RRC ID 75782
Author Naito Y, Handa O, Takagi T, Ishikawa T, Imamoto E, Nakagawa S, Yamaguchi T, Yoshida N, Matsui H, Yoshikawa T.
Title Ubiquitin-proteasome inhibitor enhances tumour necrosis factor-alpha-induced apoptosis in rat gastric epithelial cells.
Journal Aliment Pharmacol Ther
Abstract BACKGROUND:Tumour necrosis factor (TNF-alpha) is a candidate factor for involvement in inflammation-mediated gastric mucosal injury. However, the effect of this cytokine on gastric epithelial cells has been poorly investigated. In the present study, we examined whether gastric epithelial cells are resistant to TNF-alpha-induced apoptosis, and whether this resistance is related to ubiquitin-proteasome-associated nuclear factor-kappaB (NF-kappaB) activation.
METHODS:The rat gastric mucosal cell line RGM-1 was grown in DMEM/F12 medium supplemented with 10% FCS. Confluent monolayers of cells were pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant rat TNF-alpha and their viability was determined by WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342 and propidium iodide, and DNA fragmentation was determined by flow cytometry using an APO-BRDU kit. IkappaB-alpha and the p65 binding subunit of NF-kappaB were detected by Western blots.
RESULTS:Twenty-four-hour incubation with TNF-alpha alone or PSI alone did not affect the cell viability of RGM-1 cells. Pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-alpha. In RGM-1 cells treated with TNF-alpha, cytoplasmic IkappaB-alpha decreased and p65 in nuclear extracts increased markedly 30 min after cytokine stimulation. Pretreatment with PSI at 12.5 micromol/L blocked these TNF-alpha-induced changes.
CONCLUSION:PSI enhances TNF-alpha-induced apoptosis through inhibition of NF-kappaB activation in RGM-1 cells.
Volume 16 Suppl 2
Pages 59-66
Published 2002-4-1
DOI 10.1046/j.1365-2036.16.s2.30.x
PII 161
PMID 11966525
MeSH Animals Apoptosis / drug effects* Blotting, Western Calcium-Binding Proteins* Cell Line Cell Survival / drug effects Cysteine Endopeptidases DNA-Binding Proteins / metabolism Epithelial Cells / cytology Epithelial Cells / metabolism Flow Cytometry Gastric Mucosa / cytology Gastric Mucosa / metabolism* I-kappa B Proteins* Membrane Glycoproteins / metabolism Microscopy, Phase-Contrast Multienzyme Complexes / antagonists & inhibitors* NF-KappaB Inhibitor alpha NF-kappa B / metabolism* Nerve Tissue Proteins / metabolism Oligopeptides / pharmacology Proteasome Endopeptidase Complex Rats Synaptotagmin I Synaptotagmins Tumor Necrosis Factor-alpha / pharmacology* Ubiquitin / antagonists & inhibitors*
IF 7.515
Human and Animal Cells RGM1(RCB0876)