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Volume 12,Issue 3,2005 Table of Contents
2005, 12(3):167-173.
DOI: 10.3872/j.issn.1007-385X.2005.3.002
Abstract:
Objective: To establish a novel and robust hammerhead ribozyme system against the Vascular Endothelial Growth Factor (VEGF) for anti-cancer gene therapy. Methods： The structural analysis of the 2ndstructure of the VEGF RNA and the vector construction of hammerhead ribozymes (1-4) against VEGF leader region(+1 to+75); The in vitro analyses of ribozyme mediated specific cleavage; The in cell evaluation of the ribozyme mediated cleavage of the VEGF RNA. Results：Ribozymes targeting +8, +36, or +71 (Rz1,3 and 4) of the exposed region or +17 (Rz2) of the unexposed region of VEGF RNA were constructed in pGVal and pFB retroviral vector systems; Rz1,3 and 4, but not Rz2, specifically cleaved the VEGF RNA and brought the VEGF RNA level down to 61.7%, 27.6% and 44.8%(luciferase activity)as well as 66.3%, 27.0% and 30.0%(protein level) of the control; The same set of ribozymes reduced the co-transfected VEGF-LUC RNA level down to 81.4%，56.6% and 69.1% of the control in a transient transfection analysis and essentially abolished the endogenous VEGF RNA in the stable transfected setting. Conclusion: We have established three effective hammerhead ribozyme vector systems targeting +8, +36 and +71 of the VEGF RNA.
Objective: To establish a novel and robust hammerhead ribozyme system against the Vascular Endothelial Growth Factor (VEGF) for anti-cancer gene therapy. Methods： The structural analysis of the 2ndstructure of the VEGF RNA and the vector construction of hammerhead ribozymes (1-4) against VEGF leader region(+1 to+75); The in vitro analyses of ribozyme mediated specific cleavage; The in cell evaluation of the ribozyme mediated cleavage of the VEGF RNA. Results：Ribozymes targeting +8, +36, or +71 (Rz1,3 and 4) of the exposed region or +17 (Rz2) of the unexposed region of VEGF RNA were constructed in pGVal and pFB retroviral vector systems; Rz1,3 and 4, but not Rz2, specifically cleaved the VEGF RNA and brought the VEGF RNA level down to 61.7%, 27.6% and 44.8%(luciferase activity)as well as 66.3%, 27.0% and 30.0%(protein level) of the control; The same set of ribozymes reduced the co-transfected VEGF-LUC RNA level down to 81.4%，56.6% and 69.1% of the control in a transient transfection analysis and essentially abolished the endogenous VEGF RNA in the stable transfected setting. Conclusion: We have established three effective hammerhead ribozyme vector systems targeting +8, +36 and +71 of the VEGF RNA.
2005, 12(3):174-178.
DOI: 10.3872/j.issn.1007-385X.2005.3.003
Abstract:
Objectivev: To investigate whether synthetic Smac peptides containing the seven N-terminal residues essential for XIAP inactivation would increase chemo-sensitivity of pancreatic cancer cells. Methods: SmacN7 penetratin peptide was synthesized and delivered into Panc-1 cells. Interaction between SmacN7 penetratin peptide and XIAP was tested by pull-down assays. The proportions of apoptosis of Panc-1 cells induced by cisplatin or 5-fluorouracil (5-FU) in the presence and absence of SmacN7 peptides were analyzed by flow cytometry. The chemo-sensitivity of Panc-1 cells before and after treated with SmacN7 peptides was evaluated by tetrazolium bromide (MTT) assay. Results: SmacN7 penetratin peptide could successfully interact with endogenous XIAP, greatly down-regulated of XIAP expression and significantly enhanced cisplatin or 5-FU induced apoptosis of Panc-1 cells. Combining treated with SmacN7 penetratin peptide, the 50% inhibitory concentration (IC50) to cisplatin or 5-FU in Panc-1cells was markedly decreased to 1.98 and 2.62 fold respectively. Conclusion: SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor and sensitize Panc-1 cells to anticancer drug-induced apoptosis. These findings may lead to a novel approach to enhance chemotherapeutic responses in pancreatic cancer.
