Toxicoproteomic investigation of the molecular mechanisms of cycloheximide-induced hepatocellular apoptosis in rat liver.
C/EBP homologous protein (CHOP) is a transcriptional factor and is induced under conditions such as the unfolded protein response or amino acid starvation. A previous study showed that the transcriptional level of CHOP was highly increased in rat liver in which hepatocellular apoptosis was induced by cycloheximide (CHX) treatment. Here, we investigated the relationship between hepatocellular apoptosis and CHOP-mediated apoptotic pathway, and studied the mechanisms of induction of CHOP gene in the liver of rats treated with CHX. Male F344 rats were treated intravenously with 6mg/kg CHX, and sacrificed at 1, 2 and 6h after the treatment. In the gene expression assay using quantitative RT-PCR, the genes related to CHOP-mediated apoptosis such as the C/EBPbeta, ATF3 and ATF4 genes were significantly increased corresponding to the induction of hepatocellular apoptosis in rats treated with CHX. However the GRP78/Bip gene, which serves as a representative marker for the unfolded protein response, did not change after the treatment. Toxicoproteomics using two-dimensional difference gel electrophoresis and mass spectrometry indicated that GRP78/Bip was inactivated by the CHX treatment. Furthermore, the CHX-treated animals exhibited a significant decrease of phosphorylated Akt/PKB (protein kinase B). These results indicate that the protein synthesis inhibition by CHX induces the CHOP gene through a pathway similar to that of amino acid starvation, and that Akt/PKB inactivation enhances the CHOP-mediated hepatocellular apoptosis.
Kumagai
Kazuyoshi
K
Medicinal Safety Research Labs., Sankyo Co., Ltd., 717 Horikoshi, Fukuroi, Shizuoka 437-0065, Japan. kumawind@sankyo.co.jp <kumawind@sankyo.co.jp>
Ando
Yosuke
Y
Kiyosawa
Naoki
N
Ito
Kazumi
K
Kawai
Ryota
R
Yamoto
Takashi
T
Manabe
Sunao
S
Teranishi
Munehiro
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects*; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D020287:In Situ Nick-End Labeling; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051742:Transcription Factor CHOP / Q000378:metabolism; D014357:Trypsin
D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D051742:Transcription Factor CHOP; D003513:Cycloheximide; D051057:Proto-Oncogene Proteins c-akt; D014357:Trypsin; D005978:Glutathione