title: Autophagy induction contributes to the resistance to methotrexate treatment in rheumatoid arthritis fibroblast-like synovial cells through high mobility group box chromosomal protein 1.
abstract: BACKGROUND
  Rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) show resistance to methotrexate (MTX) treatment. To better understand the mechanisms of this resistance, RA-FLS and osteoarthritis fibroblast-like synovial cells (OA-FLS) were isolated and exposed to MTX. We analyzed the autophagy induced by MTX in vitro and its relationship to apoptosis.
  
  
  METHODS
  Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was detected by flow cytometry and Western blot analysis. Autophagy was determined by transmission electron microscopy as well as Western blot analysis. The expression levels of Beclin-1, LC3, Akt, p-Akt, mammalian target of rapamycin (mTOR), p-mTOR, high mobility group box chromosomal protein 1 (HMGB1), and an 85 kDa caspase cleaved fragment of poly(ADP-ribose) polymerase were measured by Western blotting.
  
  
  RESULTS
  MTX-induced apoptosis was increased in OA-FLS compared with RA-FLS. However, MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation, but not in OA-FLS. In RA-FLS, transfection with Beclin-1 small interfering RNA inhibited autophagy and increased susceptibility to MTX, which induces cell death. MTX upregulated autophagy through its ability to enhance the expression of HMGB1 and Beclin-1 rather than through the Akt/mTOR pathway.
  
  
  CONCLUSIONS
  Autophagy induction contributes to resistance to MTX treatment in fibroblasts from patients with rheumatoid arthritis.
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. santxuke1986@126.com.
authors.forename: Ke
authors.initials: K
authors.lastname: Xu
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. cys3568@163.com.
authors.forename: Yong-song
authors.initials: YS
authors.lastname: Cai
authors.affiliation: Department of Genetics and Molecular Biology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi Province, China. lushemin@mail.xjtu.edu.cn.
authors.forename: She-Min
authors.initials: SM
authors.lastname: Lu
authors.affiliation: Department of Internal Medicine, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi Province, China. xili666@126.com.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Li
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. liulin183092@163.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. lizhong0607@126.com.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. 1q2we3@163.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, 710054, Shaanxi Province, China. sousou369@163.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
mesh_terms: D000368:Aged; D018501:Antirheumatic Agents / Q000627:therapeutic use*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000502:physiology; D004351:Drug Resistance / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D024243:HMGB1 Protein / Q000096:biosynthesis*; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000627:therapeutic use*; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D034741:RNA, Small Interfering; D013583:Synovial Membrane / Q000166:cytology; D014162:Transfection
chemical_list: D018501:Antirheumatic Agents; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; D034741:RNA, Small Interfering; D008727:Methotrexate