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 s展开更多
AIM： To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation theapy in human liver cardnoma （BEL7402） model.METHODS： Recombinant plasmid pcDNA3...AIM： To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation theapy in human liver cardnoma （BEL7402） model.METHODS： Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line （BEL7402） with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells （HUVECs） were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed.RESULTS： Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis （2.1±0.3% vs 8.9±1.3%, t= 8.83, P= 0.009〈0.01） or cell cycle distribution （17.2±2.3% vs 9.8±1.2%, t = 4.94,P = 0.016〈0.05）. The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group （249±83 mm^3） was significantly different from that in the untreated group （823±148 mm^3, t= 5.86, P= 0.009〈0.01） or in the pcDNA3 group （717±94 mm^3, t= 6.46, P= 0.003〈0.01）. Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively.CONCLUSION： Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.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
The expression of therapeutic gene and its anti-tumor effects will beaugmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study wasundertaken to assess the anti-tumor effects of a...The expression of therapeutic gene and its anti-tumor effects will beaugmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study wasundertaken to assess the anti-tumor effects of a novel gene-viral therapeutic systemCNHK300-mEndostatin ( CNHK300-mE) in hepatocellular carcinoma (HCC). A novel gene-viral therapeuticsystem named CNHK300-mE was constructed using the human telomerase reverse transcriptase (hTERT)promoter to drive the expression of the adenovirus El A gene and cloning the therapeutic gene mouseendostatin into the adenovirus genome. By the tissue culture infectious dose 50 (TCID50) method andcytoviability assay, the replicative and cytolytic capabilities of CNHK300-mE in two HCC lines (HepGII and Hep3B) and one normal cell line ( MRC-5 ) were analyzed, and the transgene expressions ofmouse endostatin in vitro and in vivo were detected by Western blotting and ELISA assay. Tumorgrowth suppression and anti-angiogenesis effects in vivo were investigated using nude micexenografts model derived from SMMC-7721 HCC cells. The 3296-fold replicating capacity of CNHK300-mEin HCC cell lines versus in the normal cell line at 96 hours post infection and the 25 -foldeffective dose for killing 50% cells ( ED50) in the normal cell line versus HCC cell lines, whichwere both superior to ONYX-015, were observed. Tumor growth suppression of CNHK300-mE superior toeither Ad-mE or ONYX-015 was demonstrated (P < 0. 01) and the anti-angiogenic effects in vivosuperior to Ad-mE were also observed with immunohistochemical staining of von Willebrand factor. Incomparison with non-replicative adenovirus Ad-mE, the transgene expression of mE mediated byCNHK300- in vivo (P<0.05). Being capable of replicating in and lysing the telomerase-positive HCCcells and mediating effective expression of the therapeutic gene in vitro and in vivo, the novelgene-viral therapeutic system CNHK300-mE is potentially effective in the treatment of HCC.展开更多
AIM: To investigate the effect of adeno-associated virusmediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC).METHODS: HCC cell line Hep3B was infected with recombinantadeno-associ...AIM: To investigate the effect of adeno-associated virusmediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC).METHODS: HCC cell line Hep3B was infected with recombinantadeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined byELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk.RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P＜0.01). Intratumoral injection of rAAV2-hEndo (2×1010v.g.) led to a sustained serum endostatin level ofapproximately (86.71±5.19) ng/mL. The tumor volumeand microvessel density were less in rAAV2-hEndo group than in control groups (P＜0.01).CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.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 s
文摘AIM： To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation theapy in human liver cardnoma （BEL7402） model.METHODS： Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line （BEL7402） with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells （HUVECs） were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed.RESULTS： Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis （2.1±0.3% vs 8.9±1.3%, t= 8.83, P= 0.009〈0.01） or cell cycle distribution （17.2±2.3% vs 9.8±1.2%, t = 4.94,P = 0.016〈0.05）. The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group （249±83 mm^3） was significantly different from that in the untreated group （823±148 mm^3, t= 5.86, P= 0.009〈0.01） or in the pcDNA3 group （717±94 mm^3, t= 6.46, P= 0.003〈0.01）. Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively.CONCLUSION： Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.
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The expression of therapeutic gene and its anti-tumor effects will beaugmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study wasundertaken to assess the anti-tumor effects of a novel gene-viral therapeutic systemCNHK300-mEndostatin ( CNHK300-mE) in hepatocellular carcinoma (HCC). A novel gene-viral therapeuticsystem named CNHK300-mE was constructed using the human telomerase reverse transcriptase (hTERT)promoter to drive the expression of the adenovirus El A gene and cloning the therapeutic gene mouseendostatin into the adenovirus genome. By the tissue culture infectious dose 50 (TCID50) method andcytoviability assay, the replicative and cytolytic capabilities of CNHK300-mE in two HCC lines (HepGII and Hep3B) and one normal cell line ( MRC-5 ) were analyzed, and the transgene expressions ofmouse endostatin in vitro and in vivo were detected by Western blotting and ELISA assay. Tumorgrowth suppression and anti-angiogenesis effects in vivo were investigated using nude micexenografts model derived from SMMC-7721 HCC cells. The 3296-fold replicating capacity of CNHK300-mEin HCC cell lines versus in the normal cell line at 96 hours post infection and the 25 -foldeffective dose for killing 50% cells ( ED50) in the normal cell line versus HCC cell lines, whichwere both superior to ONYX-015, were observed. Tumor growth suppression of CNHK300-mE superior toeither Ad-mE or ONYX-015 was demonstrated (P < 0. 01) and the anti-angiogenic effects in vivosuperior to Ad-mE were also observed with immunohistochemical staining of von Willebrand factor. Incomparison with non-replicative adenovirus Ad-mE, the transgene expression of mE mediated byCNHK300- in vivo (P<0.05). Being capable of replicating in and lysing the telomerase-positive HCCcells and mediating effective expression of the therapeutic gene in vitro and in vivo, the novelgene-viral therapeutic system CNHK300-mE is potentially effective in the treatment of HCC.
基金Supported by the National Natural Science Foundation of China, No. 20074031
文摘AIM: To investigate the effect of adeno-associated virusmediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC).METHODS: HCC cell line Hep3B was infected with recombinantadeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined byELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk.RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P＜0.01). Intratumoral injection of rAAV2-hEndo (2×1010v.g.) led to a sustained serum endostatin level ofapproximately (86.71±5.19) ng/mL. The tumor volumeand microvessel density were less in rAAV2-hEndo group than in control groups (P＜0.01).CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.
