title: 5-Aza-2'-deoxycytidine enhances maspin expression and inhibits proliferation, migration, and invasion of the bladder cancer T24 cell line.
abstract: BACKGROUND
  Downregulation of maspin expression has been linked to bladder cancer development, and that DNA methylation may be important for regulating maspin gene activation in bladder cancer cells. Thus, we attempted to explore the effects of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), on the maspin expression and the biological behaviors in bladder cancer T24 cells.
  
  
  METHODS
  The methylation status of maspin in T24 cells was investigated by methylation-specific polymerase chain reaction (PCR). After treated with different concentrations of 5-Aza-CdR (0, 0.25, 0.5, 1, and 2 μM), the maspin gene mRNA expression and protein expression were examined by real-time PCR and western blotting analysis. Cell proliferations were evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Flow cytometry was used to identify the apoptosis rates. Migration and invasive ability were determined by the transwell assay. Using the western blotting analysis, the changes of Cyclin D1, VEGF-C, VEGFR-3, MMP-2, MMP-9, caspase-3 p17, Bax, and Bcl-2 expression were measured.
  
  
  RESULTS
  Promoter DNA methylation of maspin was observed in T24 cells. The expression levels of maspin mRNA and protein in T24 cells were increased in a dose manner after treatment with increasing 5-Aza-CdR (p<0.05). The proliferation, migration, and invasion of cells were significantly inhibited with increasing 5-Aza-CdR, whereas the apoptosis was greatly increased (p<0.05). These were associated with the decreased ratio of Bcl-2/Bax, activation of caspase-3, and decreased expression of Cyclin D1, VEGF-C, VEGFR-3, MMP-2 and MMP-9.
  
  
  CONCLUSIONS
  The present study demonstrates that maspin is silenced by DNA methylation in bladder T24 cells, and its expression can be reactivated by treatment with 5-Aza-CdR. 5-Aza-CdR could result in obvious inhibitions of the proliferation, migration, and invasion of T24 cells, which may serve as a potential strategy for the treatment of bladder cancer.
authors.affiliation: 1 Department of Urology, The First Affiliated Hospital of University of South China , Hengyang, Hunan Province, P.R. China .
authors.forename: Huihui
authors.initials: H
authors.lastname: Zhang
authors.forename: Fan
authors.initials: F
authors.lastname: Qi
authors.forename: Youhan
authors.initials: Y
authors.lastname: Cao
authors.forename: Xiongbing
authors.initials: X
authors.lastname: Zu
authors.forename: Minfeng
authors.initials: M
authors.lastname: Chen
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Qi
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; C085344:SERPIN-B5; D015843:Serpins; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine
keywords: 5-Aza-2′-deoxycytidine; T24; bladder cancer; maspin