Real-time analysis of membrane permeabilizing effects of oleanane saponins

Gilabert-Oriol, Roger; Mergel, Katharina; Thakur, Mayank; Mallinckrodt, Benedicta von; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander

doi:10.1016/j.bmc.2013.01.061

Cited by 49 publications

(44 citation statements)

References 37 publications

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“…Therefore, we will conduct future investigations with the aim of clinical studies with dianthin‐based products. Moreover, side effects of this therapy were not sufficiently perused to date and we therefore investigated hematological parameters after mono‐ and combination therapy of His Dianthin‐EGF in particular with regard to the known hemolytic properties of some glycosylated triterpenoids in general (Gilabert‐Oriol et al ., ,c).…”

Section: Discussionmentioning

confidence: 99%

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpeneSO1861

et al. 2017

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Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma; however, a number of protein‐based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome‐inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC‐3 and MIA PaCa‐2 revealed no significant differences to off‐target cells in the half maximal inhibitory concentration (IC 50) for the fusion protein. In contrast, combination with SO1861 decreased the IC 50 for BxPC‐3 cells from 100 to 0.17 nm, whereas control cells remained unaffected. Monotherapy of BxPC‐3 xenografts in CD‐1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm3) when compared to placebo; however, combined treatment with SO1861 resulted in a more than 13‐fold better efficacy (3.0 mm3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit, and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment.

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Section: Discussionmentioning

confidence: 99%

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpeneSO1861

et al. 2017

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“…Firstly, the permeabilizing effects of digitonin (Ysat, Wernigerode, Germany), α-hederin (Sigma-Aldrich, Steinheim, Germany) and SA1641 were measured on lysosomal membranes by the β- N -acetylglucosaminidase (NAG) assay [30]. The crude lysosomal fractions were shortly centrifuged at 1000 × g for 2 min at 4 °C to discard the aggregated organelles.…”

Section: Methodsmentioning

confidence: 99%

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Gilabert-Oriol

Thakur

Mallinckrodt

et al. 2014

Toxins

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Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM.

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“…Real-time monitoring of cell membrane permeabilization was conducted to reveal the kinetics of saponin effects [68]. In this study, oleanane saponins showed variable permeabilizing effects on the cell and lysosomal membranes at distinct concentrations of 6 µM or higher (dependent on the saponin) and hemolysis at 3 µM or higher.…”

Section: Glycosylated Triterpenoidsmentioning

confidence: 99%

“…Hemolysis, liver toxicity, and inflammation are the most prominent side effects of subcutaneously injected saponins. The hemolytic effect strongly depends on the structure of the saponins [46,68,138]. In vivo, the applied dose must be below the critical hemolytic concentration since otherwise progressive hemolysis will lead to death.…”

Section: Endosomal Escape In Animal Modelsmentioning

confidence: 99%

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

et al. 2017

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Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed.

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Real-time analysis of membrane permeabilizing effects of oleanane saponins

Cited by 49 publications

References 37 publications

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpeneSO1861

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpeneSO1861

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

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