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Yongkil Hong - One of the best experts on this subject based on the ideXlab platform.
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Integrin αvβ3 is not significantly implicated in the anti-migratory effect of anti-angiogenic urokinase Kringle Domain
Oncology Reports, 2020Co-Authors: Eunju O, Sung Hee Hong, Yongkil HongAbstract:: The recombinant Kringle Domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the Kringle Domain of uPA and integrin alphavbeta3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin alphavbeta3 is involved in tumor angiogenesis, we investigated whether integrin alphavbeta3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-alphavbeta3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-alpha2beta1 nor anti-alpha5beta1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-alphavbeta3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins alphavbeta3, alpha2beta1 and alpha5beta1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin alphavbeta3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin alphavbeta3.
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Antiangiogenic and Antitumor Activities of the Cryptic Fragments with Kringle Architecture
Biomolecules & Therapeutics, 2020Co-Authors: Byoungshik Shim, Sung Hee Hong, Dae-shik Oh, Yongkil HongAbstract:Various angiogenesis inhibitors target vascular endothelial cells and block tumor angiogenesis. Angiostatin is a specific endogenous angiogenesis inhibitor in clinical trials, which contains only the first four triple loop structures, known as Kringle Domains. Its generated by proteolytic cleavage of its parent molecule plasminogen, which itself does not exhibit antiangiogenic activity. Kringle Domains from prothrombin, apolipoprotein, hepatocyte growth factor, urokinase and tissue-type plasminogen activator also elicit anti-angiogenic or antitumor activities in several model systems, albeit low amino acid sequence identity between angiostatin and each individual Kringle. However, the differential effects of each Kringle Domain on endothelial cell proliferation, and migration observed in these Kringle Domains, suggest that the amino acid sequence of the primary structure is still important although Kringle architecture is essential for anti-mlgiogenic activity. If it is further studied as to how amino acid sequence and Kringle architecture contributes in anti-angiogenic activity, with studies on underlying mechanisms of anti-angiogenesis by Kringle-based angiogenesis inhibitors, it will provide basis for the development of new potent anti-angiogenesis inhibitors and improvement of the efficacy of angiogenesis inhibitors.
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the recombinant Kringle Domain of urokinase plasminogen activator inhibits vegf165 induced angiogenesis of huvecs by suppressing vegfr2 dimerization and subsequent signal transduction
Iubmb Life, 2012Co-Authors: Hyun Jin Choi, Yongkil Hong, Su Jin Kang, Sung Hee HongAbstract:Summary The recombinant Kringle Domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGFinduced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGFinduced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals. 2012 IUBMB IUBMB Life, 64(3): 259–265, 2012
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enhancement of anti tumor activity by low dose combination of the recombinant urokinase Kringle Domain and celecoxib in a glioma model
Cancer Letters, 2010Co-Authors: O Eunju, Sung Hee Hong, Bae Jun Oh, Yongkil HongAbstract:Abstract The Kringle Domain of urokinase-type plasminogen activator (UK1) has anti-angiogenic and anti-tumor effects. Celecoxib, an inhibitor of cyclooxygenase type 2, also suppresses angiogenesis and tumor growth. To look for potential additive effects in their activities, we examined the anti-angiogenic and anti-tumor effects of the combination of UK1 and celecoxib for malignant gliomas. In vitro , the combination of UK1 and celecoxib enhanced inhibition of proliferation, migration, and tube formation of endothelial cells, although showing no enhancement of inhibition of U87 cell growth. However, in vivo models, combination treatment of intracerebral U87 malignant glioma xenografts in nude mice with UK1 (10 mg/kg/day) and celecoxib (10 mg/kg/day) at lower doses resulted in even more potent inhibition of tumor growth than each monotherapy (by 81% compared to untreated tumors), with drastic decrease of the expression of angiogenesis-related factors and increase of apoptosis in the tumor tissues. Interestingly, UK1 inhibited VEGF or bFGF-induced phosphorylation of ERK1/2 in ECs, whereas celecoxib showed no such effects. However, celecoxib inhibited U87 cell growth and directly suppressed their VEGF production. Therefore, our data suggest that combined use at low doses of UK1 and celecoxib with different anti-angiogenic mechanisms provides a desirable strategy for anti-glioma therapy.
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the recombinant Kringle Domain of urokinase plasminogen activator inhibits in vivo malignant glioma growth
Cancer Science, 2007Co-Authors: Sung Hee Hong, Byoungshik Shim, Yongkil HongAbstract:In a previous report, the recombinant Kringle Domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, α-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis. (Cancer Sci 2007; 98: 253–258)
Francis J Castellino - One of the best experts on this subject based on the ideXlab platform.
