The Fusarium toxin Enniatin exerts p53-dependent cytostatic and p53-

independent cytotoxic activities against human cancer cells

R. DornetshuberR. Dornetshuberaa, P. Heffeter, P. Heffeterbb, M. , M. KamyarKamyaraa, W. Berger, W. Bergerbb, R. Lemmens-Gruber, R. Lemmens-Gruberaa

aaDepartment of Pharmacology and Toxicology, University of ViennaDepartment of Pharmacology and Toxicology, University of Vienna

bbInstitute of Cancer Research, Department of Medicine I, Medical University of ViennaInstitute of Cancer Research, Department of Medicine I, Medical University of ViennaThe 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.

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.

p21

ß-actin

bax

p53

p53 ko p53 (+/+)

Co Co

1µM

1µM

2.5µ

M

2.5µ

M

0.5µ

M

0.5µ

M

5µM

5µM

10µM

10µM

In Western blot analysis, induction of p53 was, as expected, only detectable in p53 wild- type cells, whereas bax activation was evident in p53 wild-type cells as well as in cells with disrupted p53.

Cell line IC50 (µM) Cell line IC50 (µM)

Melanoma Mean SD Osteosarcoma Mean SD

GUBSA 3.19 0.85 U2-OS 1.77 0.24

PNJC 2.67 0.08 OS-9 3.55 0.77

RIMA 1.75 0.15 OS-10 2.10 0.15

WUBI 1.75 0.15 SAOS 2.13 0.07

TPCK 2.72 0.11Human lung cancer

Glioblastoma A549 4.08 1.04

GBL1 2.65 0.30 A427 1.61 0.14

GBL2 2.29 0.05Diverse carcinomas

GBL3 2.55 0.14 KB-3-1 1.95 0.12

LB 2.33 0.30 MDA-MB-231 1.45 0.49

MGC 3.04 0.58 SW480 4.00 1.12

U373 4.88 0.09 CaCo2 1.99 0.09

We demonstrate that ENN exerts profound cytotoxic activity against several tested human tumor cell lines. Assays on HCT116 cells with disrupted p53, suggest that the cytostatic effects of ENN are mediated by p53-dependent mechanisms. But we also show an uncommon p53-independent induction of bax accompanied by apoptotic cell death. So further studies are underway to clarify the molecular mechanisms underlying the p53-independent cytotoxic activity of ENN.

DNA synthesis

Cell cycle distributio

nAccordingly, PI-staining revealed a more potent cell cycle arrest in GO/G1 phase following ENN treatment.

3H-thymidine incorporation revealed a significantly more efficient block of DNA synthesis by ENN in p53 wild-type as compared to corresponding knock-out cells.

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.

Summary