[Effects of over-expressed Smac gene coupling with cisplatin on proliferation and apoptosis of hepatocarcinoma cells].
OBJECTIVE
To investigate the effects of over-expressed Smac gene combined with cisplatin (CDDP) on proliferation and apoptosis of hepatic carcinoma cells.


METHODS
The recombinant plasmid pcDNA3.1+-hSmac was introduced into the human hepatic carcinoma SMMC-7721 cells using a liposome-mediated method. The expression of Smac protein was detected by Western blot and flow cytometry. The cells were treated with three different doses of CDDP, 5, 15 and 25microg/ml, for 24 hours after the transfection. MTT colorimetry was used to detect the cellular growth-inhibitory effects; acridine orange-ethidium bromide fluorescent staining (AO/EB) and flow cytometry with annexin V-PI double staining


METHODS
were used to detect the changes of cell apoptosis.


RESULTS
Western blot and flow cytometry results demonstrated that the Smac protein level in SMMC-7721 cells was significantly increased after the transfection (P less than 0.01). Compared with that of the control group, the over-expressed Smac gene inhibited the cell growth and induced cell apoptosis (P less than 0.01). After being treated with CDDP, the inhibitory rates were increased significantly with increasing concentrations of CDDP compared with that of the control group, and the inhibitory rate of the CDDP-treated plus Smac group was significantly higher than that of the CDDP-treated group (P less than 0.01). The results detected by AO/EB and flow cytometry demonstrated that the apoptotic rates of CDDP-treated plus Smac group were higher than those of the CDDP-treated group (P less than 0.01). The results demonstrated that the Smac over-expression enhanced the effects of cell growth inhibition and apoptotic promotion induced by CDDP.


CONCLUSIONS
The pro-apoptotic Smac gene could be over-expressed in hepatocarcinoma SMMC-7721 cells and inhibit cell growth and induce apoptosis. Moreover the over-expressed Smac could enhance the chemotherapeutic sensitivity of SMMC-7721 to cisplatin. This experimental work may help in further study on the regulatory mechanism of Smac in apoptosis and improve the chemotherapeutic effect on hepatoma.
Guo
Cai-xia
CX
Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, China.
Li
Yan-bo
YB
DU
Hai-ying
HY
Liu
Ying
Y
Sun
Lei
L
Jin
Ming-hua
MH
Sun
Zhi-wei
ZW
D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology*; D024101:Mitochondrial Proteins / Q000235:genetics*; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D002945:Cisplatin