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Open Access Highly Accessed Research article

Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

Daniel Habermehl1, Bernd Kammerer2, René Handrick1, Therese Eldh1, Charlotte Gruber1, Nils Cordes4, Peter T Daniel5, Ludwig Plasswilm3, Michael Bamberg1, Claus Belka1 and Verena Jendrossek1*

Author Affiliations

1 Department of Radiation Oncology, University Hospital of Tuebingen, Hoppe-Seyler-Str. 3, D-72076 Tuebingen, Germany

2 Institute of Pharmacology and Toxicology, Division of Clinical Pharmacology, University Hospital of Tuebingen, Otfried-Mueller-Str. 45, D-72076 Tuebingen, Germany

3 University Hospital, Department of Radiation Oncology, Petersgraben 4, Ch-4031 Basel, Switzerland

4 OncoRay – Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany

5 Department of Clinical and Molecular Oncology, University Medical Center Charité, Campus Buch, Humboldt University, Lindenbergerweg 80, D-13125 Berlin, Germany

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BMC Cancer 2006, 6:14  doi:10.1186/1471-2407-6-14

Published: 17 January 2006

Abstract

Background

The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry.

Methods

Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling.

Results

Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-xL and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events.

However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain.

Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation.

Conclusion

The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine.