The Experts below are selected from a list of 2352 Experts worldwide ranked by ideXlab platform
Vern L. Schramm - One of the best experts on this subject based on the ideXlab platform.
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transition state structure of rna depurination by Saporin l3
ACS Chemical Biology, 2016Co-Authors: Hongling Yuan, Christopher F Stratton, Vern L. SchrammAbstract:Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of Saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of Saporin L3. To permit KIE measurements, small stem–loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem–loop RNA (5′-GGGAGGGCCC-3′) by sa...
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Transition State Structure of RNA Depurination by Saporin L3
2016Co-Authors: Hongling Yuan, Christopher F Stratton, Vern L. SchrammAbstract:Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of Saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of Saporin L3. To permit KIE measurements, small stem–loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem–loop RNA (5′-GGGAGGGCCC-3′) by Saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates Saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for Saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known
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soapwort Saporin l3 expression in yeast mutagenesis and rna substrate specificity
Biochemistry, 2015Co-Authors: Hongling Yuan, Matthew B. Sturm, Vern L. SchrammAbstract:Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for Saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >103 times more efficient at RNA deadenylation on short GAGA stem-loops than Saporin S6, the Saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged Saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding Saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol de...
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Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity
2015Co-Authors: Hongling Yuan, Matthew B. Sturm, Vern L. SchrammAbstract:Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for Saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >103 times more efficient at RNA deadenylation on short GAGA stem-loops than Saporin S6, the Saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged Saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding Saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of Saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated Saporin L3 (amino acids 22–280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant Saporins are kinetically similar. The recombinant expression of Saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein
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transition state analogues rescue ribosomes from Saporin l1 ribosome inactivating protein
Biochemistry, 2009Co-Authors: Matthew B. Sturm, Peter C Tyler, Gary B Evans, Vern L. SchrammAbstract:Ribosome-inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of Saporin-L1 from Saponaria officinalis (soapwort) leaves and demonstrate robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of Saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (Ki*) of 2.3 to 8.7 nM at physiological conditions and bind up to 40,000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of Saporin-L1 have potential in cancer therapy that employs Saporin-L1 linked immunotoxins.
Matthias F Melzig - One of the best experts on this subject based on the ideXlab platform.
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The Endocytic Uptake Pathways of Targeted Toxins Are Influenced by Synergistically Acting Gypsophila Saponins
2016Co-Authors: Diana Bachran, Alexander Weng, Christopher Bachran, Matthias F Melzig, Stefanie Schneider, Hendrik FuchsAbstract:The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin Saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better
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triterpenoid saponin augmention of Saporin based immunotoxin cytotoxicity for human leukaemia and lymphoma cells is partially immunospecific and target molecule dependent
Immunopharmacology and Immunotoxicology, 2015Co-Authors: Suzanne E Holmes, Alexander Weng, Christopher Bachran, Hendrik Fuchs, Sopsamorn U. Flavell, Matthias F Melzig, David J FlavellAbstract:AbstractContext: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein Saporin and an EGF-Saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins.Objective: To investigate the augmentative property of SA on Saporin and Saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells.Materials and methods: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for Saporin and five Saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay.Results: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted...
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Saponins modulate the intracellular trafficking of protein toxins.
Journal of controlled release : official journal of the Controlled Release Society, 2012Co-Authors: Alexander Weng, Stefan Böttger, Matthias F Melzig, Roger Gilabert-oriol, Mayank Thakur, Benedicta Von Mallinckrodt, Figen Beceren-braun, Burkard Wiesner, Jenny Eichhorst, Hendrik FuchsAbstract:Type I ribosome inactivating proteins such as Saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of Saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of Saporin. Binding analysis demonstrated an association of the saponins with Saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a Saporin-based targeted anti-tumor toxin in combination with characterized saponins.
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enhancement of Saporin cytotoxicity by gypsophila saponins more than stimulation of endocytosis
Chemico-Biological Interactions, 2009Co-Authors: Alexander Weng, Christopher Bachran, Hendrik Fuchs, Eberhard Krause, H Stephanowitz, Matthias F MelzigAbstract:Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that Saporin exerts no cytotoxicity on seven human cell lines. However, the combination of Saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered Saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the Saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged Saporin ((his)Saporin) in ECV-304 cells. For this purpose (his)Saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)Saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)Saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)Saporin. This indicates a saponin mediated endosomal escape of Saporin.
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enhancement of toxicity of Saporin based toxins by gypsophila saponins kinetic of the saponin
Experimental Biology and Medicine, 2009Co-Authors: Alexander Weng, Cornelia Görick, Matthias F MelzigAbstract:Saponins are amphiphilic substances consisting of a hydrophobic backbone with one or two hydrophilic sugar units. Recently it was shown that saponinum album (SA) from Gypsophila paniculata L. enhan...
David J Flavell - One of the best experts on this subject based on the ideXlab platform.
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the role of cholesterol on triterpenoid saponin induced endolysosomal escape of a Saporin based immunotoxin
International Journal of Molecular Sciences, 2020Co-Authors: Wendy S Smith, Sopsamorn U. Flavell, David A Johnston, Harrison J Wensley, Suzanne E Holmes, David J FlavellAbstract:Cholesterol seems to play a central role in the augmentation of Saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled Saporin and the Saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of Saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.
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Augmentation of Saporin-Based Immunotoxins for Human Leukaemia and Lymphoma Cells by Triterpenoid Saponins: The Modifying Effects of Small Molecule Pharmacological Agents
MDPI AG, 2019Co-Authors: Wendy S Smith, Sopsamorn U. Flavell, David A Johnston, Harrison J Wensley, Suzanne E Holmes, David J FlavellAbstract:Triterpenoid saponins from Saponinum album (SA) significantly augment the cytotoxicity of Saporin-based immunotoxins but the mechanism of augmentation is not fully understood. We investigated the effects of six small molecule pharmacological agents, which interfere with endocytic and other processes, on SA-mediated augmentation of Saporin and Saporin-based immunotoxins (ITs) directed against CD7, CD19, CD22 and CD38 on human lymphoma and leukaemia cell lines. Inhibition of clathrin-mediated endocytosis or endosomal acidification abolished the SA augmentation of Saporin and of all four immunotoxins tested but the cytotoxicity of each IT or Saporin alone was largely unaffected. The data support the hypothesis that endocytic processes are involved in the augmentative action of SA for Saporin ITs targeted against a range of antigens expressed by leukaemia and lymphoma cells. In addition, the reactive oxygen species (ROS) scavenger tiron reduced the cytotoxicity of BU12-SAP and OKT10-SAP but had no effect on 4KB128-SAP or Saporin cytotoxicity. Tiron also had no effect on SA-mediated augmentation of the Saporin-based ITs or unconjugated Saporin. These results suggest that ROS are not involved in the augmentation of Saporin ITs and that ROS induction is target antigen-dependent and not directly due to the cytotoxic action of the toxin moiety
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Strategies to Improve the Clinical Utility of Saporin-Based Targeted Toxins
MDPI AG, 2018Co-Authors: Francesco Giansanti, David J Flavell, Maria Serena Fabbrini, Francesco Angelucci, Rodolfo IppolitiAbstract:Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies (chemical and genetic) to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins
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membrane cholesterol is essential for triterpenoid saponin augmentation of a Saporin based immunotoxin directed against cd19 on human lymphoma cells
Biochimica et Biophysica Acta, 2017Co-Authors: Wendy S Smith, Sopsamorn U. Flavell, David A Johnston, Suzanne E Holmes, Ella J Baker, Grielof Koster, Alan N Hunt, David J FlavellAbstract:Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of Saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-Saporin by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.
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triterpenoid saponin augmention of Saporin based immunotoxin cytotoxicity for human leukaemia and lymphoma cells is partially immunospecific and target molecule dependent
Immunopharmacology and Immunotoxicology, 2015Co-Authors: Suzanne E Holmes, Alexander Weng, Christopher Bachran, Hendrik Fuchs, Sopsamorn U. Flavell, Matthias F Melzig, David J FlavellAbstract:AbstractContext: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein Saporin and an EGF-Saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins.Objective: To investigate the augmentative property of SA on Saporin and Saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells.Materials and methods: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for Saporin and five Saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay.Results: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted...
Alexander Weng - One of the best experts on this subject based on the ideXlab platform.
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The Endocytic Uptake Pathways of Targeted Toxins Are Influenced by Synergistically Acting Gypsophila Saponins
2016Co-Authors: Diana Bachran, Alexander Weng, Christopher Bachran, Matthias F Melzig, Stefanie Schneider, Hendrik FuchsAbstract:The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin Saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better
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triterpenoid saponin augmention of Saporin based immunotoxin cytotoxicity for human leukaemia and lymphoma cells is partially immunospecific and target molecule dependent
Immunopharmacology and Immunotoxicology, 2015Co-Authors: Suzanne E Holmes, Alexander Weng, Christopher Bachran, Hendrik Fuchs, Sopsamorn U. Flavell, Matthias F Melzig, David J FlavellAbstract:AbstractContext: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein Saporin and an EGF-Saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins.Objective: To investigate the augmentative property of SA on Saporin and Saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells.Materials and methods: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for Saporin and five Saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay.Results: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted...
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Saponins modulate the intracellular trafficking of protein toxins.
Journal of controlled release : official journal of the Controlled Release Society, 2012Co-Authors: Alexander Weng, Stefan Böttger, Matthias F Melzig, Roger Gilabert-oriol, Mayank Thakur, Benedicta Von Mallinckrodt, Figen Beceren-braun, Burkard Wiesner, Jenny Eichhorst, Hendrik FuchsAbstract:Type I ribosome inactivating proteins such as Saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of Saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of Saporin. Binding analysis demonstrated an association of the saponins with Saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a Saporin-based targeted anti-tumor toxin in combination with characterized saponins.
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enhancement of Saporin cytotoxicity by gypsophila saponins more than stimulation of endocytosis
Chemico-Biological Interactions, 2009Co-Authors: Alexander Weng, Christopher Bachran, Hendrik Fuchs, Eberhard Krause, H Stephanowitz, Matthias F MelzigAbstract:Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that Saporin exerts no cytotoxicity on seven human cell lines. However, the combination of Saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered Saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the Saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged Saporin ((his)Saporin) in ECV-304 cells. For this purpose (his)Saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)Saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)Saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)Saporin. This indicates a saponin mediated endosomal escape of Saporin.
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enhancement of toxicity of Saporin based toxins by gypsophila saponins kinetic of the saponin
Experimental Biology and Medicine, 2009Co-Authors: Alexander Weng, Cornelia Görick, Matthias F MelzigAbstract:Saponins are amphiphilic substances consisting of a hydrophobic backbone with one or two hydrophilic sugar units. Recently it was shown that saponinum album (SA) from Gypsophila paniculata L. enhan...
Hendrik Fuchs - One of the best experts on this subject based on the ideXlab platform.
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The Endocytic Uptake Pathways of Targeted Toxins Are Influenced by Synergistically Acting Gypsophila Saponins
2016Co-Authors: Diana Bachran, Alexander Weng, Christopher Bachran, Matthias F Melzig, Stefanie Schneider, Hendrik FuchsAbstract:The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin Saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better
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triterpenoid saponin augmention of Saporin based immunotoxin cytotoxicity for human leukaemia and lymphoma cells is partially immunospecific and target molecule dependent
Immunopharmacology and Immunotoxicology, 2015Co-Authors: Suzanne E Holmes, Alexander Weng, Christopher Bachran, Hendrik Fuchs, Sopsamorn U. Flavell, Matthias F Melzig, David J FlavellAbstract:AbstractContext: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein Saporin and an EGF-Saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins.Objective: To investigate the augmentative property of SA on Saporin and Saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells.Materials and methods: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for Saporin and five Saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay.Results: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted...
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Saponins modulate the intracellular trafficking of protein toxins.
Journal of controlled release : official journal of the Controlled Release Society, 2012Co-Authors: Alexander Weng, Stefan Böttger, Matthias F Melzig, Roger Gilabert-oriol, Mayank Thakur, Benedicta Von Mallinckrodt, Figen Beceren-braun, Burkard Wiesner, Jenny Eichhorst, Hendrik FuchsAbstract:Type I ribosome inactivating proteins such as Saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of Saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of Saporin. Binding analysis demonstrated an association of the saponins with Saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a Saporin-based targeted anti-tumor toxin in combination with characterized saponins.
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enhancement of Saporin cytotoxicity by gypsophila saponins more than stimulation of endocytosis
Chemico-Biological Interactions, 2009Co-Authors: Alexander Weng, Christopher Bachran, Hendrik Fuchs, Eberhard Krause, H Stephanowitz, Matthias F MelzigAbstract:Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that Saporin exerts no cytotoxicity on seven human cell lines. However, the combination of Saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered Saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the Saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged Saporin ((his)Saporin) in ECV-304 cells. For this purpose (his)Saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)Saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)Saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)Saporin. This indicates a saponin mediated endosomal escape of Saporin.
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A simple method for isolation of Gypsophila saponins for the combined application of targeted toxins and saponins in tumor therapy.
Planta Medica, 2009Co-Authors: Alexander Weng, Diana Bachran, Cornelia Görick, Christopher Bachran, Hendrik Fuchs, Matthias F MelzigAbstract:: Saponinum album (SAP) is a complex mixture of triterpene saponins from Gypsophila paniculata L. Although most of the saponins from SAP are characterized, the separation of pure saponins remains time consuming and costly, involving different chromatographic techniques. Recently it was shown that SAP drastically enhanced the cytotoxicity of a chimeric toxin consisting of the N-glycosidase Saporin and human epidermal growth factor (Sap-EGF) in cell culture experiments. In view of a potential therapeutic use of the coadministration of SAP and Sap-EGF in tumor therapy, an economic and time-saving method for the isolation of pure saponins from the crude SAP mixture in high amounts is required. In this study we isolated a single saponin by a simple chromatographic method. The isolated saponin was characterized by mass spectrometry and was shown to enhance the cytotoxicity of Sap-EGF on HER14 cells.
