Ex vivo assays to detect complement activation in complementopathies.

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB++ or GVB0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Transcriptional network inference and master regulator analysis of the response to ribosome-inactivating proteins in leukemia cells.

Gene-regulatory networks reconstruction has become a very popular approach in applied biology to infer and dissect functional interactions of Transcription Factors (TFs) driving a defined phenotypic state, termed as Master Regulators (MRs). In the present work, cutting-edge bioinformatic methods were applied to re-analyze experimental data on leukemia cells (human myelogenous leukemia cell line THP-1 and acute myeloid leukemia MOLM-13 cells) treated for 6 h with two different Ribosome-Inactivating Proteins (RIPs), namely Shiga toxin type 1 (400 ng/mL) produced by Escherichia coli strains and the plant toxin stenodactylin (60 ng/mL), purified from the caudex of Adenia stenodactyla Harms. This analysis allowed us to identify the common early transcriptional response to 28S rRNA damage based on gene-regulatory network inference and Master Regulator Analysis (MRA). Both toxins induce a common response at 6 h which involves inflammatory mediators triggered by AP-1 family transcriptional factors and ATF3 in leukemia cells. We describe for the first time the involvement of MAFF, KLF2 and KLF6 in regulating RIP-induced apoptotic cell death, while receptor-mediated downstream signaling through ANXA1 and TLR4 is suggested for both toxins.

Verotoxin-1-Induced ER Stress Triggers Apoptotic or Survival Pathways in Burkitt Lymphoma Cells.

Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6-two ER stress sensors-followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic.

Acquired Resistance to Shiga Toxin-Induced Apoptosis by Loss of CD77 Expression in Human Myelogenous Leukemia Cell Line, THP-1.

Shiga toxin (Stx) is a main virulence factor of Enterohemorrhagic Escherichia coli (EHEC) that causes diarrhea and hemorrhagic colitis and occasionally fatal systemic complications. Stx induces rapid apoptotic cell death in some cells, such as human myelogenous leukemia THP-1 cells expressing CD77, a receptor for Stx internalization, and the induction of apoptotic cell death is thought to be crucial for the fatal systemic complications. Therefore, in order to suppress the fatal toxicity, it is important to understand the mechanism how cells can escape from apoptotic cell death in the presence of Stx. In this study, we isolated resistant clones to Stx-induced apoptosis from highly sensitive THP-1 cells by continuous exposure with lethal dose of Stx. All of the ten resistant clones lost the expression of CD77 as a consequence of the reduction in CD77 synthase mRNA expression. These results suggest that downregulation of CD77 or CD77 synthase expression could be a novel approach to suppress the fatal toxicity of Stx in EHEC infected patient.

Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7.

Infection with Escherichia coli O157:H7 may develop into hemorrhagic colitis, or hemolytic uremic syndrome (HUS), which usually causes kidney failure or even death. The adhesion and toxins are the important virulent factors. In this study, a novel vaccine candidate rSOBGs was constructed based on the bacterial ghost (BG). rSOBGs maintained the integrity of cellular morphology and displayed the linear Stx2Am-Stx1B antigen on the surface of outer membrane. rSOBGs induced Stxs-specific IgA/IgG antibodies and stronger intimin-specific IgA/IgG antibodies effectively in sera in this study. In vivo, the rSOBGs provided the higher protection rate (52%) than native bacterial ghost-OBGs (12%) when challenged intragastricly with high dose (500 LD50) viable E. coli O157:H7. Meanwhile, the rSOBGs provided higher protection rate (73.33%) than OBGs when challenged with 2 LD50 even to 5 LD50 lysed E. coli O157:H7. In vitro, the rSOBGs-immunized sera possessed neutralizing activity to lysed pathogenic bacteria. Furthermore, the results of histopathology also displayed that the administration of rSOBGs have the ability to reduce or inhibit the adhesion lesions and toxins damages of organs. The novel vaccine candidate rSOBGs induced both anti-toxin and anti-adhesion immune protection, suggesting the possibility to prevent the infectious diseases caused by Escherichia coli O157:H7.

Shiga toxin stimulates clathrin-independent endocytosis of the VAMP2, VAMP3 and VAMP8 SNARE proteins.

Endocytosis is an essential cellular process that is often hijacked by pathogens and pathogenic products. Endocytic processes can be classified into two broad categories, those that are dependent on clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 and VAMP8 are internalized in a clathrin-dependent manner. However, the full scope of their endocytic behavior has not yet been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 are localized on plasma membrane invaginations and very early uptake structures that are induced by the bacterial Shiga toxin, which enters cells by clathrin-independent endocytosis. We show that toxin trafficking into cells and cell intoxication rely on these SNARE proteins. Of note, the cellular uptake of VAMP3 is increased in the presence of Shiga toxin, even when clathrin-dependent endocytosis is blocked. We therefore conclude that VAMP2, VAMP3 and VAMP8 are removed from the plasma membrane by non-clathrin-mediated pathways, in addition to by clathrin-dependent uptake. Moreover, our study identifies these SNARE proteins as the first transmembrane trafficking factors that functionally associate at the plasma membrane with the toxin-driven clathrin-independent invaginations during the uptake process.

Verotoxin A subunit protects lymphocytes and T cell lines against X4 HIV infection in vitro.

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb(3) (globotriaosylceramide, Pk blood group antigen) as a natural resistance factor for HIV infection. Gb(3) is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb(3). Activated PBMCs predominantly comprise CD4+ T-cells, the primary HIV infection target. Gb(3) is the sole receptor for Escherichia coli verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb(3)+/CD4+ T-cell subset was eliminated but, surprisingly, remaining CD4+ T-cell HIV-1(IIIB) (and HIV-1(Ba-L)) susceptibility was significantly reduced. The Gb(3)-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1(IIIB) infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility.

Regulation of cytokine and chemokine expression by the ribotoxic stress response elicited by Shiga toxin type 1 in human macrophage-like THP-1 cells.

Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1ß (IL-1ß) and chemokines IL-8, growth-regulated protein-ß, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics.

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.

Shiga toxin 1 is more dependent on the P proteins of the ribosomal stalk for depurination activity than Shiga toxin 2.

Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2ßin vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.

Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways.

Diarrhea-associated hemolytic uremic syndrome (D(+)HUS) is caused by the ingestion of Escherichia coli that produce Shiga toxin (Stx), which is composed of a cytotoxic A subunit and pentameric B subunits that bind globotriaosylceramide on susceptible cells. Stx occurs in 2 types, Stx1 and Stx2. B subunits of either type stimulate von Willebrand factor (VWF) secretion from human umbilical vein endothelial cells (HUVECs), and Stx2B can cause thrombotic microangiopathy in Adamts13(-/-) mice. We have now determined that Stx1B and Stx2B activate different signaling pathways in HUVECs. VWF secretion induced by Stx1B is associated with a transient rise in intracellular Ca(2+) level that is blocked by chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, removal of extracellular Ca(2+), the phospholipase C inhibitor U73122, the protein kinase inhibitor staurosporine, or small interfering RNA knockdown of protein kinase Cα. In contrast, Stx2B-induced VWF secretion is associated with activation of protein kinase A (PKA) and is blocked by the PKA inhibitor H89 or small interfering RNA knockdown of PKA. Stx2B does not increase cAMP levels and may activate PKA by a cAMP-independent mechanism. The activation of distinct signaling pathways may be relevant to understanding why E coli that express Stx2 are more likely to cause D(+)HUS than are E coli expressing only Stx1.

Differential response of the human renal proximal tubular epithelial cell line HK-2 to Shiga toxin types 1 and 2.

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb(3) and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

Transmembrane BAX inhibitor motif containing (TMBIM) family proteins perturbs a trans-Golgi network enzyme, Gb3 synthase, and reduces Gb3 biosynthesis.

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.

Shiga toxin 1 and ricin A chain bind to human polymorphonuclear leucocytes through a common receptor.

The main cause of acute renal failure in children is HUS (haemolytic uraemic syndrome), a consequence of intestinal infections with Escherichia coli strains producing Stx (Shiga toxins). Stx released in the gut by the non-invasive bacteria reach the bloodstream and are targeted to cerebral and renal endothelium triggering HUS. PMN (polymorphonuclear leucocytes) seem to be involved in Stx delivery through an unidentified membrane receptor (Kd=10⁻8 M; 2×105 binding sites) which does not allow internalization. Some experts in the field have defined the Stx-PMN interaction as non-specific and of little biological significance. In the present study, we show that the A chain of ricin, the well-known plant RIP (ribosome-inactivating protein), interacts with PMN (Kd=10⁻9 M; 2×105 binding sites) competing for the same receptor that recognizes Stx, whereas diphtheria toxin and several agonists of TLRs (Toll-like receptors) or the mannose receptor were ineffective. No toxic effects of ricin A chain on PMN were observed, as assessed by measuring protein synthesis and the rate of spontaneous apoptosis of leucocytes. Moreover, two single-chain RIPs (gelonin and saporin S6) had the same competing effect. Thus RIPs and Stx1 share structural similarities, the same enzymatic activity and a common receptor on PMN. These observations reveal that the Stx-PMN interaction is specific, confirming that PMN recognize molecular patterns common to different foreign molecules.

