studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 463A
50% cell viability varies significantly among HCC cell lines.
We have isolated HCC cell lines that were totally resistant
to sorafenib and continuously grow in growth media supplemented
with sorafenib. The mechanism of sorafenib resistance
relates to the impaired uptake due to the reduced expression of
organic cation transporter-1 and 3 (OCT1& OCT3). The uptake
of doxorubicin is not altered in HCC cell lines. We found that
OCT1 mRNA and protein expression is reduced in all HCC
cell lines compared to primary human hepatocytes. Stable
expression of full-length OCT1 in the resistant cell line induced
cellular cytotoxicity to sorafenib and overcome the resistance.
Conversely, we showed that inhibiting OCT1 expression by
small molecule inhibitor (quinine hydrochloride) in a sensitive
cell line reduced sorafenib uptake and induces resistance.
The expression of OCT1 and OCT3 is significantly reduced
in human hepatocellular carcinoma tissues as compared to the
surrounding non-tumorous hepatocytes in the cirrhotic nodules.
Conclusions: Our results indicate that there are intrinsic differences
among HCC cell clones that affect sorafenib sensitivity.
Expression of OCT1 and OCT3 down regulation in human
samples is associated with sorafenib sensitivity. We propose
that a detailed understanding of the sorafenib sensitivity and
resistance mechanisms should allow for novel treatment options
to improve sorafenib chemotherapy response in patients with
liver cancer.
Disclosures:
The following authors have nothing to disclose: Srinivas Chava, Partha K. Chandra,
Rajesh Panigrahi, Pauline Ferraris, Fatma Aboulnasr, James Liu, Jose J.
Marin, Swan N. Thung, Tong Wu, Srikanta Dash
507
Stabilin-mediated endocytosis of antisense oligonucleotides
for modulation of liver gene expression
Colton M. Miller 1 , Aaron J. Donner 2 , Emma E. Blank 1 , Punit Seth 2 ,
Edward N. Harris 1 ; 1 Biochemistry, University of Nebraska, Lincoln,
NE; 2 ISIS Pharmaceuticals, Inc., Carlsbad, CA
Introduction: Antisense oligonucleotides (ASOs) are short
(5000-7400 Da) chemically-modified oligonucleotides that
bind to cellular RNAs and modulate their intermediary metabolism
to produce a pharmacological effect. DNA-based ASOs
down-regulate gene-expression by promoting RNase H mediated
degradation of the targeted mRNA. There are currently
over 40 ASOs in clinical development targeting genes that are
involved with metabolic diseases, cancer and inherited genetic
diseases. An important difference between natural DNA and
the ASO is that the phosphodiester (PO) backbone of DNA has
been partially or fully substituted with a phosphorothioate (PS)
backbone. The replacement of the negatively charged oxygen
for the negatively charged sulfur atom covalently bound to
phosphorus renders the short polymer both high stability in
biological fluids and a propensity to adhere to cell surfaces.
Some ASOs are further modified by 2’-O-methoxyethyl (MOE)
or 2’-O-Methyl RNA nucleotides to further enhance metabolic
stability and RNA-binding affinity. Injected ASOs accumulate
in the liver due to the scavenging function of the organ. Previous
work has shown that the liver sinusoidal endothelial cells
(SECs) and Kupffer cells (KC) internalize a much greater proportion
of ASO than hepatocytes. We have discovered that the
non-DNA binding Stabilin-2/HARE receptor specifically binds
to and internalizes several classes of ASO as this scavenger
receptor is enriched in these liver cells. Methods: In our system,
we used stable cell lines expressing recombinant human Stabilin-1
and -2 to assess internalization rates and wildtype CD-1
mice to determine 125 I-ASOs in organ distribution and uptake
in hepatic cells. Results: Cell lines expressing either Stabilin-1
or Stabilin-2 or the parental (non-Stabilin) empty vector (EV) cell
line were incubated with 125 I-ASO that was fully modified with
PS at 0°C (binding) and 37°C (endocytosis) and determined
that the amount of ASO bound did not differ, though the Stabilin-2
cells internalized 3-fold more than the Stabilin-1 cells and
7-fold more than the EV cells. Twenty percent of the retro-orbitally
injected ASO accumulated in liver with an amount of 48%,
30%, and 22% in SECs, KCs and hepatocytes, respectively.
Despite progress in the use of therapeutic oligonucleotides, the
pathways by which negatively charged ASOs enter cells and
modulate gene expression remain poorly understood. Conclusion:
In this context, our work provides fundamental insights
into cell-surface proteins which mediate ASO uptake into cells
and provide opportunities to leverage these findings to further
enhance the therapeutic properties of oligonucleotide drugs.
Disclosures:
Aaron J. Donner - Employment: Isis Pharmaceuticals; Stock Shareholder: Isis
Pharmaceuticals
Punit Seth - Employment: Isis Pharmaceuticals
The following authors have nothing to disclose: Colton M. Miller, Emma E. Blank,
Edward N. Harris
508
Shifting gut microbiota and bile acid profiles in retinoic
acid-primed mice that exhibit accelerated liver regeneration
Hui-Xin Liu, Ying Hu, Lili Sheng, Yu-Jui Yvonne Wan; Medical
Pathology and Laboratory Medicine, UC DAVIS, Sacramento, CA
Abstract Similar to bile acid (BA), all-trans retinoic acid (RA)
facilitates 2/3 partial hepatectomy (PHx)-induced liver regeneration.
RA can activate BA receptor farnesoid x receptor (FXR) to
regulate BA signaling because retinoid x receptor α (RXRα) is
a permissive partner of FXR. Our published data also showed
that RA regulates hepatic lipid homeostasis by shifting hepatic
BA profile. Since the gut microbiota plays a pivotal role in regulating
BA homeostasis and BA composition, we examined the
effect of RA in altering gut microbial and BA composition and
their relationship with increased hepatocyte proliferation in
response to liver resection. C57BL/6 mice were primed with a
single dose of RA (25 μg/g) followed by PHx. RA-primed mice
exhibited accelerated hepatocyte proliferation as indicated by
higher number of Ki67-positive cells and earlier induction of
cell cycle genes compared to untreated mice. Firmicutes and
Bacteroidetes phyla, which affect the efficiency of host energy
and are linked with adiposity in both mice and human, dominated
the gut microbial community (>85%) in both control and
RA-primed mice after PHx. RA reduced the ratio of Firmicutes
to Bacteroidetes, which is associated with a lean phenotype.
Consistently, RA-primed mice lacked transient lipid accumulation,
an energy source for proliferating hepatocytes, normally
found in regenerating mouse livers. This finding suggested that
RA-primed mice might have increased lipid metabolism. In
addition, RA altered BA signaling in the regenerating livers.
RA activated the FXR-SHP-FGF15 pathway, reduced CYP7A1
and CYP8B1 expression to inhibit BA synthesis, and increased
BA transport through Oatp2, Asbt, and Ibabp induction in the
ileum. These data suggested enhanced enterohepatic recirculation
of BAs from RA-priming. RA treatment also shifted the
BA profile by increasing the hydrophilic/hydrophobic ratio.
The ratio of taurine-conjugated cholic acid (CA) to free CA
and taurine-conjugated-β-murine CA to free β-murine CA were
elevated in RA-treated regenerating livers suggesting increased
solubility and lipid emulsification capability. Accordingly, fibroblast
growth factor 21 (FGF21) and Sirtuin1 (SIRT1), master
metabolic regulators, and their downstream targets AMPK and