Objectivev: To investigate whether synthetic Smac peptides containing the seven N-terminal residues essential for XIAP inactivation would increase chemo-sensitivity of pancreatic cancer cells. Methods: SmacN7 penetratin peptide was synthesized and delivered into Panc-1 cells. Interaction between SmacN7 penetratin peptide and XIAP was tested by pull-down assays. The proportions of apoptosis of Panc-1 cells induced by cisplatin or 5-fluorouracil (5-FU) in the presence and absence of SmacN7 peptides were analyzed by flow cytometry. The chemo-sensitivity of Panc-1 cells before and after treated with SmacN7 peptides was evaluated by tetrazolium bromide (MTT) assay. Results: SmacN7 penetratin peptide could successfully interact with endogenous XIAP, greatly down-regulated of XIAP expression and significantly enhanced cisplatin or 5-FU induced apoptosis of Panc-1 cells. Combining treated with SmacN7 penetratin peptide, the 50% inhibitory concentration (IC50) to cisplatin or 5-FU in Panc-1cells was markedly decreased to 1.98 and 2.62 fold respectively. Conclusion: SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor and sensitize Panc-1 cells to anticancer drug-induced apoptosis. These findings may lead to a novel approach to enhance chemotherapeutic responses in pancreatic cancer.
3 Changes of Lymphocyte and Its Subsets After a Single Injection of Cyclophosphamide to Balb/c Mouse
LIU Ji-yan
,
LI Yong-qiang
,
PENG Rui-qing
,
Ding Ya
,
Li Hong-li
,
CHENG Xia
,
ZHANG Nian-hua
,
ZHANG Xiao-shi
,
ZENG Yi-xin
2005, 12(3):179-182.
DOI: 10.3872/j.issn.1007-385X.2005.3.004
Abstract:
Objective : To explore the possible mechanism that single administration of low dose cyclophosphamide (CTX) has the ability to potentiate immunity in antitumor immunotherapy. Methods: Lymphocytes in peripheral blood and spleen were numerated after 50 mg/kg CTX was administrated intraperitoneally to Balb/c mice, and lymphocyte subsets were analyzed by flow cytometry on day 5 of the administration. Results: On day 1 to 5 after CTX administration, lymphocytes decreased promptly both in peripheral blood and in spleen, accompanying with the decrease in ratio of non-T lymphocyte subsets and the increase in ratio of T lymphocyte subsets in peripheral blood, and the increase in ratio of CD8+ T lymphocytes was more marked than that in ratio of CD4+ T lymphocytes. Conclusions: These changes might be associated with the immune potentiation effect of CTX, and our observation afforded some information in further investigating the mechanism of immune potentiation effect of CTX.
Objective : To explore the possible mechanism that single administration of low dose cyclophosphamide (CTX) has the ability to potentiate immunity in antitumor immunotherapy. Methods: Lymphocytes in peripheral blood and spleen were numerated after 50 mg/kg CTX was administrated intraperitoneally to Balb/c mice, and lymphocyte subsets were analyzed by flow cytometry on day 5 of the administration. Results: On day 1 to 5 after CTX administration, lymphocytes decreased promptly both in peripheral blood and in spleen, accompanying with the decrease in ratio of non-T lymphocyte subsets and the increase in ratio of T lymphocyte subsets in peripheral blood, and the increase in ratio of CD8+ T lymphocytes was more marked than that in ratio of CD4+ T lymphocytes. Conclusions: These changes might be associated with the immune potentiation effect of CTX, and our observation afforded some information in further investigating the mechanism of immune potentiation effect of CTX.
2005, 12(3):183-188.
