Saponin vermittelte intrazelluläre Liberation chimärer Toxine. Eine Basistechnologie in der zielzellgerichteten Tumortherapie
Final Report Abstract
Type I ribosome-inactivating proteins (type I RIPs) belong to a certain class of enzymes that clear a specific adenine residue from the ribosomal RNA. This is followed by an irreversible inhibition of protein synthesis leading to cell death. A prominent type I ribosome-inactivating protein is Saporin. Saporin (~30 kDa) is biosynthesized by the plant Saponaria officinalis L. and is stored in the plant seeds. Saporin exhibits high enzymatic activity along with a high structural stability. It does not contain a native cell binding domain. For these reasons Saporin is widely used as toxin moiety for the construction of chimeric anti-tumour toxins (targeted toxins). In general targeted toxins consist of a targeting domain such as growth factors or monoclonal antibodies and a toxin moiety. After binding to their target receptor the targeted toxin is internalized and delivered into the endosomal transport system (EES). The EES is a tubular vesicular network surrounded by cytosol. For exerting their anti-tumour activity targeted toxins have to escape from the EES. Otherwise they are degraded within certain cellular organelles. In previous studies it was shown that a crude mixture of low molecular weight (< 2 kDa) plant secondary metabolites (triterpensaponins) from Saponaria officinalis L. and Gypsophila paniculata L. augmented the efficacy of targeted toxins dramatically. However the molecular mechanism of this triterpensaponin-mediated efficacy increase of targeted toxins was unknown. This project was intended to investigate the underlying mechanism of this effect and to show the potential of the triterpensaponin-mediated effect for targeted tumour therapies. Here it is shown that characterized triterpensaponins, isolated from Saponaria officinalis L. and Gypsophila paniculata L. do not alter the plasma membrane integrity but selectively trigger the escape of internalized Toxins out of late endosomes and lysosomes inside the cell. The toxins are released into the cytosol in a pH-dependable manner. The systematic substitution of an epidermal growth factor (EGF) containing targeted toxin by different toxin moieties revealed that type I RIP based targeted toxins are benefitting most from the combination with triterpensaponins. This effect was less prominent for Diphtheria toxin, Pseudomonas Exotoxin A or type II RIP based targeted toxins. It is further demonstrated that the substitution of the targeting ligand by monoclonal antibodies does not negatively modulate the triterpensaponinmediated intracellular release of toxin molecules. Based on these studies triterpensaponins from Saponaria officinalis L. and Gypsophila paniculata L. can be categorized as a new class of intracellular lysogens that hold considerable therapeutic potential for anti-tumour therapies with targeted toxins.
Publications
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Chemistry and pharmacology of saponins: special focus on cytotoxic properties. Botanics: Targets and Therapy, Volume 1. 2011, pp. 19—29.
M. Thakur, MF. Melzig, H.Fuchs, A.Weng
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Electrophoretic isolation of saponin fractions from Saponinum album and their evaluation in synergistically enhancing the receptor-specific cytotoxicity of targeted toxins. Electrophoresis Vol. 32. 2011, Issue 21, pp. 3085–3089.
M. Thakur, A.Weng, D. Bachran, SB. Riese, S. Böttger, MF Melzig, H.Fuchs
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Rasayana properties of Ayurvedic herbs: Are polysaccharides a
major contributor. Carbohydrate Polymers, Vol. 87. 2012, Issue 1, pp. 3–15.
M.Thakur, A.Weng, H. Fuchs, V. Shama, CS. Bhargava, NS. Chauhan, VK. Dixit, S. Bhargava
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Real time monitoring of the cell viability during treatment
with tumor-targeted toxins and saponins using impedance measurement.
Biosensors and Bioelectronics, Vol. 35. 2012, Issue 1, pp. 503–506.
M. Thakur, K. Mergel, A. Weng, S. Frech, R. Gilabert-Oriol, D. Bachran, M.F. Melzig, and H. Fuchs
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Saponins modulate the intracellular trafficking of protein toxins.
Journal of Controlled Release, Vol. 164. 2012, Issue 1, pp. 74–86.
A. Weng, M. Thakur, B. von Mallinckrodt, F. Beceren-Braun, R. Gilabert-Oriol, B. Wiesner, J. Eichhorst, S. Bottger, M.F. Melzig, and H. Fuchs
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The toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins. Mol Oncol. 6:323-332 (2012).
Molecular Oncology, Vol. 6. 2012, Issue 3, pp. 323–332.
A. Weng, M. Thakur, F. Beceren-Braun, D. Bachran, C. Bachran, S.B. Riese, K. Jenett-Siems, R. Gilabert-Oriol, M.F. Melzig, and H. Fuchs
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Macromolecular interactions of triterpenoids and targeted toxins: Role of saponins charge. International Journal of Biological Macromolecules, Vol.61. 2013, pp. 285–294.
M. Thakur, A. Weng, A. Pieper, K. Mergel, B. von Mallinckrodt, R. Gilabert-Oriol, C. Gorick, B. Wiesner, J. Eichhorst, M.F. Melzig, and H. Fuchs
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Modified Trastuzumab and Cetuximab Mediate Efficient Toxin Delivery While Retaining Antibody-Dependent Cell-Mediated Cytotoxicity in Target Cells. Molecular Pharmaceutics, Vol.10. 2013, Issue 11, pp. 4347–4357.
R. Gilabert-Oriol, M. Thakur, B. von Mallinckrodt, T. Hug, B. Wiesner, J. Eichhorst, M.F. Melzig, H. Fuchs, and A. Weng
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Real-time analysis of membrane permeabilizing effects of oleanane saponins. Bioorganic & Medicinal Chemistry, Vol. 21. 2013, Issue 8, pp. 2387–2395.
R. Gilabert-Oriol, K. Mergel, M. Thakur, B. von Mallinckrodt, M.F. Melzig, H. Fuchs, and A. Weng.
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Small structural differences of targeted anti-tumor toxins result
in strong variation of protein expression. Protein Expression and Purification, Vol. 91. 2013, Issue 1, pp. 54–60.
R. Gilabert-Oriol, M. Thakur, C. Weise, J. Dernedde, B. von Mallinckrodt, H. Fuchs, A. Weng
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Targeted tumor therapy by epidermal growth factor appended toxin and purified saponin: an evaluation of toxicity and therapeutic potential in syngeneic tumor bearing mice. Molecular Oncology, Vol. 7. 2013, Issue 3, pp. 475–483.
M. Thakur, K. Mergel, A. Weng, B. von Mallinckrodt, R. Gilabert-Oriol, H. Durkop, M.F. Melzig, and H. Fuchs
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Triterpenoid saponins from the roots of Gypsophila trichotoma Wender.
Phytochemistry, Vol. 90. 2013, pp. 114–127.
L. Voutquenne-Nazabadioko, G. Gevrenova, N. Borie, D. Harakat, C.Sayagh, A.Weng, M.Thakur, M. Zaharieva, M. Henry
