Search

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

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.

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.

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 K(16)-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.

Neuroblastoma represents the third most common malign neoplasm occurring in children and the most common in newborn. Although mortality in childhood cancer declined in the last decade, high-risk patients have poor prospects, due to the aggressiveness of the cancer. In the recent past, we underlined the potential of sapofectosid as novel and efficient transfection enhancer, demonstrating non-toxic gene delivery, but its value in tumor therapies has yet to be elucidated. A suicide gene, coding for saporin, a ribosome-inactivating protein type I, was incorporated into targeted, peptide-based nanoplexes. The nanoplexes were characterized for their size, zeta potential and appearance by electron microscopy. Gene delivery was observed via confocal imaging. In vitro transfections were conducted to monitor the real-time cell viability. After initial tolerability studies, NMRI nu/nu-mice bearing tumors from Neuro-2A-Luc-cells (murine neuroblastoma cells, transduced with a luciferase gene), were treated with targeted nanoplexes (30 μg saporin-DNA i.v./treatment) and sapofectosid (30 μg s.c. treatment). The treatment was compared to a vehicle (PBS) control and treatment without sapofectosid in terms of body weight, tumor growth and integrated density of tumor luminescence. The study revealed an anti-tumoral effect of the sapofectosid mediated gene therapy in the Neuro-2A-tumor model. The treatments were well tolerated by the animals indicating the applicability of this approach. With these results, we were able to proof the efficacy of a therapy, consisting of targeted suicide gene nanoplexes and sapofectosid, a novel and potent transfection enhancer. This study points out the enormous value for future targeted cancer and gene therapies.

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma, however, a number of protein-based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome-inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC-3 and MIA PaCa-2 revealed no significant differences to off-target cells in the half maximal inhibitory concentration (IC50) for the fusion protein. In contrast, combination with SO1861 decreased the IC50 for BxPC-3 cells from 100 nM to 0.17 nM, whereas control cells remained unaffected. Monotherapy of BxPC-3 xenografts in CD-1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm3) when compared to placebo, however, combined treatment with SO1861 resulted in a more than 13-fold better efficacy (3.0 mm3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment. This article is protected by copyright. All rights reserved.

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.

Phytochemical analysis of the mature fruits of Furcraea tuberosa (Agavaceae) resulted in the isolation of a new bisdesmosidic spirostanol saponin (1), along with eight known steroidal glycosides (2-9), one known phenolic carboxylic acid ester (10) and three known flavonol glycosides (11-13). The structures of these compounds were assigned using a combination of ID and 2D NMR techniques including ¹H, ¹³C, COSY, TOCSY, HSQC and HMBC NMR, and confirmed by mass spectrometry. Thus the new saponin was elucidated as (25R)-6α-(β-D-glucopyranosyloxy)-5α-spirostane-3β-Ο-[(6-Ο-hexadecanoyl)-β-D- glucopyranoside]. The literature survey revealed that most of the steroidal saponins isolated have shown potent cytotoxic effects against various human cancer cell lines and the results are herein reviewed.

Transfection is the process to deliver nucleic acid into eukaryotic cells. Different transfection techniques already exist. However, they can be expensive and toxic toward subjected cells. Previous research shed light on natural occurring molecules called triterpene saponins that have great potential for the non-viral gene delivery. Using a combination of different chromatographic techniques and in vitro transfection bioassays, a new triterpenoid saponin (agrostemmoside E) from the plant Agrostemma githago L. was isolated. Agrostemmoside E was characterized by mass spectrometry, intense NMR spectroscopy and was identified as 3-{O-ß-D-Galactopyranosyl-(1→2)]-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucuronopyranosyl} quillaic acid 28-O-{[ß-D-4,6-di-(O-acetyl)-glucopyranosyl-(1→3)]-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)}-[3,4-di-(O-acetyl)-ß-D-quinovopyranosyl-(1→4)]-ß-D-fucopyranoside ester. Agrostemmoside E has a great potential for delivery of gene loaded nanoplexes and increased the transfection efficiency by 70% compared to 2% without agrostemmoside E. By comparative toxicity studies, we show that agrostemmoside E can be applied at high concentrations without toxicity, justifying its use as a new tool for gene transfections.

