[Regulatory effects on apoptotic activities of gastric cancer cell line by over-expression of Smac gene].
OBJECTIVE
To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells.


METHODS
Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity.


RESULTS
The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01).


CONCLUSIONS
Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.
Zheng
Li-Duan
LD
Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Wu
Cui-Huan
CH
Tong
Qiang-Song
QS
Wang
Liang
L
Wu
Ren-Liang
RL
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics; D016685:Mitomycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D016685:Mitomycin; D053148:Caspase 3