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

PLoS One. 2013;8(3):e59330. doi: 10.1371/journal.pone.0059330. Epub 2013 Mar 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 6 / genetics
  • Activating Transcription Factor 6 / metabolism
  • Apoptosis / drug effects*
  • Apoptosis / genetics
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Bcl-2-Like Protein 11
  • Blotting, Western
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cells, Cultured
  • Endoplasmic Reticulum Stress / drug effects*
  • Endoplasmic Reticulum Stress / genetics
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Humans
  • In Situ Nick-End Labeling
  • Lipid Peroxidation / drug effects
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mitomycin / pharmacology*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Transcription Factor CHOP / genetics
  • Transcription Factor CHOP / metabolism
  • eIF-2 Kinase / genetics
  • eIF-2 Kinase / metabolism

Substances

  • ATF6 protein, human
  • Activating Transcription Factor 6
  • Apoptosis Regulatory Proteins
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • DDIT3 protein, human
  • Heat-Shock Proteins
  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Transcription Factor CHOP
  • Mitomycin
  • ERN2 protein, human
  • EIF2AK3 protein, human
  • Protein-Serine-Threonine Kinases
  • eIF-2 Kinase
  • Endoribonucleases
  • molecular chaperone GRP78

Grant support

This work was supported by the National Natural Science Foundation of China, grants #30973058 and #81171694 (http://www.nsfc.gov.cn/Portal0/default166.htm, http://nsfc.pubmed.cn/b7c13b133, http://nsfc.pubmed.cn/042d8caa1); Jiangsu Province Nature Science Foundation (BE2010743), http://www.jsnsf.gov.cn/Index.aspxl; Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU (No. IRT-015), http://www.jsph.org.cn/art/2011/3/8/art_674_3373.html, http://www.jsph.net/; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(JX10231801). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.