Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P39060 (endostatin)
 2,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified recombinant mouse endostatin protein has been reported to regress established murine solid tumors by inhibiting the proliferation of endothelial cells. To develop a clinical gene therapy strategy with endostatin, we cloned the cDNA of human endostatin by RT-PCR from human placenta. A 150-bp sequence encoding the IgG leader peptide was fused in frame to the 5' end of the endostatin cDNA and recombinant adenoviruses, AdENDO-YFP and AdENDO, carrying endostatin gene expression cassettes were rescued. AdENDO-YFP infects cultured mammalian cells at high efficiency and expresses a biologically active human endostatin in secreted form at high levels both in vitro and in vivo. When delivered in vivo, a strain-specific expression pattern was observed, with the highest and longest endostatin expression in 129/J mice. After systemic delivery of 2 x 10(9) PFU of AdENDO-YFP into 129/J mice, human endostatin expression was achieved at a mean value of 1.34 +/- 0.42 microg/ml of serum (n = 6) and inhibition of lung metastasis was observed in an EOMA tumor model. However, high dose intravenous delivery of AdENDO-YFP and AdENDO was associated with severe acute toxicity in recipient mice that included loss of weight, bleeding, and death of animals. These events were not observed with the injection of identical doses of a control adenovirus that did not contain the endostatin gene. Because the endostatin adenovirus-associated acute toxicity was also observed in immunodeficient NCRNU-M nude mice, the toxicity does not appear to be the result of the immunogenicity against human endostatin or the EYFP protein.
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PMID:Adenovirus-mediated human endostatin gene delivery demonstrates strain-specific antitumor activity and acute dose-dependent toxicity in mice. 1124 27

The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.
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PMID:High-level expression and secretion of recombinant mouse endostatin by Escherichia coli. 1192 62

Restin, a homologous protein of endostatin (62% homology), is the NC domain of collagen XV at C-terminal. The recombinant restin expressed in E. coli had the ability to suppress the proliferation of bovin aortic endothelial cells and cause apoptosis. In this report, mouse restin gene was fused with a sequence of human plasminogen signal peptide by PCR and cloned into eukaryotic expression vector pCDNA3. The plasmid containing restin gene was named pCDNAXV and was transfected into human hepatoma cell line Bel7404. Stable transfected clones were screened and expression of restin was confirmed by RT-PCR and Western blot. The proliferated cells were injected subcutaneusly into nude mice. The growth of tumors formed by cells transfected with restin gene was much slower than that of control group. These results indicated that the expressed restin in vivo could suppress the growth of tumor, and this suppression might be achieved by restraining angiogenesis since the restin had no effect on the proliferation of tumor cells. At the same time, this report provided a new method to investigate the effect of anti-angiogenetic proteins on the tumor growth.
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PMID:[Restin expressed in vivo suppresses the growth of tumors in nude mice]. 1219 58

Endostatin can inhibit angiogenesis and tumor growth in mice. A potential limitation of endostatin as an antitumor agent in humans is the short serum half-life of the protein that may decrease effective concentration at the site of tumor and necessitate frequent dosing. In an effort to improve antitumor activity, endostatin was fused to an antibody specific for the tumor-selective HER2 antigen to create an antibody-endostatin fusion protein (anti-HER2 IgG3-endostatin). Normal endostatin rapidly cleared from serum in mice (T(1/2)(2), = 0.6-3.8 hours), whereas anti-HER2 IgG3-endostatin had a prolonged half-life (90% intact; T(1/2)(2), 40.2-44.0 hours). Antigen-specific targeting of anti-HER2 IgG3-endostatin was evaluated in BALB/c mice implanted with CT26 tumors or CT26 tumors engineered to express the HER2 antigen (CT26-HER2). Radio-iodinated anti-HER2 IgG3-endostatin preferentially localized to CT26-HER2 tumors relative to CT26 tumors. Administration of anti-HER2 IgG3-endostatin to mice showed preferential inhibition of CT26-HER2 tumor growth compared with CT26. Anti-HER2 IgG3-endostatin also markedly inhibited the growth of human breast cancer SK-BR-3 xenografts in severe combined immunodeficient mice. Anti-HER2 IgG3-endostatin inhibited tumor growth significantly more effectively than endostatin, anti-HER2 IgG3 antibody, or the combination of antibody and endostatin. CT26-HER2 tumors treated with the endostatin fusion protein had decreased blood vessel density and branching compared with untreated CT26-HER2 or CT26 treated with the fusion protein. The enhanced effectiveness of anti-HER2 IgG3-endostatin may be due to a longer half-life, improved serum stability, and selective targeting of endostatin to tumors, resulting in decreased angiogenesis. Linking of an antiangiogenic protein, such as endostatin, to a targeting antibody represents a promising and versatile approach to antitumor therapy.
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PMID:Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein. 1595 53

