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Clifford A Lingwood - One of the best experts on this subject based on the ideXlab platform.
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detergent resistant globotriaosyl ceramide may define Verotoxin glomeruli restricted hemolytic uremic syndrome pathology
Kidney International, 2009Co-Authors: Fahima Khan, Francois Proulx, Clifford A LingwoodAbstract:Verotoxin binding to its receptor, globotriaosyl ceramide(Gb 3 ) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb 3 is expressed in both tubular and glomerular cells. Gb 3 within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that Verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong Verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against Verotoxin glomerular binding. Mouse glomeruli remained Verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong Verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb 3 in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb 3 ligand) was detergent sensitive, tubule-restricted, and inhibited by Verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb 3 organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome.
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differential intracellular transport and binding of Verotoxin 1 and Verotoxin 2 to globotriaosylceramide containing lipid assemblies
Journal of Cellular Physiology, 2008Co-Authors: Patty Tam, Radhia Mahfoud, Anita Nutikka, Aye Aye Khine, Beth Binnington, Paul Paroutis, Clifford A LingwoodAbstract:Although Verotoxin-1 (VT1) and Verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb3), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb3 assemblies. At 4°C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb3 microdomains. After 10 min at 37°C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3–6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential ‘raft’ association. >90% 125I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb3-containing sucrose gradient ‘insoluble’ fraction, whereas only 30% 125I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb3/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb3 ratios. VT2 competed less effectively for 125I-VT1/Gb3 DRM-binding but only VT2-Gb3/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb3 raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology. J. Cell. Physiol. 216: 750–763, 2008, © 2008 Wiley-Liss, Inc.
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membrane cytosolic translocation of Verotoxin a1 subunit in target cells
Microbiology, 2007Co-Authors: Patricia J Tam, Clifford A LingwoodAbstract:In sensitive cells, Verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [125I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA1, was demonstrated for the first time by monitoring [125I]VTA1 release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA1 and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA1 detection. Brefeldin A and 16 °C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA1 cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA1 values indicates that about one molecule of translocated VTA1 per cell is sufficient to inhibit protein synthesis and kill a cell.
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brefeldin a and filipin distinguish two globotriaosyl ceramide Verotoxin 1 intracellular trafficking pathways involved in vero cell cytotoxicity
Glycobiology, 2004Co-Authors: Aye Aye Khine, Anita Nutikka, Patty Tam, Clifford A LingwoodAbstract:In Verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb 3 ) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transportof VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to <50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb 3 .
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Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity
Cellular Microbiology, 2003Co-Authors: D Elaine E Hoey, Clifford A Lingwood, Carol G Currie, David L Gally, Linda Sharp, David SmithAbstract:Summary Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceram- ide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells con- trasts with the absence of Gb3 on human intestinal epithelium in vivo . Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithe- lial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abro- gation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl- b -cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies dem- onstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.
Parviz Behnammotlagh - One of the best experts on this subject based on the ideXlab platform.
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acquired cisplatin resistance in malignant pleural mesothelioma cells is reversed by both bh3 mimetic obatoclax and iap inhibitor at 406
2016Co-Authors: Andreas Tyler, Anders Johansson, Parviz Behnammotlagh, Camilla Sandberg, Amanda Blom, Veronica Rondahl, Kjell GrankvistAbstract:Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for Verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin.Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells.Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.
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cisplatin induced expression of gb3 enables Verotoxin 1 treatment of cisplatin resistance in malignant pleural mesothelioma cells
British Journal of Cancer, 2010Co-Authors: David Johansson, Carola Andersson, Jasmin Moharer, Anders Johansson, Parviz BehnammotlaghAbstract:Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glyc ...
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expression of Verotoxin 1 receptor gb3 in breast cancer tissue and Verotoxin 1 signal transduction to apoptosis
BMC Cancer, 2009Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz BehnammotlaghAbstract: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. 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. 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 μg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 μmol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (≤ 0.1 μg/L VT-1) or reduced (0.5 – 5 μg/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 μg/L VT-1), -8, and -9 (1 and 5 μg/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 μg/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. 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.
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expression of Verotoxin 1 receptor gb3 in breast cancer
2008Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz BehnammotlaghAbstract:Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and Verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of Verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.
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Verotoxin 1 induction of apoptosis in gb3 expressing human glioma cell lines
Cancer Biology & Therapy, 2006Co-Authors: David Johansson, Anders Johansson, Thomas Brannstrom, Kjell Grankvist, Ulrika Andersson, Roger Henriksson, Per Bergstrom, Parviz BehnammotlaghAbstract:The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of Verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by ...
James L Brunton - One of the best experts on this subject based on the ideXlab platform.
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tumour necrosis factor alpha is not an essential component of Verotoxin 1 induced toxicity in mice
Microbial Pathogenesis, 2002Co-Authors: Vince M Wolski, James L Brunton, Anna M SoltykAbstract:Abstract Previous studies have shown that tumour necrosis factor α (TNF-α) genetranscription is induced in the mouse kidney in response to Shiga-like toxin 1 (Stx 1, or Verotoxin 1, VT1) administration, suggesting that local TNF-α expression plays a role in renal pathogenesis caused by the toxin. Further, TNF-α neutralizing antibody pretreatment of mice orally infected with VT-producing Escherichia coli (VTEC) protected the animals from disease development and death. We examined the role of TNF-α release in response to parenteral challenge with purified VT1. Mice injected with 10- and 100-fold the 50% lethal dose (LD50) of VT1 showed a weak, transient elevation of serum TNF-α only at the higher toxin dose. TNF-α was not detected in the urine of mice at either dose. Treatment of BALB/c mice with a neutralizing anti-TNF-α antibody prior to administration of 3 LD50 of toxin failed to protect the mice from VT1-mediated toxicity. Further, TNF-α knock-out mice administered 3 LD50 of VT1 were not protected against the lethal effects of the toxin relative to the wild-type animals. These findings suggest that VT1 is a poor inducer of TNF-αin vivo and that the low levels of the cytokine released in response to toxin challenge do not play a direct role in potentiating the toxicity of VT1 in mice. Strong toxin accumulation in the kidney but not in the brain was demonstrated by immunohistochemistry after intraperitoneal administration of VT1. Tubular damage and extensive apoptosis in the kidney, together with a 10-fold increase in levels of blood urea nitrogen, suggest that mice died of acute renal failure.
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mouse toxicity and cytokine release by Verotoxin 1 b subunit mutants
Infection and Immunity, 2001Co-Authors: James L Brunton, Vince M Wolski, Anna M SoltykAbstract:The crystal structure of the Verotoxin 1 (VT1) B subunit complexed with a globotriaosylceramide (Gb(3)) analogue showed the presence of three receptor binding sites per monomer. We wished to study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro. We found that while the site 1 and 2 mutants were modestly (two- to sevenfold) reduced in their ability to cause disease in BALB/c mice, the site 3 mutant, W34A, was as toxic as VT1. However, all the double-mutant proteins, irrespective of which two sites were mutated, exhibited approximately a 100-fold reduction in their 50% lethal doses for mice. These results suggest that multivalent receptor binding is important in vivo and that all three binding sites make a similar contribution to the latter process. The triple-mutant holotoxin, F30A G62T W34A, administered intraperitoneally without adjuvant, stimulated a strong antibody response in BALB/c mice, and the immune sera neutralized the activity of VT1 in vitro. Induction of tumor neurosis factor alpha release from differentiated human monocytes (THP-1 cells) was relatively impaired for site 1 and site 2 but not site 3 mutants, suggesting an auxiliary role for the latter site in mediation of cytokine release in vitro. Cytotoxicity assays on undifferentiated THP-1 cells have also demonstrated the importance of sites 1 and 2 and the relatively small role played by site 3 in causing cell death. These data suggest an association between the cytotoxicity of the protein and its ability to induce cytokine release.
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the identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 b subunit
Molecular Microbiology, 1999Co-Authors: Randy J Read, James L Brunton, Clifford G Clark, Darrin J Bast, L BanerjeeAbstract:The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor-binding specificity is associated with the terminally linked Galα(1–4) Galβ disaccharide sequence of the receptor. Recently, three globotriose (Galα[1–4] Galβ [1–4] Glcβ) binding sites per B-subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine-30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N-terminal end of the α-helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation-binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D-17E) or site II (G-62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC-5 cell cytotoxicity (by seven and five logs, respectively, for G-62T and by four and two logs, respectively, for D-17E). In contrast, the substitution of alanine for W-34 at site III did not reduce the high-affinity binding of the B subunit, despite causing a fourfold reduction in the receptor-binding capacity. The corresponding mutant W-34A holotoxin had a two-log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC-5 cells. We conclude that the high-affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W-34 for binding at this site.
