Aim: To investigate the altered expression of apoptosis pathway genes of prostate cancer cells treated by radiation and androgen withdrawal and whether the combined treatment may induce additive apoptosis. Methods: An...Aim: To investigate the altered expression of apoptosis pathway genes of prostate cancer cells treated by radiation and androgen withdrawal and whether the combined treatment may induce additive apoptosis. Methods: Androgen sensitive prostate cancer cell line LNCaP was cultured and treated by radiation, androgen withdrawal and combination of the two. Apoptosis was determined using apoptotic cells staining and mononuclear cell direct cytotox-icity assay. The total RNA were extracted and harvested. cDNA probes were prepared and labeled with biotin-16-dUTP and then hybridized to commercially available cDNA arrays, including apoptosis pathway-specific genes. The expression of important gene was further determined using RT-PCR. Results: Radiation induced additive apoptosis of prostate cancer cells; androgen withdrawal exhibited synergetic action. TNFRSF8 variant 2, DFFA, LTbR, mdm2, Myd88, TNFRSF14 and TNFSF4 mRNA were up regulated by radiation, while Survivin and Bar mRNA were down regulated. Mcl-1, TNFRSF14, MyD88 and TNFSF4 mRNA were up regulated by androgen withdrawal, while Bar, Survivin and TRAIL-R3 mRNA were down regulated. Conclusion: Radiation and androgen withdrawal altered the expression of apoptosis pathway genes of prostate cancer cells in different patterns, which may contribute to the additive apoptotic effect induced by the combined treatment.展开更多
To investigate the effects of down-regulation of prostate androgen regulated （PAR） expression on proliferation of PC3 cells by using RNA interference （RNAi）, suppression of PAR expression was achieved by transfect...To investigate the effects of down-regulation of prostate androgen regulated （PAR） expression on proliferation of PC3 cells by using RNA interference （RNAi）, suppression of PAR expression was achieved by transfection of PC3 cells with short hairpin RNA （shRNA） expression vectors against PAR, designated as psiRNA-PAR1, psiRNA-PAR2 and psiRNA-PAR3. The inhibitory effects were confirmed by RT-PCR. The growth features of PC3 transfectants were analyzed by cell counts, colon formation in soft agar and flow cytometry. The expression of PAR was suppressed by the three shRNA expression vectors, psiRNA-PAR1 was shown to inhibit the PAR expression most efficiently, with the inhibitory rate reaching a peak at （81.18±1.68）% 48 h after the transfection. PC3 transfectants exhibited a decreased proliferation in cell culture and a low efficiency of colon formation in soft agar. Flow cytometry revealed a G2/M arrest and induced apoptosis. Down-regulated PAR expression inhibited the growth of PC3 cells by inducing G2/M arrest and activating apoptotic pathway. As a potential proto-oncogene that triggers and/or has persistent malignant proliferation, PAR may serves as a very target for the gene therapy.展开更多
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
Objective： To investigated the antitumor effects of PUMA gene transfection on pancreatic cancer Aspc-1 cells. Methods： Plasmid pGFP-PUMA-C1 and pGFP-C1 was introduced into the pancreatic cancer ASPC-1 cells by Lipof...Objective： To investigated the antitumor effects of PUMA gene transfection on pancreatic cancer Aspc-1 cells. Methods： Plasmid pGFP-PUMA-C1 and pGFP-C1 was introduced into the pancreatic cancer ASPC-1 cells by LipofectinamineTm 2000 transfection. 24 h and 48 h after transfection, these cells were collected, PUMA protein and PUMA mRNA expression in ASPC-1 cells were detected by Western blot and semiquantitative reverse transcription polymerase chain reaction （RT-PCR） methods, respectively. Cell apoptosis was examined by flow cytometry and terminal deoxynucleotidyl transferase（TdT）-mediated dUTP nick end labeling （TUNEL）. Growth inhibition of Aspc-1 cells was determined by the colorimetric MTT cell Viability/proliferation assay. Results： Transfection of pGFP-PUMA-C 1 into Aspc-1 cells resulted in the upregulation of the corresponding mRNA and PUMA protein, which was associated with a reduced number of viable cells and increased number of apoptosis cells, but the mRNA and PUMA protein and the corresponding viable cells and apoptosis cells had no significant differences in Aspc-1 cells/pGFP-C1 compared to control cells. Conclusion： Re-expression of PUMA gene, which is lost in human pancreatic cancer cells, can induce apoptosis, resulting in inhibition of tumor growth.展开更多
