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1.
Cloning, expression, and in vitro activity of human endostatin. 总被引:57,自引:0,他引:57
M Dhanabal R Volk R Ramchandran M Simons V P Sukhatme 《Biochemical and biophysical research communications》1999,258(2):345-352
Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we have expressed human endostatin in a yeast expression system (10 mg/L). The recombinant protein was expressed in a soluble form and purified to homogeneity. It specifically inhibited the proliferation and migration of endothelial cells. In addition, we report for the first time that endostatin caused G1 arrest of endothelial cells. Also, we show that endostatin treatment resulted in apoptosis of HUVE and HMVE cells and that all of these effects do not occur in nonendothelial cells. Collectively, these findings demonstrate the expression of a biologically active form of human endostatin in yeast and provide important mechanistic insight into endostatin action on endothelial cells. 相似文献
2.
Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1 总被引:40,自引:0,他引:40
Kim YM Hwang S Kim YM Pyun BJ Kim TY Lee ST Gho YS Kwon YG 《The Journal of biological chemistry》2002,277(31):27872-27879
Endostatin, a fragment of collagen XVIII, is a potent anti-angiogenic protein, but the molecular mechanism of its action is not yet clear. We examined the effects of endostatin on the biological and biochemical activities of vascular endothelial growth factor (VEGF). Endostatin blocked VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein endothelial cells. Endostatin also inhibited the binding of VEGF(165) to both endothelial cells and purified extracellular domain of KDR/Flk-1. Moreover, the binding of VEGF(121) to KDR/Flk-1 and VEGF(121)-stimulated ERK activation were blocked by endostatin. The direct interaction between endostatin and KDR/Flk-1 was confirmed by affinity chromatography. However, endostatin did not bind to VEGF. Our findings suggest that a direct interaction of endostatin with KDR/Flk-1 may be involved in the inhibitory function of endostatin toward VEGF actions and responsible for its potent anti-angiogenic and anti-tumor activities in vivo. 相似文献
3.
Hanai J Dhanabal M Karumanchi SA Albanese C Waterman M Chan B Ramchandran R Pestell R Sukhatme VP 《The Journal of biological chemistry》2002,277(19):16464-16469
4.
Digtyar AV Pozdnyakova NV Feldman NB Lutsenko SV Severin SE 《Biochemistry. Biokhimii?a》2007,72(3):235-246
Endogenous inhibitors of angiogenesis are proved to be a major factor preventing the emergence of clinically manifested stages of human cancer. The protein endostatin, a 20-kD proteolytic fragment of type XVIII collagen, is one of the most active natural inhibitors of angiogenesis. Endostatin specifically inhibits the in vitro and in vivo proliferation of endothelial cells, inducing their apoptosis through inhibition of cyclin D1. On the surface of endothelial cells, endostatin binds with the integrin alpha(5)beta(1) that activates the Src-kinase pathway. The binding of endostatin with integrins also down-regulates the activity of RhoA GTPase and inhibits signaling pathways mediated by small kinases of the Ras and Raf families. All these events promote disassembly of the actin cytoskeleton, disorders in cell-matrix interactions, and decrease in endotheliocyte mobility, i.e., promote the suppression of angiogenesis. Endostatin displays a high antitumor activity in vivo: it inhibits the progression of more than 60 types of tumors. This review summarizes results of numerous studies concerning the biological activity and action mechanism of endostatin. 相似文献
5.
Cell surface glypicans are low-affinity endostatin receptors 总被引:16,自引:0,他引:16
Karumanchi SA Jha V Ramchandran R Karihaloo A Tsiokas L Chan B Dhanabal M Hanai JI Venkataraman G Shriver Z Keiser N Kalluri R Zeng H Mukhopadhyay D Chen RL Lander AD Hagihara K Yamaguchi Y Sasisekharan R Cantley L Sukhatme VP 《Molecular cell》2001,7(4):811-822
Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities. 相似文献
6.
Antiangiogenesis signals by endostatin. 总被引:49,自引:0,他引:49
7.
Endostatin inhibits adhesion of endothelial cells to collagen I via alpha(2)beta(1) integrin, a possible cause of prevention of chondrosarcoma growth 总被引:10,自引:0,他引:10
Furumatsu T Yamaguchi N Nishida K Kawai A Kunisada T Namba M Inoue H Ninomiya Y 《Journal of biochemistry》2002,131(4):619-626
Endostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of alpha(2), alpha(5)beta(1), and alpha(V)beta(3) integrins but not that of alpha(1) integrin. Our results suggest that endostatin might suppress the alpha(2)beta(1) integrin function of endothelial cells via alpha(5)beta(1) or alpha(V)beta(3) integrin. We propose here that endostatin might be effective for anti-angiogenic therapy for human chondrosarcomas through the suppression of alpha(2)beta(1) integrin functions in endothelial cells. 相似文献
8.
