Apoptosis-inducing effect of recombinant Caspase-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901

Abstract

Aim: 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.

Conclusion: 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.

Figure 1

Schematic map of constructions of recombinant Caspases-3 and eukaryotic expression plasmid pcDNA/Rev-Caspases-3.

Figure 2

Identification of plasmid pBS/Rev-Caspase-3 by PCR with SS-forward and LS-reverse as primers. M: 100 bp ladder DNA marker, 1: PCR product, 882 bp.

Figure 3

Enzyme digestion analysis of plasmid pcDNA/Rev-Caspase-3. M: 100 bp ladder DNA marker, 1: pcDNA/Rev-Caspase-3 cut with Stu I, 2: pcDNA/Rev-Caspase-3 cut with

Figure 4

Comparison of cell count between SGC7901 cells trans-fected with pcDNA3.1 (+) and pcDNA/Rev. Caspase.

Figure 5

Comparison of MTT assays between SGC7901 cells transfected with pcDNA3.1 (+) and pcDNA/Rev.Caspase-3.

Figure 6

Electron microscopy of SGC7901 cells transfected with pcDNA3.1 (+) or pcDNA/Rev.Caspases-3. A: SGC7901 trans-fected with pcDNA/Rev. Caspases-3. Notice the chromatin condensation, crescent formation, original margination (× 5000); B: SGC7901 transfected with pcDNA3.1 (+), Notice no characteristics of apoptosis (× 5000).