The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.
Animals ; Anti-HIV Agents/pharmacology ; Bone Marrow Cells/cytology ; Chemokine CXCL12/biosynthesis ; Endothelial Progenitor Cells/*cytology ; Hematopoietic Stem Cells/*cytology ; Heterocyclic Compounds/*pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Male ; Neovascularization, Physiologic ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4/antagonists & inhibitors ; Surgical Flaps/*blood supply/surgery ; Tissue Expansion/*methods ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis/metabolism

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

OBJECTIVETo express granzyme B-vascular endothelial growth factor (VEGF) receptor-binding peptide (GrB-VRB) fusion protein in Bifidobacteria longum (B. longum) and investigate the effects of this fusion protein on the proliferation and apoptosis of cells expressing VEGF receptor II, the kinase domain receptor (KDR).

METHODSThe recombinant expression vectors pBBADx-VRB, pBBADx-GrB and pBBADx-GrB-VRB were separately transformed into B. longum cells by electroporation. The expressed products were identified by enzyme-linked immunosorbent assay and Western blotting, and their effects on KDR-positive cells were analyzed using proliferation assay and TUNEL assay.

RESULTSThe expressed products were detected in both the supernatant and cellular fractions of B. longum cells. The recombinant GrB-VRB fusion protein reacted with such KDR-positive cells as human umbilical vein endothelial cells (HUVEC) and mouse colon cancer cell line CT-26, and caused obvious cell proliferation inhibition, cytotoxicity and cell apoptosis in these cells.

CONCLUSIONThe recombinant GrB-VRB fusion protein secreted by the engineered B. longum cells can induce KDR-positive cell death as the result of GrB-induced cell apoptosis following the cell recognition by VRB.

Animals ; Apoptosis ; Bifidobacterium ; metabolism ; Carrier Proteins ; Cell Line, Tumor ; Cell Proliferation ; Granzymes ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-2 ; 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

OBJECTIVETo study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.

METHODSSGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.

RESULTSThe infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.

CONCLUSIONLentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.

Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cytosine Deaminase ; biosynthesis ; genetics ; Cytotoxins ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stomach Neoplasms ; genetics ; pathology ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo study the inhibitory effect of adenovirus-mediated fusion gene system driven by KDR promoter on the proliferation of human gastric adneocarcinoma SCG7901 cells and observe the bystander effect in vitro.

METHODSSCG7901, ECV304 and HepG2 cells were infected with Ad-KDR-CDglyTK and Ad-CMV-CDglyTK at a multiplicity of infection (MOI) of 100, and the infection efficiency and the mRNA expressions of the transferred fusion gene were investigated. GCV and/or 5-FC at different concentrations were added into the culture medium of the infected cells to observe the targeted antitumor effect and bystander effect of CDglyTK suicide gene driven by KDR promoter.

RESULTSWith the MOI of the adenovirus of 100, the fluorescence emitted by green fluorescent protein (GFP) was observed in 95% of the infected SCG7901, ECV304 and HepG2 cells. All the cells infected by Ad-CMV-CDglyTK and SCG7901 and ECV304 cells infected by Ad-KDR-CDglyTK were highly sensitive to the prodrugs. In comparison, HepG2 cells infected with Ad-KDR-CDglyTK did not show much sensitivity to the two prodrugs. Following treatment with the prodrugs at the same concentration, the infected SCG7901 and ECV304 cells exhibited gradually lowered survival rates as the culture time was prolonged, whereas the transgenic HepG2 cells showed no such time-dependent changes. When the non-infected cells were cocultured with the transgenic cells, the bystander effect of CDglyTK gene was observed, which increased with the ratio of the transgenic cells. In these mixed cell culture systems, GCV and 5-FC showed obvious synergetic effect in suppressing the cell survival.

CONCLUSIONThe CDglyTK fusion gene system driven by KDR promoter can inhibit the proliferation of SCG7901 and ECV304 cells with obvious bystander effect in vitro. The combination of the prodrugs produces obvious synergetic effect against the cell survival.

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

Animals ; Bile Ducts ; cytology ; Cell Transdifferentiation ; Hepatocytes ; cytology ; Liver ; embryology ; metabolism ; Mice ; Mice, Inbred C57BL ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis

The catalytic domain of KDR kinase (KDR-CD) was amplified from RNA of HUVCEs cells with RT-PCR and expressed in E. coli BL21(DE3) by plasmid pET30a as vector. The recombinant protein was purified with affinity chromatography (Ni-NTA). Western blotting showed that the recombinant KDR-CD was phosphorylated in E. coli BL21(DE3). The recombinant KDR-CD was identified to have kinase activity catalyzing the substrate phosphorylated with ATP in the enzymatic reaction.
Catalytic Domain ; genetics ; Cloning, Molecular ; Endothelial Cells ; cytology ; enzymology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessel. We prepared recombinant extracellular domain 3 (KDR3) of human vascular endothelial growth factor receptor-2 in Escherichia coli and studied its specific binding activity with its ligand. The target DNA was synthesized by overlapping PCR, ligated with expression vector p-ET32a and transformed into E. coli Rosetta (DE3). The soluble fusion protein Trx-KDR3 was expressed in cytoplasm, which was up to 20% of total soluble protein in cytoplasm after having been induced by 1 mmol/L IPTG for 5 h at 30 degrees C. It was characterized to be target protein by Western blotting. The product was purified by CM cation exchange resin and immobilized metal affinity chromatography(IMAC). Its VEGF-binding activity was determined by ELISA assay and its influence on the propagation of HUVEC induced by VEGF. The protein product showed high ligand binding activity in the ELISA and HUVEC propagation study compared to the control. Therefore, ligand binding active, soluble recombinant extracellular domain 3 of VEGFR-2 KDR was successfully expressed and purified in E. coli, which would be applied to anti-angiogenesis anti-tumor therapy and anti-KDR antibody development.
Cell Proliferation ; Cells, Cultured ; Cloning, Molecular ; Endothelial Cells ; cytology ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Humans ; Ligands ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics ; metabolism

The extracellular domain 2-4 loop cDNA of quail vascular endothelial growth factor receptor quek1 (qVEGFR2) was obtained from plasmid carrying qVEGFR2 by PCR. Then it was cloned into expression vector pPICZalphaA of Pichia pastoris. To construct recombinant expression plasmid pPICZalphaA-qVEGFR2, linearized pPICZalphaA-qVEGFR2 with SacI was transformed to electroporated Pichia pastoris GS115. Subsequently, positive clone was selected by PCR and its phenotype was determined. SDSPAGE and Western blot assays of culture medium from a methanol-induced expression strain demonstrated that recombinant qVEGFR2 proteins were expressed and secreted into the culture medium. These results could provide a basis for further researches on tumor protein vaccine as well as for the preparation of tumor protein vaccine with Pichia pastoris.
Animals ; Cancer Vaccines ; DNA, Complementary ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Quail ; Receptors, Neurotransmitter ; biosynthesis ; genetics ; Recombinant Proteins ; analysis ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics