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1.
Int J Oncol ; 55(1): 309-319, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180549

RESUMO

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Exotoxinas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Albuminas/administração & dosagem , Albuminas/química , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Linhagem Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Camundongos , Neoplasias/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
2.
J Biol Chem ; 292(43): 17668-17680, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28882889

RESUMO

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.


Assuntos
ADP Ribose Transferases , Toxinas Botulínicas , Integrina beta1 , Neurônios/metabolismo , Oligopeptídeos , Sinaptossomos/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/farmacocinética , ADP Ribose Transferases/farmacologia , Motivos de Aminoácidos , Animais , Toxinas Botulínicas/química , Toxinas Botulínicas/farmacocinética , Toxinas Botulínicas/farmacologia , Linhagem Celular , Integrina beta1/química , Integrina beta1/genética , Integrina beta1/metabolismo , Camundongos , Vimentina/química , Vimentina/genética , Vimentina/metabolismo
3.
J Control Release ; 164(1): 58-64, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23075769

RESUMO

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Citosol/metabolismo , Portadores de Fármacos/administração & dosagem , Exotoxinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Técnicas de Cultura de Células , Rastreamento de Células , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacocinética , Portadores de Fármacos/farmacocinética , Exotoxinas/genética , Exotoxinas/farmacocinética , Citometria de Fluxo , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
4.
J Biol Chem ; 287(10): 7367-73, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22228764

RESUMO

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.


Assuntos
Abrina/farmacocinética , Substâncias para a Guerra Química/farmacocinética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Ricina/farmacocinética , Tiorredoxinas/metabolismo , ADP Ribose Transferases/farmacocinética , ADP Ribose Transferases/farmacologia , Abrina/farmacologia , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/farmacologia , Substâncias para a Guerra Química/farmacologia , Retículo Endoplasmático/genética , Exotoxinas/farmacocinética , Exotoxinas/farmacologia , Humanos , Células Jurkat , Proteínas de Membrana/genética , Oxirredução/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacocinética , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacocinética , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Ricina/farmacologia , Saporinas , Tiorredoxinas/genética , Fatores de Virulência/farmacocinética , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosa
5.
Mol Cancer Ther ; 10(10): 1829-38, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21862685

RESUMO

A novel anticancer agent was constructed by fusing a gene encoding the scFV that targets both glycosylated and unglycosylated forms of CD133 to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, was studied to determine its ability to bind and kill tumor-initiating cells in vitro and in vivo. The anti-CD133 scFV selectively bound HEK293 cells transfected with the CD133 receptor gene. Time course viability studies showed that dCD133KDEL selectively inhibited NA-SCC and UMSCC-11B, 2 head and neck squamous cell carcinomas that contain a CD133 expressing subpopulation. Importantly, the drug did not inhibit the viability of hematopoietic lineages measured by long-term culture-initiating cell and colony-forming assays from sorted human CD34+ progenitor cells. In addition to in vitro studies, in vivo tumor initiation experiments confirmed that CD133-sorted cells implanted into the flanks of nude mice grew faster and larger than unsorted cells. In contrast, cells that were pretreated with dCD133KDEL before implantation showed the slowest and lowest incidence of tumors. Furthermore, UMSCC-11B-luc tumors treated with multiple intratumoral injections of dCD133KDEL showed marked growth inhibition, leading to complete degradation of the tumors that was not observed with an irrelevant control-targeted toxin. Experiments in immunocompetent mice showed that toxin deimmunization resulted in a 90% reduction in circulating antitoxin levels. These studies show that dCD133KDEL is a novel anticancer agent effective at inhibiting cell proliferation, tumor initiation, and eliminating established tumors by targeting the CD133 subpopulation. This agent shows significant promise for potential development as a clinically useful therapy.


Assuntos
Antígenos CD/imunologia , Carcinoma de Células Escamosas/tratamento farmacológico , Glicoproteínas/imunologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Imunotoxinas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Antígeno AC133 , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , ADP Ribose Transferases/farmacologia , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Exotoxinas/genética , Exotoxinas/farmacocinética , Exotoxinas/farmacologia , Feminino , Glicoproteínas/biossíntese , Glicoproteínas/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/farmacologia , Imunotoxinas/genética , Imunotoxinas/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Transfecção , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Fatores de Virulência/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosa
6.
J Neurotrauma ; 28(5): 787-96, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21381984

