Anticancer Activity of the Fusarium Toxin Enniatin

1. ACKNOWLEDGEMENTS We are indebt 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 BCRP overexpressing subline vs parental line MRP1 overexpressing subline vs parental line Dose response curve after 72 hrs treatment with Enniatin was determined by MTT. KB3-1 control apoptotic bodies Cytospin and DAPI staining after treatment with 5 µM Enniatin for 24 hrs. Parp cleavage after 24 hrs treatment with Enniatin PARP cl. PARP 1 µM 2,5 µM 5 µM 10 µM Co 0,5 µM Impact of ABC transporters 2,5 µM Enniatin control 1 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 Dose response curves of several ABC transporter-overexpressing cell lines after 72 hrs treatment with Enniatin was determined by MTT. Influence on cell cycle distribution 3H-Thymidineincorporation after 24 hrs treatment with severel concentrations of Enniatin Induction of cell cycle arrest in G2/M Phase KB3-1 control 2,5 µM Enniatin G0-G1: 45% S: 34,3% G2-M: 20,7% G0-G1: 30,6% S: 48,2% G2-M: 21,2% after 24hrs 5 µM Enniatin 10 µMEnniatin G0-G1: 27,4% S: 44,3% G2-M: 28,2% G0-G1: 29,29% S: 36,6% G2-M: 34,1% Anticancer Activity of the Fusarium Toxin Enniatin R. Dornetshubera, P. Heffeterb, L. Elblingb, M. Mickscheb, W. Bergerb, R. Lemmens-Grubera aInstitute of Pharmacology and Toxikology, University of Vienna bInstitute of Cancer Research, Medical University of Vienna 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 IC50 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 3H-thymidine into DNA was measured. Enniatin treatment led to cell cycle arrest which was further characterized as occuring in G2-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 G2-M phase. Although further experiments with this substance have to be conducted the results up to now show promise for enniatin in cancer therapy. Apoptotic cell death Conclusion • 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 G2-M phase.