title: Development of TRAIL resistance by radiation-induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma.
abstract: OBJECTIVE
  There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis.
  
  
  METHODS
  Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line.
  
  
  RESULTS
  HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine.
  
  
  CONCLUSIONS
  Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.
authors.affiliation: Department of Otorhinolaryngology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.
authors.forename: Jong Cheol
authors.initials: JC
authors.lastname: Lee
authors.affiliation: Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.
authors.forename: Won Hyeok
authors.initials: WH
authors.lastname: Lee
authors.affiliation: Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Min
authors.affiliation: Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.
authors.forename: Hee Jeong
authors.initials: HJ
authors.lastname: Cha
authors.affiliation: Department of Otorhinolaryngology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.
authors.forename: Myung Woul
authors.initials: MW
authors.lastname: Han
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Hyo Won
authors.initials: HW
authors.lastname: Chang
authors.affiliation: Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Sun-A
authors.initials: SA
authors.lastname: Kim
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Seung-Ho
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. Electronic address: swhokim@gmail.com.
authors.forename: Seong Who
authors.initials: SW
authors.lastname: Kim
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea. Electronic address: sykim3715@gmail.com.
authors.forename: Sang Yoon
authors.initials: SY
authors.lastname: Kim
mesh_terms: D000368:Aged; D017209:Apoptosis / Q000528:radiation effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry; D002294:Carcinoma, Squamous Cell / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D018899:CpG Islands / Q000528:radiation effects*; D019175:DNA Methylation / Q000528:radiation effects*; D000077209:Decitabine; D004307:Dose-Response Relationship, Radiation; D044127:Epigenesis, Genetic; D005260:Female; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D007822:Laryngeal Neoplasms / Q000532:radiotherapy*; D008297:Male; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000532:radiotherapy; D020411:Oligonucleotide Array Sequence Analysis; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry* / Q000235:genetics
chemical_list: D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505653:TNFSF10 protein, human; D000077209:Decitabine; D001374:Azacitidine