title: High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis.
abstract: OBJECTIVE
  To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters.
  
  
  METHODS
  uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay.
  
  
  RESULTS
  The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA.
  
  
  CONCLUSIONS
  uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Parichut
authors.initials: P
authors.lastname: Thummarati
authors.forename: Sitsom
authors.initials: S
authors.lastname: Wijitburaphat
authors.forename: Aruna
authors.initials: A
authors.lastname: Prasopthum
authors.forename: Apaporn
authors.initials: A
authors.lastname: Menakongka
authors.forename: Banchob
authors.initials: B
authors.lastname: Sripa
authors.forename: Rutaiwan
authors.initials: R
authors.lastname: Tohtong
authors.forename: Tuangporn
authors.initials: T
authors.lastname: Suthiphongchai
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000378:metabolism* / Q000473:pathology*; D005260:Female; D006801:Humans; D053208:Kaplan-Meier Estimate; D008207:Lymphatic Metastasis / Q000473:pathology*; D008297:Male; D046228:Microarray Analysis; D008875:Middle Aged; D009361:Neoplasm Invasiveness / Q000473:pathology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism; D012189:Retrospective Studies; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator