title: Methotrexate induces apoptosis of human choriocarcinoma cell line JAR via a mitochondrial pathway.
abstract: OBJECTIVE
  To investigate methotrexate (MTX)-induced apoptosis and the involved pathways in human choriocarcinoma cells.
  
  
  METHODS
  MTX-induced apoptosis of human choriocarcinoma cell line JAR was examined using a PI/Annexin V stain with flow cytometer (FCM). Mitochondrial apoptosis was detected by fluorescence microscopy, and analyzed by FCM using a MitoCapture mitochondrial apoptosis detection kit. The activities of caspase-8 and caspase-9 were quantified by microtiter plate reader at 405 nm using FLICE/Caspase-8 colorimetric assay kit and Caspase-9/Mch6 colorimetric assay kit. The changes in Bax and Bcl-2 expression were detected during apoptosis using immunocytochemistry and Western blot analysis.
  
  
  RESULTS
  JAR cells underwent apoptosis after exposure to 0.1-2.5 microg/ml MTX for 48 h. Decreased mitochondrial membrane potential was observed both by fluorescence microscopy and FCM. The activation of caspase-9 was increased 4.35+/-0.76-fold in MTX-incubated JAR, while there was no obvious change in the activation of caspase-8. When JAR cells underwent apoptosis, the expression of Bcl-2 was decreased and the expression of Bax was increased; both were detected by immunocytochemistry assay.
  
  
  CONCLUSIONS
  Methotrexate in lower concentrations induces apoptosis of human choriocarcinoma cells via mitochondrial-initiated pathways, including reduction of mitochondrial membrane potential, activation of caspase-9, and up-regulation of Bax/Bcl-2 expression.
authors.affiliation: Women's Hospital, School of Medicine, Zhejiang University, No. 2 Xueshi Road, Hangzhou 310006, China. chenyax@zju.edu.cn
authors.forename: Ya-Xia
authors.initials: YX
authors.lastname: Chen
authors.forename: Wei-Guo
authors.initials: WG
authors.lastname: Lv
authors.forename: Huai-Zeng
authors.initials: HZ
authors.lastname: Chen
authors.forename: Feng
authors.initials: F
authors.lastname: Ye
authors.forename: Xing
authors.initials: X
authors.lastname: Xie
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000378:metabolism / Q000473:pathology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008727:Methotrexate / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D011247:Pregnancy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D014594:Uterine Neoplasms / Q000378:metabolism / Q000473:pathology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D053453:Caspase 9; D008727:Methotrexate