RESUMO
The ability of certain triterpenoid saponins to modulate the endosomal release during the process of endocytosis and to ensure a nontoxic and efficient transfection recently led to an exceptional interest in the field of nonviral gene delivery. In vitro and in vivo studies demonstrated promising results in terms of tumor growth inhibition after the delivery of a suicide gene such as saporin and dianthin. With that, the question arises which structural features are necessary or advantageous to achieve an effective endosomal escape. Former studies described certain important characteristics a potent saponin should have. Particularly SA1641 (Gypsophila paniculata) and SO1861 (Saponaria officinalis) played an utmost important role to get a first insight into the structure-activity relationship. However, a number of issues such as the purpose of functional groups on the aglycon and the substitution of sugars and their modification remain unsolved and their value needs to be specified. By conducting a screening of several diverse saponins in terms of their transfection improving ability, we aimed to examine these questions in more detail and get a better understanding of the relevant features. The transfection of Neuro-2A-cells with GFP-DNA containing peptide-based nanoplexes provided a reliable method in order to compare the activity of the saponins. With that, we were able to provide new and essential insights regarding the structure-activity relationship of transfection-modulating saponins and give an idea of how a highly potent saponin for future gene therapies may look like.
Assuntos
Técnicas de Transferência de Genes , Saponinas/farmacologia , Transfecção , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Camundongos , Nanoestruturas , Saponinas/química , Relação Estrutura-Atividade , Transfecção/métodosRESUMO
We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptores da Calcitonina/antagonistas & inibidores , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Humanos , Receptores da Calcitonina/imunologia , Células Tumorais CultivadasRESUMO
Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.
Assuntos
Vacinas Anticâncer/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Saponinas/farmacologia , Neoplasias Cutâneas/terapia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígeno CD11b/metabolismo , Vacinas Anticâncer/química , Linhagem Celular Tumoral , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Feminino , Humanos , Imunidade Celular/efeitos dos fármacos , Inflamassomos/imunologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/imunologia , Saponinas/imunologia , Neoplasias Cutâneas/imunologiaRESUMO
Triterpenoidal saponins are synthesized in the roots of Saponaria officinalis L. The same plant is also a source for the toxin Saporin, which is a ribosome-inactivating protein. Triterpenoidal saponins are known to increase the cytotoxicity of Saporin by modulating its intracellular trafficking. Here, we investigated if the combinatorial effects elicited by purified saponins and Saporin can be applied to increase the therapeutic efficacy of the immunotoxin Saporin-Rituximab. First, saponins were purified by high-performance liquid chromatography. Thereafter, their intrinsic cytotoxicity was evaluated on Ramos cells with no observed effect up to 5 µg/mL, however, saponins increased the cytotoxicity of Saporin, while no influence was observed on its N-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI50 (concentration inhibiting cell growth to 50â%) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI50 of Saporin-Rituximab was 0.01 nM, a nearly 700-fold increase in efficacy. Moreover, two further immunotoxins, namely Saporin-anti-CD22 and Saporin-anti-CD25, were tested in combination with saponins yielding enhancement factors of 170-fold and 25-fold, respectively. All three receptors are present in Ramos cells and the differences in cytotoxicity enhancement may be explained by the differing expression levels of the cellular receptors. The application of purified saponins from S. officinalis L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.
Assuntos
Imunotoxinas/farmacologia , Linfoma de Células B/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Rituximab/farmacologia , Saponaria/química , Saponinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Humanos , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/química , Saponinas/isolamento & purificação , SaporinasRESUMO
The therapeutic relevance of immunotoxins is based on the conjugation of monoclonal antibodies to toxins. In cancer therapies, the conjugated antibodies not only direct the binding of immunotoxins to cancer-specific receptors and mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. In the present study, the therapeutic antibodies Cetuximab (anti-EGFR, Erbitux(®)), Panitumumab (anti-EGFR, Vectibix(®)) and Trastuzumab (anti-HER2, Herceptin(®)) were chemically conjugated to the toxin dianthin. In the first instance, recombinant dianthin was characterized by mass spectrometry and its stability was analyzed by circular dichroism. Dianthin showed increased cytotoxicity on MCF-7 cells when tested in combination with a glycosylated triterpenoid (SO1861) in a real-time impedance-based cytotoxicity assay. In data obtained by live cell imaging, SO1861 specifically mediated the endo/lysosomal escape of dianthin without disrupting the plasma membrane. The purity of immunotoxins was confirmed by SDS-PAGE and Western blot. Their cytotoxicity was evaluated in the presence of SO1861 and dianthin-Cetuximab presented a GI50 (50% growth inhibition) of 5.3pM, dianthin-Panitumumab of 1.5pM, and dianthin-Trastuzumab of 23pM. Finally, the specificity of these immunotoxins was validated in a fluorescence-based real-time assay, where their binding to target cells was prevented by preincubation with an excess of label-free unconjugated antibody. Based on these data, we propose the use of dianthin and SO1861 as a new platform technology to enhance the efficacy of therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cetuximab/farmacologia , Imunotoxinas/farmacologia , Saponinas/farmacologia , Trastuzumab/farmacologia , Sequência de Aminoácidos , Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/administração & dosagem , Reagentes de Ligações Cruzadas/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Células HCT116 , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/genética , Imunotoxinas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Dados de Sequência Molecular , Panitumumabe , Estabilidade Proteica , Proteínas Recombinantes , Saponinas/administração & dosagem , Trastuzumab/administração & dosagemRESUMO
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
Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Glicosídeos/química , Lipídeos/química , Peptídeos/química , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Nanopartículas/química , Saponaria/química , TransfecçãoRESUMO
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
The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success.
