title: Stable transfection of extrinsic Smac gene enhances apoptosis-inducing effects of chemotherapeutic drugs on gastric cancer cells.
abstract: OBJECTIVE
  To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene.
  
  
  METHODS
  After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry.
  
  
  RESULTS
  When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12+/-0.21 vs 0.82+/-0.14, t = 7.52, P<0.01) and protein levels (4.02+/-0.24 vs 0.98+/-0.11, t = 8.32, P<0.01). After treatment with 10 microg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8+/-1.2-54.8+/-2.9%) was significantly higher than that of MKN-45 (5.8+/-0.4- 24.0+/-1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4+/-2.1%, which was significantly higher than that of MKN-45 (15.2+/-0.8%, t = 9.25, P<0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39+/-0.42 vs 0.96+/-0.14, t = 8.63, P<0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364+/-0.010 vs 0.112+/-0.007, t = 6.34, P<0.01).
  
  
  CONCLUSIONS
  Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.
authors.affiliation: Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
authors.forename: Li-Duan
authors.initials: LD
authors.lastname: Zheng
authors.forename: Qiang-Song
authors.initials: QS
authors.lastname: Tong
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Qian
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000235:genetics*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins / Q000235:genetics*; D016685:Mitomycin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013274:Stomach Neoplasms; D014162:Transfection
chemical_list: D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D016685:Mitomycin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases