The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure.

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Combined application of saponin and chimeric toxins drastically enhances the targeted cytotoxicity on tumor cells.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

Cytotoxic activity of the seeds from Agrostemma githago var. githago.

In our investigations aqueous extracts of the seeds from Agrostemma githago L. proved to be remarkably more cytotoxic in comparison with isolated agrostemmasaponins in equal concentrations. A combination of agrostemmasaponin 1 with a formyl function attached to triterpene position 4 together with agrostin, a ribosome-inactivating protein (RIP type 1; M (r): 27 kDa) which we isolated from the seed material, resulted in a markedly increased cytotoxicity. In analogy to the well-known lectin-like mechanism of action which can be inhibited by different monosaccharides, we tested seven different sugars known for their affinity to prevent cell recognition and thereby detoxify lectins. None of the tested monosaccharides diminished toxicity values significantly. This suggests that agrostin, combined with agrostemmasaponin 1, has a different mode of action to penetrate through the cell membrane than lectins. On the basis of these facts we concluded that both compounds work as a functional unity showing a mechanism of "cooperative toxicity".