Apoptosis-Inducing Effect of Caspase-3 Over-Expressed on Gastric Cancer Cell Line SGC7901

Abstract

To investigate the apoptosis-inducing effect of Caspases-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.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.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 1.8times106, 1.55times106, 2.0times106, and 3.1times106 in the experimental group and 2.5times106, 3.1times106, 4.0times106, and 5.7times106 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.The expression of Rev-Caspase-3 by the apoptosis of gastric cancer cell line SGC7901, which may exhibit a potential way in gastric cancer gene therapy.