Apoptosis-inducing effect of recombinant Caspase-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.
OBJECTIVE
To investigate the apoptosis-inducing effect of Caspases-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.


METHODS
PCR was employed to amplify the sequences of both small and large subunits of Caspases-3. Its products were separately cloned into the Sma I site of pBluescript KS(+) to generate both plasmids pBS/SS and pBS/LS. The small subunit fragment was excised from plasmid pBS/SS with BamH I and then inserted into the BamH I site of plasmid pBS/LS preceding that of the large subunit to yield plasmid pBS/Rev-Caspase-3. Rev-Caspase-3 cDNA was excised with Kpn I+Xba I and then subcloned into plasmid pcDNA3.1 (+) to construct Rev-Caspase-3 eukaryotic expression vector pcDNA/Rev-Caspase-3, which was used to transiently transfect SGC7901 cell line. Cell count, MTT assay and electron microscopy were used to confirm the antiproliferation and apoptosis-inducing effect of Rev-Caspase-3 expression on gastric cancer cells.


RESULTS
Plasmid pBS/Rev-Caspase-3 and eukaryotic expression vector pcDNA/Rev-Caspase-3 were successfully constructed. SGC7901 cells were transiently transfected by either pcDNA/Rev-Caspase-3 or pcDNA3.1 (+) for 24, 48, 72, and 96 h respectively. Cell growth was measured by cell count and MTT assay. In cell count assay, the cell numbers were 1.8 X 10(6), 1.55 X 10(6), 2.0 X 10(6), and 3.1 X 10(6) in the experimental group and 2.5 X 10(6), 3.1 X 10(6), 4.0 X 10(6), and 5.7 X 10(6) in the control group at 24, 48, 72 and 96 h respectively. The growth of SGC7901 cells was suppressed by Rev-Caspase-3 in a time-dependent manner (P<0.05). The results of MTT assay were similar to that of cell count (P<0.05). The characteristics of apoptosis such as chromatin condensation, crescent formation and margination were seen and more obvious with time in the given-experimental period in the experimental group, but not easily observed in the control group.


CONCLUSIONS
The expression of Rev-Caspase-3 by the constructed eukaryotic vector can significantly induce apoptosis of gastric cancer cell line SGC7901, which may exhibit a potential way in gastric cancer gene therapy.
Fu
Yuan-Gen
YG
Department of Biochemistry and Molecular Biology, Medical College, Shantou University, Guangdong Province, China.
Qu
Yao-Jun
YJ
Wu
Kai-Chun
KC
Zhai
Hui-Hong
HH
Liu
Zhi-Guo
ZG
Fan
Dai-Ming
DM
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000494:pharmacology*; D005057:Eukaryotic Cells; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000494:pharmacology; D013274:Stomach Neoplasms / Q000473:pathology / Q000503:physiopathology*; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases