ABSTRACT
BACKGROUND & AIMS: Mucins are high-molecular-weight glycoproteins that protect the gastric epithelium. Previous data suggested that gastric surface-type mucin is decreased in Helicobacter pylori-infected patients and restored after eradication of the infection. Our aim was to determine the effect of H. pylori on mucin synthesis in cultured gastric epithelial cells. METHODS: Mucin synthesis was measured by labeling with [(3)H]glucosamine and size-exclusion chromatography. Expression of MUC5AC and MUC1 mucin protein antigens was quantitated by Western blot analysis. RESULTS: Mucin synthesis was inhibited more than 80% when KATO III cells were incubated with H. pylori, with no effect on mucin secretion or degradation. Inhibition was rapid (4 hours), partially reversible, dependent on concentration of bacteria, and associated with the insoluble membrane fraction. H. pylori decreased levels of MUC5AC and MUC1 mucins. MUC1 inhibition was half-maximal by 4 hours and partially reversed by 24 hours, but the decrease in MUC5AC was less rapid and not reversible within 24 hours. CONCLUSIONS: H. pylori inhibits total mucin synthesis in vitro and decreases the expression of MUC5AC and MUC1. A decrease in gastric mucin synthesis in vivo may disrupt the protective surface mucin layer.
Subject(s)
Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Helicobacter Infections/metabolism , Helicobacter pylori , Mucins/biosynthesis , Blotting, Western , Cell Fractionation , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gastric Mucosa/cytology , Humans , Mucin 5AC , Mucin-1/analysis , Mucin-1/biosynthesis , Mucins/analysis , Peptide Fragments/analysis , Peptide Fragments/biosynthesis , Stomach Neoplasms , Tumor Cells, CulturedABSTRACT
BACKGROUND & AIMS: Alterations in the production of epithelial mucins have been correlated with advanced tumor stage in the colon, but direct evidence for a role of specific mucin genes in liver metastasis is lacking. The current study was designed to establish more directly the role of MUC2 in colon cancer metastasis. METHODS: MUC2 levels were manipulated in highly metastatic human colon cancer cells using eukaryotic expression constructs designed to express a portion of MUC2 complementary DNA in antisense orientation. To assess the effect of MUC2 levels on metastatic potential, liver colonization was assessed in athymic mice after splenic-portal inoculation. RESULTS: Stable integration of the MUC2 antisense construct into metastatic colon cancer cells (LS LiM6) resulted in an 80% reduction in MUC2-specific messenger RNA and a concomitant decrease in MUC2 apomucin protein. This reduction was associated with a 50% reduction in synthesis of mature glucosamine-labeled mucin, almost complete inhibition of secretion of sialyl-LeX and sialyl-Tn antigens, and a 40% decrease in binding of colon cancer cells to E-selectin. Reduction in MUC2 levels was associated with a marked decrease in liver colonization. CONCLUSIONS: This study provides direct evidence that MUC2 plays an important role in colon cancer metastasis.
Subject(s)
Biomarkers, Tumor/biosynthesis , Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Liver Neoplasms/metabolism , Mucins/biosynthesis , Neoplasm Proteins/biosynthesis , Animals , Biomarkers, Tumor/antagonists & inhibitors , Blotting, Western , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA Primers , DNA, Antisense/metabolism , DNA, Complementary/metabolism , DNA, Neoplasm/metabolism , E-Selectin/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Mice , Mice, Nude , Mucin-2 , Mucins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND & AIMS: Galectin 3 is a beta-galactoside-binding protein whose expression has been correlated with advanced tumor stage in the colon, but direct evidence for a role in metastasis is lacking. The current study was designed to more directly establish the role of galectin 3 in colon cancer metastasis. METHODS: Galectin 3 levels were manipulated in human colon cancer cells using eukaryotic expression constructs designed to express the complete galectin 3 complementary DNA in either the sense or antisense orientation. Liver colonization was assessed in athymic mice after splenic-portal inoculation or after spontaneous metastasis during cecal growth. RESULTS: Introduction of galectin 3 antisense into metastatic colon cancer cells (LSLiM6, HM7) resulted in a significant reduction in galectin 3-specific messenger RNA and total and cell surface galectin 3 protein. Conversely, stable integration of galectin 3 in the sense orientation resulted in an increase in cellular and cell surface galectin 3 in cells of low metastatic potential (LS174T). Reduction in galectin 3 levels was associated with a marked decrease in liver colonization and spontaneous metastasis by LSLiM6 and HM7 cells, whereas up-regulation of galectin 3 resulted in increased metastasis by LS174T cells. CONCLUSIONS: This study provides direct evidence that galectin 3 plays an important role in colon cancer metastasis.
Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Antigens, Differentiation/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Galactosides/metabolism , Animals , Antisense Elements (Genetics)/genetics , Cell Line, Transformed , Cell Membrane/metabolism , Galectin 3 , Humans , Mice , Mice, Nude , Tissue Distribution , Transfection/genetics , Tumor Cells, CulturedABSTRACT
Mucin production by human colon cancer cells correlates with liver metastasis in animal models, but it is not known which steps in metastasis depend on specific alterations in mucin synthesis. Clonal variants of cell line LS174T selected for differences in mucin core carbohydrate expression have been further characterized biochemically, and tested for their ability to participate in metastasis-related events. LS-C mucin contains truncated carbohydrates enriched for sialyl Tn and these cells bind to basement membrane matrix to a greater extent than LS-B cells. This binding is partially inhibitable by antibody to sialyl Tn. LS-B produces more fully glycosylated mucin and preferentially binds to hepatic sinusoidal endothelial cells and E-selectin through sialylated peripheral mucin-associated carbohydrate structures. Adhesion of LS-B to endothelial cells is inhibited by neutralizing antibody to E-selectin, and inhibition of glycosylation or desialylation of LS-B mucin abrogates binding to E-selectin in vitro. LS-B cells spontaneously metastasized from cecum to liver and colonized the liver of athymic mice after splenic-portal injection to a significantly greater extent than LS-C, suggesting that expression of peripheral mucin carbohydrate structures is most important for metastasis of human colon cancer cells.
Subject(s)
Carbohydrates/chemistry , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Liver Neoplasms/secondary , Mucins/chemistry , Animals , Antigens, Tumor-Associated, Carbohydrate/metabolism , Basement Membrane/metabolism , Cell Adhesion , E-Selectin/metabolism , Endothelium/metabolism , Humans , Liver/cytology , Mice , Neoplasm Metastasis , Tumor Cells, CulturedABSTRACT
BACKGROUND & AIMS: Helicobacter pylori is found within the gastric mucous gel layer and in association with the epithelial surface. The aim of this study was to determine the effect of H. pylori infection on mucin gene expression (MUC6, MUC5, and MUC1) in the gastric epithelium. METHODS: Gastric biopsy specimens were examined by immunohistochemistry and in situ hybridization for mucin gene expression. RESULTS: MUC6 was limited to mucous glands of H. pylori-negative patients, but 72% of H. pylori-positive patients also expressed MUC6 in surface mucous cells. In contrast, MUC5 mucin was found in significantly fewer surface mucous cells of H. pylori-positive specimens. Carbohydrates recognized by Lewis (Le) X and paradoxical concanavalin A (con A) staining were aberrantly expressed in the surface mucous cells of 16 of 27 and 17 of 23 H. pylori-positive tissues, respectively. Surface Le(b) was present in 26 of 27 H. pylori-positive and 19 of 30 H. pylori-negative tissues. Eradication of H. pylori resulted in reversal of surface MUC5 and MUC6 expression toward normal patterns. Purified gastric mucins of H. pylori-positive patients bound more anti-MUC6 and anti-Le(b) than mucins from H. pylori-negative patients. CONCLUSIONS: The type of mucin that is normally limited to mucous glands is aberrantly expressed in surface mucous cells of H. pylori-positive patients, whereas less of the surface epithelium expresses normal surface-type gastric mucin.