文摘Aim: To investigate the altered expression of apoptosis pathway genes of prostate cancer cells treated by radiation and androgen withdrawal and whether the combined treatment may induce additive apoptosis. Methods: Androgen sensitive prostate cancer cell line LNCaP was cultured and treated by radiation, androgen withdrawal and combination of the two. Apoptosis was determined using apoptotic cells staining and mononuclear cell direct cytotox-icity assay. The total RNA were extracted and harvested. cDNA probes were prepared and labeled with biotin-16-dUTP and then hybridized to commercially available cDNA arrays, including apoptosis pathway-specific genes. The expression of important gene was further determined using RT-PCR. Results: Radiation induced additive apoptosis of prostate cancer cells; androgen withdrawal exhibited synergetic action. TNFRSF8 variant 2, DFFA, LTbR, mdm2, Myd88, TNFRSF14 and TNFSF4 mRNA were up regulated by radiation, while Survivin and Bar mRNA were down regulated. Mcl-1, TNFRSF14, MyD88 and TNFSF4 mRNA were up regulated by androgen withdrawal, while Bar, Survivin and TRAIL-R3 mRNA were down regulated. Conclusion: Radiation and androgen withdrawal altered the expression of apoptosis pathway genes of prostate cancer cells in different patterns, which may contribute to the additive apoptotic effect induced by the combined treatment.
文摘To investigate the effects of down-regulation of prostate androgen regulated （PAR） expression on proliferation of PC3 cells by using RNA interference （RNAi）, suppression of PAR expression was achieved by transfection of PC3 cells with short hairpin RNA （shRNA） expression vectors against PAR, designated as psiRNA-PAR1, psiRNA-PAR2 and psiRNA-PAR3. The inhibitory effects were confirmed by RT-PCR. The growth features of PC3 transfectants were analyzed by cell counts, colon formation in soft agar and flow cytometry. The expression of PAR was suppressed by the three shRNA expression vectors, psiRNA-PAR1 was shown to inhibit the PAR expression most efficiently, with the inhibitory rate reaching a peak at （81.18±1.68）% 48 h after the transfection. PC3 transfectants exhibited a decreased proliferation in cell culture and a low efficiency of colon formation in soft agar. Flow cytometry revealed a G2/M arrest and induced apoptosis. Down-regulated PAR expression inhibited the growth of PC3 cells by inducing G2/M arrest and activating apoptotic pathway. As a potential proto-oncogene that triggers and/or has persistent malignant proliferation, PAR may serves as a very target for the gene therapy.
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.
文摘Objective： To investigated the antitumor effects of PUMA gene transfection on pancreatic cancer Aspc-1 cells. Methods： Plasmid pGFP-PUMA-C1 and pGFP-C1 was introduced into the pancreatic cancer ASPC-1 cells by LipofectinamineTm 2000 transfection. 24 h and 48 h after transfection, these cells were collected, PUMA protein and PUMA mRNA expression in ASPC-1 cells were detected by Western blot and semiquantitative reverse transcription polymerase chain reaction （RT-PCR） methods, respectively. Cell apoptosis was examined by flow cytometry and terminal deoxynucleotidyl transferase（TdT）-mediated dUTP nick end labeling （TUNEL）. Growth inhibition of Aspc-1 cells was determined by the colorimetric MTT cell Viability/proliferation assay. Results： Transfection of pGFP-PUMA-C 1 into Aspc-1 cells resulted in the upregulation of the corresponding mRNA and PUMA protein, which was associated with a reduced number of viable cells and increased number of apoptosis cells, but the mRNA and PUMA protein and the corresponding viable cells and apoptosis cells had no significant differences in Aspc-1 cells/pGFP-C1 compared to control cells. Conclusion： Re-expression of PUMA gene, which is lost in human pancreatic cancer cells, can induce apoptosis, resulting in inhibition of tumor growth.
