title: [Apoptosis of human lung carcinoma cell line EBC-1 induced by N, N'-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide and its molecular mechanism].
abstract: OBJECTIVE
  To study whether N, N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) inhibits proliferation and induces apoptosis in human lung carcinoma cell line EBC-1 cells and its molecular mechanism.
  
  
  METHODS
  Different concentrations of ZGDHu-1 and different times of culture were used to treat EBC-1 cells in vitro. The inhibition of proliferation was measured by BrdU-ELISA. Cell apoptosis was detected by Annexin V/PI staining and cellular DNA fragmentation ELISA. Phosphorylated p38MAPK and STAT3 were examined by flow cytometry. The protein expressions of bcl-2, bax, p53, Fas, and caspase-3 were detected by Western blot analysis.
  
  
  RESULTS
  ZGDHu-1 inhibited EBC-1 cell proliferation within a certain range of treating times and does, with a 24 h IC(50) of (295 ± 25) ng/ml, 48 h of (112 ± 8) ng/ml and 72 h of (23 ± 2) ng/ml. The EBC-1 cell apoptosis was confirmed by Annexin V/PI labeling and cellular DNA fragmentation ELISA in a dose-related manner. When EBC-1 cells were treated with 50, 200, and 500 ng/ml ZGDHu-1 for 48 h, the expression rates of phosphor-p38MAPK protein were 67.4%, 88.2%, 91.1%, respectively, and that of the control was 10.6%. That of STAT3 protein were 56.5%, 43.6% and 34.6%, respectively, and that of the control was 89.1%. The expression of bax, p53 and Fas protein was significantly increased, that of bcl-2 was not changed, and that of caspase-3 was significantly decreased by the ZGDHu-1 treatment.
  
  
  CONCLUSIONS
  ZGDHu-1 can inhibit proliferation and induce apoptosis in EBC-1 cells. The mitochondrial pathway mediated by Fas may be one of its mechanisms. The apoptosis of EBC-1 cells may associate with up-regulation of phosphor-p38MAPK and down-regulation of phosphor-STAT3 in the cells.
authors.affiliation: Central Laboratory, Zhejiang Provincial People's Hospital, Hangzhou 310014, China. lab_zyl@126.com
authors.forename: Yong-Lie
authors.initials: YL
authors.lastname: Zhou
authors.forename: Wu-Lin
authors.initials: WL
authors.lastname: Xu
authors.forename: Zhen-Ni
authors.initials: ZN
authors.lastname: Wang
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Lü
authors.forename: Wei-Xiao
authors.initials: WX
authors.lastname: Hu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation; D006573:Heterocyclic Compounds, 1-Ring / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: C491901:FAS protein, human; D006573:Heterocyclic Compounds, 1-Ring; C522668:N,N-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxamide; D019253:Proto-Oncogene Proteins c-bcl-2; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D048051:p38 Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3