Impact of fragile histidine triad gene transfection on the proliferation and apoptosis of human colorectal cancer cell.
- Author:
Jie CAO
1
;
Li-yuan LIANG
;
Ping YANG
;
Yue-jun QIAN
;
Hui WANG
;
Zheng SUN
;
Wang-lin LI
;
Ming-hua TAN
Author Information
- Publication Type:Journal Article
- MeSH: Acid Anhydride Hydrolases; genetics; Apoptosis; Caspase 8; metabolism; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; genetics; pathology; Humans; Neoplasm Proteins; genetics; RNA, Messenger; genetics; Transfection
- From: Chinese Journal of Gastrointestinal Surgery 2010;13(9):691-694
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression.
METHODSThe 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.
RESULTSAt 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.
CONCLUSIONSFHIT 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.