title: Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G₂/M arrest.
abstract: OBJECTIVE
  To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.
  
  
  METHODS
  The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis.
  
  
  RESULTS
  Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 μmol/L. Genipin 200-400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.
  
  
  CONCLUSIONS
  These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.
authors.affiliation: Key Laboratory of Bio-resources and Eco-environment of the Ministry of Education, College of Science, Sichuan University, Chengdu, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Feng
authors.forename: Hou-li
authors.initials: HL
authors.lastname: Cao
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Xiao-rong
authors.initials: XR
authors.lastname: Li
authors.forename: Yan-qin
authors.initials: YQ
authors.lastname: Ren
authors.forename: Lin-fang
authors.initials: LF
authors.lastname: Du
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division; D016195:G2 Phase; D006801:Humans; D057889:Iridoid Glycosides / Q000494:pharmacology*; D039823:Iridoids; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020014:K562 Cells; D007938:Leukemia / Q000473:pathology*
chemical_list: D057889:Iridoid Glycosides; D039823:Iridoids; C007834:genipin; D048031:JNK Mitogen-Activated Protein Kinases