Transcript The Fusarium toxin Enniatin exerts p53

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
Cell cycle
distribution
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.
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.