title: FH535 increases the radiosensitivity and reverses epithelial-to-mesenchymal transition of radioresistant esophageal cancer cell line KYSE-150R.
abstract: BACKGROUND
  Acquired radioresistance has significantly compromised the efficacy of radiotherapy for esophageal cancer. The purpose of this study is to investigate the roles of epithelial-mesenchymal transition (EMT) and the Wnt/β-catenin signaling pathway in the acquirement of radioresistance during the radiation treatment of esophageal cancer.
  
  
  METHODS
  We previously established a radioresistant cell line (KYSE-150R) from the KYSE-150 cell line (a human cell line model for esophageal squamous cell carcinoma) with a gradient cumulative irradiation dose. In this study, the expression of EMT phenotypes and the Wnt/β-catenin signaling pathway proteins were examined by real-time PCR, western blot and immunofluorescence in the KYSE-150R cells. The KYSE-150R cells were then treated with a β-Catenin/Tcf inhibitor FH535. The expressions of nuclear and cytoplasmic β-catenin and EMT markers in KYSE-150R cells were assessed at both mRNA and protein level after FH535 treatment. The radiosensitization effect of FH535 on KYSE-150R was evaluated by CCK8 analysis and a colony forming assay. DNA repair capacities was detected by the neutral comet assays.
  
  
  RESULTS
  KYSE-150R cell line displayed obvious radiation resistance and had a stable genetic ability. EMT phenotype was presented in the KYSE-150R cells with decreased E-cadherin and increased snail and twist expressions. The up-regulated expressions of Wnt/β-catenin signaling pathway proteins (Wnt1, FZD1-4, GSK3β, CTNNB1 and Cyclin D1), the increased phosphorylation of GSK3β, and the decreased phosphorylation of β-catenin were observed in KYSE-150R cells compared with KYSE-150 cells, implicating the activation of the Wnt pathway in KYSE-150R cells. The expression of nuclear β-catenin and nuclear translocation of β-catenin from the cytoplasm was decreased after FH535 treatment. FH535 also reversed EMT phenotypes by increasing E-cadherin expression. The cell proliferation rates of KYSE-150R were dose-dependent and the radiation survival fraction was significantly decreased upon FH535 treatment. Neutral comet assays indicated that FH535 impairs DNA double stranded break repair in KYSE-150R cells.
  
  
  CONCLUSIONS
  Acquisition of radioresistance and EMT in esophageal cancer cells is associated with the activation of the Wnt/β-catenin pathway. EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting the Wnt/β-catenin pathway with FH535 treatment.
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. 12602639@qq.com.
authors.forename: Huafang
authors.initials: H
authors.lastname: Su
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. jinxc1979@hotmail.com.
authors.forename: Xiance
authors.initials: X
authors.lastname: Jin
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. baxuza@126.com.
authors.forename: Xuebang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. zlh0577@gmail.com.
authors.forename: Lihao
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. chaichaiwz@163.com.
authors.forename: Baochai
authors.initials: B
authors.lastname: Lin
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. lovelydowndown@126.com.
authors.forename: Lili
authors.initials: L
authors.lastname: Li
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. hui-y@163.com.
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Fei
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. 337612340@qq.com.
authors.forename: Lanxiao
authors.initials: L
authors.lastname: Shen
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. fangyafy@126.com.
authors.forename: Ya
authors.initials: Y
authors.lastname: Fang
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. phlphh@163.com.
authors.forename: Huanle
authors.initials: H
authors.lastname: Pan
authors.affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. wzxiecongying@163.com.
authors.forename: Congying
authors.initials: C
authors.lastname: Xie
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D004938:Esophageal Neoplasms / Q000473:pathology*; D000077277:Esophageal Squamous Cell Carcinoma; D006801:Humans; D010641:Phenotype; D021381:Protein Transport / Q000187:drug effects; D011836:Radiation Tolerance / Q000187:drug effects*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology*; D060449:Wnt Signaling Pathway / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism
chemical_list: C575430:FH535; D011838:Radiation-Sensitizing Agents; D013449:Sulfonamides; D051176:beta Catenin