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PubMed2006

Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome.

Circulating autoantibodies to self-antigens overexpressed by cancer cells are common in cancer patients. As specific proteins are expressed during neoangiogenesis, a similar phenomenon might occur with particular antigens of tumour vessels. Collagen XVIII, from which endostatin is cleaved, is highly expressed in the perivascular basement membrane of tumour-associated blood vessels and autoantibodies to endostatin have been reported in cancer patients. The present study analyses the incidence of naturally occurring autoantibodies to endostatin in the sera of breast cancer patients and their relation to endostatin serum levels and patient clinical outcome. Serum samples from 36 patients with localised breast cancer and 59 patients with a fully documented history of metastatic breast cancer were used. The immunoreactivity of serum samples was tested against purified recombinant human endostatin and endostatin levels were determined by immunoassay. We could detect anti-endostatin antibodies in the sera of 66% of the patients with localised disease and 42% of the patients with metastatic disease (P=0.03). There was no correlation between the presence of antibodies to endostatin and circulating levels of endostatin. The detection of autoantibodies to endostatin was associated with better prognosis in metastatic breast cancer patients (median survival time: 20 vs 8 months, P = 0.03), as was the presence of low levels of serum endostatin (median survival time: 20 vs 9 months, P = 0.007). These results show that a natural immune reaction against endostatin can occur in breast cancer patients. This could have important therapeutic implications with regard to endostatin therapy and raises the question of a possible role of this humoral reaction against endostatin in the neoplastic process.

Adult↗
PubMed2004

Inhibition of tumor growth in xenografted nude mice with adenovirus-mediated endostatin gene comparison with recombinant endostatin protein.

BACKGROUND: Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a large-scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endostatin protein. METHODS: Hepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice. Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra-tumoral injections of Ad/hEndo of 5 x 10(8) pfu (low-dose group) and 1 x 10(9) pfu (high-dose group) at intervals of six days, respectively. Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg.kg(-1).d(-1) at a site nearby the tumor for ten days. The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) after Ad/hEndo injection. Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme-linked immunosorbent assay (ELISA). RESULTS: After 4 courses of treatment, the tumor growth rates of high-dose treated group with 1 x 10(9) pfu of Ad/hEndo were inhibited by 42.26% compared with the Ad/LacZ control group (P = 0.001) and by 46.26% compared with the NIH buffer control group (P = 0.003), respectively. However, in this study, Ad/hEndo at low dose of 5 x 10(8) pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1 x 10(9) pfu and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later. CONCLUSIONS: Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 x 10(9) pfu compared with other groups. The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high-level, relatively long-term expression in vivo and bioactivity capability.

Adenoviridae↗
PubMed2001

Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin.

The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as an Endostatin-binding protein. We show that recombinant human Endostatin binds tropomyosin in vitro and to tropomyosin-associated microfilaments in a variety of endothelial cell types. The most compelling evidence that tropomyosin modulates the activity of Endostatin was demonstrated when E37 blocked greater than 84% of the tumor-growth inhibitory activity of Endostatin in the B16-BL6 metastatic melanoma model. We conclude that the E37 peptide mimics the Endostatin-binding epitope of tropomyosin and blocks the antitumor activity of Endostatin by competing for Endostatin binding. We postulate that the Endostatin interaction with tropomyosin results in disruption of microfilament integrity leading to inhibition of cell motility, induction of apoptosis, and ultimately inhibition of tumor growth.

Actin Cytoskeleton↗
PubMedDate not supplied

Endostatin down-regulates soluble guanylate cyclase (sGC) in endothelial cells in vivo: influence of endostatin on vascular endothelial growth factor (VEGF) signaling.