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murine antibody responses to the Verotoxin 1 b subunit demonstration of major histocompatibility complex dependence and an immunodominant epitope involving phenylalanine 30
Infection and Immunity, 1997Co-Authors: Darrin J Bast, J Sandhu, N Hozumi, B Barber, James L BruntonAbstract:Structurally conserved Verotoxin 1 (VT1) mutant derivatives, showing reduced receptor binding and cytotoxicity, may serve as natural toxoids to protect against VT-mediated disease. In this study, the antibody responses to the wild-type VT1 B subunit, a B-subunit mutant (Phe30Ala B), and the corresponding holotoxin (Phe30Ala HT) were examined in three inbred mouse strains. BALB/c (H-2d) and CBA (H-2k) mice produced strong antibody responses to both wild-type and mutant B subunits. VT1 B-raised sera reacted more strongly with VT1 B than with Phe30Ala B in enzyme-linked immunosorbent assays, while Phe30Ala B-raised sera reacted equally with VT1 B and Phe30Ala B. C57BL/6 (H-2b) and congenic BALB/c (BALB x B [H-2b]) mice produced no detectable antibody response to either VT1 B or Phe30Ala B. However, an anti-VT1 B antibody response was detected in H-2b mice immunized with biologically active Phe30Ala HT. Based on these observations, we conclude that the VT1 B subunit possesses a B-cell immunodominant epitope formed partly by phenylalanine 30 and that the B-subunit antibody response is dependent on the H-2 haplotype of the mouse strain. Our results also support a potential role for the A subunit in providing the T-cell help necessary to overcome a deficient B-subunit antibody response in H-2b mice.
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phenylalanine 30 plays an important role in receptor binding of Verotoxin 1
Molecular Microbiology, 1996Co-Authors: Clifford G Clark, Randy J Read, Penelope E Stein, Darrin J Bast, Allan M Sharp, Phaedria M St Hilaire, Rummana Agha, Eric J Toone, James L BruntonAbstract:The homopentameric B subunit of Verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.
Anders Johansson - One of the best experts on this subject based on the ideXlab platform.
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ral ssBioMed CentBMC Cancer Open AcceResearch article Expression of Verotoxin-1 receptor Gb3 in breast cancer tissue and
2016Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz Behnam-motlaghAbstract:Verotoxin-1 signal transduction to apoptosi
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acquired cisplatin resistance in malignant pleural mesothelioma cells is reversed by both bh3 mimetic obatoclax and iap inhibitor at 406
2016Co-Authors: Andreas Tyler, Anders Johansson, Parviz Behnammotlagh, Camilla Sandberg, Amanda Blom, Veronica Rondahl, Kjell GrankvistAbstract:Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for Verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin.Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells.Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.
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Verotoxin-1 Treatment or Manipulation of its Receptor Globotriaosylceramide (Gb3) for Reversal of Multidrug Resistance to Cancer Chemotherapy
Toxins, 2010Co-Authors: Parviz Behnam-motlagh, Kjell Grankvist, Andreas Tyler, Anders JohanssonAbstract: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.
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cisplatin induced expression of gb3 enables Verotoxin 1 treatment of cisplatin resistance in malignant pleural mesothelioma cells
British Journal of Cancer, 2010Co-Authors: David Johansson, Carola Andersson, Jasmin Moharer, Anders Johansson, Parviz BehnammotlaghAbstract:Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glyc ...
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expression of Verotoxin 1 receptor gb3 in breast cancer tissue and Verotoxin 1 signal transduction to apoptosis
BMC Cancer, 2009Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz BehnammotlaghAbstract: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. 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. 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 μg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 μmol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (≤ 0.1 μg/L VT-1) or reduced (0.5 – 5 μg/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 μg/L VT-1), -8, and -9 (1 and 5 μg/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 μg/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. 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.
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ral ssBioMed CentBMC Cancer Open AcceResearch article Expression of Verotoxin-1 receptor Gb3 in breast cancer tissue and
2016Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz Behnam-motlaghAbstract:Verotoxin-1 signal transduction to apoptosi
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cisplatin induced expression of gb3 enables Verotoxin 1 treatment of cisplatin resistance in malignant pleural mesothelioma cells
British Journal of Cancer, 2010Co-Authors: David Johansson, Carola Andersson, Jasmin Moharer, Anders Johansson, Parviz BehnammotlaghAbstract:Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glyc ...
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expression of Verotoxin 1 receptor gb3 in breast cancer tissue and Verotoxin 1 signal transduction to apoptosis
BMC Cancer, 2009Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz BehnammotlaghAbstract: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. 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. 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 μg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 μmol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (≤ 0.1 μg/L VT-1) or reduced (0.5 – 5 μg/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 μg/L VT-1), -8, and -9 (1 and 5 μg/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 μg/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. 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.
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expression of Verotoxin 1 receptor gb3 in breast cancer
2008Co-Authors: David Johansson, Jasmin Moharer, Anders Johansson, Eldina Kosovac, Ingrid Ljuslinder, Thomas Brannstrom, Parviz BehnammotlaghAbstract:Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and Verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of Verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.
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Verotoxin 1 induction of apoptosis in gb3 expressing human glioma cell lines
Cancer Biology & Therapy, 2006Co-Authors: David Johansson, Anders Johansson, Thomas Brannstrom, Kjell Grankvist, Ulrika Andersson, Roger Henriksson, Per Bergstrom, Parviz BehnammotlaghAbstract:The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of Verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by ...
