International Journal of Biological Macromolecules 61 (2013) 285–294 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo ur nal homep age: www.elsevier.com/locate/ijbiomac Macromolecular interactions of triterpenoids and targeted toxins: Role of saponins charge Mayank Thakur a,∗,1 , Alexander Weng a,1 , Alexandra Pieper a , Katharina Mergel a , Benedicta von Mallinckrodt a , Roger Gilabert-Oriol a , Cornelia Görick b , Burkhard Wiesner c , Jenny Eichhorst c , Matthias F. Melzig b , Hendrik Fuchs a a Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany b Institute of Pharmacy, Free University Berlin, Koenigin-Luise Str. 2+4, D-14195 Berlin, Germany c Leibnitzinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany a r t i c l e i n f o Article history: Received 21 May 2013 Received in revised form 10 July 2013 Accepted 12 July 2013 Available online 22 July 2013 Keywords: Endosomal escape Saponaria saponins Real time evaluation Tumor targeted toxins a b s t r a c t Macromolecular interaction of protein toxins with certain plant triterpenoids holds potential for appli- cation 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 con- served 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 experi- ments 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 spe- cific 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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Tumor cell-targeted toxins are recombinant proteins that con- sist of a targeting domain and an enzymatically active domain. They are capable of identifying the tumor cells and thereafter enabling the entry of protein into the target cells along with a bacterial or plant toxin thereby killing the tumor cells. An array of toxins have been a matter of research in the recent years and some are now under clinical approval. The apoptotic pathways induced by differ- ent toxins are varied in most cases. Many toxins are restricted in ∗ Corresponding author at: Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: +49 30 8445 2507; fax: +49 30 8445 4152. E-mail addresses: mayank.thakur@charite.de, mayankthakur25@gmail.com (M. Thakur). 1 Contributed equally. their efficacy because of lysosomal degradation and in this context plant triterpenoids (saponins) have shown great promise [1,2]. Amongst various secondary metabolites obtained from plants, saponins possess potent pharmacological and biomedical appli- cations [3,4]. Medicinal properties attributed to saponins include their anti-inflammatory, hypo-lipidemic, expectorant, antiulcer, androgenic and anabolic properties; other attributes of saponin include their applications as vaccine adjuvants and nutritional supplements [5,6]. Presence of saponins in variable quantities is reported in more than 90 plant families [7,8]. While there are some generalized effects elicited by saponins due to their detergent- like properties, the majority of functions ascribed to saponins are clearly structure-dependent as has been shown for instance in case of their ability to enhance the effect of targeted toxins in tumor therapy [9]. Saponins have also shown effectiveness in reducing oxida- tive and nitrosative stress and therefore the ability to suppress the development of malignancies and related diseases [10]. In our working group we have been evaluating the role of saponins 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.07.008