Endostatin was suggested to be an antiangiogenic agent with the potential for clinical use in cancer therapy. Unfortunately, up to now no antiangiogenic effect was seen in clinical trials using this substance. The lack of response might be caused by an incomplete understanding of endostatin signaling. Endostatin is known to influence the vascular endothelial growth factor (VEGF) signaling pathway. It has been reported to bind to the VEGF receptor KDR directly and to decrease the phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 via the protein phosphatase 2A (PP2A). But so far no details of endostatin signaling with regard to NO downstream effectors have been revealed. In the present work the authors demonstrate that endostatin down-regulates the protein level of soluble guanylate cyclase (sGC) in endothelial cells of newly formed blood vessels in 5 day-old wounds (control: 62.5 +/- 33 vessels/mm2, endostatin: 9.2 +/- 3.2 vessels/mm2). This was confirmed in experiments with endothelial tubes of embryoid bodies and endothelial cells derived from embryonic stem cells (eESCs; control: 126 +/- 20, endostatin: 58 +/- 10). The decrease of sGC protein levels in response to endostatin was abolished after preincubation with the PP2A inhibitor okadaic acid. No alterations of sGC mRNA levels could be found under endostatin treatment in eESC. The authors conclude that endostatin affects VEGF signaling in endothelial cells by a post-transcriptional PP2A-dependent down-regulation of sGC protein levels.

Angiogenesis Inhibitors↗
PubMed1999

Novel glycosylated forms of human plasma endostatin and circulating endostatin-related fragments of collagen XV.

Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117)HLRPAR. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII, respectively. These polypetides are found to be O-glycosylated at T(125) (residue 9) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate. These polypeptides exhibit the N-terminal sequences P(66)HLLPPP. and Y(81)EKPALH. of the collagen XV NC-1 domain. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments.

Amino Acid Sequence↗
PubMed2004

Anti-endostatin monoclonal antibody enhances growth of human hepatocellular carcinoma cells by inhibiting activity of endostatin secreted by the transplanted cells in nude mice.

Endostatin, a fragment of collagen XVIII, inhibits angiogenesis in tumors, and is expected to become a new anticancer drug. However, its effectiveness is still controversial, because some researchers failed to reproduce the same marked regression of tumors by the peptide. We gave anti-endostatin monoclonal antibody, designated as CH18B, to nude mice transplanted with human hepatocellular carcinoma cells (JHH-1 line) that endogenously produced endostatin from collagen XVIII secreted by the cells themselves. As a result, CH18B promoted tumor angiogenesis by inhibiting endostatin activity in the tumor and subsequently increased tumor mass by preventing cancer cells from undergoing apoptosis. But the antibody itself did not stimulate proliferation of the tumor cells. Our present experimental procedure, the use of anti-endostatin antibody, definitely solved the question whether endostatin might exert its anticancer activity.

Animals↗
PubMed2004

Endostatin's endpoints-Deciphering the endostatin antiangiogenic pathway.

Up until now, the precise mechanism for endostatin's antiangiogenesis action was not known. In a recent report, have taken advantage of gene array and proteomic analysis to map the antiangiogenic pathways turned on by endostatin. This study resolves some of the controversies surrounding endostatin's biology, and provides a new direction to help dissect the molecular pathways involved in endostatin's selective tumor antiangiogenic effects.

Angiogenesis Inhibitors↗
PubMed2003

Studies on the structure-activity relationship of endostatin: synthesis of human endostatin peptides exhibiting potent antiangiogenic activities.

The aim of the present research was to study the relationship between chemical structure and antiangiogenic activity of endostatin. Four peptides, containing about 40 amino acid residues, designed to cover nearly the whole sequence of endostatin, were synthesized by the solid-phase method. They were termed Fragment I (sequence 6-49), II (sequence 50-92), III (sequence 93-133), and IV (sequence 134-178), with the latter bearing the original disulfide bond Cys135-Cys165. These peptides were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis assays in matrigel. Fragments I and IV inhibited cell proliferation and cell migration with a potency and an efficacy higher than that of the full length endostatin. Fragment I was also active in inhibiting in vitro the formation of tubules and in vivo the vascularization of the matrigel. Fragments II and III were devoid of antiangiogenic activity. We propose to use the peptides 6-49 and 134-178 as angiogenesis inhibitors in substitution of full length endostatin, in therapeutic applications for cancer, rheumatoid arthritis, and retinopathies.

