AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer ...AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.展开更多
目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),...目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib+300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.展开更多
Objective To evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cell...Objective To evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cells in vitro and in vivo. Methods Cells were transfected with Ad-p53 (100 MOI), and PCNA-ASO (1.6 μmol/L) was then introduced into the cells using a cationic lipid (lipofectamine, 20 μl/ml). In vitro and in vivo antitumor effects of combining PCNA-ASO with Ad-p53 were measured using the MTT assay, flow cytometry, clone formation, and a nude mice model. Results The combination of PCNA-ASO and Ad-p53 inhibited cell viability in both the EJ (89.3%) and BIU-87 (78.6%) cell lines. The ability of the cells to form foci was also reduced by 74.8% in EJ cells and by 67.5% in BIU-87 cells (P<0.01). A significant decrease of cells in the S phase (11.4% in EJ cells, 14.6% in BIU-87 cells) and a significant increase of cells in G1 phase (62.2% in EJ, 56.8% in BIU-87) were noted. The mean tumor volume after 7 days of treatment with PCNA-ASO or Ad-p53 in combination decreased to 47.6% or 36.4% of the initial tumor size in the two cell lines respectively. Conclusion These results indicate that combined PCNA-ASO and Ad-p53 in the treatment of bladder cancer with mutant p53 has important therapeutic potential, significantly suppressing the growth of human bladder cancer both in vitro and in vivo.展开更多
文摘AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.
文摘目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib+300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.
文摘Objective To evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cells in vitro and in vivo. Methods Cells were transfected with Ad-p53 (100 MOI), and PCNA-ASO (1.6 μmol/L) was then introduced into the cells using a cationic lipid (lipofectamine, 20 μl/ml). In vitro and in vivo antitumor effects of combining PCNA-ASO with Ad-p53 were measured using the MTT assay, flow cytometry, clone formation, and a nude mice model. Results The combination of PCNA-ASO and Ad-p53 inhibited cell viability in both the EJ (89.3%) and BIU-87 (78.6%) cell lines. The ability of the cells to form foci was also reduced by 74.8% in EJ cells and by 67.5% in BIU-87 cells (P<0.01). A significant decrease of cells in the S phase (11.4% in EJ cells, 14.6% in BIU-87 cells) and a significant increase of cells in G1 phase (62.2% in EJ, 56.8% in BIU-87) were noted. The mean tumor volume after 7 days of treatment with PCNA-ASO or Ad-p53 in combination decreased to 47.6% or 36.4% of the initial tumor size in the two cell lines respectively. Conclusion These results indicate that combined PCNA-ASO and Ad-p53 in the treatment of bladder cancer with mutant p53 has important therapeutic potential, significantly suppressing the growth of human bladder cancer both in vitro and in vivo.