L Taddei P Chiarugi L Brogelli P Cirri L Magnelli G Raugei M Ziche H J Granger V Chiarugi G Ramponi 《Biochemical and biophysical research communications》1999,263(2):340-345
Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy. 相似文献
9.
Protein kinase B inhibits endostatin-induced apoptosis in HUVECs 总被引:10,自引:0,他引:10
Kang HY Shim D Kang SS Chang SI Kim HY 《Journal of biochemistry and molecular biology》2006,39(1):97-104
Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8. 相似文献
10.
目的:探讨内皮抑素对人脐静脉内皮细胞(HUVEC)及体外微血管模型的作用及其可能的机制。方法:1.MTT法检测不同浓度(10~50μg/ml)内皮抑素作用72h和30μg/ml内皮抑素作用不同时间(24~72h)对HUVEC细胞的影响;2、电镜观察HUVEC细胞超微结构的变化;3.光镜下观察内皮抑素(30μg/ml)对体外人造血管模型的影响。结果:1.MTT检测显示,内皮抑素(20~50μg/ml)能抑制HUVEC细胞的增殖(P〈0.05,P〈0.01),具有剂量-时间依赖性。2.电镜观察,HUVEC细胞内皮抑素作用组均出现凋亡改变。3.光镜观察,内皮抑素能抑制新生血管的形成,并能破坏新生的血管网。结论:内皮抑素能抑制人脐静脉血管内皮细胞HUVEC的增殖,并具有时间一剂量依赖性,机制可能为诱导细胞凋亡。提示,内皮抑素可能通过诱导HUVEC的凋亡抑制其增殖,并能破坏新生的血管。内皮抑素可能以此抑制机体肿瘤的生长与转移。 相似文献
11.
12.
The morphogenic properties of oligomeric endostatin are dependent on cell surface heparan sulfate 总被引:1,自引:0,他引:1
Clamp A Blackhall FH Henrioud A Jayson GC Javaherian K Esko J Gallagher JT Merry CL 《The Journal of biological chemistry》2006,281(21):14813-14822
Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface. 相似文献
13.
Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble alpha5beta1 integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of alpha5beta1 integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through alpha5beta1 integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo. 相似文献
14.
Heljasvaara R Nyberg P Luostarinen J Parikka M Heikkilä P Rehn M Sorsa T Salo T Pihlajaniemi T 《Experimental cell research》2005,307(2):292-304
Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis. 相似文献
15.
Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. 总被引:64,自引:0,他引:64
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N Yamaguchi B Anand-Apte M Lee T Sasaki N Fukai R Shapiro I Que C Lowik R Timpl B R Olsen 《The EMBO journal》1999,18(16):4414-4423
Endostatin, produced as recombinant protein in human 293-EBNA cells, inhibits the migration of human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor (VEGF) in a dose-dependent manner and prevents the subcutaneous growth of human renal cell carcinomas in nude mice at concentrations and in doses that are from 1000- to 100 000-fold lower than those previously reported. The inhibition of migration is not affected by mutations which eliminate Zn or heparin binding and inhibition of tumor growth does not depend on Zn binding. The results of the migration assays suggest that endostatin causes a block at one or more steps in VEGF-induced migration, while VEGF in turn can cause a block of the inhibition by endostatin of VEGF-induced migration of HUVECs. 相似文献
16.
17.
Cirri L Donnini S Morbidelli L Chiarugi P Ziche M Ledda F 《The International journal of biological markers》1999,14(4):263-267
Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size. Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor. Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors. Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not. Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis. The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown. Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclinical and clinical trials. 相似文献
18.
Chura-Chambi RM Tornieri PH Spencer PJ Nascimento PA Mathor MB Morganti L 《Protein expression and purification》2004,35(1):11-16
Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF). 相似文献
19.
Reijerkerk A Mosnier LO Kranenburg O Bouma BN Carmeliet P Drixler T Meijers JC Voest EE Gebbink MF 《Molecular cancer research : MCR》2003,1(8):561-568
Endostatin is a fragment of collagen XVIII that acts as an inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study, we have investigated the cellular effects of insoluble endostatin. We previously found that insoluble endostatin shows all the hallmarks of amyloid aggregates and potently stimulates tissue plasminogen activator-mediated formation of the serine protease plasmin. We here show that amyloid endostatin induces plasminogen activation by endothelial cells, resulting in vitronectin degradation and plasmin-dependent endothelial cell detachment. Endostatin-mediated stimulation of plasminogen activation, vitronectin degradation, and endothelial cell detachment is inhibited by carboxypeptidase B, indicating an essential role for carboxyl-terminal lysines. Our results suggest that amyloid endostatin may inhibit angiogenesis and tumor growth by stimulating the fibrinolytic system. 相似文献
20.
Kheirollah Yari Saboor Afzali Hadi Mozafari Kamran Mansouri Ali Mostafaie 《Molecular biology reports》2013,40(2):1027-1033
Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni–NTA (Ni2+-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml. 相似文献