RESUMO

Multiple lines of evidence have validated the Rho pathway as important in controlling the neuronal response to growth inhibitory proteins after central nervous system (CNS) injury. A drug called BA-210 (trademarked as Cethrin(®)) blocks activation of Rho and has shown promise in pre-clinical animal studies in being used to treat spinal cord injury (SCI). This is a report of a Phase I/IIa clinical study designed to test the safety and tolerability of the drug, and the neurological status of patients following the administration of a single dose of BA-210 applied during surgery following acute SCI. Patients with thoracic (T2-T12) or cervical (C4-T1) SCI were sequentially recruited for this dose-ranging (0.3 mg to 9 mg Cethrin), multi-center study of 48 patients with complete American Spinal Injury Association assessment (ASIA) A. Vital signs; clinical laboratory tests; computed tomography (CT) scans of the spine, head, and abdomen; magnetic resonance imaging (MRI) of the spine, and ASIA assessment were performed in the pre-study period and in follow-up periods out to 1 year after treatment. The treatment-emergent adverse events that were reported were typical for a population of acute SCI patients, and no serious adverse events were attributed to the drug. The pharmacokinetic analysis showed low levels of systemic exposure to the drug, and there was high inter-patient variability. Changes in ASIA motor scores from baseline were low across all dose groups in thoracic patients (1.8±5.1) and larger in cervical patients (18.6±19.3). The largest change in motor score was observed in the cervical patients treated with 3 mg of Cethrin in whom a 27.3±13.3 point improvement in ASIA motor score at 12 months was observed. Approximately 6% of thoracic patients converted from ASIA A to ASIA C or D compared to 31% of cervical patients and 66% for the 3-mg cervical cohort. Although the patient numbers are small, the observed motor recovery in this open-label trial suggests that BA-210 may increase neurological recovery after complete SCI. Further clinical trials with Cethrin in SCI patients are planned, to establish evidence of efficacy.


Assuntos
ADP Ribose Transferases/uso terapêutico , Toxinas Botulínicas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , ADP Ribose Transferases/farmacocinética , Adulto , Toxinas Botulínicas/farmacocinética , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Fármacos Neuroprotetores/farmacocinética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Recuperação de Função Fisiológica
7.
PLoS One ; 6(1): e16465, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21304583

RESUMO

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming.


Assuntos
Bactérias/metabolismo , Reprogramação Celular , Sistemas de Liberação de Medicamentos/métodos , Proteínas Nucleares/administração & dosagem , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/administração & dosagem , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/farmacocinética , Animais , Sistemas de Secreção Bacterianos , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/farmacocinética , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Integrases , Camundongos , Sinais de Localização Nuclear , Proteínas Nucleares/farmacocinética , Pseudomonas aeruginosa/química , Recombinação Genética , Fatores de Transcrição/farmacocinética
8.
FEBS J ; 277(18): 3735-49, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20718861

RESUMO

To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell-free hepatic endosomes, and pure in vitro protease assays. ETA taken up into rat liver in vivo was rapidly associated with plasma membranes (5-30 min), internalized within endosomes (15-60 min), and later translocated into the cytosolic compartment (30-90 min). Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA-A subunit and low molecular mass ETA-A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA-degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA-A. Cell-free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA-A in vitro in a pH-dependent and ATP-dependent manner. Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activation, and DNA fragmentation. In an in vitro assay, intact ETA induced ADP-ribosylation of EF-2 and mitochondrial release of cytochrome c, with the former effect being efficiently increased by a cathepsin B/cathepsin D pretreatment. The data show a novel processing pathway for internalized ETA, involving cathepsins B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction.


Assuntos
ADP Ribose Transferases/metabolismo , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Catepsinas/metabolismo , Exotoxinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/farmacocinética , Animais , Toxinas Bacterianas/farmacocinética , Catepsina B/metabolismo , Catepsina D/metabolismo , Endocitose , Endossomos/enzimologia , Endossomos/metabolismo , Exotoxinas/farmacocinética , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
9.
Int J Pharm ; 374(1-2): 145-52, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19446771

RESUMO

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab' of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab') covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab' to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab' took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab'. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.


Assuntos
ADP Ribose Transferases/farmacocinética , Toxinas Bacterianas/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Exotoxinas/farmacocinética , Fatores de Virulência/farmacocinética , ADP Ribose Transferases/imunologia , ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/farmacologia , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Exotoxinas/imunologia , Exotoxinas/farmacologia , Feminino , Citometria de Fluxo , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Lipossomos , Tamanho da Partícula , Polietilenoglicóis/química , Receptor ErbB-2/imunologia , Compostos de Sulfidrila/química , Fatores de Virulência/imunologia , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosa
10.
Cell Microbiol ; 11(5): 780-95, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19159389

RESUMO

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.