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 50 %) 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.

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.

Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.

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.

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.

International audience; Saponins enhance the cytotoxicity of the type I ribosome-inactivating protein (RIP-I) saporin. In the present study, the synergistic cytotoxicity of nine newly isolated gypsogenin-containing saponins from Gypsophila trichotoma in combination with RIP-I was evaluated in vitro and used to derive a quantitative structure – activity relationship (QSAR). The QSAR model distinguished two important structural features of the studied saponins necessary for their cytotoxicity enhancing activity: the branched trisaccharide moiety attached to C-3 should contain one xylose residue instead of an arabinose and the branched tetrasaccharide at C-28 should contain one acetyl group attached to the glucose residue.

ABSTRACT 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 18F-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.

Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain. Different methods have been developed in order to determine the N-glycosidase activity of RIPs and RIP domain containing anti-tumor toxins. However the existing methods are elaborate and include radioassays, HPLC and enzymatic conversion assays. Here, a simple and cost effective N-glycosidase assay is presented, which is based on the direct determination of the released adenine by thin-layer chromatography (TLC) and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. Following TLC development the released adenine is quantified on silica glass plates by UV absorbance at 260 nm.

This thesis treats the relation between integration as a political practice of appropriation and attachment as a personal and intimate process of longing and belonging. It qualitatively investigates how a select group of Syrian refugees develop a sense of belonging in Denmark after having fled from Syria due to the outbreak of the country’s civil war. In general, the refugee stream has led to a political crisis in Europe, which has witnessed tightenings in national integration policies as well in the entire European Union. This political development challenges the possibilities for refugees from non-Western countries and cultures to develop a sense of belonging in Denmark and ‘feel at home’ - even those who have fled from Syria prior to the Arab Spring in 2015. We find that issues of ethnicity play a central role in the refugees’ possibilities for belonging. The notion of ‘home’ is of great empirical significance as well as theoretical interest in this thesis. Drawing on inspiration from Marco Antonsich (2009; 2010) primarily, the thesis argues how complex, personal processes of attachment can be studied within an analytical framework of belonging. A key factor in the new lives of the Syrian informants is voluntarism, which we argue also plays a significant role in their possibilities for belonging. To enrich the thesis’ discussion, it also treats the subject from a postcolonial perspective, and ultimately concludes that the narrow political practices of integration in Denmark, which focuses primarily on labor, challenges the Syrians’ possibilities of becoming integrated fellow citizens. The thesis also includes a suggestion for political integration practices, which might benefit from widening its perspective to also attend to issues of parenthood. Enabling opportunities for future fellow citizens to support their children’s participation in society might open new possibilities for refugees to be able to feel at home in Denmark.

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.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.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.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.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.

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.

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 β-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.

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.

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.

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody- targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

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.

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.

Rasayana constitutes a very important class of Ayurvedic herbs, which acts as rejuvenators and tonic. Despite intensive research on evaluating the medicinal basis for the purported medical benefits of Rasayana herbs, still a specific chemical guiding principle for the characterization of these herbs in one single category is unclear. Here we explore the possibility that polysaccharides play a key role in Rasayana properties exhibited by medicinal plants. Further on this could be a possible field for exploration for a common factor present in some of the Ayurvedic herbs. The present review covers a literature spanning from 1956 to 2011. Some translations of traditional Ayurvedic texts dating back to the first century AD have been referred to as well. In our assessment of the present literature and studies carried out it is presupposed that the presence of benevolent plant polysaccharides must be one of the important features symbolizing a common effectiveness in most of the Ayurvedic Rasayana herbs. Of the many plant metabolites polysaccharides have not so far been considered and studied effectively compared to other secondary metabolites like saponins, alkaloids, etc. Although all the results reported until now suggest a major contribution of polysaccharide towards the maintenance of physiological homeostasis, which is the guiding principle of Rasayana therapy. The present review is an attempt to find a connective link between the concept of Rasayana and well-being; and the role of plant polysaccharides. Lack of clinical information on number of polysaccharides showing promise is a limiting factor for a complete understanding. It is also important to carry out a molecular interaction study to understand the behavior of polysaccharides discussed in the present review.

