Abstract 4797: Continuous hepatocyte apoptosis accelerates diethylnitrosamine-induced liver tumor development
Authors: Nozaki Yasutoshi , Hikita Hayato , Tanaka Satoshi , Myojin Yuta , Sakane Sadatsugu , Murai Kazuhiro , Shiode Yuto , Kai Yugo , Makino Yuki , Nakabori Tasuku , Saito Yoshinobu , Kodama Takahiro , Sakamori Ryotaro , Tatsumi Tomohide , Miyoshi Eiji , Takehara Tetsuo 
Published: July 2017

Abstract
Background and Aim: Apoptosis serves as an important mechanism for removing DNA damaged-cells and is considered to inhibit carcinogenesis. Hepatocyte apoptosis is a key feature of chronic liver disease, as established precancerous condition for liver cancer. The present study examined the impact of continuous hepatocyte apoptosis on liver tumor development.
Methods: We used male hepatocyte-specific knockout (KO) mice of Mcl-1, one of anti-apoptotic proteins, as a model of continuous hepatocyte apoptosis, and hepatocyte-specific single or double KO mice of Bak and Bax, proapoptotic proteins, as a model of apoptosis-resistant liver. To induce liver carcinogenesis those mice were intraperitoneally administered 20 mg/kg diethylnitrosamine (DEN) at 2 weeks.
Results: Among control group treated with PBS, neither Mcl-1 KO nor wild-type (WT) mice developed macroscopically and microscopically any liver tumors at age of 6 months. On the other hand, while only 8% (2/26) of DEN-treated WT mice developed macroscopic liver tumors, 100% (15/15) of Mcl-1 KO mice developed (p&lt;0.05). Microscopically, 57% (15/26) of DEN-treated WT mice developed liver tumors, 100% (15/15) of Mcl-1 KO mice developed (p&lt;0.05). These tumors were histologically classified as well-differentiated HCC. The average of maximum tumor size (12.4 vs. 6.5 mm), tumor numbers (21.4 vs. 4.0) and liver weight to body weight ratio (12.3 vs. 4.2 %) in Mcl-1 KO mice were significantly higher than those in WT mice. At 6 weeks, serum ALT levels and caspase 3/7 activity of DEN-treated Mcl-1 KO mice were significantly higher than those of DEN-treated WT mice. Immunohistochemistry revealed that PCNA, Ki67, phospho-H2AX and 8-OHdG positive cells significantly increased in DEN-treated Mcl-1 KO mice compared with DEN-treated WT mice. The mRNA expression levels of TNF-alfa and Mcp-1 in DEN-treated Mcl-1 KO mice were significantly higher than those of DEN-treated WT mice.In contrast, macroscopic liver tumor incidence rate in WT, Bak KO, Bax KO and Bak/Bax double KO mice treated with DEN at 9 months was 46 (6/13), 40 (6/15), 35 (7/20) and 52% (11/21), respectively. Microscopic liver tumor incidence rate in WT, Bak KO, Bax KO and Bak/Bax double KO mice was 100 (13/13), 80 (12/15), 70 (14/20) and 81% (17/21), respectively. There was no statistically significant difference in incidence rate of both macroscopic and microscopic tumors among 4 groups. At age of 6 weeks, there was no significant difference in serum ALT levels and caspase 3/7 activity among 4 groups.
Conclusion: Continuous hepatocyte apoptosis is suggested to promote DEN-induced liver tumor development. Increased liver damage, compensative hepatocyte proliferation, genomic instability and inflammation may accelerate the incidence of DEN-induced liver tumorigenesis. In contrast, inhibition of apoptosis does not make much impact on DEN-induced liver tumor development.
Citation Format: Yasutoshi Nozaki, Hayato Hikita, Satoshi Tanaka, Yuta Myojin, Sadatsugu Sakane, Kazuhiro Murai, Yuto Shiode, Yugo Kai, Yuki Makino, Tasuku Nakabori, Yoshinobu Saito, Takahiro Kodama, Ryotaro Sakamori, Tomohide Tatsumi, Eiji Miyoshi, Tetsuo Takehara. Continuous hepatocyte apoptosis accelerates diethylnitrosamine-induced liver tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2017-4797