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.
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PMID:Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells. 1639 52

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N(2) and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0 degrees C and for several months at -80 degrees C. The enzyme was purified using (NH(4))(2)SO(4) fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO(3). About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO(2) (-) per minute per milligram protein). A sequential elution with NADH followed by KNO(3) (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO(3) (-)-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.
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PMID:Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme. 1666 12

There is a great demand for the improvement of mammalian cell production systems such that they can compete economically with their prokaryotic counterparts. Of a number of parameters that need to be explored to accomplish this we have tested the effects of different signal peptides on the synthesis and secretion of Gaussia princeps luciferase in mammalian cells. A series of plasmids were transfected into CHO cells where the coding region for the marine luciferase was fused to the signal peptide coding regions derived from different sources. Both cell extracts and medium samples were analysed for luciferase activity. When the native Gaussia luciferase signal sequence in the vector was substituted by that from human interleukin-2 or albumin then the amount of active recombinant protein produced was substantially reduced, both in transiently and stably transfected cells. Western blotting showed that enzyme activity and protein levels mirrored one another. The major decrease in luciferase activity was shown not to be a result of decreased mRNA levels, indicating the involvement of a post-transcriptional event. When the coding region of human endostatin was fused to that of the Gaussia luciferase signal peptide then an elevated level of secreted endostatin was observed compared to when that of the albumin signal peptide was used. Stable transfection of HepG2 cells with the different signal peptide constructs gave essentially the same results as seen in CHO cells. The overall results indicate that the choice of signal peptide can be imperative to ensure an optimal synthesis and secretion of a recombinant protein in a mammalian cell culture system.
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PMID:The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide. 1731 61

The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were "seamlessly" fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2-7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall findings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the post-transcriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.
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PMID:Vectors encoding seven oikosin signal peptides transfected into CHO cells differ greatly in mediating Gaussia luciferase and human endostatin production although mRNA levels are largely unaffected. 1993 96

Angiogenesis is one of the key acquired characteristics or "hallmarks" essential for the growth and development of all solid tumor types. The antiangiogenic agent vascular endothelial growth factor-Trap (VEGF-Trap) (aflibercept), which is a composite decoy receptor based on VEGF receptor-1 and VEGF receptor-2 fused to an Fc segment of immunoglobulin G1 that binds specifically to VEGF, has demonstrated preclinical efficacy in a range of different tumor types. VEGF-Trap exerts its antiangiogenic effects through regression of tumor vasculature, remolding or normalization of surviving vasculature, and inhibition of new tumor vessel growth. Preclinical and clinical studies have reported that VEGF-Trap can be combined effectively with both chemotherapy and radiotherapy. This review examines the main effects of VEGF-Trap on tumor vasculature and on different types of solid tumors, and explores the preclinical and clinical benefits of incorporating VEGF-Trap into anticancer treatment strategies.
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PMID:Clinical applications of VEGF-trap (aflibercept) in cancer treatment. 2087 16

The first generation of clinically useful antiangiogenic agents focused on VEGF and targets in the VEGF pathway. The strengths and limitations of these therapeutics are now clear. Some tumors do not respond to VEGF-directed therapies de novo and others become non-responsive or resistant over time by switching to other angiogenic pathways. The next generation of angiogenesis-directed therapeutics will expand the field beyond the VEGF pathway and become more disease selective. Placental growth factor, a protein closely related to VEGF, is induced as tumors lose responsiveness to VEGF-directed therapies. Angiopoietins 1 and 2 are being targeted with a unique peptibody, a human recombinant Fc constant region fused to peptides, in clinical trials. The HGF/c-Met pathway is upregulated in some tumors as an alternate angiogenic pathway. The CXCL12 (SDF-1)/CXCR4 pathway represents a stromal chemokine axis involved in tumor angiogenesis. CXCR2 is a G-protein coupled receptor with several ligands including interleukin-8 and other angiogenic cytokines and may represent a useful target for antiangiogenic agents. The notch pathway is being investigated as a target in the setting of tumor angiogenesis. Sphingosine-1-phosphate is a bioactive lipid that can be neutralized with a monoclonal antibody. The anti-S-1-P antibody is under investigation as an antiangiogenic agent. Finally, several multi-targeted kinase inhibitors each with a unique pattern of inhibitory potency are in clinical trial with a focus on antiangiogenic activity. The expansion of the scope of potential antiangiogenic agents in or entering clinical trial should allow the development of antiangiogenic combination regimens and single agents that address diseases refractory to VEGF-directed therapeutics.
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PMID:Antiangiogenic agents and targets: A perspective. 2092 Apr 81


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