title: Endoplasmic reticulum stress signaling is involved in mitomycin C (MMC)-induced apoptosis in human fibroblasts via PERK pathway.
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.
authors.affiliation: Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Shi
authors.forename: Daode
authors.initials: D
authors.lastname: Wang
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Cao
authors.forename: Yingbin
authors.initials: Y
authors.lastname: Ge
mesh_terms: D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects* / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D015227:Lipid Peroxidation / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D051742:Transcription Factor CHOP; D016685:Mitomycin; C403982:ERN2 protein, human; C460640:EIF2AK3 protein, human; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D004722:Endoribonucleases