title: [Mechanisms of Apoptosis Induced by FTY720 in Multiple Myeloma Cell Line U266].
abstract: OBJECTIVE
  To investigate the effects of FTY720 on apoptosis in multiple myeloma cell line U266 and to clarify the molecular mechanism of apoptosis induced by FTY720.
  
  
  METHODS
  U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the apoptotic rates were tested by flow cytometry with Annexin-V-FITC/PI staining. Then U266 cells were treated with 20 µmol/L FTY720 for 0, 6, 16 and 24 hours, the apoptotic rates were tested. U266 cells were treated with DMSO and FTY720 separately and then were stained with DAPI for 5 min. Drop the cells to the slides and cover the slide with the glass. The cells were observed by fluorescence microscopy. U266 cells were treated with 5 µmol/L FTY720 or together with different doses of Z-VAD-fmk (12.5, 25, 50 µmol/L), a pancaspase inhibitor, for 24 hours, then the cell viability was tested by CCK-8. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expression of cleaved caspase-3 was tested by Western blot. U266 cells were treated with 0, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expressions of MCL-1, survivin, BCL-2, BID, BAX, BAK, P-ERK were tested by Western blot.
  
  
  RESULTS
  The apoptotic rate increased in U266 cells treated with FTY720 and showed the characteristic of time-dependent and dose-dependent manner. Karyopyknosis and nuclearfragmentation could be observed in U266 cells treated with FTY720 after being stained with DAPI under fluorescent microscope. The same effect was not observed in the cells treated with DMSO. Z-VAD-fmk could rescue the apoptosis in U266 cells treated with FTY720 in dose-dependent manner. The expression of MCL-1, survivin and BCL-2 decreased in U266 cells treated with FTY720. The cleavage of BID could be observed in U266 cells treated with FTY720. FTY720 had no effect on the expression of BAX, BAK and P-ERK.
  
  
  CONCLUSIONS
  FTY720 can induce the apoptosis in U266 cells, the apoptosis was Caspase-3-depended. The apoptosis induced by FTY720 is due to the decrease of MCL-1, survivin and BCL-2, which are the inhibitors of apoptosis. Meanwhile, the apoptosis was also due to the activation of BID, which is pro-apoptotic protein.
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China. E-mail: liaoaj@sj-hospital.org.
authors.forename: Ai-Jun
authors.initials: AJ
authors.lastname: Liao
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Shu-Chen
authors.initials: SC
authors.lastname: Li
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Hu
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Ying-Chun
authors.initials: YC
authors.lastname: Li
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Yao
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Hematology, Shenjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China.
authors.forename: Zhuo-Gang
authors.initials: ZG
authors.lastname: Liu
mesh_terms: D000590:Amino Acid Chloromethyl Ketones; D017209:Apoptosis; D053148:Caspase 3; D045744:Cell Line, Tumor; D002470:Cell Survival; D000068876:Fingolimod Hydrochloride; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D009101:Multiple Myeloma
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D051034:Inhibitor of Apoptosis Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505407:CASP3 protein, human; D053148:Caspase 3; D000068876:Fingolimod Hydrochloride