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The Fusarium toxin Enniatin exerts p53dependent cytostatic and p53-independent cytotoxic activities against human cancer cells R. a Dornetshuber , P. b Heffeter , aDepartment bInstitute M. a Kamyar , W. b Berger , R. a Lemmens-Gruber of Pharmacology and Toxicology, University of Vienna of Cancer Research, Department of Medicine I, Medical University of Vienna The tumor-suppressor gene p53, the “guardian of the genome“, is mediating cell cycle regulating- as well as apoptosis-inducing properties. The major mechanism to induce apoptosis is believed to work transcriptionally via p53 binding to the promoter region of bax, a proapoptotic member of the bcl-2 protein family. But there also exists a less common, p53-mediated way of inducing apoptosis, believed to be independent of transcription regulation. Enniatin (ENN) is a cyclic hexadepsipeptide, produced by the genus Fusarium, which is well known as an inhibitor of mammalian cholesterol acyl-transferase. ENN possesses antibiotic, immunomodulatory, and ionophoric activities. Here we demonstrate that ENN exerts profound cytotoxic activity against several human tumor cells. Consequently, we further investigated the mechanisms underlying ENN-induced cell death with a focus on apoptosis- and cell cycle-regulating proteins. 0.30 0.05 0.14 0.30 0.58 0.09 IC50 (µM) Mean SD 1.77 0.24 3.55 0.77 2.10 0.15 2.13 0.07 4.08 1.61 1.04 0.14 1.95 1.45 4.00 1.99 0.12 0.49 1.12 0.09 1.5 p21(+/+) p21(-/-) 1.0 0.5 2.5 5.0 7.5 ENN (µM) 10.0 12.5 1.5 foldgrowth foldgrowth foldgrowth 1.5 0.0 0.0 2.0 2.0 bax(+/+) bax(-/-) 1.0 0.5 0.0 0.0 2.5 5.0 7.5 ENN (µm) 10.0 12.5 p53 p53/het p53/ko 1.0 DNA synthesis 3H-thymidine 1.0 P53 p53/ko 0.5 0.0 0.0 Measured by MTT assays ENN showed profound cytotoxic activity against several human tumor cell lines. The IC50 values were calculated from whole dose response curves and given as means ± SD from at least two independent experiments performed in triplicates. 2.0 1.5 2.5 5.0 7.5 10.0 12.5 ENN (µM) p53 incorporation revealed a significantly more efficient block of DNA synthesis by ENN in p53 wildtype as compared to corresponding knock-out cells. 80 70 60 G0-G1 G2-M S Apoptosis 50 40 30 20 10 0 0.0 2.5 5.0 7.5 10.0 12.5 ENN (µM) Accordingly, PI-staining revealed a more potent cell cycle arrest in GO/G1 phase following ENN treatment. Cell cycle distribution p53/ko 80 70 60 G0-G1 G2-M S Apoptosis 50 % 2.65 2.29 2.55 2.33 3.04 4.88 Cell line Osteosarcoma U2-OS OS-9 OS-10 SAOS Human lung cancer A549 A427 Diverse carcinomas KB-3-1 MDA-MB-231 SW480 CaCo2 -fold radioactivity IC50 (µM) Mean SD 3.19 0.85 2.67 0.08 1.75 0.15 1.75 0.15 2.72 0.11 % Cell line Melanoma GUBSA PNJC RIMA WUBI TPCK Glioblastoma GBL1 GBL2 GBL3 LB MGC U373 40 30 20 10 0 0.0 2.5 5.0 7.5 10.0 12.5 ENN (µM) 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 ENN (µM) HCT116 cells with p53, p21 or bax genes disrupted by targeted homologous recombination were used to study ENN-induced cytotoxicity. In MTT assays, no significant influences of these proteins were detected, resulting for all HCT116 subclones at IC50 values in the low µM range at a 72 h drug exposure. Summary We demonstrate that ENN exerts profound cytotoxic activity against several tested human tumor cell lines. p53 ko p53 (+/+) Assays on HCT116 cells with disrupted p53, suggest that the p53 cytostatic effects of ENN are mediated p21 by p53-dependent mechanisms. bax But we also show an uncommon p53-independent induction of bax ß-actin accompanied by apoptotic cell death. So further studies are underway to clarify the molecular mechanisms In Western blot analysis, induction of p53 was, as expected, only detectable in p53 wild- type cells,underlying whereas bax activation evident in p53 wild-type c the wasp53-independent cells with disrupted p53. cytotoxic activity of ENN.