studies

1168A AASLD ABSTRACTS HEPATOLOGY, October, 2015
The following authors have nothing to disclose: Taro Yamashita, Mitsumasa
Kondo, Naoki Oishi, Masao Honda
1971
Development of HCC is dependent upon hepatocellular
apoptosis in a murine model of NASH associated tumoriogenesis
Casey Johnson 1 , Alexander Wree 1,2 , Joan Font-Burgada 3 , Akiko
Eguchi 1 , Davide Povero 1 , Michael Karin 3 , Ariel E. Feldstein 1 ;
1 Pediatrics, UCSD, La Jolla, CA; 2 Department of Internal Medicine
III, University Hospital RWTH-Aachen, Aachen, Germany; 3 Laboratory
of Gene Regulation and Signal Transduction, Departments of
Pharmacology and Pathology, UCSD, La Jolla, CA
Background: Nonalcoholic steatohepatitis (NASH) is increasingly
associated with the development of hepatocellular carcinoma
(HCC), even in patients without established liver cirrhosis.
While the oncogenic mechanisms of blocking persistent hepatocyte
cell death, a common feature among various chronic liver
diseases, remain unclear it nevertheless presents as a logical
treatment modality. Therefore, we aimed at investigating the
influence of hepatocyte specific Bid depletion – a BH3-only
Bcl-2 family member that amplifies apoptotic death signals –
on tumor development in a murine model of NASH induced
HCC. Methods: Hepatocyte-specific conditional Bid-knockout
mice (Bid Δhep ) and functional Bid-expressing control mice (Bidflo/flo
) were injected with 100mg/Kgr streptozotocin (STZ) at
2-days of age. Mice were weaned onto a 60% Kcal from fat
diet and liver tumorigenesis was investigated 5 months later.
Results: Western Blot analysis confirmed hepatocyte specific
Bid depletion in Bid Δhep mice. Survival rate was impaired in
Bid flo/flo when compared to Bid Δhep mice (Bid flo/flo 3/12 vs.
Bid Δhep 0/12; p < 0.05). Bid flo/flo mice exhibited higher incidences
of liver tumors larger than 5mm when compared to
Bid Δhep mice, which translated into an increased tumor load
(Bid flo/flo 10/10 vs. Bid Δhep 4/10; p < 0.05). Liver transaminase
levels remained normal within Bid Δhep mice, while Bid flo/
flo
mice were elevated (Bid Δhep 249 IU/l ±12 IU/l (mean ±
SEM) vs. Bid flo/flo 84±12; p < 0.05). Tissue section quantification
revealed an increased rate of hepatocellular apoptosis
in Bid flo/flo mice when compared to Bid Δhep mice. Quantitative
PCR analysis of mRNA levels of AFP (alphafetoprotein), HGF
(hepatocyte growth factor), Cyclin D1 and PCNA were significantly
increased in Bid flo/flo mice as compared to Bid Δhep
animals indicating cellular proliferation. Liver sections of Bid flo/
flo
mice also exhibited increased infiltration of inflammatory
cells and concomitant upregulation of mRNA levels of F4/80,
Ly6c, TNFa (tumor necrosis factor), and IL-1beta (interleukin
1 beta). Liver fibrosis developed in both mouse groups, while
mRNA levels indicating hepatic stellate cell activation (aSMA
– smooth muscle actin; and TIMP1 – tissue inhibitor of metalloproteinase)
were significantly increased in Bid flo/flo mice when
compared to Bid Δhep mice. Conclusion: Our study demonstrates
that blocking hepatic apoptosis ameliorated HCC development
in a NASH-related tumorigenesis model. These results suggest
that reducing hepatocyte cell death, liver inflammation and
compensatory proliferation presents a strong beneficial effect
that supersedes the potential side effect of enhancing tumor
cell survival.
Disclosures:
Akiko Eguchi - Grant/Research Support: Gilead, Conatus
The following authors have nothing to disclose: Casey Johnson, Alexander Wree,
Joan Font-Burgada, Davide Povero, Michael Karin, Ariel E. Feldstein
1972
Cytotoxic synergy between sorafenib and cyclooxygenase
2 inhibitor celecoxib in vitro: induction of cell death
of hepatocellular carcinoma through ER stress mediated
apoptosis
Sera Yang 1 , SoHee Kang 1 , Soohyun Park 2 , Jonghwa Kim 1 , Yong
Han Paik 1,2 ; 1 Department of Medicine, Samsung Medical Center,
Sungkyunkwan University School of Medicine, Seoul, Korea
(the Republic of); 2 Department of Health Science and Technology,
Samsung Advanced Institute for Health Science and Technology,
Sungkyunkwan University, Seoul, Korea (the Republic of)
Background/Aim: Sorafenib, a multi-kinase inhibitor, is currently
recommended for the treatment of advanced hepatocellular
carcinoma (HCC). However, the response rate of sorafenib
in advanced HCC treatment are not satisfactory and there is
a rationale for investigating its use in combination with other
agents. Celecoxib is known as a selective cyclooxygenase-2
(COX-2) inhibitor and has been known to exhibit anti-tumor
effects in HCC cells but the mechanism is still largely unknown.
The aim of this study was to investigate the synergistic effect of
celecoxib on the anti-tumor activity of sorafenib in HCC and
to determine the underlying molecular mechanism. Methods:
HCC cell line PLC/PRF/5 were treated with sorafenib and/
or celecoxib, and then proliferation was analyzed by MTS
assay. Cell death was evaluated by annexin staining and
FACS analysis. Western blot was also used to study the mechanism
of the induced cell death. Results: MTS assay revealed
that each of sorafenib and celecoxib alone marginally inhibited
HCC cell proliferation. However, the combination of
sorafenib and celecoxib showed a synergistic effect to inhibit
cell proliferation, while the effect was less strongly observed
with 2,5-dimethyl-celecoxib (DMC, a derivative of celecoxib
that lacks COX2-inhibitory function). Using flow cytometry, we
found that the combined treatment synergistically increased the
population of Annexin + /PI + at 48h, indicating the increased
apoptosis. The increased cleavage of caspase-8 and PARP was
also demonstrated in Western blot. In addition, we also found
that ER stress marker proteins, such as GRP78/Bip, CHOP,
and spliced XBP-1, were dramatically increased in the combined
treatment of sorafenib and celecoxib. The activation of
caspases and cell death induced by sorafenib plus celecoxib
was significantly inhibited by pretreatment with 4μ8C, a ER
stress inhibitor. These data indicated that sorafenib and celecoxib
synergistically induce cell death of HCC cell line through
ER-stress-mediated apoptosis. Conclusion: Combined treatment
of sorafenib and celecoxib synergistically inhibit the growth
of HCC cells via inducing ER-stress mediated apoptosis. This
finding raises the possibility of the combined treatment using
sorafenib and celecoxib for the augmentation of anticancer
effect of sorafenib in HCC.
Disclosures:
The following authors have nothing to disclose: Sera Yang, SoHee Kang,
Soohyun Park, Jonghwa Kim, Yong Han Paik