RESUMO
The Chinese yam (Dioscorea polystachya, DP) is known for the nutritional value of its tuber. Nevertheless, DP also has promising pharmacological properties. Compared with the tuber, the leaves of DP are still very little studied. However, it may be possible to draw conclusions about the plant quality based on the coloration of the leaves. Magnesium, as a component of chlorophyll, seems to play a role. Therefore, the aim of this research work was to develop an atomic absorption spectrometry-based method for the analysis of magnesium (285.2125 nm) in leaf extracts of DP following the graphite furnace sub-technique. The optimization of the pyrolysis and atomization temperatures resulted in 1500 °C and 1800 °C, respectively. The general presence of flavonoids in the extracts was detected and could explain the high pyrolysis temperature due to the potential complexation of magnesium. The elaborated method had linearity in a range of 1-10 µg L-1 (R2 = 0.9975). The limits of detection and quantification amounted to 0.23 µg L-1 and 2.00 µg L-1, respectively. The characteristic mass was 0.027 pg, and the recovery was 96.7-102.0%. Finally, the method was applied to extracts prepared from differently colored leaves of DP. Similar magnesium contents were obtained for extracts made of dried and fresh leaves. It is often assumed that the yellowing of the leaves is associated with reduced magnesium content. However, the results indicated that yellow leaves are not due to lower magnesium levels. This stimulates the future analysis of DP leaves considering other essential minerals such as molybdenum or manganese.
Assuntos
Dioscorea , Magnésio , Espectrofotometria Atômica , Clorofila , FlavonoidesRESUMO
While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-ß-glucosidases, pectinesterases, polygalacturonases, ß-galactosidases and ß-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.
Assuntos
Vesículas Extracelulares/ultraestrutura , Magnoliopsida/metabolismo , Metabolismo Secundário , Técnicas de Cultura de Células , Células Cultivadas , Craterostigma , Fosfolipídeos/metabolismo , ProteomaRESUMO
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
Herbal medicine has been used to treat diseases for centuries; however, the biological active components and the mechanistic understanding of actions of plant-derived drugs are permanently discussed. MicroRNAs are a class of small, non-coding RNAs that play crucial roles as regulators of gene expression. In recent years, an increasing number of reports showed that microRNAs not only execute biological functions within their original system, they can also be transmited from one species to another, inducing a posttranscriptional repression of protein synthesis in the recipient. This cross-kingdom regulation of microRNAs provides thrilling clues that small RNAs from medicinal plants might act as new bioactive components, interacting with the mammalian system.In this article, we provide an overview of the cross-kingdom communication of plant-derived microRNAs. We summarize the microRNAs identified in medicinal plants, their potential targets in mammals, and discuss several recent studies concerning the therapeutic applications of plant-based microRNAs. Health regulations of herbal microRNAs in mammals are a new concept. Continuing efforts in this area will broaden our understanding of biological actions of herbal remedies, and will open the way for the development of new approaches to prevent or treat human diseases.
Assuntos
MicroRNAs/farmacologia , Plantas Medicinais/química , RNA de Plantas/farmacologia , Animais , Humanos , MicroRNAs/isolamento & purificação , Plantas Medicinais/genética , RNA de Plantas/isolamento & purificaçãoRESUMO
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
CONTEXT: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein saporin and an EGF-saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins. OBJECTIVE: To investigate the augmentative property of SA on saporin and saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells. MATERIALS AND METHODS: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for saporin and five saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay. RESULTS: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted for the non-immunospecific effects of SA on an off-target IT, the true augmentative effects of SA were found to be largely non-immunospecific. Antibody blocking studies demonstrated that the augmentative effect of SA was only partially immunospecific. Separate exposure of target cells to IT and SA at different times demonstrated that immunospecific augmentation of IT by SA could be achieved but only if cells were exposed to IT first and SA second. CONCLUSIONS: SA significantly, although variably, augments the cytotoxicity of saporin and saporin-based immunotoxins. Concomitant exposure to both IT and SA can result in non-immunospecific cytotoxicity that can be overcome by temporally separating exposure to each.
Assuntos
Apoptose/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Antígenos de Diferenciação de Linfócitos T/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/imunologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Esquema de Medicação , Citometria de Fluxo , Humanos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/administração & dosagem , Saponinas/isolamento & purificação , Saporinas , Triterpenos/administração & dosagem , Triterpenos/isolamento & purificaçãoRESUMO
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
Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.
Assuntos
Endocitose , Saponinas , Animais , Camundongos , Saponinas/metabolismo , Arginina , Endossomos/metabolismo , Linhagem Celular Tumoral , Mutação , Análise Mutacional de DNA , Sobrevivência Celular/efeitos dos fármacosRESUMO
Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.
Assuntos
Saponinas , Animais , Humanos , Camundongos , Saponinas/química , Saponinas/farmacologia , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Peptídeos/química , Transfecção/métodos , Saponaria/química , Saporinas/química , Saporinas/farmacologia , Terapia Genética , Sobrevivência Celular/efeitos dos fármacos , Cátions/químicaRESUMO
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
The authors wish to make corrections to their paper [...].
RESUMO
Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.
Assuntos
Saponaria , Vaccaria , N-Glicosil Hidrolases/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Isoformas de Proteínas , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Ribossomos/metabolismo , Saponaria/química , Saponaria/metabolismo , Sementes/químicaRESUMO
Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt's lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.
Assuntos
Imunotoxinas , Linfoma de Células B , Linfoma , Neoplasias , Animais , Anticorpos Monoclonais Humanizados , Humanos , Imunotoxinas/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Camundongos , SaponinasRESUMO
Due to the lower risks of adverse effects, nonviral gene therapy is a suitable alternative to transfect cancer cells with a suicide gene to let them kill themselves by expressing toxic ribosome-inactivating proteins. Plasmids are stable and easy-to-produce vectors, but they have some disadvantages due to the bacterial backbone. Applying the minicircle technology, this problem can be solved with manageable effort in a well-equipped laboratory. With the described methodology, minicircle-DNA can be produced at low costs. The cell killing properties are monitored following transfection using the CytoSMART® Omni system-a camera based live cell imaging device.
Assuntos
Vetores Genéticos , Proteínas Inativadoras de Ribossomos , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , Ribossomos/genética , TransfecçãoRESUMO
Conventional eukaryotic expression plasmids contain a DNA backbone that is dispensable for the cellular expression of the transgene. In order to reduce the vector size, minicircle DNA technology was introduced. A drawback of the minicircle technology are considerable production costs. Nanoplasmids are a relatively new class of mini-DNA constructs that are of tremendous potential for pharmaceutical applications. In this study we have designed novel suicide nanoplasmid constructs coding for plant derived ribosome-inactivating proteins. The suicide-nanoplasmids were formulated with a targeted K16-lysine domain, analyzed for size, and characterized by electron microscopy. The anti-proliferative activity of the suicide-nanoplasmids was investigated in vitro by real time microscopy and the expression kinetic was determined using an enhanced green fluorescent protein nanoplasmid variant. In an aggressive in vivo neuroblastoma tumor model, treated mice showed a reduced tumor growth whereby the therapy was well tolerated.
Assuntos
Vetores Genéticos , Proteínas Inativadoras de Ribossomos , Animais , Camundongos , Plasmídeos , RibossomosRESUMO
Saponinum album (SA) is a commercial mixture of saponins isolated from Gypsophila species. In the previously published work, we reported that SA dramatically improves the inhibition of tumor growth by targeted toxins in mice in a synergistic way. Here we report a simplified electrophoretic method for the isolation of a highly effective fraction of SA with a relative electrophoretic mobility to the dye front (R(f) ) of 0.63 from the mixture. In total, four different fractions were separated at a preparative scale, and evaluated by ESI-MS, HPLC and TLC analysis. Electrophoretic mobility and electrochemical properties of the different fractions of saponins from SA were set into relation to their ability to enhance the cytotoxicity of epidermal growth factor (EGF)-based targeted toxins. We here treated HER-14 cells, which are NIH-3T3 Swiss mouse embryo cells transfected with the human EGF receptor. Untransfected NIH-3T3 cells served as control. The major bulk of SA (72.3%) (R(f) =0.78) migrated the farthest and was found to be significantly ineffective (p<0.05) in enhancing the cytotoxicity of the targeted toxin, while the second fraction (R(f) =0.63) showed an enhancement of 9800-fold. The third (R(f) =0.56) had an enhancement factor of 3200, the fourth (R(f) =0.08) was again significantly ineffective (p<0.05) in exhibiting any enhancement of cytotoxicity.
Assuntos
Eletroforese/métodos , Imunotoxinas/metabolismo , Saponinas/isolamento & purificação , Animais , Caryophyllaceae/química , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Saponinas/química , Saponinas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better.