Heat-shock protein 70-overexpressing gastric epithelial cells are resistant to indomethacin-induced apoptosis.
OBJECTIVE
Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress.


METHODS
HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0-500 microM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis.


RESULTS
Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells.


CONCLUSIONS
Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell injury.
Hirata
Ikuhiro
I
Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan.
Naito
Yuji
Y
Handa
Osamu
O
Hayashi
Natsuko
N
Mizushima
Katsura
K
Adachi
Satoko
S
Omatsu
Tatsushi
T
Okayama
Tetsuya
T
Kishimoto
Etsuko
E
Ichikawa
Hiroshi
H
Takagi
Tomohisa
T
Kokura
Satoshi
S
Otaka
Michiro
M
Yoshikawa
Toshikazu
T
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000009:adverse effects* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005753:Gastric Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis*; D007213:Indomethacin / Q000009:adverse effects* / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats
D000894:Anti-Inflammatory Agents, Non-Steroidal; D018840:HSP70 Heat-Shock Proteins; D007213:Indomethacin