RESUMO
AIMS: The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models. MATERIALS & METHODS: Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using (18)F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography. RESULTS: In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05). CONCLUSION: This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.
Assuntos
Carcinoma/patologia , Neoplasias do Colo/patologia , Imunotoxinas/farmacologia , Saponinas/farmacologia , Animais , Carcinoma/diagnóstico , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/tratamento farmacológico , Dianthus/química , Modelos Animais de Doenças , Quimioterapia Combinada , Fator de Crescimento Epidérmico , Hemólise/efeitos dos fármacos , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/efeitos adversos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Proteínas Inativadoras de Ribossomos Tipo 1 , Saponinas/administração & dosagem , Saponinas/efeitos adversos , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Humanos , Imunotoxinas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponinas/química , Saporinas , Trastuzumab , Triterpenos/químicaRESUMO
Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to ß-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins.
Assuntos
Antineoplásicos/metabolismo , Imunotoxinas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Imunotoxinas/genética , Imunotoxinas/metabolismo , Imunotoxinas/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Reprodutibilidade dos Testes , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Saponins are a group of plant and marine derived glycosides with numerous biological functions. Two important characteristics of certain plant saponins are their ability to enhance cytotoxicity of type I ribosome inactivating proteins and stimulation of the immune system. The main objective of the present study was to investigate in real-time the permeabilizing effects of saponins on cell membrane. A set of oleanane saponins (glycyrrhizinic acid, Gypsophila, Saponaria and Quillaja saponins) and a steroid saponin (digitonin) were tested. The effects of these saponins on lysosomal membranes and hemolysis, along with their charge were also studied. Real-time monitoring of cell membrane permeabilization facilitated a highly sensitive analysis of the cellular kinetics. Saponins showed variable permeabilizing effects on cellular and lysosomal membranes at concentrations from 6 µM and hemolysis from 3 µM. Further, the results suggest that charge of the saponin may be relevant for permeabilizing effects of oleanane saponins.
Assuntos
Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Eletroforese , Humanos , Ácido Oleanólico/farmacologiaRESUMO
Quillaja saponins are used as adjuvants in animal vaccines but their application in human vaccination is still under investigation. Isolation and characterization of adjuvant saponins is very tedious. Furthermore, standardization of Quillaja saponins is critical pertaining to its application in humans. In this study, a convenient method based on agarose gel electrophoresis was developed for the separation of Quillaja saponins. Six different commercial Quillaja saponins were segregated by size/charge into numerous fractions. Each of the fractions was characterized by ESI-TOF-MS spectroscopy and thin layer chromatography. Real-time impedance-based monitoring and red blood cell lysis assay were used to evaluate cytotoxicity and hemolytic activities respectively. Two specific regions in the agarose gel (delimited by specific relative electrophoretic mobility values) were identified and characterized by exclusive migration of acylated saponins known to possess immune adjuvant properties (0.18-0.58), and cytotoxic and hemolytic saponins (0.18-0.94). In vivo experiments in mice with the isolated fractions for evaluation of adjuvant activity also correlated with the relative electrophoretic mobility. In addition to the separation of specific Quillaja saponins with adjuvant effects as a pre-purification step to HPLC, agarose gel electrophoresis stands out as a new method for rapid screening, separation and quality control of saponins.
Assuntos
Adjuvantes Imunológicos/isolamento & purificação , Saponinas de Quilaia/isolamento & purificação , Adjuvantes Imunológicos/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ensaio de Desvio de Mobilidade Eletroforética , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Quillaja/química , Saponinas de Quilaia/química , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.
Assuntos
Endossomos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Toxinas Biológicas/farmacologia , Linhagem Celular , Endossomos/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Lisossomos/metabolismo , Toxinas Biológicas/uso terapêuticoRESUMO
Targeted toxin-based therapeutics are hindered by poor intracellular uptake, limited stability and non-specific immune stimulation. To address these problems, ligand-targeted toxins in combination with low dose saponin mixtures have been adapted and tested in vivo in the past, however, undefined saponin raw mixtures are not suitable for use in clinical development. In the present work we therefore used a targeted toxin (Sap3-EGF, i.e. saporin fused to epidermal growth factor) in combination with a structurally defined isolated saponin m/z 1861 (SO-1861). In vitro evaluation confirmed a 6900-fold enhancement in the cytotoxic efficacy of Sap3-EGF against TSA-EGFR target cells. The required dose of the targeted toxin was appreciably reduced and there was a highly synergistic effect observed. An ex vivo hemolysis assay showed no or very less hemolysis up to 10 µg/mL of SO-1861. In the acute toxicity studies SO-1861 was found to be non-toxic up to a dose of 100 µg/treatment. The enzymes aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase did not show any statistically significant liver damage, which was further confirmed by histological examination. Additionally, creatinine was also similar to the control group thus ruling out damage to kidney. In vivo studies in a syngeneic BALB/c tumor model characterized by EGFR overexpression were done by applying 30 µg SO-1861 and 0.1 µg Sap3-EGF per treatment. A more than 90% reduction (p < 0.05) in the average tumor volume was observed by this combined therapy.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Fator de Crescimento Epidérmico/uso terapêutico , Imunotoxinas/uso terapêutico , Veículos Farmacêuticos/metabolismo , Saponinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacocinética , Receptores ErbB/metabolismo , Feminino , Humanos , Imunotoxinas/metabolismo , Imunotoxinas/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Veículos Farmacêuticos/química , Veículos Farmacêuticos/isolamento & purificação , Saponaria/química , Saponinas/química , Saponinas/isolamento & purificaçãoRESUMO
Macromolecular interaction of protein toxins with certain plant triterpenoids holds potential for application in tumor therapy. The ability of only certain saponins to enhance the endosomal escape of toxins specifically in tumor cells was evaluated and set into correlation with the electrophoretic mobility. Saponins from Saponaria officinalis Linn, were selected as a lead to understand this evolutionarily conserved principle in detail. Agarose gel electrophoresis was utilized to procure pure saponin fractions with different electrophoretic mobility, which were tested for their ability to enhance the toxicity by live cell monitoring. Five fractions (SOG1-SOG5) were isolated with a relative electrophoretic mobility of (-0.05, 0.41, 0.59, 0.75 and 1.00) and evaluated using thin layer chromatography, HPLC, and mass spectroscopic analysis. Cytotoxicity experiments revealed highest effectiveness with SOG3. Live cell imaging experiments with SOG3 revealed that this saponin with a specific REM of 0.59 could assist in the lyso/endosomal release of the toxic payload without affecting the integrity of plasma membrane and could lead to the induction of apoptosis. This charge dependent enhancement was also found to be highly specific to type I ribosome inactivating proteins compared to bacterial toxins. Charge interaction of plant toxins and saponins with tumor cells, plays a major role in toxin specific modulation of response. The finding opens up newer ways of finding protein saponin interaction conserved evolutionarily and to test their role in endosomal escape of therapeutic molecules.
Assuntos
Saponinas/química , Toxinas Biológicas/química , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Camundongos , Extratos Vegetais/química , Saponaria/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.