Transcript Anticancer Activity of the Fusarium Toxin Enniatin

Anticancer Activity of the Fusarium Toxin Enniatin
R.
a
Dornetshuber ,
P.
b
Heffeter ,
aInstitute
L.
b
Elbling ,
M.
b
Micksche ,
W.
b
Berger ,
R.
a
Lemmens-Gruber
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
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
PGP overexpressing
subline vs parental line
BCRP overexpressing
subline vs parental line
1.5
KBC-1
KB-3-1
0.5
0.0
0.0
2.5
5.0
7.5
10.0
12.5
MDA-MB-231
MDA-MB-231/BCRP
1.0
0.5
0.0
0.0
0,5 µM 1 µM 2,5 µM 5 µM 10 µM
1.5
fold growth
1.0
foldgrowth
fold growth
1.5
Co
MRP1 overexpressing
subline vs parental line
2.5
5.0
7.5
10.0
0.5
0.0
0.0
12.5
GLC-4/adr
GLC-4
1.0
2.5
Enn (µM)
Enniatin (µM)
5.0
7.5
10.0
12.5
Enniatin (µM)
Dose response curve after 72 hrs treatment with Enniatin was determined by MTT.
KBC-1
HL60 adr
HL60 vinc
GLC4 adr
MDA BCRP
1.0
Impact of ABC transporters
control
1 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
0.5
0.0
0.0
2.5
5.0
7.5
10.0
12.5
Enniatin (µM)
Dose response curves of several ABC transporteroverexpressing cell lines after 72 hrs treatment with
Enniatin was determined by MTT.
7500
radioactivity (cpm)
foldgrowth
1.5
KB-3-1
5000
0
0.0
Influence on cell cycle distribution
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%
5 µM Enniatin
G0-G1: 27,4%
S: 44,3%
G2-M: 28,2%
G0-G1: 30,6%
S: 48,2%
G2-M: 21,2%
after 24hrs
Conclusion
2500
10 µMEnniatin
G0-G1: 29,29%
S: 36,6%
G2-M: 34,1%
2.5
5.0
7.5
10.0
12.5
Enniatin (µM)
3H-Thymidineincorporation
after
24 hrs treatment with severel
concentrations of 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 G2-M phase.
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.