3 H-Thymidineincorporation after 24 hrs treatment with severel concentrations of Enniatin Anticancer Activity of the Fusarium Toxin Enniatin Enniatin, a cyclic hexadepsipeptide, is a secondary metabolite, produced by various strains of the genus Fusarium. It is reported to have antibiotic, insecticidal and ionophoric activity. In this study we investigated whether enniatin also has anticancer activity. For this purpose we used several human cancer cell models. The IC 50 of enniatin against all tested cell lines was in low µM range. Moreover, ABC-transporter overexpression (PGP, MRP1 and BCRP) had no influence on the anticancer activity of enniatin. Cell shrinkage, chromatin condensation, and apoptotic bodies, all indicating apoptosis, were shown after 24 hrs treatment with 5µM and 10µM enniatin by DAPI staining. Correspondingly, PARP cleavage was detectable in Western blot analysis. This was accompanied by the loss of mitochondrial membrane potential (JC-1 staining). Additionally no signs of necrosis like release of lactate dehydrogenase (LDH) were detectable after treatment with enniatin for 24 hrs. To monitor effects of enniatin on the cell cycle progression, the incorporation of the radioactive 3 H-thymidine into DNA was measured. Enniatin treatment led to cell cycle arrest which was further characterized as occuring in G 2 -M phase (probidium iodide staining, FACS analysis). In summary, the Fusarium toxin enniatin has anticancer activity in vitro, which is not influenced by ABC-transporter overexpression. The cytotoxic activity of this natural drug is based on the induction of apoptosis and an arrest in G 2 -M phase. Although further experiments with this substance have to be conducted the results up to now show promise for enniatin in cancer therapy. R. Dornetshuber a, P. Heffeter b, L. Elbling b, M. Micksche b, W. Berger b, R. Lemmens-Gruber a a Institute of Pharmacology and Toxikology, University of Vienna b Institute of Cancer Research, Medical University of Vienna apoptotic bodies KB3-1 control Cytospin and DAPI staining after treatment with 5 µM Enniatin for 24 hrs. control1 µM Enniatin 2,5 µM Enniatin Loss of mitochondrial membrane potential was measured with FACS analyses of JC-1 stainded KB3-1 cells after treatment with 1 µM and 2,5 µM Enniatin for 24 hrs Co0,5 µM 1 µM2,5 µM5 µM10 µM PARP cl. PARP Parp cleavage after 24 hrs treatment with Enniatin  Enniatin shows anticancer activity which is not reduced by ABC transporter overexpression.  Exposure to Enniatin leads to loss of mitochondrial membrane potential and accordingly to apoptosis.  Cells exposed to Enniatin are arrested at low concentrations in S- and higher in G 2 -M phase. Conclusion Dose response curves of several ABC transporter- overexpressing cell lines after 72 hrs treatment with Enniatin was determined by MTT. Apoptotic cell death KB3-1 control 2,5 µM Enniatin 5 µM Enniatin10 µMEnniatin Induction of cell cycle arrest in G2/M Phase G0-G1: 45% S: 34,3% G2-M: 20,7% G0-G1: 30,6% S: 48,2% G2-M: 21,2% G0-G1: 27,4% S: 44,3% G2-M: 28,2% G0-G1: 29,29% S: 36,6% G2-M: 34,1% after 24hrs Influence on cell cycle distribution ACKNOWLEDGEMENTS We are in debt to Marlies Spannberger and Vera Bachinger for the skillful handling of cell culture, Pakiza Rawnduzi, Berger Barbara, Peter Höflich, Elisabeth Rabensteiner, Rosa-Maria Weiss, and Christian Balcarek for competent technical assistance and Irene Herbakec for FACS analysis. Thanks belongs also to Prof. Christian Studenik for interest and advise. PGP overexpressing subline vs parental line MRP1 overexpressing subline vs parental line BCRP overexpressing subline vs parental line Dose response curve after 72 hrs treatment with Enniatin was determined by MTT. Impact of ABC transporters