studies

536A AASLD ABSTRACTS HEPATOLOGY, October, 2015
prevent the recurrence and development of HCC after surgical
removal of the primary tumors. Previous in vitro experiments
showed that ACR selectively suppresses the growth of human
HCC cells (JHH7, HuH7, and HepG2) but not normal human
hepatic cells (Hc). In the present study, the genome-wide transcriptome
analysis was performed to identify a biomarker for
ACR treatment of HCC. Method: Differential gene expression
profiles of JHH7 and Hc cells treated with ACR were measured
using the next-generation sequencing-based Cap Analysis
Gene Expression (CAGE) analysis. Inhibitory effect of ACR on
MYCN expression was confirmed by real-time PCR in culture
cells, animal models and liver biopsy of HCC patients administered
with ACR (n = 6). MYCN-dependent signaling pathways
underlying the growth suppression by ACR were explored
using knowledge-based ingenuity pathway analysis (IPA),
RNA interference, chemical inhibitors and luciferase assays.
Relationship between MYCN levels and tumor recurrence in
HCC patients (n = 102) was evaluated using the log-rank test.
Correlations between MYCN and liver cancer stem cell (CSC)
markers were determined using Pearson correlation coefficients
based on microarray database or in patients. Results: MYCN
was expressed in JHH7 cells but not in Hc cells, and inhibited
by ACR but not by its ethyl analogues that showed no growth
suppression. ACR also selectively suppressed the growth of
MYCN-positive neuroblastoma cells, but not that negative for
MYCN. Four out of 6 liver biopsies of HCC patients (66.7%)
who had received 8 weeks of ACR (600 mg/day) after definitive
treatment showed decreased MYCN expression (< 0.5-
fold). The result of IPA suggested a Sp1/MYCN/caspase-8
signaling pathway underlying ACR’s anticancer effect. Upon
knockdown of MYCN, HCC cells showed suppressed cell
growth and increased caspase-8 activity. An Sp1 inhibitor
suppressed MYCN expression, whereas a caspase-8 inhibitor
restored ACR-induced growth suppression. Finally, clinical
analysis indicated that MYCN expression in HCC tumors
was significantly correlated with CSC markers AFP, EpCAM
and CD133 but not CD90, and negatively associated with
recurrence of early-stage HCC. Conclusion: MYCN is a new
biomarker of eligibility for ACR treatment that correlates with
liver CSC with progenitor features and poor prognosis of HCC.
ACR suppressed HCC cell growth through a Sp1/MYCN/
caspase-8 dependent signaling pathway.
Disclosures:
Naoto Ishibashi - Employment: KOWA Company, LTD.
The following authors have nothing to disclose: Soichi Kojima, Xian-Yang Qin,
Harukazu Suzuki, Masao Honda, Goshi Shiota, Hisataka Moriwaki, Masahito
Shimizu
658
Farnesoid X Receptor (FXR) Activation by Bile Salts and
FXR Agonists Increases the Hepatic Expression of the
Unfolded Protein Response Regulator X-box Binding
Protein 1 Spliced (XBP1s)
Xiaoying Liu, David Hilburn, Seong W. Park, Brian E. LeCuyer,
Richard M. Green; Northwestern University, Chicago, IL
The Unfolded Protein Response (UPR) is an adaptive response
to endoplasmic reticulum stress that occurs in cholestatic and
other forms of liver disease. X-box binding protein 1 spliced
(XBP1s) is a highly conserved regulator of the UPR, and is
formed by an unconventional splicing mechanism that removes
26 nucleotides from the unspliced XBP1 mRNA. The farnesoid
X receptor (FXR) is a nuclear bile acid receptor which regulates
bile acid metabolism, and FXR agonists may provide
novel therapies for cholestatic and fatty liver diseases. In this
study, we demonstrate a novel role of FXR in regulating liver
XBP1 splicing and XBP1s expression. Methods: Male FVB mice
(8-12 weeks) were fed chow supplemented with 0.3% sodium
deoxycholate (DCA), an endogenous FXR bile salt ligand, for
up to 7 days. Huh7/NTCP or HepG2 cells were treated with
either bile salts (GCDCA, TCDCA, TDCA and TUDCA) or the
synthetic FXR agonists obeticholic acid (OCA) and GW4064
for 4-8 hours. These cells were also co- treated with the FXR
antagonist guggulsterone, and either FXR siRNA or control
siRNA. Gene and protein expression was analyzed by qPCR
and Western blotting. XBP1 splicing activity was measured
using an XBP1-luciferase reporter construct that assays the 26-nt
atypical splicing of the XBP1 mRNA. Results: Hepatic XBP1s
and its downstream target ERdj4 mRNA expression increased
6.5-fold (P < 0.05) and 1.7-fold (P < 0.05) in mouse livers after
DCA feeding for 1 and 3 days, respectively. GCDCA (50 μM),
TCDCA (10 μM) and TDCA (10 μM) treatment increased XBP1s
protein expression in Huh7/NTCP cells, while TUDCA (10
μM) had no effect. The synthetic FXR agonists OCA (2.5 μM)
and GW4064 (2.5 μM) also induced XBP1s mRNA and protein
expression. XBP1s protein induction by FXR agonists was
blocked by the FXR antagonist guggulsterone (50 μM) and by
FXR siRNA. The FXR agonists TCDCA (100 μM) and GW4064
(10 μM) also increased XBP1 splicing-luciferase activity 2 and
4-fold, respectively compared to controls (P < 0.001). Conclusions:
DCA bile salt feeding to mice increases the expression
of hepatic XBP1s and its downstream target ERdj4. FXR bile
salt ligands and synthetic agonists increase XBP1s expression
in Huh7/NTCP or HepG2 cells, while TUDCA has no effect.
These effects are due, at least in part, to enhanced XBP1 splicing
into XBP1s. Increased XBP1s expression is blocked by the
FXR antagonist guggulsterone and by FXR knockdown. We
demonstrate the novel finding that a FXR-mediated pathway
increases the expression of liver XBP1s. We speculate that this
may be important in the pathogenesis of cholestatic and fatty
liver diseases.
Disclosures:
Richard M. Green - Consulting: McNeil
The following authors have nothing to disclose: Xiaoying Liu, David Hilburn,
Seong W. Park, Brian E. LeCuyer
659
Cerium oxide nanoparticle treatment enhances liver
regeneration after two-thirds partial hepatectomy in
rats
Manuel Morales-Ruiz 1,3 , Altamira Arce-Cerezo 1,5 , Montserrat
Pauta 1,5 , Jordi Ribera 1,3 , Denise Oró 1,3 , Gregori Casals 1,3 , Guillermo
Fernández-Varo 1,3 , Tetyana Yudina 4 , Víctor Puntes 4 , Wladimiro
Jiménez 1,2 ; 1 Biochemistry and Molecular Genetics, Hospital
Clinic, Barcelona, Spain; 2 Department of Physiological Sciences I,
IDIBAPS, CIBERehd, University of Barcelona School of Medicine,
Barcelona, Spain; 3 IDIBAPS, CIBERehd, Barcelona, Spain; 4 Catalan
Institute of Nanoscience and Nanotechnology, UAB, Bellaterra,
Spain; 5 IDIBAPS, Barcelona, Spain
Introduction and aim: Several studies have shown that oxidative
stress impairs hepatic regeneration in mice. Therefore,
testing new anti-oxidant drugs to improve liver regeneration
has clinical interest. Recently, considerable attention has been
paid to cerium oxide nanoparticles (CeO 2
NPs) as a treatment
for oxidative stress-related diseases. This interest relies on the
multi-enzyme mimetic properties of CeO 2
NPs that turn these
nanoparticles into an effective scavenger of reactive oxygen
species. Consequently, we aimed to investigate the effect of
CeO 2
NPs treatment on hepatic regeneration after partial hepatectomy.
Methods: CeO 2
NPs (4 nm in size), were synthesized
by precipitation of a cerium (III) salt by the addition of ammo-