[Impact of fragile histidine triad gene transfection on the proliferation and apoptosis of human colorectal cancer cell].
OBJECTIVE
To investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression.


METHODS
The eukaryotic expression plasmid containing FHIT, pRc/CMV2-FHIT was prepared and purified, and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively.


RESULTS
At 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was (63.2±3.5)% and (50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant (P<0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group.


CONCLUSIONS
FHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.
Cao
Jie
J
Department of Gastrointestinal Surgery, Guangzhou First Municipal People's Hospital, Affiliated to Guangzhou Medical College, Guangzhou 510180, China. czhongt@126.com
Liang
Li-yuan
LY
Yang
Ping
P
Qian
Yue-jun
YJ
Wang
Hui
H
Sun
Zheng
Z
Li
Wang-lin
WL
Tan
Ming-hua
MH
D017766:Acid Anhydride Hydrolases / Q000235:genetics*; D017209:Apoptosis; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D053181:Caspase 8; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000099170:Bis(5'-adenosyl)triphosphatase; C505530:CASP8 protein, human