regulate the expression of sialyl-Lewis a and sialyl-Lewis x surface antigens and consequently could play an important role in metastatic properties of human pancreatic cancer cells.

Topics: Animals; CHO Cells; Cricetinae; E-Selectin; Female; Fucosyltransferases; Humans; Mice; Mice, Nude; Oligosaccharides; Pancreatic Neoplasms; Peritoneal Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Sialyl Lewis X Antigen; Transcription, Genetic; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Carbohydrate Sequence; Cattle; Epitopes; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Molecular Sequence Data; Mucin-1; Mucins; N-Acetylglucosaminyltransferases; N-Acetylneuraminic Acid; Oligosaccharides; Pancreatic Neoplasms; Peptides; Polysaccharides; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured

Increased expression of sialyl Lewis antigens, sLe(a) and sLe(x), is frequent during malignant transformation and tumor progression of gastrointestinal cancers and it is assumed to be correlated with the increased metastatic potential of tumor cells and, consequently, with poor patient survival. To determine the influence of the increased expression of these antigens in disease progression of ductal carcinoma of the pancreas, immunohistochemical expressions of sLe(x) and sLe(a) in 51 ductal carcinomas of the pancreas were examined. We also examined the expression of glycosyltransferase genes, which are involved in the synthetic pathways of these antigens to understand the molecular mechanism involved in the increased expression of these antigens. Of the 51 primary ductal carcinomas of the pancreas, 40 (78.4%) were sLe(a)-positive and 11 (21.6%) were sLe(a)-negative; 16 (31.4%) were sLe(x)-positive and 35 (68.6%) were sLe(x)-negative. Although there were no significant differences in any examined clinicopathological factors such as age, sex, histological type, tumor size, presence of lymph node metastasis, or presence of vessel invasion between the positive and negative groups with both the sLe(a) and sLe(x) antigens, patient survival tended to be worse in the antigens-positive group than in the antigens-negative group. Increased expression, however, was not dependent on the increased expression of a single glycosyltransferase gene examined among five such genes, which are postulated to be responsible for the synthesis of the sLe(a) and sLe(x) epitopes in the glycosylation pathway. Furthermore, the increased expression of these antigens was not closely associated with mutations status of the K-ras or p53 genes. These findings suggested that increased expression of sialyl Lewis antigens are involved in pancreatic tumorigenesis and that the accumulation of genetic alterations or epigenetic changes is responsible for the molecular mechanisms of increased expression of the sLe(a) and sLe(x) antigens in ductal carcinomas of pancreas.

Topics: Adenocarcinoma; Adult; Aged; Antigens, Neoplasm; CA-19-9 Antigen; Cell Transformation, Neoplastic; DNA Mutational Analysis; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Glycosylation; Glycosyltransferases; Humans; Male; Middle Aged; Neoplasm Proteins; Pancreatic Neoplasms; Prognosis; Protein Processing, Post-Translational; Sialyl Lewis X Antigen; Survival Analysis

