@Article{096504017X15005102445191,
AUTHOR = {Sanae Haga, Akira Kanno, Takeaki Ozawa, Naoki Morita, Mami Asano,¶ and Michitaka Ozaki},
TITLE = {Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury  of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting  Protein (RIP)1/RIP3 Binding},
JOURNAL = {Oncology Research},
VOLUME = {26},
YEAR = {2018},
NUMBER = {3},
PAGES = {503--513},
URL = {http://www.techscience.com/or/v26n3/56662},
ISSN = {1555-3906},
ABSTRACT = {Liver injury is often observed in various pathological conditions including posthepatectomy state and 
cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell 
death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated 
necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 
(RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 
cells, TNF-a/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by 
the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by 
necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved 
by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is 
inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, 
induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was 
significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H<sub>2</sub>O<sub>2</sub>, by itself, 
induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding 
after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/
reoxygenation (H/R) was also improved by Nec-1. Similar to H<sub>2</sub>O<sub>2</sub>, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligandinduced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional 
caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury 
induced by oxidative stress and FasL in the absence of apoptosis.},
DOI = {10.3727/096504017X15005102445191}
}