Assuntos
Toxinas Botulínicas/farmacocinética , Ciclosporina/farmacologia , Endossomos/metabolismo , ADP Ribose Transferases/farmacocinética , ADP Ribose Transferases/farmacologia , ADP Ribose Transferases/fisiologia , Animais , Toxinas Botulínicas/farmacologia , Toxinas Botulínicas/fisiologia , Células CACO-2 , Chlorocebus aethiops , Citosol/metabolismo , Células HT29 , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , Transporte Proteico/efeitos dos fármacos , Células Vero
11.
Blood ; 113(16): 3792-800, 2009 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-18988862

RESUMO

Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.


Assuntos
ADP Ribose Transferases/farmacologia , Anticorpos Monoclonais/farmacologia , Toxinas Bacterianas/farmacologia , Exotoxinas/farmacologia , Região Variável de Imunoglobulina/farmacologia , Imunotoxinas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Fatores de Virulência/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , ADP Ribose Transferases/efeitos adversos , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Toxinas Bacterianas/efeitos adversos , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Ensaios Clínicos como Assunto , Endossomos/metabolismo , Exotoxinas/efeitos adversos , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Região Variável de Imunoglobulina/efeitos adversos , Região Variável de Imunoglobulina/genética , Imunotoxinas/efeitos adversos , Imunotoxinas/genética , Imunotoxinas/farmacocinética , Leucemia Linfocítica Crônica de Células B/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Fatores de Virulência/efeitos adversos , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
12.
Basic Clin Pharmacol Toxicol ; 99(6): 398-404, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17169119

RESUMO

LHRH-PE40, a recombinant DNA-derived protein composed of LHRH and Pseudomonas aeruginosa exotoxin A, is being developed for the treatment of malignant tumours. This experiment was designed to assess its preclinical safety. Reproductive toxicity studies, pharmacokinetic studies, single- and repeat-dose intraperitoneal or intravenous toxicity studies in mice, rats and monkeys were conducted to assess the toxicity of LHRH-PE40. In intravenous single-dose studies in mice, the LD50 was 731.26 microg/kg and 676.03 microg/kg in male and female mice respectively. In single-dose studies and repeat-dose range-finding studies in rats, dose-limited severe vascular leakage syndromes occurred. In repeat-dose long-term studies, except drug-related vascular leakage syndromes, other drug-related changes included decreased testis weight and testis atrophy. In single-dose and repeat-dose studies in monkeys, dose-limited acute tubular necrosis of the kidneys was the chief finding. In reproductive studies, drug-related changes were decreased food intakes, decreased testis weight and uterus weight, decreased foetus weight and increased foetus mortality, increased maternal and F1 offspring mortality and decreased maternal and F1 offspring body weight. Pharmacokinetic studies showed a similar half-time of distribution and clearance in mice and monkeys. Tissue distribution showed a high concentration in the kidneys and a low concentration in the brain. LHRH-PE40 induced vascular leak syndromes in rats and acute tubular necrosis in monkeys. It also led to testicle atrophy in rats and overt productive toxicity to parents and F1 generations in mice. Because of these findings, it should be monitored carefully in human clinical trials for things such as respiratory, urinary and reproductive toxicities.


Assuntos
ADP Ribose Transferases/toxicidade , Antineoplásicos/toxicidade , Toxinas Bacterianas/toxicidade , Exotoxinas/toxicidade , Hormônio Liberador de Gonadotropina/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Fatores de Virulência/toxicidade , ADP Ribose Transferases/imunologia , ADP Ribose Transferases/farmacocinética , Animais , Anticorpos/sangue , Antineoplásicos/imunologia , Antineoplásicos/farmacocinética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Exotoxinas/imunologia , Exotoxinas/farmacocinética , Feminino , Hormônio Liberador de Gonadotropina/imunologia , Hormônio Liberador de Gonadotropina/farmacocinética , Rim/efeitos dos fármacos , Rim/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Neoplasias/tratamento farmacológico , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Reprodução/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testículo/patologia , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimento , Fatores de Virulência/imunologia , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
13.
Clin Cancer Res ; 12(10): 3145-51, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16707614

RESUMO

PURPOSE: To determine if the tumor-targeted cytotoxin interleukin 13 bound to Pseudomonas exotoxin (IL13-PE) could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution in real-time using a surrogate magnetic resonance imaging tracer, we used convection-enhanced delivery to perfuse rat and primate brainstems with IL13-PE and gadolinium-bound albumin (Gd-albumin). EXPERIMENTAL DESIGN: Thirty rats underwent convective brainstem perfusion of IL13-PE (0.25, 0.5, or 10 microg/mL) or vehicle. Twelve primates underwent convective brainstem perfusion of either IL13-PE (0.25, 0.5, or 10 microg/mL; n = 8), co-infusion of 125I-IL13-PE and Gd-albumin (n = 2), or co-infusion of IL13-PE (0.5 microg/mL) and Gd-albumin (n = 2). The animals were permitted to survive for up to 28 days before sacrifice and histologic assessment. RESULTS: Rats showed no evidence of toxicity at all doses. Primates showed no toxicity at 0.25 or 0.5 microg/mL but showed clinical and histologic toxicity at 10 microg/mL. Quantitative autoradiography confirmed that Gd-albumin precisely tracked IL13-PE anatomic distribution and accurately showed the volume of distribution. CONCLUSIONS: IL13-PE can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and over clinically relevant volumes using convection-enhanced delivery. Moreover, the distribution of IL13-PE can be accurately tracked by co-infusion of Gd-albumin using real-time magnetic resonance imaging.


Assuntos
ADP Ribose Transferases/farmacocinética , Toxinas Bacterianas/farmacocinética , Barreira Hematoencefálica , Tronco Encefálico/química , Exotoxinas/farmacocinética , Interleucina-13/farmacocinética , Fatores de Virulência/farmacocinética , ADP Ribose Transferases/efeitos adversos , Animais , Autorradiografia , Toxinas Bacterianas/efeitos adversos , Neoplasias do Tronco Encefálico/tratamento farmacológico , Meios de Contraste/administração & dosagem , Convecção , Relação Dose-Resposta a Droga , Exotoxinas/efeitos adversos , Gadolínio/administração & dosagem , Glioma/tratamento farmacológico , Interleucina-13/efeitos adversos , Macaca mulatta , Substâncias Macromoleculares , Imageamento por Ressonância Magnética , Masculino , Ratos , Ratos Sprague-Dawley , Albumina Sérica/administração & dosagem , Fatores de Virulência/efeitos adversos , Exotoxina A de Pseudomonas aeruginosa
14.
World J Gastroenterol ; 10(19): 2870-3, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15334689

RESUMO

AIM: To explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) and LHRH binding to LHRH receptor (LHRHR) on the membrane surface of human liver cancer HEPG cells. METHODS: LHRH was labeled by using (125)I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay. RESULTS: The specific activity of LHRH labeled with (125)I was 2.7 x 10(4) kBq/microL, and its radiochemical purity reached to 99.2-99.7%. The binding of (125)I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of (125)I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of (125)I-LHRH binding to the membrane LHRHR of HEPG cells, (125)I-LHRH of 1 x 10(5) cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40 and LHRH binding to the membrane LHRHR of HEPG cells were 0.43+/-0.12 nmol/L and 4.86+/-1.47 nmol/L, respectively, and their receptor volumes were 0.37+/-0.15 micromol/g and 0.42+/-0.13 micromol/g, respectively. The binding of LHRH-PE40 to the membrane protein of normal liver cells was not observed. CONCLUSION: The recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.


Assuntos
ADP Ribose Transferases/farmacocinética , Antineoplásicos/farmacocinética , Toxinas Bacterianas/farmacocinética , Exotoxinas/farmacocinética , Hormônio Liberador de Gonadotropina/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores LHRH/metabolismo , Fatores de Virulência/farmacocinética , ADP Ribose Transferases/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Exotoxinas/farmacologia , Humanos , Cinética , Neoplasias Hepáticas/patologia , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosa
15.
Mol Cancer Ther ; 1(2): 79-84, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12467225

RESUMO

We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.


Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Dissulfetos/química , Exotoxinas/farmacologia , Imunotoxinas/farmacologia , Mesotelioma/imunologia , Neoplasias Ovarianas/imunologia , Fatores de Virulência/farmacologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Animais , Anticorpos Monoclonais , Antígenos de Neoplasias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Proteínas Ligadas por GPI , Expressão Gênica , Humanos , Fragmentos de Imunoglobulinas/imunologia , Imunotoxinas/genética , Imunotoxinas/farmacocinética , Glicoproteínas de Membrana/metabolismo , Mesotelina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Plasmídeos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
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