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Genotoxicity and Cellular Uptake of Cyclotides: Evidence for Multiple Mode of Action
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2012 (English)In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 747, no 2, 176-181 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are a family of ultra stable, head-to-tail cyclic plant mini-proteins with each member comprising about 30 amino acid residues. Their stability is associated with the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring which is knotted by a third disulfide bond. The cyclotides find potential applications in drug industry as a drug scaffolds for unstable drugs and also as medicinal agents due to the wide range of inherent pharmacological activities they possess. However, there is a lack of fundamental toxicological studies on these classes of compounds. The current study determined a possible DNA damaging effect of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1 in human lymphoma cells using the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sublethal concentration, cycloviolacin O2 and vaby D gave a bell shaped dose-response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub cytotoxic concentrations. Single cell microautoradiography was carried out on tritium labeled cycloviolacin O2 in order to understand the mechanism behind the dose-response curve. The results revealed that the peptide is taken up into the cell, at both cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms/targets still remain unknown, the current study has unequivocally demonstrated that these compounds also must have other dose-dependent modes of action. 

Place, publisher, year, edition, pages
2012. Vol. 747, no 2, 176-181 p.
Keyword [en]
cyclotide, cycloviolacin O2, vaby D, kalata B1, comet assay, DNA damage, microautoradiography, mode of action, viola odorata, viola abyssinica, oldenlandia affinis, cell penetrating, radiolabeling
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-171150DOI: 10.1016/j.mrgentox.2012.05.006ISI: 000307148600003OAI: oai:DiVA.org:uu-171150DiVA: diva2:510262
Available from: 2012-03-15 Created: 2012-03-15 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Cyclotides: Tuning Parameters Toward Their Use in Drug Design
Open this publication in new window or tab >>Cyclotides: Tuning Parameters Toward Their Use in Drug Design
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned.

An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method.

Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m.

One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile.

Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 81 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 156
Keyword
cyclotide, Viola abyssinica, Viola odorata, Extraction, Violaceae, vaby, cytotoxic, genotoxic, cell penetrating, microautoradiography, LC-MS, comet assay, cycloviolacin O2
National Category
Medical and Health Sciences Natural Sciences
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-169712 (URN)978-91-554-8307-4 (ISBN)
Public defence
2012-04-26, B41, Husargatan 3, Biomedical Center, Uppsala University, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2012-04-04 Created: 2012-03-05 Last updated: 2012-04-19Bibliographically approved

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Yeshak, Mariamawit YGöransson, UlfBurman, RobertHellman, Björn

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