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.

Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved.

Background and purpose: Certain saponins synergize with antitumour drugs to enhance their efficacy, but the mechanisms underlying this synergy in vivo are not well studied. Here, we describe the distribution of Saponinum album (Spn) from Gypsophila paniculata L. in mice after subcutaneous injection. Experimental approach: The [3H]-labelled Spn used for in vivo experiments was biologically active, as it still increased the cytotoxicity of a chimeric toxin in vitro. Distribution of [3H]-Spn was measured in BALB/c mice, with or without subcutaneous tumours in the flank. Labelled Spn was subcutaneously injected in the neck, and samples of organs, blood, urine and tumour tissue were analysed for radioactivity, 5–240 min after the injection. Key results: The majority of [3H]-Spn distributed within 10 min throughout the entire animal, with high levels of radioactivity in the urine by 30 min. No preferential accumulation in tumour tissue or other organs was observed. In tumour-bearing mice, using a sequential combination of Spn (given first) and a chimeric toxin against the epidermal growth factor receptor, ErbB1, we tested two different pretreatment times for Spn. There was high antitumour efficacy (66% inhibition of tumour growth) after 60 min pre treatment with Spn, but no significant inhibition after 10 min pre treatment with Spn. Conclusions and implications: [3H]-Spn was rapidly cleared from the mice after s.c. injection, and antitumour synergy with chimeric toxins was correlated with the removal of excess Spn from tissues. Disposition of Spn in vivo may critically determine antitumour synergy with chimeric toxins.

Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin ((his)saporin) in ECV-304 cells. For this purpose (his)saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)saporin. This indicates a saponin mediated endosomal escape of saporin.

Saponins are amphiphilic substances consisting of a hydrophobic backbone with one or two hydrophilic sugar units. Recently it was shown that saponinum album (SA) from Gypsophila paniculata L. enhanced cytotoxicity of a saporin-based chimeric toxin (up to 385,000-fold) as well as the toxicity of saporin (up to 100,000-fold) with N-glycosidase activity. Previously we have shown that toxicity of other N-glycosidases such as ricin A-chain, nigrin b, and toxins such as diphtheria toxin or microcystin-LR was not enhanced by SA. This points to a specific SA-dependent mechanism of toxicity enhancement on saporin and saporin-based toxins. Although the cytotoxicity enhancing effect was observed in up to 10 different cell lines, nothing is known about the kinetic of SA under cell culture conditions. Therefore SA was titrated, and the uptake respective liberation profile of SA was investigated in ECV-304 cells. Treatment of cells with [3H]-SA leads to an immediate uptake of saponin molecules. After cells were saturated with [3H]-SA, a first equilibrium (first eq.) was reached. The first eq. was disturbed by washing until a second equilibrium was reached between the activity observed within cells and that seen in the supernatant. After a further extensive washing, a small portion of saponin molecules remained durable associated with the cell. This portion was sufficient to induce a drastic toxicity enhancement on saporin indicating a long-lasting sensitization of cells against the toxin.

Saporin, a type I ribosome-inactivating protein (RIP), removes an adenine residue from the 28S ribosomal RNA as part of a process that leads to inhibition of protein synthesis. As a result, saporin has been shown to exert a strong toxicity in the immune system against macrophages. However, studies have shown that macrophage-like U937 cells are fully resistant against low concentrations of unmodified and his-tagged saporin solutions (6 pM) that normally kill macrophages and APC. In the studies reported here, we noted that with these cells, these RIP solutions only became highly cytotoxic when they were used in a combined treatment with Gypsophila saponins. We determined that this cytotoxicity was the result of an induction of apoptosis triggered by the now-internalized saporin molecules that had previously remained outside of these cells. The results here indicate that the Gypsophila saponins induce, in some manner yet to be fully defined, a stimulation of the endocytosis of saporin by these cells.

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.