DOI: 10.3872/j.issn.1007-385X.2005.3.005
Abstract:
Objective : To construct a recombinant adenovirus(rAdCDES) which is capable of both direct and indirect treatment to mammary cancer and enhancement to the antitumor effect of radiation. Methods： A method of homologous recombination in bacteria was used to construct prAdCDglyES. The recombination adenovirus was transfected to 293 cells by liposome， in which rAdCDES was packaged and generated. The growth curve and MTT methods was used to detect the growth inhibition effect of rAdCDES on MCF-7；rAdCDES was directly injected into established MA737 tumors-bearing mice for observing difference in tumor size and survival days of mice and enhancement of the antitumor effect of radiation. Results: The inhibiting rate of rAdCDES on MCF-7 cell was (83.1± 8.1)% and had significant difference compared with control was (19.2± 7.8)% (P<0.01).We observed also that there was a significant difference in tumor size and survival days in mice between the therapy group and control group and rAdCDES had enhancement of the antitumor effect of radiation. Conclusion: rAdCDES is capable of growth inhibition effect on mammary cancer in vivo and vitro and enhancement of the antitumor effect of radiation.
Objective : To construct a recombinant adenovirus(rAdCDES) which is capable of both direct and indirect treatment to mammary cancer and enhancement to the antitumor effect of radiation. Methods： A method of homologous recombination in bacteria was used to construct prAdCDglyES. The recombination adenovirus was transfected to 293 cells by liposome， in which rAdCDES was packaged and generated. The growth curve and MTT methods was used to detect the growth inhibition effect of rAdCDES on MCF-7；rAdCDES was directly injected into established MA737 tumors-bearing mice for observing difference in tumor size and survival days of mice and enhancement of the antitumor effect of radiation. Results: The inhibiting rate of rAdCDES on MCF-7 cell was (83.1± 8.1)% and had significant difference compared with control was (19.2± 7.8)% (P<0.01).We observed also that there was a significant difference in tumor size and survival days in mice between the therapy group and control group and rAdCDES had enhancement of the antitumor effect of radiation. Conclusion: rAdCDES is capable of growth inhibition effect on mammary cancer in vivo and vitro and enhancement of the antitumor effect of radiation.
2005, 12(3):189-192.
DOI: 10.3872/j.issn.1007-385X.2005.3.006
Abstract:
Objective : To construct RNAi adenovirus targeting both VEGF and p53 in the same time. Methods: The interfering plasmids targeting VEGF and p53 were constructed respectively. Then the H1 promoter and RNAi sequence were cut and ligated to pAdTrack successively to generate pAdTrack/VEGF/p53, which was transfected into BJ5183 cells containing pAdEasy-1 to generate pAd/VEGF/p53 after homologous recombination, harvesting the adenovirus after infecting 293 cells. The knockdown of VEGF and p53 by pAd/VEGF/p53 was detected through real-time PCR after infecting MCF-7 cells. Results:The constructed pAd/VEGF/p53 containing being inserted two H1 promoters and RNAi sequences was identified by BamH digesting. The VEGF and p53 mRNA level of MCF-7 cells decreased obviously after being infected by pAd/VEGF/p53. Conclusions: The RNAi adenovirus targeting both VEGF and p53 in the same time was successfully constructed and it can silence both genes obviously.
Objective : To construct RNAi adenovirus targeting both VEGF and p53 in the same time. Methods: The interfering plasmids targeting VEGF and p53 were constructed respectively. Then the H1 promoter and RNAi sequence were cut and ligated to pAdTrack successively to generate pAdTrack/VEGF/p53, which was transfected into BJ5183 cells containing pAdEasy-1 to generate pAd/VEGF/p53 after homologous recombination, harvesting the adenovirus after infecting 293 cells. The knockdown of VEGF and p53 by pAd/VEGF/p53 was detected through real-time PCR after infecting MCF-7 cells. Results:The constructed pAd/VEGF/p53 containing being inserted two H1 promoters and RNAi sequences was identified by BamH digesting. The VEGF and p53 mRNA level of MCF-7 cells decreased obviously after being infected by pAd/VEGF/p53. Conclusions: The RNAi adenovirus targeting both VEGF and p53 in the same time was successfully constructed and it can silence both genes obviously.
