globotriaosylceramide and Neoplasms

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Drug Delivery Systems; Humans; Neoplasms; Ribosomes; Shiga Toxins; Signal Transduction; Trihexosylceramides

Topics: Animals; CRISPR-Cas Systems; Escherichia coli Infections; Hemolytic-Uremic Syndrome; Host-Pathogen Interactions; Humans; Immunotoxins; Models, Molecular; Neoplasms; Protein Conformation; Shiga Toxins; Shiga-Toxigenic Escherichia coli; Structure-Activity Relationship; Trihexosylceramides

Verotoxin (VT) is involved in the etiology of both hemorrhagic colitis and the hemolytic uremic syndrome which are microvasculopathies of the colon and pediatric renal glomerulus respectively. Thus, VT can be considered a vasotoxin. Cell sensitivity in vitro varies according to the receptor glycolipid (globotriaosyl ceramide-Gb3) expression and also to intracellular trafficking of the receptor/toxin complex, such that in highly sensitive cells, the toxin is targeted to the endoplasmic reticulum and nuclear envelope. Such cells include tumor cells which have become drug resistant. Thus Gb3 is upregulated in certain tumors and when such tumor cells become drug resistant, their sensitivity to verotoxin increases. This may be due to a direct role of the MDRI drug efflux pump in glycolipid biosynthesis. In addition to the tumor tissue, the toxin receptor may also be expressed in the tumor neovasculature suggesting that activated endothelial cells may be verotoxin sensitive. Thus VT may have both a direct and indirect antineoplastic potential. VT has proved highly effective in a xenograft cancer model and the possible therapeutic use of VT is discussed.

Topics: Animals; Antineoplastic Agents; Bacterial Toxins; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Neoplasms; Neovascularization, Pathologic; Receptors, Cell Surface; Shiga Toxin 1; Trihexosylceramides

Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant in cancer cells can be valuable new tools for selective cancer cell killing. The lectin Shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in Escherichia coli, isolated them by Gb3-affinity chromatography, and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labeled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid N

Topics: Antineoplastic Agents; Escherichia coli; HeLa Cells; Humans; Lectins; Neoplasms; Peptides

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

Topics: Biological Assay; Biological Transport, Active; Drug Carriers; HT29 Cells; Humans; K562 Cells; Neoplasms; Shiga Toxins; Trihexosylceramides

Glycosphingolipids are amphipathic molecules composed of hydrophilic oligosaccharide chain and a hydrophobic ceramide part, located primarily in the membrane microdomains of animal cells. Their oligosaccharide chains make them excellent candidates for the cell surface recognition molecules. Natural glycosphingolipid, globotriaosylceramide (Gal α1-4, Gal β1-4, Glc β1-1, ceramide), is also called CD77 and its expression was previously associated with proliferating centroblasts undergoing somatic hypermutation, but it has been demonstrate that globotriaosylceramide is not a reliable marker to discriminate human centroblasts from centrocytes. Globotriaosylceramide constitutes rare P k blood group antigen on erythrocytes, and it is also known as Burkitt's lymphoma antigen. On endothelial cells, globotriaosylceramide plays as the receptor for bacterial toxins of the Shiga family, also called verotoxins. Precise biological function and significance of globotriaosylceramide expression on endothelial cells remains to be the subject of many studies and it is believed globotriaosylceramide represents an example of a glycolipid antigen able to transduce a signal leading to apoptosis. In past decade, cancer researches put a great afford in determining new therapeutic agents such as bacterial toxins against tumor malignancies. Reports have demonstrated that verotoxin-1 induces apoptosis in solid tumor cell lines expressing globotriaosylceramide such as astrocytoma, renal cell carcinoma, colon cancer and breast cancer due to verotoxin-1 high specificity and apoptosis-inducing properties, and therefore, it is suggested to be an anticancer agent. Verotoxins have been investigated weather they could reduce treatment side-effects and toxicity to normal tissues and become a new oncological tool in cancer labeling.

Topics: Animals; Biomarkers, Tumor; Humans; Neoplasms; Shiga Toxin 1; Trihexosylceramides

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.

