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 i...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 G_(418) 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-FTTC 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 vs0.98±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 vs0.96±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 vs0.112±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.展开更多
To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme fo...To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.展开更多
The second mitochondria-derived activator of caspases (Smac) is a novelproapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation ontumor cells. The purpose of this study was to ...The second mitochondria-derived activator of caspases (Smac) is a novelproapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation ontumor cells. The purpose of this study was to investigate the effects of extrinsic Smac genetransfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. Afterthe Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtainedby persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Westernblot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatmentwith X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazoliumbromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy,annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities ofcellular caspase-3 were assayed by Western blot and colorimetry. Smac mRNA and protein levels inHeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higherthan those of HeLa (P <0. 01). There was no significant difference in cellular viabilities betweenthem (P > 0. 05 ) . However, after irradiation with 8 Gy X-ray, growth activities of HeLa/ Smac werereduced by 22.42% (P < 0. 01). When compared with those of HeLa, partial HeLa/Smac cells presentedcharacteristic morphological changes of apoptosis under electronic microscope, with higher apoptosisrates (16. 4% vs. 6. 2% , P < 0. 01 ) ; the caspase-3 expression levels in HeLa/Smac cells wereimproved significantly (P <0. 01) , while its activities were increased by 3. 42 times (P <0. 01).Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell linecould significantly enhance the expression and activities of cellular caspase-3 and ameliorateapoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improveradiotherapeutic effects on cervical cancer.展开更多
目的:探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法:取构建好的耐药A549细胞,将其分为A549细胞(LC)组、A549细胞+Smac-NC(SN)组、A549细胞+Smac抑制剂(SI)组、A549细胞+Smac激动...目的:探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法:取构建好的耐药A549细胞,将其分为A549细胞(LC)组、A549细胞+Smac-NC(SN)组、A549细胞+Smac抑制剂(SI)组、A549细胞+Smac激动剂(SM)组、A549细胞+Caspase-3-NC(CN)组、A549细胞+Caspase-3抑制剂(CI)组、A549细胞+Caspase-3激动剂(CM)组、A549细胞+Smac激动剂+Caspase-3激动剂(MM)组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果:肺腺癌细胞系中的Smac、Caspase-3 m RNA表达量显著低于正常肺上皮细胞系BEAS-2B (P<0.05),其中A549的Smac、Caspase-3 m RNA值最小(P<0.05),因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);Smac与Caspase-3呈现正相关(r=0.470,P=0.002),组间具有显著差异。结论:Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。展开更多
BACKGROUND:T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI(Smac/DIABLO)was recently identified as a protein that is released from mitochondria in respons...BACKGROUND:T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI(Smac/DIABLO)was recently identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins.Furthermore,Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis.However,little is known about the clinical significance of Smac/DIABLO in various cancers including hepatocellular carcinoma(HCC).This study was undertaken to investigate the expression of Smac and Survivin and their relationship with the apoptosis in primary HCC.METHODS:The expression of Smac and Survivin proteins was evaluated by immunohistochemistry.The mRNA expression of Smac and Survivin was evaluated by reverse transcription-polymerase chain reaction(RT-PCR)in HCC tissues of 50 patients,para-carcinoma tissues of 20 patients,and normal liver tissues of 15 patients.RESULTS:Smac mRNA was detected by RT-PCR in HCC tissues of 21(42.0%)of the 50 patients,para-carcinoma tissues of 19(95.0%)of the 20 patients,and normal liver tissues of 15(100%)of the 15 patients.Survivin mRNA was found in HCC tissues of 46 of the 50 patients,para-carcinoma tissues of 2 of the 20 patients,and normal liver tissues of 0 of 15 patients.Immunohistochemistry revealed Smac protein in HCC tissues of 20 patients(40.0%),in para-carcinoma tissues of 18 patients(90.0%),and normal liver tissues of 15 patients(100.0%).The expression of Smac was significantly different in HCC tissues and non-HCC tissues.Survivin protein was found in HCC tissuesin 45 patients,para-carcinoma tissues in 2 patients,and normal liver tissues in none of the patients.The expression of Survivin was significantly different in HCC tissues and non-HCC tissues.CONCLUSION:Smac inhibits apoptosis of HCC cells by suppression of Survivin,and the two genes probably form an important link in the signal pathway of HCC cells.展开更多
文摘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 G_(418) 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-FTTC 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 vs0.98±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 vs0.96±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 vs0.112±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.
