Endoplasmic reticulum stress signaling is involved in mitomycin C (MMC)-induced apoptosis in human fibroblasts via PERK pathway

• Published in: PloS one 2013-03, Vol.8 (3), p.e59330
• Main Authors: Shi, Kun, Wang, Daode, Cao, Xiaojian, Ge, Yingbin
• Format: Article
• Language: English
• Subjects: Activating transcription factor 6; Activating Transcription Factor 6 - genetics; Activating Transcription Factor 6 - metabolism; Adenosine diphosphate; Analysis; Annexin V; Apoptosis; Apoptosis - drug effects; Apoptosis - genetics; Apoptosis Regulatory Proteins - genetics; Apoptosis Regulatory Proteins - metabolism; BAX protein; Bcl-2 protein; Bcl-2-Like Protein 11; BIM protein; Biology; Blotting, Western; Caspase; Caspase-3; CCAAT/enhancer-binding protein; Cell culture; Cell cycle; Cell proliferation; Cell Survival - drug effects; Cell Survival - genetics; Cells, Cultured; DNA nucleotidylexotransferase; eIF-2 Kinase - genetics; eIF-2 Kinase - metabolism; Endoplasmic reticulum; Endoplasmic Reticulum Stress - drug effects; Endoplasmic Reticulum Stress - genetics; Endoribonucleases - genetics; Endoribonucleases - metabolism; Esophagus; Fibroblasts; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; Gastric cancer; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Homology; Humans; In Situ Nick-End Labeling; Inositol; Iodides; Kinases; Lipid Peroxidation - drug effects; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mitomycin; Mitomycin - pharmacology; Mitomycin C; Orthopedics; Oxidative stress; Oxygen; Poly(ADP-ribose) polymerase; Propidium iodide; Protein folding; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Reactive oxygen species; Reverse Transcriptase Polymerase Chain Reaction; Ribose; Rodents; Scars; Signal transduction; Signal Transduction - drug effects; Signal Transduction - genetics; Signaling; Staining; Stomach cancer; Stresses; Surgery; Transcription Factor CHOP - genetics; Transcription Factor CHOP - metabolism; Western blotting
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0059330

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.