Abstract 5624: Saponins from Gypsophila paniculata L significantly potentiate the immunospecific cytotoxic activity of anti-CD19 and CD38 saporin-based immunotoxins for a human lymphoma cell line
Authors: Flavell David J. , Holmes Suzanne , Gibbs Emily , Fuchs Hendrik , Bachran Christopher , Melzig Matthias F. , Bachran Diana , Flavell Sopsamorn U. 
Published: February 2011

Saponins are a highly diverse group of glycosides of plant origin containing either a steroidal or triterpenoid aglycone to which one or more sugar chains are attached. They have been variously described as enhancing the cytotoxic activity of ribosome-inactivating proteins (RIPs) and ligand-directing conjugates constructed with these for eukaryotic cells. Saponins are thought to facilitate the entry of RIPs into the cytosol across the membranes of intracellular vesicular structures. This characteristic of saponins therefore makes them of interest as molecules that could be useful in improving the therapeutic index of immunotoxins (IT) in a clinical setting.

In the current study we used a protein synthesis inhibition assay based on leucine incorporation together with cell proliferation studies in culture to demonstrate that saponins from Gypsophila paniculata L. potentiate the cytotoxicity of the anti-CD19 and CD38 saporin-based immunotoxins (BU12-SAPORIN and OKT10-SAPORIN, respectively) by several thousand-fold for the human lymphoma cell line Daudi. Various experiments revealed that the enhancing effect of saponins on immunotoxin activity did not appear to be dependent on extra cellular pH in the assay system we used. Kinetic studies further revealed that saponin at a predetermined optimal concentration of 2 μg/ml reduced the t10 (time taken to reduce protein synthesis to 10% of the level seen in control Daudi cells) for OKT10-SAPORIN at 0.1μg/ml from 50 hours without saponin to 12.5 hours with saponin. In blocking experiments using OKT10-SAPORIN IT at the IC50 concentration in combination with saponin together with a gross excess of native OKT10 antibody we established that the immunospecific cytotoxic activity of OKT10-SAPORIN was fully retained when leucine incorporation was used as the surrogate measure of cytotoxicity but only partially so when thymidine incorporation was used. This observation of a partial uncoupling of protein and DNA synthesis as a measure of cytotoxicity was further supported by cell proliferation experiments in culture which were undertaken in parallel.

If saponins are to be of any clinical value as candidate molecules for improving the therapeutic index of immunotoxins it is imperative that the immunospecificity of IT activity is fully retained. Whilst this study clearly demonstrates that saponins from G. paniculata L. do indeed significantly potentiate IT activity it seems that this effect may only be partially immunospecific. Nevertheless, the dramatic improvements that saponins confer on IT activity argue in favor of in vivo experiments in transgenic animal models of human lymphoma combined with additional studies to explore other strategies that might be employed to improve the immunospecific effect achievable.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5624.