Caspase-8-mediated cleavage of Bid and protein phosphatase 2A-mediated activation of Bax are necessary for Verotoxin-1-induced apoptosis in Burkitt's lymphoma cells.

Verotoxin (VT-1) is a cytotoxin, produced by Shigella dysenteriae type 1 or by Shiga toxin-producing Escherichia coli, which binds specifically to globotriaosylceramide (Gb3). This glycosphingolipid is a B cell differentiation antigen (Gb3/CD77) strongly expressed on Burkitt's lymphoma cells. We have previously shown that, in these cells, VT-1 induces apoptosis via a caspase- and mitochondria-dependent pathway. In this report, we provide new insights into this signal transduction pathway. First, we demonstrate that VT-1-induced apoptosis requires degradation of the caspase-8 inhibitory molecule c-FLIPL and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we show that mitochondrial activation is mainly due to i) cleavage and activation of the pro-apoptotic Bcl-2 family member Bid by caspase-8 and ii) Bax relocalization to mitochondrial membranes which lead to cytochrome c release. However, tBid is not involved in Bax relocalization, and relocalization is most likely controlled by the extent of Bax phosphorylation: in non-treated BL cells, p38 MAPK participates in the retention of Bax in the cytoplasm in an inactive form whereas in VT-1 treated cells, protein phosphatase 2A is activated and induces Bax relocalization to mitochondria.

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3.

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

Verotoxin-1 treatment or manipulation of its receptor globotriaosylceramide (gb3) for reversal of multidrug resistance to cancer chemotherapy.

A major problem with anti-cancer drug treatment is the development of acquired multidrug resistance (MDR) of the tumor cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the globotriaosylceramide membrane receptor (Gb3), a glycolipid associated with multidrug resistance. Gb3 is overexpressed in many human tumors and tumor cell lines with inherent or acquired MDR. Gb3 is co-expressed and interplays with the membrane efflux transporter P-gp encoded by the MDR1 gene. P-gp could act as a lipid flippase and stimulate Gb3 induction when tumor cells are exposed to cancer chemotherapy. Recent work has shown that apoptosis and inherent or acquired multidrug resistance in Gb3-expressing tumors could be affected by VT-1 holotoxin, a sub-toxic concentration of the holotoxin concomitant with chemotherapy or its Gb3-binding B-subunit coupled to cytotoxic or immunomodulatory drug, as well as chemical manipulation of Gb3 expression. The interplay between Gb3 and P-gp thus gives a possible physiological approach to augment the chemotherapeutic effect in multidrug resistant tumors.

Expression of verotoxin-1 receptor Gb3 in breast cancer tissue and verotoxin-1 signal transduction to apoptosis.

BACKGROUND: The prerequisite for the potential use of the bacterial toxin verotoxin-1 in the treatment of breast cancer was investigated by first determining the expression of its receptor Gb3 (CD77) in clinical breast cancer tissue specimens. We then examined the cytotoxicity and mechanism of apoptosis induction of Escherichia coli verotoxin-1 (VT-1) in two human breast cancer cell lines. METHODS: Immunohistochemistry for Gb3 expression was performed on cryostat section from 25 breast cancer specimens. The human breast cancer cell lines T47D and MCF-7 were screened for Gb3 expression by flow cytometry. Fluorescein diacetate and LDH release was used to determine cell viability after VT-1 exposure. Apoptosis was studied by measuring caspase activity and DNA-fragmentation. Signal transduction studies were performed on T47D cells with immunoblotting. RESULTS: Gb3 expression was detected in the vascular endothelial cells of all tumours specimens, and in tumour cells in 17 of the specimens. We found no associations between tumour cell Gb3-expression and age, tumour size, TNM-classification, histological type, hormone receptor expression, or survival time. T47D cells strongly expressed Gb3 and were sensitive to the cytotoxicity, caspase activation and DNA fragmentation by VT-1, whereas MCF-7 cells with faint Gb3-expression were insensitive to VT-1. VT-1 (0.01 - 5 microg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 micromol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (< or = 0.1 microg/L VT-1) or reduced (0.5 - 5 microg/L VT-1). VT-1 did not cause cellular LDH-release or cell cycle arrest. VT-1 induction of caspase-3 (0.1, 1, and 5 microg/L VT-1), -8, and -9 (1 and 5 microg/L VT-1) activity and DNA fragmentation of T47D cells was blocked by PPMP. Key components of MAP kinase signalling pathways that control mitochondrial function were investigated. VT-1 0.1 - 5 microg/L induced phosphorylation of JNK as well as MKK3/6 suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. CONCLUSION: The high specificity and apoptosis-inducing properties of verotoxin-1 indicates that the toxin potentially may be used for treatment of Gb3-expressing breast cancer.

Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity.

Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD(50) > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD(50) of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.