Alexander Weng

Chemistry and pharmacology of saponins: special focus on cytotoxic properties

Correspondence: Alexander weng institut fur Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charite Universitatsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany Tel +49 30 8445 3097 Fax +49 30 8445 4152 email alexander.weng@charite.de Abstract: Saponins are bioactive compounds produced mainly by plants but also by some marine organisms and insects. In the recent past, there has been unforeseen interest in the clinical utilization of saponins as chemotherapeutic agents. The research on saponins in various forms as a treatment for cancer has generated a lot of potential. The advent of nanotechnology and the cytotoxicity enhancing properties of saponins are some of the highlights of the current decade. This review gives an updated overview of the clinical potential that saponins hold as cytotoxic agents, and covers the literature for 1957–2011, with the main focus on research conducted in the last decade. It is conceivable that saponins hold a lot of therapeutic potential and could be a lead for identification of synthetic or semisynthetic molecules for the treatment of cancer via membrane-mediated or transport-mediated pathways.

Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

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.

Inhibition of Tumor Growth by Targeted Toxins in Mice is Dramatically Improved by Saponinum Album in a Synergistic Way

The application of targeted toxins in cancer therapy remains a challenge due to the severe side effects as a consequence of the high systemic doses required. Here, we describe the combined application of a glycosylated triterpenoid (Spn) and epidermal growth factor receptor (EGFR)-targeted chimeric toxins (SA2E). The cytotoxicity of SA2E on murine TSA tumor cells transfected with human EGFR was enhanced 20,000-fold by low nonpermeabilizing Spn concentrations in a synergistic manner. Subcutaneous application of Spn and SA2E in BALB/c mice bearing a solid TSA cells transfected with epidermal growth factor receptor tumor resulted in 94% tumor volume reduction with a 50-fold lower chimeric toxin concentration compared with pure SA2E treatment. Side effects as monitored by observable complications, body weight, blood parameters; histologic analyses and antibody responses were only moderate and usually reversible.

Real time monitoring of the cell viability during treatment with tumor-targeted toxins and saponins using impedance measurement.

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.

Ribosome-Inactivating and Related Proteins

Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs.

Saponins modulate the intracellular trafficking of protein toxins

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 toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins.

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.

Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells

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.

A convenient method for saponin isolation in tumour therapy.

Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.

Targeted tumor therapy by epidermal growth factor appended toxin and purified saponin: An evaluation of toxicity and therapeutic potential in syngeneic tumor bearing mice

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.