studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1173A
hyperinsulinaemia rather than hyperglycemia can accelerate
the progression of HCC.
Disclosures:
The following authors have nothing to disclose: Koichi Tsuneyama, Ryosuke
Bessho, Masahiro Miki, Hayato Baba, Tetsuyuki Takahashi, Hirohisa Ogawa,
Hisanori Uehara
Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD
K.K., Bristol-Meyer Squibb, Mitsubishi Tanabe Pharma Corparation, Toray Industories
Inc. ; Speaking and Teaching: MSD K.K., Bristol-Meyer Squibb, Janssen
Pharmaceutical Companies
The following authors have nothing to disclose: Yoshiki Onishi, Tomohide Tatsumi,
Satoshi Aono, Seiichi Tawara, Akira Nishio, Atsuo Takigawa, Takahiro
Suda, Tadashi Kegasawa, Shigeki Suemura, Minoru Shigekawa, Ryotaro Sakamori,
Naoki Hiramatsu
1982
Branched chain amino acids promote sorafenib-mediated
apoptosis in hepatocellular carcinoma via
down-regulation of Mcl-1
Yoshiki Onishi, Tomohide Tatsumi, Satoshi Aono, Seiichi Tawara,
Akira Nishio, Atsuo Takigawa, Takahiro Suda, Tadashi Kegasawa,
Shigeki Suemura, Minoru Shigekawa, Hayato Hikita,
Ryotaro Sakamori, Naoki Hiramatsu, Tetsuo Takehara; Department
of Gastroenterology and Hepatology, Osaka University Gradute
School of Medicine, Suita, Japan
Background&aim: Sorafenib is an only effective chemotherapeutic
agent in the treatment of hepatocellular carcinoma
(HCC). However, the survival benefit is limited. Branched
chain amino acids (BCAAs) promote albumin synthesis through
the activation of mTOR pathway. BCAAs treatment has been
reported to reduce the incidence of HCC in patients with liver
cirrhosis and to reduce the recurrence of HCC after curative
treatment. We previously reported a retrospective study that
the combined use of sorafenib and BCAAs prolonged the
overall survival in HCC patients. In this study, we examined
the mechanism of anticancer effect of the combination therapy.
Method: Human HCC cell lines, HepG2, PLC/PRF/5 and
Huh7, were treated with sorafenib and BCAAs. Cell viabiliy
was evaluated by WST assay. To evaluate apoptosis of HCC
cells, we measured caspase3/7 activities in the culture supernatants
and Annexin-V positive cells by flow cytometry. Growth
signals and regulatory proteins of apoptosis were analyzed
by western blotting. mRNA levels were evaluated by using
quantitative RT-PCR. Result: The combined use of sorafenib and
BCAAs significantly decreases in cell viability of all treated
HCC cells. Western blotting analysis revealed that addition
of BCAAs enhanced inhibition of ERK pathway in sorafenibtreated
HCC cells. Sorafenib treatment induced Akt phosphorylation
in HCC cells as previously reported. However, addition
of BCAAs antagonized the Akt phosphorylation. The combined
use of sorafenib and BCAAs resulted in significant increase
in caspase 3/7 activities and Annexin-V positive cell rates in
HCC cells compared with sorafenib alone. We next examined
pro-and anti-apoptotic proteins in HCC cells treated with
sorafenib and BCAAs. Addition of BCAAs enhanced reduction
of anti-apoptotic protein Mcl-1 expression in sorafeib-treated
HCC cells. In contrast, both pro-apoptotic proteins Bak/Bax
and anti-apoptotic protein Bcl-xL did not changed. mRNA
expression levels of Mcl-1 were significantly lower in HCC cells
treated by sorafenib with BCAAs than those without BCAAs.
Furthermore, we evaluated Mcl-1 degradation in the HCC cells
by using cycloheximide, a protein synthesis inhibitor. After inhibition
of protein synthesis, Mcl-1 levels were decreased more in
sorafenib-treated cells with BCAAs than those without BCAAs.
These results indicate that BCAAs reduce Mcl-1 expression at
both transcriptional and post-transcriptional levels in sorafenibtreated
HCC cells. Conclusion: BCAAs enhanced sorafenib-mediated
apoptosis in HCC cells via down regulation of Mcl-1.
These results suggest that the combined use of sorafenib and
BCAAs is effective in the treatment of advanced HCC patients.
Disclosures:
Hayato Hikita - Grant/Research Support: Bristol-Myers Squibb
1983
DNA methylome and cancer-specific expression change
of clustered miRNAs in non-B non-C hepatocellular carcinoma
Takeshi Matsui 2,1 , Masanori Nojima 8 , Etsuko Iio 2 , Akihiro Tamori 3 ,
Shoji Kubo 4 , Ken Shirabe 5 , Koichi Kimura 9 , Mitsuo Shimada 9 ,
Tohru Utsunomiya 6 , Yasuteru Kondo 7 , Yasuhito Tanaka 2 ; 1 Center
for Gastroenterology, Teine-Keijinkai Hospital, Sapporo, Japan;
2 Department of Virology unit, Nagoya City University Graduate
School of Medical Sciences, Nagoya, Japan; 3 Department of
Hepatology, Osaka City University Graduate School of Medicine,
Osaka, Japan; 4 Department of Hepato-Biliary-Pancreatic Surgery,
Graduate School of Medicine, Osaka City University, Osaka,
Japan; 5 Department of Surgery and Science, Graduate School
of Medical Sciences, Kyushu University, Fukuoka, Japan; 6 Oita
prefectual hospital, Oita, Japan; 7 Division of Gastroenterology,
Tohoku University Hospital, Sendai, Japan; 8 Institute of Medical
Science Hospital, the University of Tokyo, Tokyo, Japan; 9 Department
of Surgery, the University of Tokushima, Tokushima, Japan
[Background] While HBV and HCV infection have been suppressed
with development of public health measures and
medical treatment, non-B non-C hepatocellular carcinoma
(NBNC-HCC) is considered increasing based on increase of
nonalcoholic fatty liver disease (NAFLD) in Japan. Molecular
biological mechanism should be pursued against this disease
structure change. [Aim] To explore critical changes of DNA
methylation in micro RNA (miRNA) coding regions and its
expression in tumorigenesis of NBNC-HCC. [Methods] Infinium
HumanMethylation450 BeadChip Kit (Illumina) and micro
3D-Gene miRNA Oligo Chip (Toray) were applied in 26 pairs
of tumor and non-tumor background tissue samples. Pooled
analysis using the data from Gene Expression Omnibus (GEO,
NCBI) was also conducted to strengthen generalizability, and
to seek common DNA methylation changes across infection
status (HBV, HCV or NBNC). Gene ontology analysis were
applied for gene clusters defined by clustering and other statistical
characteristics. [Results and plan] Hierarchical clustering
of the DNA methylome status demonstrates clear discrimination
between tumor and non-tumor samples. While approx. 20% of
CpG islands in promoters were dominantly hypermethylated
in the tumor, other regions were globally hypomethylated in
the tumor. The mean difference in methylation levels of total
probes was -10.5% in non-CpG islands, and 0.68% in CpG
islands (tumor vs. non-tumor). It suggests that the change of
methylation status was totally different between CpG and non-
CpG islands. Next, to investigate the molecular biological significance
of DNA hypomethylation in the tumor, we determine
the characteristics of hypomethylated sites, and then found several
miRNA clusters were markedly hypomethylated (-30% to
-40%) in tumor tissues (e.g. miR-493 in 14q32.2, miR-154 in
14q32.31, miR-518F in 19q13.41, miR-888 in Xq27.3). Average
methylation level in miRNA coding region were lower than
other regions. In detail, 52.8% of total miRNA probes were significantly
hypomethylated, and mean difference in methylation
levels were -14.4% in non-CpG islands, and 0.14% in CpG
islands. Consistent with the hypomethylation tendency, average
miRNA expression level was 28.0% up-regulated. In particular,
miRNA expression significantly around 80% to 100%