Li-Duan Zheng1, Qiang-Song Tong, Liang Wang, Jun Liu, Wei Qian. 1. Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
Abstract
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 microg/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.
AIM: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on humangastric 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 microg/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/Smaccancer 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.
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