BACKGROUNDHuman sulfatase-1 (Hsulf-1) is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans (HSPGs), altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation, cell motility, and apoptosis. We investigated the role of combined cancer gene therapy with Hsulf-1 and cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene on a hepatocellular carcinoma (HCC) cell line, HepG2, in vitro and in vivo.

METHODSReverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC. Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy. We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.

RESULTSA significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system. A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed. Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene. In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone, and the combined treatment resulted in a significant increase in survival.

CONCLUSIONSHsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.

Animals ; Apoptosis ; drug effects ; genetics ; Carcinoma, Hepatocellular ; enzymology ; metabolism ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; genetics ; Cytosine Deaminase ; genetics ; metabolism ; Flucytosine ; pharmacology ; Genetic Therapy ; Hep G2 Cells ; Humans ; Liver Neoplasms ; enzymology ; metabolism ; Sulfatases ; genetics ; metabolism

OBJECTIVETo evaluate the killing effect of adenovirus(Ad)-mediated double suicide gene driven by kinase domain-containing receptor (KDR) promoter on gastric cancer MGC-803 cells.

METHODSThe 293 packaging cells were transfected by the plasmids pAdEasy-KDR-CDglyTK to generate infectious viruses. The gastric cancer MGC-803 cells were infected by the Ad followed by treatment with 5-FC and/or ganciclovir at different concentrations. The cell-killing effects were evaluated and the bystander effects analyzed after coculture of the cells without AdKDR-CDglyTK infection with the infected cells at different ratios. The cell cycle distribution was detected by flow cytometry and the pathological changes of the cells were observed by electron microscopy.

RESULTSThe infection rate of the resultant recombinant Ad in the cells increased gradually with increment of the multiplicity of infection (MOI) of the Ads. The killing effect of CD/TK fusion gene on the MGC-803 cells was much stronger than that of either of the single suicide gene (P<0.001), and considerable bystander effect was observed. The Ad infection caused MGC-803 cell growth arrest at G(1) phase with onset of apoptotic and necrotic morphologies of the cells as seen under electron microscope.

CONCLUSIONThe CD/TK fusion gene system driven by the KDR promoter possesses effective killing effect on the KDR-expressing gastric cancer MGC-803 cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Promoter Regions, Genetic ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; Stomach Neoplasms ; pathology

OBJECTIVETo elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.

METHODK562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.

RESULTSAt the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.

CONCLUSIONYCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Animals ; Cytosine Deaminase ; Flucytosine ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Humans ; K562 Cells ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; therapy ; Nucleoside Deaminases ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

OBJECTIVETo evaluate the effect of adenovirus-mediated CD/TK double suicide gene system on tumor growth and cytokine levels in the tumor microenvironment in mice bearing transplanted colorectal cancer.

METHODSCT26 cells were implanted subcutaneously into 30 Balb/c mice, which were subsequently randomized into the control (n=15) and experimental group (n=15). After the tumor formation, CD/TK double suicide gene system was administered for tumor treatment, and the changes in the tumor volume, tumor inhibition rate, and levels of cytokines in the tumor microenvironment were investigated.

RESULTSCD/TK double suicide gene system resulted in a significant inhibition of the tumor growth and significantly increased levels of such cytokines as IL-2, IL-10, TNFalpha and IFNgamma in the tumor microenvironment.

CONCLUSIONCD/TK double suicide gene system produces significant tumor inhibition effect and causes obvious cytokine changes in the tumor microenvironment in mice bearing transplanted colorectal cancer.

Adenoviridae ; genetics ; metabolism ; Animals ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; therapy ; Cytokines ; metabolism ; Cytosine Deaminase ; genetics ; metabolism ; Female ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo investigate the therapeutic effect and metabolism of 5-fluorocytosine (5-FC) in human tongue squamous carcinoma cells after treatment with adenovirus-mediated cytosine deaminase (AdCMVCD)/5-FC system.

METHODSHuman tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5-FC system. The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay. Tumor inhibition effect and histopathological changes were observed in vivo. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of 5-FC in vitro and in vivo.

RESULTSAdCMVCD/5-FC system had strong killing effect and bystander effect on Tca8113 cells. Both condition media and cell extracts showed two peaks identified as 5-FC and 5-fluorouracil (5-FU) by HPLC and a time-dependent generation of 5-FU and concomitant time-dependent decreases of 5-FC. Compared to the control groups, mice treated with AdCMVCD/5-FC system demonstrated significant tumor regression (P < 0.001); the tumor doubling time prolonged and inhibition rate was 92.62%. There were substantial tumor necrotic areas and infiltrative lymphocytes around necrotic areas in the AdCMVCD/5-FC treated group under light microscope. There was a significantly low concentration of 5-FC and high concentration of 5-FU in tumor tissue, but only 5-FC was found in blood.

CONCLUSIONAdCMVCD/5-FC suicide gene system had significant in vitro and in vivo anti-tumor effect on human tongue squamous cell carcinoma due to convert 5-FC into 5-FU.

