Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-in...Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon(IFN)-γ- endostatin and studied the anti-tumor effect of pEgr-IFN-γ-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.Methods Gene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-γ-endostatin, and single-gene expression plasmid pEgr-IFN-γ and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-α secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.Results The tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-α secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-γgene-radiotherapy group. Conclusion The anti-tumor effect of dual-gene-radiotherapy was significantly better than that of single-gene-radiotherapy by combining the enhancement of anti-tumor immunologic function induced by IFN-γ with the anti-angiogenesis function of endostatin.展开更多
Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice However, its widespread application has been hampered by difficulties in a large scale productio...Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice However, its widespread application has been hampered by difficulties in a large scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration These limitations could be resolved by in vivo delivery and expression of the endostatin gene In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL 7402 xenografted tumors, comparison with recombinant endostatin protein Methods Hepatocellular carcinoma BEL 7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra tumoral injections of Ad/hEndo of 5×10 8 pfu (low dose group) and 1×10 9 pfu (high dose group) at intervals of six days, respectively Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg·kg 1 ·d 1 at a site nearby the tumor for ten days The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription polymerase chain reaction (RT PCR) after Ad/hEndo injection Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme linked immunosorbent assay (ELISA) Results After 4 courses of treatment, the tumor growth rates of high dose treated group with 1×10 9 pfu of Ad/hEndo were inhibited by 42 26% compared with the Ad/ LacZ control group ( P =0 001) and by 46 26% compared with the NIH buffer control group ( P =0 003), respectively However, in this study, Ad/hEndo at low dose of 5×10 8 pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47% However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50% The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1×10 9 pfu and gradually dropped undetectable by day 7 Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later Conclusions Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL 7402 xenografted tumors at a high dose of 1×10 9 pfu compared with other groups The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high level, relatively long term expression in vivo and bioactivity capability展开更多
Objective To investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization. Methods pBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Ⅰand Sal Ⅰ, by PCR re...Objective To investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization. Methods pBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Ⅰand Sal Ⅰ, by PCR reaction, by sequence, and then by alignment of PCR products with the geneBank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO 3 and 25% KNO 3 cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs. Results pBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.Conclusions The plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.展开更多
This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellu-lar growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and en...This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellu-lar growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egr1-shTRAIL-shES and X-ray irradiation. Then MTT assay was used for determining the cellular proliferation, and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression. The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshut-tle-Egr1-shTRAIL-shES transfection in conjunction with irradiation. In the TRAIL-endostatin-based single- or double-gene-radiotherapy, the cell viability declined in a time- and dose-dependent manner, the percentage of cells at G2/M phase and apoptotic rate was increased, and the percentage of cells at G0/G1 phase was lowered as compared with those receiving radiotherapy alone. Moreover, TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition, promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than single-gene-radiotherapy.展开更多
Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line,LoVo.Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and p...Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line,LoVo.Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2×10 5 CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.Results After transfection and superinfection,amphotropic retrovirus was collected,and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited endothelial cell growth up to 67% ( P <0.001),compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group,accompanied by a reduction in vessels,compared with the control group ( P <0.01). Conclusion Retroviruses can allow functional expression of the endostatin gene in human colon tumors,showing promise for an antitumor strategy using antiangiogenesis.展开更多
文摘Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon(IFN)-γ- endostatin and studied the anti-tumor effect of pEgr-IFN-γ-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.Methods Gene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-γ-endostatin, and single-gene expression plasmid pEgr-IFN-γ and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-α secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.Results The tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-α secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-γgene-radiotherapy group. Conclusion The anti-tumor effect of dual-gene-radiotherapy was significantly better than that of single-gene-radiotherapy by combining the enhancement of anti-tumor immunologic function induced by IFN-γ with the anti-angiogenesis function of endostatin.
文摘Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice However, its widespread application has been hampered by difficulties in a large scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration These limitations could be resolved by in vivo delivery and expression of the endostatin gene In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL 7402 xenografted tumors, comparison with recombinant endostatin protein Methods Hepatocellular carcinoma BEL 7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra tumoral injections of Ad/hEndo of 5×10 8 pfu (low dose group) and 1×10 9 pfu (high dose group) at intervals of six days, respectively Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg·kg 1 ·d 1 at a site nearby the tumor for ten days The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription polymerase chain reaction (RT PCR) after Ad/hEndo injection Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme linked immunosorbent assay (ELISA) Results After 4 courses of treatment, the tumor growth rates of high dose treated group with 1×10 9 pfu of Ad/hEndo were inhibited by 42 26% compared with the Ad/ LacZ control group ( P =0 001) and by 46 26% compared with the NIH buffer control group ( P =0 003), respectively However, in this study, Ad/hEndo at low dose of 5×10 8 pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47% However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50% The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1×10 9 pfu and gradually dropped undetectable by day 7 Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later Conclusions Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL 7402 xenografted tumors at a high dose of 1×10 9 pfu compared with other groups The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high level, relatively long term expression in vivo and bioactivity capability
基金ThisstudywassupportedbyGuangdongProvinceMedicalScienceandTechnologyResearchFund (No .B2 0 0 0 0 5 1)
文摘Objective To investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization. Methods pBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Ⅰand Sal Ⅰ, by PCR reaction, by sequence, and then by alignment of PCR products with the geneBank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO 3 and 25% KNO 3 cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs. Results pBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.Conclusions The plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.
基金supported by agrant from the National Natural Science Foundation of China(No.30570546)
文摘This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellu-lar growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egr1-shTRAIL-shES and X-ray irradiation. Then MTT assay was used for determining the cellular proliferation, and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression. The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshut-tle-Egr1-shTRAIL-shES transfection in conjunction with irradiation. In the TRAIL-endostatin-based single- or double-gene-radiotherapy, the cell viability declined in a time- and dose-dependent manner, the percentage of cells at G2/M phase and apoptotic rate was increased, and the percentage of cells at G0/G1 phase was lowered as compared with those receiving radiotherapy alone. Moreover, TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition, promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than single-gene-radiotherapy.
文摘Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line,LoVo.Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2×10 5 CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.Results After transfection and superinfection,amphotropic retrovirus was collected,and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited endothelial cell growth up to 67% ( P <0.001),compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group,accompanied by a reduction in vessels,compared with the control group ( P <0.01). Conclusion Retroviruses can allow functional expression of the endostatin gene in human colon tumors,showing promise for an antitumor strategy using antiangiogenesis.