Amino Acid Sequence↗
PubMed2005

[Optimization of fermentation of recombinant human Endostatin (rh-Endostatin) expression in Escherichia coli].

The fermentation process of recombinant human Endostatin expression in Escherichia coli BL21 (DE3) was studied. The effects of factors such as concentration of IPTG, induction time, cultivation temperature and feeding strategies were investigated. Beside that, by changing the temperature to 40 degrees C after induction, the high-density cultivation finished in a much shorter period. After 9 hours cultivation, the optical density (OD) at 600 nm reached 140 and the yield of inclusion body was 3 g/L. While E. coli system was used, protein with better activity and stability was obtained. The cost was much lower and the producing process was much steadier. It will meet the demands of the industrial production.

Endostatins↗
PubMed2002

Assessment of antiangiogenic effect using 99mTc-EC-endostatin.

PURPOSE: Tumor vascular density may provide a prognostic indicator of metastatic potential or survival. The purpose of this study was to develop 99mTc-ethylenedicysteine-endostatin (99mTc-EC-endostatin) for the evaluation of anti-angiogenesis therapy. METHOD: 99mTc-EC-endostatin was prepared by conjugating ethylenedicysteine (EC) to endostatin, followed by adding pertechnetate and tin chloride. Radiochemical purity was > 95%. In vitro cell viability, affinity and TUNEL assays were performed. Tissue distribution and planar imaging of radiolabeled endostatin were determined in tumor-bearing rats. To assess anti-angiogenic treatment response, rats were treated with endostatin, paclitaxel and saline, followed by imaging with 99mTc-EC-endostatin. Tumor response to endostatin therapy in tumor-bearing animal models was assessed by correlating tumor uptake dose with microvessel density, VEGF, bFGF and IL-8 expression during endostatin therapy. RESULTS: In vitro cell viability and TUNEL assays indicated no marked difference between EC-endostatin and endostatin. Cellular uptake assay suggests that endostatin binds to endostatin receptor. Biodistribution of 99mTc-EC-endostatin in tumor-bearing rats showed increased tumor-to-tissue count density ratios as a function of time. Tumor uptake (%ID/g) of 99mTc-EC-endostatin was 0.2-0.5. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-endostatin. The optimal time for imaging using radiolabeled endostatin was 2 hrs. 99mTc-EC-endostatin could assess treatment response. There was a correlation between tumor uptake and cellular targets expression. CONCLUSION: The results indicate that it is feasible to use 99mTc-EC-endostatin to assess efficiency of anti-angiogenesis therapy.

Angiogenesis Inhibitors↗
PubMed2000

Endostatin (EntreMed).