Adenoviridae ; genetics ; Animals ; Carcinoma, Squamous Cell ; pathology ; therapy ; Cytosine Deaminase ; Female ; Flucytosine ; metabolism ; therapeutic use ; Genetic Therapy ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Nucleoside Deaminases ; genetics ; Tongue Neoplasms ; pathology ; therapy ; Transplantation, Heterologous ; Tumor Cells, Cultured

Enzyme/prodrug approach is one of the actively developing areas for cancer therapy. In an effort to develop more effective enzyme/prodrug systems, cell-permeable cytosine deaminase was produced by fusing yeast cytosine deaminase (yCD) in frame with RKKRRQRRR domain of HIV-1 Tat which is an efficient delivery peptide of the foreign proteins into cells. The purified Tat-yCD fusion protein expressed in Escherichia coli was readily transduced into mammalian cells in a time- and dose-dependent manner. A significant level of the transduced Tat-yCD protein was recovered in the cell and was stable for 24 h as indicated by both results of the enzymatic assay of 5-fluorocytosine (5-FC) conversion to 5-fluorouracil (5-FU) and Western blot analysis. The cells transduced with Tat-yCD become highly sensitive to the cytotoxicity of 5-FC, while cells treated with yCD are unaffected by 5-FC. In addition, a strong bystander effect was observed with conditioned media from cells transduced with Tat-yCD added to non-transduced cells. Tat-yCD fusion protein demonstrated here for its ability to transduce into cells and convert nontoxic prodrug 5-FC to the toxic antimetabolite 5-FU, may be a useful approach for cancer therapy.
Animals ; Antimetabolites/*metabolism/pharmacology ; Bystander Effect ; Cytosine Deaminase/genetics/*metabolism ; Flucytosine/*metabolism/pharmacology ; Gene Products, tat/chemistry/genetics/*metabolism ; Genetic Vectors/genetics/metabolism ; HIV-1/metabolism ; Hela Cells/drug effects/physiology ; Humans ; Prodrugs/metabolism/therapeutic use ; Recombinant Fusion Proteins/genetics/*metabolism ; Research Support, Non-U.S. Gov't ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; *Transduction, Genetic

OBJECTIVETo study the effect of adenovirus (Ad)-mediated fusion gene system driven by the KDR promoter on the proliferation of human colon adenocarcinoma SW620 cells.

METHODSThe KDR-expressing SW620 cells and LS174T cells not expressing KDR were both infected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or ganciclovir at different concentrations. The effect of the transfection on the cell proliferation was evaluated.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SW620 and LS174T cells with a multiplicity of infection (MOI) of 100. Significant difference was not founded in the growth of SW620 and LS174T cells with or without the transfection. The infected SW620 cells exhibit high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). The CDglyTK fusion gene produced much stronger killing effect of on the target cells than either of the single suicide genes (P<0.01).

CONCLUSIONCDglyTK fusion gene system driven by the KDR promoter selectively kills the KDR-CDglyTK SW620 cells and inhibits the cell proliferation.

Adenocarcinoma ; pathology ; Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; pathology ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.

METHODS293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.

RESULTSThe recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).

CONCLUSIONThe double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.

Antimetabolites ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytosine Deaminase ; genetics ; metabolism ; Flow Cytometry ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

OBJECTIVETo study the selective killing effects of adenovirus (Ad)-mediated double suicide gene system driven by KDR promoter (KDR-CdglyTK) on the human hepatic carcinoma cells and human umbilical vein endothelial cells (HUVECs).

METHODSKDR-expressing BEL-7402 and HUVECs and HepG2 cells that did not express KDR were infected by KDR-CdglyTK, and the infection efficiency and the expression of CdglyTK in the cells was detected by RT-PCR. The infected cells were treated with the the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method.

RESULTSAt the multiplicity of infection (MOI) of 100, the recombinant AdKDR-CDglyTK showed similar infection efficiency in the 3 cell lines. RT-PCR demonstrated CDglyTK expression in the recombinant adenovirus and the 3 infected cell lines. BEL-7402 and HUVECs infected by the KDR-CdglyTK, but not the HepG2 cells, were highly sensitive to the prodrugs (P<0.001). Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected BEL-7402 and HUVECs.

CONCLUSIONThe double suicide gene system driven by KDR promoter has specific killing effect on KDR-expressing hepatocellular carcinoma cells and HUVECs.

Adenoviridae ; genetics ; Apoptosis ; genetics ; Cells, Cultured ; Cytosine Deaminase ; genetics ; metabolism ; Endothelial Cells ; cytology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Neoplasms ; pathology ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo study the selective killing effect of adenovirus (Ad)-mediated double suicide gene system driven by the KDR promoter (KDR-CDglyTK) on human colon adneocarcinoma SW480 cells.

METHODSKDR-expressing SW480 cells and LS174T cells that did not express KDR were infected by KDR-CDglyTK, and the infection efficiency and the expression of CDglyTK in the cells were detected by RT-PCR. The infected cells were treated with the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method. DNA content and the cell cycle changes in SW480 cells were detected by flow cytometry.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SW480 and LS174T cells with a multiplicity of infection (MOI) of 100. RT- PCR demonstrated that the product of CD/TK gene existed in SW480 cells infected by Ad- KDR- CD/TK, but not in infected LS174 cells. The infected SW480 cells exhibited high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected SW480 cells. At the MOI of 100, treatment of the infected cells with the prodrugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase and the prodrug-treated cells showed an apoptotic peak in flow cytometry.

CONCLUSIONCDglyTK fusion gene system driven by the KDR promoter selectively kills and induces the apoptosis of the KDR-CDglyTK SW480 cells.

Adenocarcinoma ; genetics ; pathology ; Adenoviridae ; genetics ; metabolism ; Apoptosis ; genetics ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; pathology ; Cytosine Deaminase ; biosynthesis ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism