Saponins from Saponaria officinalis L. Augment the Efficacy of a Rituximab-Immunotoxin

Abstract

Triterpenoidal saponins are synthesized in the roots of 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-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI (concentration inhibiting cell growth to 50 %) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI 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 L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

“…The remaining saponin containing aqueous phase can be processed differently. Reducing the polarity of the solution, by adding acetonitrile or cold acetone, results in precipitation of the more polar triterpene saponins [44,45]. Alternative approaches comprise agarose gel-electrophoresis of the aqueous phase, resulting in pre-purified agarose-gel fractions that contain charged triterpene saponins [46,47,48].…”

“…Many other reports emphasize numerous biological functions [19] such as fungicidal [53], anti-microbial [54], insecticidal [55], and molluscicidal [56] activity and the enhancement of the endosomal escape and therefore their importance for targeted toxins [44,57]. …”

“…While independent of the targeting moiety of the targeted toxin, the saponin-mediated enhancement factor of the cytotoxic effect is dependent on the cell line and on the expression and internalization level of the target molecule (Table 1) [33,38,40,41,44,110,133]. The enhancement factor is defined as the ratio of the concentrations in the absence and presence of saponins required for half maximal growth inhibition compared to untreated controls.…”

“…The enhancement factor is defined as the ratio of the concentrations in the absence and presence of saponins required for half maximal growth inhibition compared to untreated controls. The enhancement factor is different for varying targets (CD20, CD22, CD25) on the same cell line [44] and for the same target on a number of different cell lines [110]. The latter effect appears to be mostly dependent on target receptor expression, however, the enhancement factor decreased at very high amounts of the receptor.…”

Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells

“…In the last few years, several anti-CD20 and anti-CD22 ITs composed of mAbs linked to RIPs [15,16,29], bacterial toxins [30], ribonucleases [31], drugs [32,33], or radioisotopes [1,2] have demonstrated potent anti-tumor effects in vitro and in vivo in animal models and, in some cases, also in clinical trials.…”