hepatocellular carcinoma is mediated by...
TRANSCRIPT
57
ESP
E
Poster
presented at:
This project was funded by the
European Union
H2020 grant EXNADMINA
705869
P2-P170 Development of resistance to sorafenib, a multikinase inhibitor, in
hepatocellular carcinoma is mediated by SIRTAntje Garten1,2, Theresa Grohmann1, Anja Barnikol-Oettler1, Katarina Kluckova2, Gareth Lavery2, Wieland Kiess1, Melanie Penke1
1 University Hospital for Child and Adolescent Medicine, Center for Pediatric Research Leipzig, Germany
2 Institute of Metabolism and Systems Research, University of Birmingham, UK
contact: [email protected]
Sorafenib decreases phosphorylation of ERK and
induces apoptosis in hepatocarcinoma cell lines
Background
Results
Figure 3 Oxygen flow measured by high resolution respirometry (Oxygraph2K, Oroboros) in A) permeabilised
HUH7 cells after stimulation with 1 µM sorafenib and B) after direct addition of sorafenib to isolated mitochondria
(10 µM). Values are normalised to maximal O2 flux (uncoupling conditions). O2 consumption is measured after
addition of substrates malate and glutamate (MGL), ADP (MGP), succinate (MGSP), uncoupling by FCCP, complex
I inhibitor rotenone and complex III inhibitor Antimycin A. C) ATP levels were measured by CellTiter-Glo
Luminescent Cell Viability Assay after stimulation with sorafenib [1µM; 2.5µM and 5µM] for 24h. Serum free
medium [0 µM] was set one. Data are shown as mean ± SEM (n≥3). con: solvent control (DMSO); *p<0.05
Sorafenib regulates phosphorylation of the AMPK/mTOR
pathway in HUH7 cells
Sorafenib decreases NAD levels in HUH7 cells which is
not mediated by NAMPT or SIRT1.
Sorafenib® - induced apoptosis is counteracted by SIRT1
overexpression and NAD supplementation
A)
Figure 2 For all experiments, HUH7 and Hep3B cells were stimulated with sorafenib [1 µM; 2.5µM and 5
µM] for 24h A) Intracellular NAD levels were measured by gradient HPLC /UV analysis (n=3). B) NAMPT
and SIRT1 protein were measured by Western blot analysis (n=3). Data are shown as mean ± SEM. con:
solvent control (DMSO°p<0.05 (HUH7); *p<0.05 (Hep3B).
Conclusion
Sorafenib® targets multiple cellular pathways including the SIRT1/AMPK axis and that overexpression of SIRT1 could be an underlying
mechanism of resistance to sorafenib treatment in HCC.
A)
0 2 4 6 8 1 0
0 .0
0 .5
1 .0
1 .5
S o ra fe n ib [µ M ]
ce
ll v
iab
ilit
y
[fo
ld o
ve
r c
on
tro
l]
H e p 3BH U H 7
°*
°*
c o n
S o ra fe n ib [µ M ]
ap
op
tos
is
[fo
ld o
ve
r c
on
tro
l]
0 2 4 6
0 .0
2 .0
4 .0
6 .0
8 .0
H U H 7
H e p3B
c o n
°
*
B) C)
0 1 2.5 5 con
Sorafenib [µM]
phERK
ERK
HUH7 Hep3B
0 1 2.5 5 con
Sorafenib [µM]
Figure 1 For all experiments,
HUH7 and Hep3B cells were
stimulated with sorafenib [0 -10 µM]
for 24h. A) Western Blot analysis of
ph-ERK and total ERK after
stimulation with sorafenib for 24h
(n=3). B) WST Assay was
perfomed to measure cell viablitaty.
C) Apoptotic cell number was
measured by flow cytometry (n=3).
