title: Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation.
abstract: OBJECTIVE
  To investigate the role of endoplasmic reticulum (ER) stress in cancer radiotherapy and its molecular mechanism.
  
  
  METHODS
  Tunicamycin (TM) was applied to induce ER stress in human esophageal cancer cell line EC109, and the radiosensitization effects were detected by acute cell death and clonogenic survival assay. Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide. Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase. Autophagic response was determined by acridine orange (AO) staining and Western blotting of microtubule-associated protein-1 light chain-3 (LC3) and autophagy related gene 5 (ATG5). In order to test the biological function of autophagy, specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy, and its effect on cell apoptosis was thus detected. Additionally, involvement of the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway was also detected by Western blotting. Finally, male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations.
  
  
  RESULTS
  Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation (IR), which suggested an obvious radiosensitization effect of TM. Moreover, TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. We also observed an increase of AO positive cells, and the protein level of LC3-II and ATG5 was induced by TM treatment, which suggested an autophagic response in EC109 cells. However, inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability, which suggested a cytoprotective role of autophagy in stressed EC109 cells. Furthermore, TM treatment also activated mTORC1, and in turn reduced Akt phosphorylation, which suggested the PI3K/Akt/mTOR signal pathway was involved in the TM-induced autophagic response in EC109 cells. Tumor xenograft results also showed synergistic retarded tumor growth by TM treatment and IR, as well as the involvement of the PI3K/Akt/mTOR pathway.
  
  
  CONCLUSIONS
  Our data showed that TM treatment sensitized human esophageal cancer cells to radiation via apoptosis and autophagy both in vitro and in vivo.
authors.affiliation: Department of Oncology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Xue-Li
authors.initials: XL
authors.lastname: Pang
authors.forename: Gang
authors.initials: G
authors.lastname: He
authors.forename: Yang-Bo
authors.initials: YB
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000528:radiation effects; D000071186:Beclin-1; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D046912:Multiprotein Complexes / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D011838:Radiation-Sensitizing Agents; D014415:Tunicamycin; D058539:Phosphatidylinositol 3-Kinase; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases
keywords: Apoptosis; Autophagy; Endoplasmic reticulum stress; Esophageal cancer; Radiosensitivity; Tunicamycin
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