Human pancreatic cancer is characterized by an alteration in fucose-containing surface blood group antigens such as H antigen, Lewis b, Lewis y, and sialyl-Lewis. These carbohydrate determinants can be synthesized by sequential action of alpha(2,3) sialyltransferases or alpha(1,2) fucosyltransferases (Fuc-T) and alpha(1,3/1,4) fucosyltransferases on (poly)N-acetyllactosamine chains. Therefore, the expression and the function of seven fucosyltransferases were investigated in normal and cancer pancreatic tissues and in four pancreatic carcinoma cell lines. Transcripts of FUT1, FUT2, FUT3, FUT4, FUT5, and FUT7 were detected by RT-PCR in carcinoma cell lines as well as in normal and tumoral tissues. Interestingly, the FUT6 message was only detected in tumoral tissues. Analysis of the acceptor substrate specificity for fucosyltransferases indicated that alpha(1,2) Fuc-T, alpha(1,3) Fuc-T, and alpha(1,4) Fuc-T were expressed in microsome preparations of all tissues as demonstrated by fucose incorporation into phenyl beta-d-galactoside, 2'-fucosyllactose, N-acetyllactosamine, 3'-sialyl-N-acetyllactosamine, and lacto-N-biose. However, these fucosyltransferase activities varied between tissues. A substantial decrease of alpha(1,2) Fuc-T activity was observed in tumoral tissues and cell lines compared to normal tissues. Conversely, the activity of alpha(1,4) Fuc-T, which generates Lewis a and sialyl-Lewis a structures, and that of alpha(1,3) Fuc-T, able to generate a lactodifucotetraose structure, were very important in SOJ-6 and BxPC-3 cell lines. These increases correlated with an enhanced expression of Lewis a, sialyl-Lewis a, and Lewis y on the cell surface. The activity of alpha(1,3) Fuc-T, which participates in the synthesis of the sialyl-Lewis x structure, was not significantly modified in cell lines compared to normal tissues. However, the sialyl-Lewis x antigen was expressed preferentially on the surface of SOJ-6 and BxPC-3 cell lines but was not detected on Panc-1 and MiaPaca-2 cell lines suggesting that several alpha(1,3) Fuc-T might be involved in sialyl-Lewis x synthesis.

Topics: Aged; Carbohydrates; Epitopes; Female; Fucosyltransferases; Gene Expression; Humans; Isoenzymes; Lewis X Antigen; Male; Middle Aged; Oligosaccharides; Pancreas; Pancreatic Neoplasms; RNA, Messenger; Sialyl Lewis X Antigen; Tumor Cells, Cultured

We have shown previously that the mucins of the human pancreatic cancer cell line, SW1990, have both sialyl-Lewis(a) and sialyl-Lewis(x) carbohydrate ligands that are implicated in tumor cell metastasis. In the present study, we undertook to identify the protein core of these mucins. SW1990 mucins that carry sialyl-Lewis(a) and sialyl-Lewis(x) bound to the MUC1 peptide-specific mAb 139H2. Removal of most of the sialic acids from SW1990 mucins by neuraminidase greatly enhanced binding of two other MUC1 peptide specific antibodies, HMFG-2 and SM-3. After removal of sialic acids, most of the mucins rich in sialyl-Lewis(a) and sialyl-Lewis(x) oligosaccharides no longer bound to a DEAE-cellulose column at pH 8.0. These results indicate that at least part of the sialyl-Lewis(a) and sialyl-Lewis(x) in SW1990 cells is associated with the MUC1 polypeptide. Moreover, sialic acids play an important role in determining the net negative charge of sialyl-Lewis(a) and sialyl-Lewis(x) rich mucins and in obscuring MUC1 peptide regions.

Topics: Amino Acid Sequence; Antibodies, Neoplasm; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; CA-19-9 Antigen; Carcinoma, Ductal, Breast; Epitope Mapping; Gangliosides; Humans; In Vitro Techniques; Membrane Glycoproteins; Molecular Sequence Data; Mucin-1; Mucins; Neuraminidase; Pancreatic Neoplasms; Peptides; Sialyl Lewis X Antigen; Tumor Cells, Cultured

The sialyl Lewis antigens, the ligands of endothelial leukocyte adhesion molecule-1 (ELAM-1), have been used as important tumor markers for the digestive system. The expression of two sialyl Lewis antigens, sialyl Lewis A antigen (SLea) and sialyl Lewis X antigen, was examined in human pancreatic cancer cells. The expression of SLea was dominant, as shown by flow cytometric analysis. Furthermore, suppressive effects of anti-SLea antibody and anti-ELAM-1 antibody on human pancreatic cancer cell vasoinvasion were demonstrated using a new assay system, the vasoinvasion chamber. These results suggest the critical involvement of SLea and ELAM-1 molecules in human pancreatic cancer cell vasoinvasion.

Topics: Adenocarcinoma; Biomarkers, Tumor; CA-19-9 Antigen; E-Selectin; Gangliosides; Humans; Neoplasm Invasiveness; Oligosaccharides; Pancreatic Neoplasms; Sialyl Lewis X Antigen; Tumor Cells, Cultured