2005, 12(3):193-196.
DOI: 10.3872/j.issn.1007-385X.2005.3.007
Abstract:
Objective : To investigate the mechanism on cytotoxicity of human hepatoma cells SMMC7721 induced by NDV HN gene. Methods: 24 h after transfected with the mixture of NDV HN gene and liposome in vitro, SMMC7721 cell mortality rate was measured through MTT staining; the content of cell surface sialic acid was measured; the death pattern of SMMC7721 cells was detected by staining with AO/EB; expressions of HN gene and HLA-A,B,C was measured by FCM. Results: Transfected into SMMC7721 cells 24 h later, pVHN reduced the surface sialic acid of SMMC7721 cells（P<0.05）and killed the cells significantly in vitro; the difference of HN gene expression was significantly between experimental groups and control groups, and the HLA-A,B,C expression was upregulated. Conclusions: The HN gene plays an important role in the reduction of sialic acid of SMMC7721 cells and caused the cell death. The expressions of HN gene was significant and HLA-A,B,C expression was upregulated after HN gene tansfected into SMMC7721 in virto. Therefore, immunogenicity and immunoregonization of tumor cells might be increased, so the SMMC7721 cells was killed.
Objective : To investigate the mechanism on cytotoxicity of human hepatoma cells SMMC7721 induced by NDV HN gene. Methods: 24 h after transfected with the mixture of NDV HN gene and liposome in vitro, SMMC7721 cell mortality rate was measured through MTT staining; the content of cell surface sialic acid was measured; the death pattern of SMMC7721 cells was detected by staining with AO/EB; expressions of HN gene and HLA-A,B,C was measured by FCM. Results: Transfected into SMMC7721 cells 24 h later, pVHN reduced the surface sialic acid of SMMC7721 cells（P<0.05）and killed the cells significantly in vitro; the difference of HN gene expression was significantly between experimental groups and control groups, and the HLA-A,B,C expression was upregulated. Conclusions: The HN gene plays an important role in the reduction of sialic acid of SMMC7721 cells and caused the cell death. The expressions of HN gene was significant and HLA-A,B,C expression was upregulated after HN gene tansfected into SMMC7721 in virto. Therefore, immunogenicity and immunoregonization of tumor cells might be increased, so the SMMC7721 cells was killed.
SUN Tao
,
HUANG You-tian
,
YANG Hong-yan
,
YANG Yue-jing
,
YUAN Yi-ming
,
ZHAO Ming-yao
,
DONG Zi-ming
2005, 12(3):197-201.
DOI: 10.3872/j.issn.1007-385X.2005.3.008
Abstract:
Objective:To investigate the adjuvant effects of CpG motif-containing oligodeoxynucleotide (CpG) in vitro on human esophageal cancer vaccine.Methods:Matured dendritic cells (DC) activated by peptides on Eca-109 cell membranes were used for human esophageal tumor vaccine and matured DC activated by peptides on HL-60 cell membranes were used for control vaccine. With adjuvant of CpG or the cell wall skeleton of coryne bacterium parvum (CBPw), we analyzed each group's T-cell proliferation and cytotoxic T lymphocyte (CTL) response. Results:The adjuvant of CpG or CBPw markedly enhanced T-cell proliferation and CTL response（P＜0.01）; The groups with human esophageal tumor vaccine had markedly greater T-cell proliferation and CTL response than other groups（P＜0.05）; The HLA gene locus alleles to match Eca-109 cell gene affected the activity of T cells.Conclusion:CpG may be the optimal adjuvant of human esophageal tumor vaccine and the HLA gene locus may affect T cells' function.