EntreMed has licensed the worldwide rights to the angiogenesis inhibitor Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, from the Children's Hospital of Boston, a teaching affiliate of Harvard Medical School. It is being developed as a potential cancer treatment and may also be useful in certain types of blindness and arthritis [227427]. EntreMed filed an IND for Endostatin in June 1999 [334125] and as of September 1999, phase I trials were underway [341462]. As of April 2000, the company had initiated plans for testing low doses of Endostatin in cancer patients using continuous infusion and sc administration in a further phase I study to be conducted in Europe [361594]. A phase I trial of Endostatin which will evaluate the safety and efficacy of Endostatin at a range of doses in no more than 100 cancer patients has been initiated. The trial will take place at the University of Texas MD Anderson Medical Center and the University of Wisconsin Cancer Center in Madison. The National Cancer Center will be sponsoring the trial, which is expected to be completed in late 2000. As of March 2000, there had been no serious adverse events attributable to Endostatin administration. The first report from this trial is expected in autumn 2000 [341462], [366312]. The mechanism of action for Endostatin remains unclear, although reports from the 91st AACR Meeting in April 2000 showed that recombinant human endostatin bound to a number of tropomyosin cDNAs in a library screen [362039]. In preclinical studies, repeated administration of Endostatin consistently shrank primary tumors and did not produce any drug resistance. In mice, a variety of tumors which had progressed to 1 to 2% of total body weight, regressed to microscopic, dormant lesions following Endostatin treatment [231418], [231470], [270673]. Types of cancers which respond to Endostatin include lung, skin, vascular and fibrosarcomas. Toxicology studies in cynomolgus monkeys showed that bolus injections of human endostatin at doses up to 300 mg/kg produced no toxic effects [317916]. Research presented at the 91st Annual Meeting of the AACR (San Francisco, April 2000), demonstrated that peritumoral administration of Endostatin, at concentrations 2000-fold less than that required to inhibit tumor progression in previously published reports using systemic dosing, significantly deters the development of a variety of tumors in mice [361584]. Other research presented at the meeting demonstrated a role for tropomysin as a molecular target for Endostatin [361680]. In August 1997, EntreMed and the NCI signed letters of intent to commence research collaborations for developing anti-angiogenic therapies, including Endostatin, in preclinical and clinical studies [258354], while in August 1998, EntreMed entered into an agreement with Covance Biotechnology Services for the production of Endostatin [304919]. In August 1999, Entremed and Cell Genesys entered a collaboration to combine Entremed's Endostatin genes with Cell Genesys's gene delivery system as a possible treatment for cancer [336909]. In December 1999, EntreMed signed an agreement with Chiron for the production of bulk Endostatin for phase II clinical trials [350397]. In April 1999, analysts at First Security Van Kasper predicted a US launch for Endostatin in 2004, with first-year revenues of US 320 million dollars, including 70 million dollars in royalties to EntreMed. European launch was predicted for 2005, with total revenues from Europe and the US for that year of US 760 milion dollars (160 million dollars royalties to EntreMed), rising to 1600 million dollars (335 million dollars royalties to EntreMed) in 2007 [370813].

Journal Article↗
PubMed2005

Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein.

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.

Angiogenesis Inhibitors↗
PubMed2004

Acid-induced unfolding mechanism of recombinant human endostatin.

Endostatin is a potent angiogenesis inhibitor. The structure of endostatin is unique in that its secondary structure is mainly irregular loops and beta-sheets and contains only a small fraction of alpha-helices with two pairs of disulfide bonds in a nested pattern. We choose human endostatin as a model system to study the folding mechanism of this kind. Nuclear magnetic resonance (NMR), tryptophan emission fluorescence, and circular dichroism (CD) were used to monitor the unfolding process of endostatin upon acid titration. Urea-induced unfolding was used to measure the stability of endostatin under different conditions. Our results show that endostatin is very acid-resistant; some native structure still remains even at pH 2 as evidenced by (1)H NMR. Trifluoroethanol (TFE) destabilizes native endostatin, while it makes endostatin even more acid-resistant in the low pH region. Stability measurement of endostatin suggests that endostatin is still in native structure at pH 3.5 despite the decreased stability. Acid-induced unfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3. Surprisingly, the alpha-helical content of endostatin is increased when it is unfolded at pH 1.6, and the alpha-helical content of the polypeptide chain of unfolded endostatin increases linearly with TFE concentration in the range of 0-30%. This observation indicates that the polypeptide chain of unfolded endostatin has an intrinsic alpha-helical propensity. Our discoveries may provide clues for refolding endostatin more efficiently. The acid-resistance property of endostatin may have biological significance in that it cannot be easily digested by proteases in an acidic environment such as in a lysosome in the cell.

Acetic Acid↗
PubMedDate not supplied

Antitumor interaction of short-course endostatin and ionizing radiation.

PURPOSE: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. METHODS: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. RESULTS: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. DISCUSSION: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.

Animals↗
PubMed2006

[Antisense oligonucleotide targeting endostatin enhances hematopoiesis reconstitution in BMT mice].