基金This work was supported by National Natural Science Foundation of China(30271301)
文摘To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.
文摘The second mitochondria-derived activator of caspases (Smac) is a novelproapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation ontumor cells. The purpose of this study was to investigate the effects of extrinsic Smac genetransfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. Afterthe Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtainedby persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Westernblot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatmentwith X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazoliumbromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy,annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities ofcellular caspase-3 were assayed by Western blot and colorimetry. Smac mRNA and protein levels inHeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higherthan those of HeLa (P <0. 01). There was no significant difference in cellular viabilities betweenthem (P > 0. 05 ) . However, after irradiation with 8 Gy X-ray, growth activities of HeLa/ Smac werereduced by 22.42% (P < 0. 01). When compared with those of HeLa, partial HeLa/Smac cells presentedcharacteristic morphological changes of apoptosis under electronic microscope, with higher apoptosisrates (16. 4% vs. 6. 2% , P < 0. 01 ) ; the caspase-3 expression levels in HeLa/Smac cells wereimproved significantly (P <0. 01) , while its activities were increased by 3. 42 times (P <0. 01).Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell linecould significantly enhance the expression and activities of cellular caspase-3 and ameliorateapoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improveradiotherapeutic effects on cervical cancer.
文摘目的:探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法:取构建好的耐药A549细胞,将其分为A549细胞(LC)组、A549细胞+Smac-NC(SN)组、A549细胞+Smac抑制剂(SI)组、A549细胞+Smac激动剂(SM)组、A549细胞+Caspase-3-NC(CN)组、A549细胞+Caspase-3抑制剂(CI)组、A549细胞+Caspase-3激动剂(CM)组、A549细胞+Smac激动剂+Caspase-3激动剂(MM)组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果:肺腺癌细胞系中的Smac、Caspase-3 m RNA表达量显著低于正常肺上皮细胞系BEAS-2B (P<0.05),其中A549的Smac、Caspase-3 m RNA值最小(P<0.05),因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);Smac与Caspase-3呈现正相关(r=0.470,P=0.002),组间具有显著差异。结论:Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。
文摘BACKGROUND:T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI(Smac/DIABLO)was recently identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins.Furthermore,Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis.However,little is known about the clinical significance of Smac/DIABLO in various cancers including hepatocellular carcinoma(HCC).This study was undertaken to investigate the expression of Smac and Survivin and their relationship with the apoptosis in primary HCC.METHODS:The expression of Smac and Survivin proteins was evaluated by immunohistochemistry.The mRNA expression of Smac and Survivin was evaluated by reverse transcription-polymerase chain reaction(RT-PCR)in HCC tissues of 50 patients,para-carcinoma tissues of 20 patients,and normal liver tissues of 15 patients.RESULTS:Smac mRNA was detected by RT-PCR in HCC tissues of 21(42.0%)of the 50 patients,para-carcinoma tissues of 19(95.0%)of the 20 patients,and normal liver tissues of 15(100%)of the 15 patients.Survivin mRNA was found in HCC tissues of 46 of the 50 patients,para-carcinoma tissues of 2 of the 20 patients,and normal liver tissues of 0 of 15 patients.Immunohistochemistry revealed Smac protein in HCC tissues of 20 patients(40.0%),in para-carcinoma tissues of 18 patients(90.0%),and normal liver tissues of 15 patients(100.0%).The expression of Smac was significantly different in HCC tissues and non-HCC tissues.Survivin protein was found in HCC tissuesin 45 patients,para-carcinoma tissues in 2 patients,and normal liver tissues in none of the patients.The expression of Survivin was significantly different in HCC tissues and non-HCC tissues.CONCLUSION:Smac inhibits apoptosis of HCC cells by suppression of Survivin,and the two genes probably form an important link in the signal pathway of HCC cells.