Topics: Animals; Contrast Media; Drug Delivery Systems; Flow Cytometry; HT29 Cells; Humans; Mice; Microbubbles; Microscopy, Fluorescence; Neoplasms; Nonlinear Dynamics; Protein Binding; Protein Subunits; Shiga Toxin; Trihexosylceramides; Ultrasonography; Xenograft Model Antitumor Assays

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

Topics: Adamantane; Animals; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Availability; Caco-2 Cells; Calcium Channel Blockers; Cell Membrane; Cell Polarity; Cyclosporine; Dogs; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Endocytosis; Enzyme Inhibitors; Fluorescent Dyes; Golgi Apparatus; Humans; Intestinal Mucosa; Neoplasms; Protein Binding; Protein Structure, Secondary; Protein Synthesis Inhibitors; Protein Transport; Rhodamine 123; Shiga Toxins; Time Factors; Trihexosylceramides; Up-Regulation; Verapamil; Vinblastine

Rhamnose-binding lectins are widely found in fish eggs. However, their biologic effects on cultured cells are still unknown. Since catfish (Silurus asotus) egg lectin (SAL) bound to globotriaosylceramide (Gb3) expressed on the surface of cells, we analyzed the relationship between Gb3 expression and SAL binding in tumor cell lines using Raji, Daudi, ACHN, P388, and K562 cells. Gb3 was highly expressed on Raji cells but not on K562 cells. SAL bound abundantly to Raji cells but not to K562 cells, and SAL binding depended on the amount of Gb3 on the cell surface. SAL caused a reduction in cell size and increased annexin-V binding to and propidium iodide (PI) incorporation into Raji cells. Although this effect on Raji cells might represent damage at the late apoptosis or necrosis stage, SAL-treated Raji cells remained alive. Thus SAL enhanced PI incorporation into Raji cells without induction of cell death. We examined whether the effects of chemotherapeutic agent(s) are influenced by SAL. SAL increased the incorporation of doxorubicin (Dox) into Raji cells and consequently enhanced the cytotoxic effects of Dox. These results indicate that SAL may induce cell permeability without cytotoxity.

Topics: Animals; Annexin A5; Antibiotics, Antineoplastic; Apoptosis; Cell Membrane; Cell Membrane Permeability; Doxorubicin; Drug Synergism; Fish Proteins; Humans; Lectins; Mice; Neoplasms; Propidium; Rabbits; Trihexosylceramides; Tumor Cells, Cultured

The most devastating aspect of cancer is the emergence of metastases. Thus, identification of potentially metastatic cells among a tumor cell population and the underlying molecular changes that switch cells to a metastatic state are among the most important issues in cancer biology. Here we show that, although normal human colonic epithelial cells lack the glycosphingolipid globotriaosylceramide (Gb(3)), this molecule is highly expressed in metastatic colon cancer. In addition, a subpopulation of cells that are greatly enriched in Gb(3) and have an invasive phenotype was identified in human colon cancer cell lines. In epithelial cells in culture, Gb(3) was necessary and sufficient for cell invasiveness. Transfection of Gb(3) synthase, resulting in Gb(3) expression in noncancerous polarized epithelial cells lacking endogenous Gb(3), induced cell invasiveness. Furthermore, Gb(3) knockdown by small inhibitory RNA in colon cancer epithelial cells inhibited cell invasiveness. Gb(3) is the plasma membrane receptor for Shiga toxin 1. The noncatalytic B subunit of Shiga toxin 1 causes apoptosis of human colon cancer cells expressing Gb(3). Injections of the B subunit of Shiga toxin 1 into HT29 human colon cancer cells engrafted into the flanks of nude mice inhibited tumor growth. These data demonstrate the appearance of a subpopulation of Gb(3) containing epithelial cells in the metastatic stage of human colon cancer and suggest their possible role in colon cancer invasiveness.

Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Epithelial Cells; Galactosyltransferases; Glycosphingolipids; Humans; Lasers; Mice; Mice, Nude; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Phenotype; Pseudopodia; Shiga Toxins; Trihexosylceramides

To assess the relationship between venous thrombosis and plasma glucosylceramide (GlcCer) or phosphatidylethanolamine (PE), plasma levels of GlcCer and PE were determined for 70 venous thrombosis patients referred for evaluation and 70 healthy blood donors. The mean GlcCer level, but not the PE level, was lower in patients versus controls (4.9 vs 6.5 microg/mL [P =.0007] and 66 vs 71 microg/mL [P =.48], respectively). As a measure of relative risk, the odds ratio for deep vein thrombosis in subjects with GlcCer levels below the 10th percentile of controls was 5.7 (95% CI, 2.3-14). To assess the influence of glycolipids on anticoagulant response to activated protein C (APC):protein S in modified prothrombin time assays, the effects of depleting endogenous plasma GlcCer by glucocerebrosidase treatment or of adding exogenous purified GlcCer or other neutral glycolipids to plasma were tested. Glucocerebrosidase treatment reduced plasma sensitivity to APC:protein S in parallel with GlcCer reduction. Exogenously added GlcCer and the homologous Glc-containing globotriaosylceramide (Gb3Cer), but not galactosylceramide, dose-dependently prolonged clotting times of normal plasma in the presence, but not absence, of APC:protein S, which suggests that GlcCer or Gb3Cer can enhance protein C pathway anticoagulant activity. In studies using purified proteins, inactivation of factor Va by APC:protein S was enhanced by GlcCer alone and by GlcCer in multicomponent vesicles containing phosphatidylserine and phosphatidylcholine. These results suggest that the neutral glycolipids GlcCer and Gb3Cer may directly contribute to the anticoagulant activity of the protein C pathway and that deficiency of plasma GlcCer may be a risk factor for venous thrombosis. (Blood. 2001;97:1907-1914)

Topics: Activated Protein C Resistance; Adult; Aged; Aged, 80 and over; Blood Coagulation; Chromatography, High Pressure Liquid; Comorbidity; Contraceptives, Oral, Hormonal; Factor Va; Female; Galactosylceramides; Glucosylceramidase; Glucosylceramides; Humans; Male; Middle Aged; Neoplasms; Odds Ratio; Phosphatidylethanolamines; Postoperative Complications; Protein C; Protein S; Pulmonary Embolism; Risk; Thrombophilia; Trihexosylceramides; Venous Thrombosis; White People; Wounds and Injuries

The verotoxin receptor globotriaosyl ceramide (Gb3) is overexpressed in an ovarian tumour resistant to chemotherapy. An overlay of frozen tumour sections shows extensive staining of the tumour cells with verotoxin B subunit. In addition, blood vessels within the tumour mass are stained. The sensitivity of ovarian tumour cells in vitro to verotoxin can be modulated by culturing the cells in sodium butyrate to obtain an approximately 5000-fold increase in susceptibility. This increased susceptibility is correlated with the intracellular targeting of verotoxin as monitored by using FITC-VT B subunit, in that prior to sodium butyrate treatment the toxin is internalized to a juxtanuclear (likely) Golgi location whereas, following butyrate treatment the intracellular toxin is distributed around the nucleus, consistent with endoplasmic reticulum and nuclear envelope location. This perinuclear location is similar to that found for drug-resistant variants of ovarian tumour cell lines. These results suggest that intracellular targeting of verotoxin to the perinuclear area results in increased cytotoxicity. Potentially such targeting may also occur in other human tumours.

Topics: Biological Transport; Cell Membrane; Cell Nucleus; Humans; Neoplasms; Receptors, Cell Surface; Trihexosylceramides; Tumor Cells, Cultured

The purpose of this study was to isolate mouse monoclonal antibodies (MAbs) recognizing Gal alpha 1-4Gal beta 1-4Glc (Pk, CD77), which has been described as a Burkitt's-lymphoma-associated antigen. Three IgGI MAbs reactive with Pk were developed using a synthetic glycoconjugate as immunogen and a filter immunoplaque screening assay. One MAb (PK67) was characterized by immuno-thin-layer chromatography, ELISA and competition assays using neutral glycolipids from various sources and a variety of carbohydrates and glycoproteins. Epitope analysis showed that all 3 carbohydrates moieties are required in PK67 binding. No cross-reactivity was observed with closely related carbohydrate structures, with the exception of Gal alpha 1-4Gal beta 1-4GlcNAc. However, the reactivity with this structure was several orders of magnitude lower than with Pk. Immunohistochemical analysis of fresh-frozen tissue specimens showed that Gb3 is widely expressed: prominent staining was observed in many normal tissues, e.g., kidney and gastric tissue and on capillary endothelial cells. In lymph nodes, very weak staining of a few B cells was observed. Flow cytometric analysis of hematopoietic neoplasms showed Gb3 expression on B-cell neoplasms, particularly those with committed B-cell phenotype indicating that, in addition to earlier reports of Gb3 expression on Burkitt's lymphoma, Gb3 is present on other committed B-cell neoplasms.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Carbohydrate Sequence; Carbohydrates; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Glycolipids; Glycoproteins; Humans; Hybridomas; Immunoenzyme Techniques; Leukemia; Lymphoma; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Organ Specificity; Reference Values; Trihexosylceramides