Objective:To investigate the adjuvant effects of CpG motif-containing oligodeoxynucleotide (CpG) in vitro on human esophageal cancer vaccine.Methods:Matured dendritic cells (DC) activated by peptides on Eca-109 cell membranes were used for human esophageal tumor vaccine and matured DC activated by peptides on HL-60 cell membranes were used for control vaccine. With adjuvant of CpG or the cell wall skeleton of coryne bacterium parvum (CBPw), we analyzed each group's T-cell proliferation and cytotoxic T lymphocyte (CTL) response. Results:The adjuvant of CpG or CBPw markedly enhanced T-cell proliferation and CTL response（P＜0.01）; The groups with human esophageal tumor vaccine had markedly greater T-cell proliferation and CTL response than other groups（P＜0.05）; The HLA gene locus alleles to match Eca-109 cell gene affected the activity of T cells.Conclusion:CpG may be the optimal adjuvant of human esophageal tumor vaccine and the HLA gene locus may affect T cells' function.
8 The Role of JNK/SAPK Signaling-Transduction Pathway in the Effect of Elemene Against Hepatocarcinoma
2005, 12(3):202-205.
DOI: 10.3872/j.issn.1007-385X.2005.3.009
Abstract:
Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene.Methods:The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively.Results:Gas chomatography showed that elemene was detected at 8.42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion:Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.
Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene.Methods:The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively.Results:Gas chomatography showed that elemene was detected at 8.42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion:Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.
2005, 12(3):206-210.
DOI: 10.3872/j.issn.1007-385X.2005.3.010
Abstract:
Objective:To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPA1 active center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E.coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test.Results:Human carboxypeptidase A1 and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells.Conclusions:Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.
Objective:To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPA1 active center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E.coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test.Results:Human carboxypeptidase A1 and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells.Conclusions:Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.
2005, 12(3):211-214.
DOI: 10.3872/j.issn.1007-385X.2005.3.011
Abstract:
Objective:To investigate the immunological mechanism of influenza A virus for murine S180 ascites sarcoma.Methods:After inocutation with S180 sarcoma cells, mice were i.p. injected with influenza A virus or vehicle 15 days. The average living time and survival rate of the mice were examined. The levels of IL-2, IL-6 and TNF-α were detected. The sarcoma cell′s apoptosis was detected by DNA ladder, flow cytometry (FMC), fluorescent microscope and electron microscope (EM). Results: The average living time and survival rate of the mice injected with Influenza A virus were significantly longer or higher than that of the controls. The levels of IL-2, IL-6 and TNF-α also had the same differences. The apoptosis cells were detected by EM and fluorescent microscope. Sub-diploid peaks were observed by FCM analysis and DNA ladder was seen after electrophoresis in the ascites cells. Conclusion:Our results demonstrated that the feasibility and potential of delivery of influenza A virus as a general means for the treatment of S180 ascites sarcoma.
Objective:To investigate the immunological mechanism of influenza A virus for murine S180 ascites sarcoma.Methods:After inocutation with S180 sarcoma cells, mice were i.p. injected with influenza A virus or vehicle 15 days. The average living time and survival rate of the mice were examined. The levels of IL-2, IL-6 and TNF-α were detected. The sarcoma cell′s apoptosis was detected by DNA ladder, flow cytometry (FMC), fluorescent microscope and electron microscope (EM). Results: The average living time and survival rate of the mice injected with Influenza A virus were significantly longer or higher than that of the controls. The levels of IL-2, IL-6 and TNF-α also had the same differences. The apoptosis cells were detected by EM and fluorescent microscope. Sub-diploid peaks were observed by FCM analysis and DNA ladder was seen after electrophoresis in the ascites cells. Conclusion:Our results demonstrated that the feasibility and potential of delivery of influenza A virus as a general means for the treatment of S180 ascites sarcoma.
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.