OBJECTIVE: To explore the effect of antisense oligonucleotide targeting endostatin (endostatin-ASON) transfecting bone marrow stromal cells ( BMSC) on hematopoiesis reconstitution in BMT mice. METHODS: Inhibition of endostatin / VCAM-1 protein and mRNA expression was investigated by transfection of antisense oligonucleotide targeting endostatin with confocal microscopy, Western blot and RT-PCR. Bone marrow stromal cells were cultured and divided into 4 groups: group (1) without any treatment; group (2) BMT only; group (3) BMT + endostatin-ASON transfection; group (4) BMT + endostatin scrambled sequence transfection. RESULTS: (1) Endostatin-ASON was successfully introduced into BMSC in vitro, and the transfecting rate was 86% ;(2) After Endostatin-ASON transfected into BMSC, the expression of Endostatin mRNA and its protein on the BMSC was signficantly inhibited at different time point after BMT [the grey value of Endostatin was (0.09 +/- 0.03) - (1.44 +/- 1.19) and (0.02 + 0.02) - (0.14 +/- 0.05), respectively] (P < 0.01 and P < 0.05); (3) Transfecting with Endostatin-ASON effectively promoted the expression of VCAM-1 mRNA and its protein on the BMSC [the gray value of VCAM-1 was (1.60 +/- 0. 92) - (8.05 +/- 0.87) and (0.07 +/- 0.02) - (0.67 +/- 0.09) , respectively] (P <0.01 and P <0.05) ; (4) There was no effects of transfecting Endostatin scrambled sequence on the expression of Endostatin and VCAM-1 on the BMSC (P > 0.05). CONCLUSION: Endostatin-ASON could inhibit Endostatin expression and enhance VCAM-1 expression in BMSC after syngeneic-BMT in mice, which might be one of the mechanisms underlying the endostatin-ASON accelerating hematopoiesis reconstitution after allogeneic-BMT.

Animals↗
PubMed2004

Improved biological activity of a mutant endostatin containing a single amino-acid substitution.

Human endostatin has an internal Asn-Gly-Arg (NGR) motif at position 126-128 following a proline at position 125. Asn-Gly-Arg-containing peptides have been shown to target tumour vasculature and inhibit aminopeptidase N activity. We previously compared the in vitro and in vivo biological activities of native endostatin and endostatin with a proline to alanine mutation (P125A-endostatin). P125A-endostatin exhibited greater inhibition of both endothelial cell proliferation and human ovarian cancer growth compared to native endostatin. Here we explore further the effects on biological activity of the P125A mutation, and show that aminopeptidase N is not involved. To determine whether the increased biological activity of the mutant was due to unmasking of downstream NGR-sequence, effect of endostatin on aminopeptidase N activity was investigated. Neither the native nor the P125A-endostatin inhibited aminopeptidase N. However, synthetic peptides consisting of the S118-T131 region of endostatin inhibited aminopeptidase N. These results suggest that the internal NGR site in native or mutant endostatin is not accessible to aminopeptidase N, and that this activity is not involved in the enhanced biological activity of the P125A form. P125A-endostatin bound to endothelial cells more efficiently than native endostatin and exhibited greater inhibition of not only proliferation but also migration of endothelial cells. P125A-endostatin also localised into tumour tissue to a higher degree than the native protein, and displayed greater inhibition of growth of colon cancer in athymic mice. Both proteins inhibited tumour cell-induced angiogenesis effectively. Real-time PCR analysis showed that both native and P125A-endostatin decreased expression of key proangiogenic growth factors. Vascular endothelial growth factor and angiopoietin 1 were downregulated more by the mutant. These studies suggest that the region around P125 can be modified to improve the biological activity of endostatin.

Amino Acid Substitution↗
PubMed2006

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function.

PURPOSE: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF165-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. METHODS: C57/Bl6 mice were injected with VEGF165 and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF165 and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. RESULTS: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF165. It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF165-mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF165-mediated permeability compared with control (untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF165. CONCLUSIONS: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF165-induced paracellular permeability.

Angiogenesis Inhibitors↗
PubMed2002

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro.

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P<0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.

Animals↗