Published online Jan 7, 2005. doi: 10.3748/wjg.v11.i1.79
Revised: March 8, 2004
Accepted: April 5, 2004
Published online: January 7, 2005
AIM: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene.
METHODS: After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry.
RESULTS: When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12±0.21 vs 0.82±0.14, t = 7.52, P<0.01) and protein levels (4.02±0.24 vs 0.98±0.11, t = 8.32, P<0.01). After treatment with 10 μg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8±1.2-54.8±2.9%) was significantly higher than that of MKN-45 (5.8±0.4- 24.0±1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4±2.1%, which was significantly higher than that of MKN-45 (15.2±0.8%, t = 9.25, P<0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39±0.42 vs 0.96±0.14, t = 8.63, P<0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364±0.010 vs 0.112±0.007, t = 6.34, P<0.01).
CONCLUSION: Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.