Assuntos
Imunotoxinas/química , Imunotoxinas/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos/metabolismo , Animais , Humanos , Imunotoxinas/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
Saponaria officinalis L. (Caryophyllaceae), also known as fuller's herb or soapwort is a medicinal plant, which grows from Europe to Central Asia. Medicinal properties attributed to this plant include its antitussive and galactogogue properties. Recently, bisdesmodic saponins with very specific structural features from S. officinalis have been shown to strongly enhance the efficacy of specific targeted toxins (anti-tumor antibodies connected to protein toxins) in-vitro and in-vivo in a synergistic manner. In the presently reported novel approach we used preparative all-liquid high-speed countercurrent chromatography (HSCCC) to recover a total of 22 fractions using biphasic solvent system tert-butylmethylether/n-butanol/acetonitrile/water 1:3:1:5 (v/v/v/v) from a complex precipitated crude saponin mixture. Out of these 22 fractions, 3 fractions had the enhancer effect on anti-tumor toxins out of which one fraction (F7) was further tested elaborately in different cell lines. The molecular weight distribution and compound profiles of separated saponins were monitored by off-line injections of the sequentially collected fractions to an electrospray ion-trap mass-spectrometry system (ESI-IT-MS). The functional saponin fractions were mainly bisdesmosidc and contained saponin m/z 1861 amongst other. Using the bio-assay guided monitoring, the highly active fractions containing 2 to 3 bisdesmodic saponins (5µg/mL) were screened for their effectiveness in enhancing the anti-tumor activity of targeted toxin Sap3-EGF, which was determined using the impedance based real-time cell cytotoxicity evaluation. This novel combination of HSCCC fractionation, MS-target-guided profiling procedure and bio-assay guided fractionation yielded 100mg of functional saponins from a 60g crude drug powder in a rapid and convenient manner.
Assuntos
Distribuição Contracorrente/métodos , Extratos Vegetais/química , Saponaria/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Antineoplásicos , Sinergismo Farmacológico , Imunotoxinas , Saponinas/análise , Saponinas/química , Saponinas/isolamento & purificaçãoRESUMO
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
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
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
Eleven triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wender. (G. trichotoma Wender. var. trichotoma) (Caryophyllaceae), together with one known compound. The structures were established on the basis of extensive NMR analysis ((1)H, (13)C NMR, COSY, TOCSY, ROESY, HSQC, and HMBC), completed by analysis of HR-ESI-MS and ESI-MS(n). The saponins have the commonly found gypsogenin as the aglycone substituted at C-3 with trisaccharide and at C-28 with oligosaccharide through a fucose residue, as saponins isolated from Gypsophila perfoliata L. originated from China. The oligosaccharide attached to C-28 is substituted with acetyl and (or) sulfate groups. Тhe cytotoxicity of the saponin extract from G. trichotoma was evaluated against a rat alveolar macrophage-like cell line NR8383 and human leukemia cell lines U937 and BV-173. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.
Assuntos
Caryophyllaceae/química , Raízes de Plantas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Conformação Molecular , Ratos , Saponinas/química , Relação Estrutura-Atividade , Triterpenos/química , Células U937RESUMO
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
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
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.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Citosol/metabolismo , Portadores de Fármacos/química , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Saponinas/química , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos Fitogênicos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Rastreamento de Células , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/toxicidade , Endocitose , Fator de Crescimento Epidérmico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Ligação Proteica , Transporte Proteico , Proteínas Inativadoras de Ribossomos Tipo 1/farmacocinética , Proteínas Inativadoras de Ribossomos Tipo 1/uso terapêutico , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Saponinas/farmacocinética , Saponinas/toxicidade , Saporinas , Análise de Célula Única , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Testes de Toxicidade Aguda , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This work describes the application of an impedance-based measurement for the real time evaluation of targeted tumor therapies in cell culture (HeLa cells). We used a treatment procedure that is well established in cells and mice. Therein, tumor cells are treated with a combination of an epidermal growth factor-based targeted toxin named SE and particular plant glycosides called saponins. In the present study HeLa cells were seeded in different numbers onto interdigitated electrode structures integrated into the bottom of a 96 well plate. The cells were treated with SE in the presence and absence of the saponin SpnS-1 (isolated from Saponaria officinalis roots). The impedance was directly correlated with the viability of the cells. As expected from known end point measurements, a concentration dependent enhancement of toxicity was observed; however, with the impedance measurement we were for the first time able to trace the temporal changes of cell death during the combination treatment. This substantially added to the understanding of initial cellular mechanisms in the augmentation of the toxicity of targeted toxins by saponins and indicated the superiority of real time monitoring over end point assays. The method is less labor intensive and label-free with ease of monitoring the effects at each time point.
Assuntos
Técnicas Biossensoriais/métodos , Sobrevivência Celular/efeitos dos fármacos , Saponinas/administração & dosagem , Toxinas Biológicas/administração & dosagem , Antineoplásicos/administração & dosagem , Sistemas Computacionais , Sinergismo Farmacológico , Impedância Elétrica , Receptores ErbB/antagonistas & inibidores , Células HeLa , HumanosRESUMO
Tumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects. This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR. We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells. We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity. Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641. We further demonstrate that SA1641 did not influence the plasma membrane permeability, indicating an intracellular interaction of SA1641 and the toxin components of targeted toxins. Surface plasmon resonance measurements point to a transient binding of SA1641 to the toxin components of targeted toxins.
Assuntos
Receptores ErbB/metabolismo , Imunotoxinas/química , Imunotoxinas/farmacologia , Saponinas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Humanos , Camundongos , Células NIH 3T3 , Ressonância de Plasmônio de SuperfícieRESUMO
Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC-FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2â1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7-8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2-50 µg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9±4.4% at 5.6 µg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8±1.2% at 25 µg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the 'Rasayana' properties of this plant.