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Cyclotides: a novel type of cytotoxic agents
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.
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2002 (English)In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 1, no 6, 365-369 p.Article in journal (Refereed) Published
Abstract [en]

Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin O2, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin O2 was the most potent in all cell lines (IC50 0.1– 0.3 _M), followed by varv A (IC50 2.7–6.35 _M) and varv F (IC50 2.6 –7.4 _M), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.

Place, publisher, year, edition, pages
2002. Vol. 1, no 6, 365-369 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-92805ISI: 000178770300001PubMedID: 12477048OAI: oai:DiVA.org:uu-92805DiVA: diva2:166103
Note

De två första författarna delar på förstaförfattarskapet.

Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2013-06-10Bibliographically approved
In thesis
1. Cytotoxic Compounds of Plant Origin – Biological and Chemical Diversity
Open this publication in new window or tab >>Cytotoxic Compounds of Plant Origin – Biological and Chemical Diversity
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Cytotoxiska föreningar från växter – biologisk och kemisk diversitet
Abstract [en]

The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms.

The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage.

The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species.

In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 7
Keyword
Pharmacognosy, pharmacognosy, cytotoxicity, antitumour, Nuphar alkaloid, cyclotide, thionin, cardiac glycoside, Farmakognosi
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-5728 (URN)91-554-6197-2 (ISBN)
Public defence
2005-04-22, Room C8:301, BMC, Husargatan 1, Uppsala, 09:15
Opponent
Supervisors
Available from: 2005-04-01 Created: 2005-04-01Bibliographically approved
2. Macrocyclic polypeptides from plants
Open this publication in new window or tab >>Macrocyclic polypeptides from plants
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential.

A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (Viola arvensis), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides.

Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes.

Cyclotides were found to be common in the plant family Violaceae; with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and Hybanthus parviflorus. For six members of the genus Viola, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus.

To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds.

Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 59 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 270
Keyword
Pharmaceutical chemistry, Farmaceutisk kemi
National Category
Medicinal Chemistry
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-1956 (URN)91-554-5279-5 (ISBN)
Public defence
2002-04-26, BMC sal B22, Uppsala, 10:15
Opponent
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2013-06-10Bibliographically approved

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Göransson, UlfGullbo, JoachimLarsson, RolfBohlin, LarsBacklund, Anders

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