Serum free medium [0 µM] was set
one. Data are shown as mean ±
SEM. con: solvent control (DMSO);
*p<0.05 (HUH7); °p<0.05 (Hep3B)
B) HUH7
Hep3B
NAMPT
SIRT1
NAMPT
SIRT1
0 1 2.5 5 con
Sorafenib [µM]S o ra fe n ib [µ M ]
NA
D c
on
ce
ntr
ati
on
[nm
ol/
mg
pro
tein
]
0 2 4 6
0
5
1 0
1 5
H U H 7 H e p 3B
c o n
*
°
Sorafenib incubation induces mitochondrial dysfunction
S o ra fe n ib [µ M ]
AT
P [
fold
ov
er
co
ntr
ol]
0 2 4 6
0 .0
0 .5
1 .0
1 .5
c o n
*
0
2 0 0
4 0 0
6 0 0
8 0 0
O2
flo
w (
pm
ol/
s*1
0e
6 c
ell
s)
S o ra fe n ib [1 µ M ]c o n
MG LMG P M G S P E T S E T S CII
**
*
A) B) C)
Ph-AMPK
Ph-mTOR
AMPK
mTOR
4EBP1
Ph-4EBP1
p70S6k
Ph-p70S6k
Figure 4 HUH7 cells cells were stimulated with sorafenib [1 µM; 2.5µM and 5 µM] for 24h. Western
blot analysis was performed to determine phosphorylation status of AMPK, mTOR, 4EBP1 and
p70S6K.
0 1 2.5 5 con
Sorafenib [µM]
0 1 2.5 5 con
Sorafenib [µM]
S o ra fe n ib [µ M ]
ap
op
tos
is
[fo
ld o
ve
r c
on
tro
l]
0 2 4 6
0
2
4
6
8
1 0
c o n
* + F K 8 6 6
S o ra fe n ib
D M S O
P a lm ita te
Inhibition of SIRT1 does not sensitize hepatocarcinoma
cells to sorafenib (24h)
ce
ll c
yc
le [
%]
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
G 1
S
G 2/M
s ub G 1
S o ra fe n ib [5 µ M ] - + - +
s iR N A S IR T 1 - - + +
A)
Figure 5 A) Cells were stimulated with sorafenib alone or in combination with FK866 [10 nM] for
24h. A) Apoptosis was determined by flow cytometry (n=3). DMSO served as solvent control and
palmitate [0.5 mM] as positive control. Serum free medium [0 µM] was set one. B) SIRT1 protein
levels were downregulated by SIRT1 siRNA using electroporation. Cell cycle distribution was
analyzed by PI staining and flow cytometry. Data are shown as mean ± SEM. *p<0.05.
B)
An
+ +
An
/PI+
po
sit
ive
ce
lls
[fo
ld o
ve
r c
on
tro
l]
0
1
2
3
4
5p E C E
S IR T 1*
N M N - - + +
S o ra fe n ib - + - +
*
*
Ph-AMPK
Tubulin
AMPK
NMN - - + +
Sorafenib - + - +
NA
D
[nm
ol/
mg
pro
tein
]
0
2
4
6
8*
*
N M N - - + +
S o ra fe n ib - + - +
*
A) B)
Figure 6 HUH7 cells were stimulated with sorafenib
[5µM], NMN [250 µM] or a combination of sorafenib
and NMN. A) SIRT1 was overexpressed in HUH7
cells using electroporation (n=3). B) Intracellular
NAD levels were measured by gradient HPLC /UV
analysis (n=3). C) Western Blot analysis was
performed to determine phosphorylation status of
AMPK (n=3). Data are shown as mean ± SEM. con:
solvent control (DMSO); *p<0.05.
C)*
*
Sorafenib is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs).
Resistance to sorafenib develops frequently and could be mediated by the NAD dependent deacetylase sirtuin (SIRT) 1, a master regulator of
cellular energy metabolism and stress responses. We aimed to find out if sorafenib effects depend on changes in cellular NAD levels as well as
activity of SIRT1 and the cellular energy sensor adenosine monophosphate kinase (AMPK).
0 .0
0 .5
1 .0
1 .5
flu
x c
on
tro
l ra
tio
D M S O S o ra fe n ib
*
*
MG LMG P M G S P E T S E T S CII
R O X
170--P2Melanie Penke DOI: 10.3252/pso.eu.57ESPE.2018
Fat, metabolism and obesity