AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.展开更多
AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the im...AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.展开更多
The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect...The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P展开更多
AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by i...AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.展开更多
目的:探讨外源性p53AIP1(p53-regulated apoptosis-inducing protein 1)基因抗肿瘤效应及其作用机制,以评估p53AIP1基因在肿瘤基因治疗中应用的可行性。方法:构建携带p53AIP1基因的重组复制缺陷型腺病毒Ad-p53AIP1,感染人肝癌细胞HepG2...目的:探讨外源性p53AIP1(p53-regulated apoptosis-inducing protein 1)基因抗肿瘤效应及其作用机制,以评估p53AIP1基因在肿瘤基因治疗中应用的可行性。方法:构建携带p53AIP1基因的重组复制缺陷型腺病毒Ad-p53AIP1,感染人肝癌细胞HepG2,应用MTT比色法、Western blotting、流式细胞术、罗丹明染色以及电镜等观察外源性p53AIP1基因表达对肿瘤细胞的作用及其相关机制。小鼠皮下接种感染Ad-p53AIP1的小鼠乳腺癌4T1细胞,观察Ad-p53AIP1对肿瘤细胞体内成瘤性的影响;建立4T1细胞皮下移植瘤小鼠模型,直接瘤内多点注射重组腺病毒,观察Ad-p53AIP1的抗肿瘤疗效。结果:感染Ad-p53AIP1的HepG2细胞能够高效表达P53AIP1蛋白。MTT检测显示Ad-p53AIP1对人肝癌细胞HepG2的生长抑制率达50%以上;流式细胞术分析证实p53AIP1使肿瘤细胞周期阻滞于G_2/M期;罗丹明染色、电镜观察以及凋亡相关蛋白PARP表达的检测均证实p53AIP1能诱导肿瘤细胞发生明显凋亡。感染Ad-p53AIP1的肿瘤细胞体内成瘤受到非常明显的抑制(P<0.01),Ad-p53AIP1瘤体注射对移植瘤生长也有明显的抑制作用(P<0.05)。Western blotting以及RT-PCR检测证实Ad-p53AIP1对p53 mRNA表达无影响,能下调mdm2基因的表达;p53AIP1能上调P53蛋白的表达、下调MDM2蛋白的表达水平,同时还影响P21等细胞周期相关蛋白和凋亡相关蛋白的表达。结论:p53AIP1有明显的体内外抗肿瘤作用,其作用机制与其反向调控P53蛋白、调控多种细胞周期和细胞凋亡相关蛋白、诱导细胞周期阻滞和细胞凋亡有关,该基因在肿瘤基因治疗中具有潜在的应用前景。展开更多
OBJECTIVE: To investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. METHODS: HRz was designed by computer programs. HRz's activity was identified b...OBJECTIVE: To investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. METHODS: HRz was designed by computer programs. HRz's activity was identified by cleavage experiments in vitro. HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot. Cell growth curves and soft agar forming ability were studied. The ability of each cell line to form tumors was assessed in nude mice. Apoptosis rates and expression of c-myc, bcl-2, p53 and Fas were detected by flow cytometry (FCM). Antigens of tumor cells, HLA-1, HLA-2, B7-1 and B7-2 were also detected. NK, LAK, and CD(3)AK cells were induced. Their cytotoxicities were detected in CaSKi-R, CaSKi-P, and CaSKi cells. RESULTS: In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site-specific manner. HRz could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA levels were lower in CaSKi-R than in CaSKi. The growth rate of CaSKi-R was slower than those of CaSKi and CaSKi-P. The soft agar-forming rate of CaSKi-R was lower compared with those of CaSKi and CaSKi-P cells. The ability of CaSKi-R to form tumors in nude mice was also poor. The apoptosis rate of CaSKi-R cells was much higher than those of CaSKi and CaSKi-P. HRz could reduce the expression of E6, c-myc and bcl-2 proteins, and increase the expression of p53 as well. HRz could increase the expression of HLA-2, B7-1 and B7-2 antigens. The cytotoxicity of NK, LAK and CD(3)AK cells was much higher in CaSKi-R than in CaSKi-P and CaSKi cells. CONCLUSION: HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.展开更多
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.
基金Supported by a faculty research grant of Yonsei University College of Medicine for 2002,No.2002-06
文摘AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.
文摘The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P
基金Supported by the National Postdoctoral Science Foundation of China,No.199711.
文摘AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.
文摘OBJECTIVE: To investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. METHODS: HRz was designed by computer programs. HRz's activity was identified by cleavage experiments in vitro. HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot. Cell growth curves and soft agar forming ability were studied. The ability of each cell line to form tumors was assessed in nude mice. Apoptosis rates and expression of c-myc, bcl-2, p53 and Fas were detected by flow cytometry (FCM). Antigens of tumor cells, HLA-1, HLA-2, B7-1 and B7-2 were also detected. NK, LAK, and CD(3)AK cells were induced. Their cytotoxicities were detected in CaSKi-R, CaSKi-P, and CaSKi cells. RESULTS: In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site-specific manner. HRz could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA levels were lower in CaSKi-R than in CaSKi. The growth rate of CaSKi-R was slower than those of CaSKi and CaSKi-P. The soft agar-forming rate of CaSKi-R was lower compared with those of CaSKi and CaSKi-P cells. The ability of CaSKi-R to form tumors in nude mice was also poor. The apoptosis rate of CaSKi-R cells was much higher than those of CaSKi and CaSKi-P. HRz could reduce the expression of E6, c-myc and bcl-2 proteins, and increase the expression of p53 as well. HRz could increase the expression of HLA-2, B7-1 and B7-2 antigens. The cytotoxicity of NK, LAK and CD(3)AK cells was much higher in CaSKi-R than in CaSKi-P and CaSKi cells. CONCLUSION: HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.