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The importance of the hydrophobic components of the binding energies in the interaction of ω‐amino acid ligands with isolated Kringle polypeptide Domains of human plasminogen
International Journal of Peptide and Protein Research, 2009Co-Authors: Nick Menhart, Francis J CastellinoAbstract:: Three of the five Kringle Domains of human plasminogen (HPg), viz the first, fourth and fifth, exhibit significantly strong binding to omega-amino acids, such as epsilon-aminocaproic acid (EACA) and transaminomethylcyclohexane-1-carboxylic acid (AMCHA). In all cases, ligand stabilization is due to ion dipole attractions of its charged groups with polypeptide side chains, as well as hydrophobic clustering of the ligand methylene groups with appropriate hydrophobic residues within the Kringle Domain. In order to estimate the significance of the hydrophobic components of ligand stabilization, we have sought a more detailed description of these binding interactions. The standard thermodynamic binding parameters, delta G degrees, delta H degrees and delta S degrees, for association of EACA and AMCHA with isolated recombinant Kringle regions of HPg have been determined at several temperatures to evaluate the changes in standard heat capacities (delta C degrees p) accompanying these interactions. In each case, the delta C degrees p values of binding were negative and in the range -36 to -91 cal mol -1 K -1, reflective of the importance of the hydrophobic components of the binding process and their probable effects on surrounding water structure.
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a fast acting modular structured staphylokinase fusion with Kringle 1 from human plasminogen as the fibrin targeting Domain offers improved clot lysis efficacy
Journal of Biological Chemistry, 2003Co-Authors: Sauching Wu, Francis J Castellino, Suilam WongAbstract:Abstract To develop a fast-acting clot dissolving agent, a clot-targeting Domain derived from the Kringle-1 Domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein inBacillus subtilis and Pichia pastoris was examined. The Kringle Domain in the fusion protein produced fromB. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase Domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each Domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.
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an internal histidine residue from the bacterial surface protein pam mediates its binding to the Kringle 2 Domain of human plasminogen
Journal of Peptide Research, 2000Co-Authors: M M Schenone, Mary Prorok, Scott E Warder, J A Martin, Francis J CastellinoAbstract:: The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a Kringle Domain were investigated using an up-regulated lysine binding Kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant Kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.
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High‐level secretion in Pichia pastoris and biochemical characterization of the recombinantKringle 2 Domain of tissue‐type plasminogen activator
Biotechnology and Applied Biochemistry, 1997Co-Authors: Stephanie L Nilsen, Melanie E Deford, Mary Prorok, Bakshy A K Chibber, Roger K Bretthauer, Francis J CastellinoAbstract:The Kringle 2 (K2) Domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI15 and KM71. This construct contained a hexahistidine sequence at the C-terminus of the Kringle to aid in purification by immobilized metal-ion-affinity chromatography. The exact amino acid sequence of the isolated Kringle was EAEAYV-[K2 tPA ]SR(H) 6 , where [K2 tPA ] represents amino acid sequence residues C 1 -C 82 of the Kringle Domain (residues 180-261 of tPA). The clones of the yeast transformants provided large amounts of the recombinant (r)-[K2 tPA ]-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter. Purification of the Kringle Domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni + -affinity chromatography or lysine-Sepharose affinity chromatography. N-linked glycans were present on approx. 30% of this yeast-expressed material, at N 5 of the Kringle (corresponds to N 11 of the particular construct, N 184 of full-length tPA). The expressed recombinant Kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 Domain of the protein, interacted properly with various ω-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1 H-NMR. The results demonstrate that the P. pastoris system can be employed to obtain large amounts of secreted and properly folded Kringle Domains.
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role of tryptophan 63 of the Kringle 2 Domain of tissue type plasminogen activator in its thermal stability folding and ligand binding properties
Biochemistry, 1997Co-Authors: Yuan Chang, Jaroslav Zajicek, Francis J CastellinoAbstract:: Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W63, of the isolated recombinant Kringle 2 Domain of tissue-type plasminogen activator (r-K2tPA), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties of this isolated protein Domain. The variants were expressed in Pichia pastoris cells. The binding constants of epsilon-aminocaproic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrations of the alterations in intrinsic fluorescence of the variant Kringles with the ligands. As compared to wild-type r-K2tPA, increases in the Kd (dissociation) values of approximately 15-fold and 20-200-fold were found for the W63F and W63Y mutants, respectively, toward these three ligands. Neither the W63H nor the W63S variant interacted with these same ligands. Differential scanning calorimetric analyses were also performed on each of the peptides to determine whether the alterations affected the conformational stability of wtr-K2tPA. The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (Tm) values that were 12-20 degrees C lower than that of wtr-K2tPA. Addition of EACA resulted in increases (approximately 12 degrees C) in the Tm values of r-[W63F]-K2tPA and r-[W63Y]K2tPA, a result showing that EACA stabilized the native conformations adopted by these Kringle Domains. As expected from its greatly diminished binding to r-[W63H]K2tPA and r-[W63S]-K2tPA, high concentrations of EACA had little effect on the Tm of thermal denaturation of these latter mutants. 1H-NMR analysis of the two aromatic mutant Kringles was employed to assess their overall comparative folding properties. The high upfield chemical shifts (-0.98 ppm) of the CH3(delta') protons of L47, a major signal of proper Kringle folding, were slightly lowered to -0.83 to -0.86 ppm in the cases of all of the mutants. This is due to alterations in the W25-L47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distance relationships of these two amino acid side chains. Assignments of nearly all of the protons of the aromatic residues in the W63F and W63Y mutants were accomplished, and few additional differences from their wild-type counterpart were noted. Reactivities of the mutants against four different monoclonal antibodies directed to wtr-K2tPA revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W63. We conclude that W63 possesses an important direct role in the ligand binding properties of r-K2tPA. This residue also contributes significantly to the stability of the native conformation of this Kringle Domain and perhaps to maintenance of local conformations.
Richard M Lawn - One of the best experts on this subject based on the ideXlab platform.
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Loss of a Splice Donor Site at a ‘Skipped Exon’ in a Gene Homologous to Apolipoprotein(a) Leads to an mRNA Encoding a Protein Consisting of a Single Kringle Domain
Arteriosclerosis Thrombosis and Vascular Biology, 1995Co-Authors: Christopher D Byrne, Karen Schwartz, Richard M LawnAbstract:Abstract Apolipoprotein(a) [apo(a)] and plasminogen are located in a gene cluster on chromosome 6 together with two other genes that share highly homologous 5′ flanking regions. We have isolated the human liver transcript derived from one of these genes, designated apo(a)-related gene C, that encodes a polypeptide of 132 amino acids composed of a secretion signal and a single Kringle Domain. Although the gene encodes several additional Kringle Domains, sequence analysis shows that the second Kringle is incomplete in the derived mRNA because it lacks an apparent exon present in the gene. Analysis of genomic sequence shows that the predicted exon at this site lacks a canonical splice donor site. This results in “exon skipping” during maturation of the mRNA, causing a coding frame shift and the presence of premature stop codons.
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loss of a splice donor site at a skipped exon in a gene homologous to apolipoprotein a leads to an mrna encoding a protein consisting of a single Kringle Domain
Arteriosclerosis Thrombosis and Vascular Biology, 1995Co-Authors: Christopher D Byrne, Karen Schwartz, Richard M LawnAbstract:Abstract Apolipoprotein(a) [apo(a)] and plasminogen are located in a gene cluster on chromosome 6 together with two other genes that share highly homologous 5′ flanking regions. We have isolated the human liver transcript derived from one of these genes, designated apo(a)-related gene C, that encodes a polypeptide of 132 amino acids composed of a secretion signal and a single Kringle Domain. Although the gene encodes several additional Kringle Domains, sequence analysis shows that the second Kringle is incomplete in the derived mRNA because it lacks an apparent exon present in the gene. Analysis of genomic sequence shows that the predicted exon at this site lacks a canonical splice donor site. This results in “exon skipping” during maturation of the mRNA, causing a coding frame shift and the presence of premature stop codons.
Sung Hee Hong - One of the best experts on this subject based on the ideXlab platform.
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Antiangiogenic and Antitumor Activities of the Cryptic Fragments with Kringle Architecture
Biomolecules & Therapeutics, 2020Co-Authors: Byoungshik Shim, Sung Hee Hong, Dae-shik Oh, Yongkil HongAbstract:Various angiogenesis inhibitors target vascular endothelial cells and block tumor angiogenesis. Angiostatin is a specific endogenous angiogenesis inhibitor in clinical trials, which contains only the first four triple loop structures, known as Kringle Domains. Its generated by proteolytic cleavage of its parent molecule plasminogen, which itself does not exhibit antiangiogenic activity. Kringle Domains from prothrombin, apolipoprotein, hepatocyte growth factor, urokinase and tissue-type plasminogen activator also elicit anti-angiogenic or antitumor activities in several model systems, albeit low amino acid sequence identity between angiostatin and each individual Kringle. However, the differential effects of each Kringle Domain on endothelial cell proliferation, and migration observed in these Kringle Domains, suggest that the amino acid sequence of the primary structure is still important although Kringle architecture is essential for anti-mlgiogenic activity. If it is further studied as to how amino acid sequence and Kringle architecture contributes in anti-angiogenic activity, with studies on underlying mechanisms of anti-angiogenesis by Kringle-based angiogenesis inhibitors, it will provide basis for the development of new potent anti-angiogenesis inhibitors and improvement of the efficacy of angiogenesis inhibitors.
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Integrin αvβ3 is not significantly implicated in the anti-migratory effect of anti-angiogenic urokinase Kringle Domain
Oncology Reports, 2020Co-Authors: Eunju O, Sung Hee Hong, Yongkil HongAbstract:: The recombinant Kringle Domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the Kringle Domain of uPA and integrin alphavbeta3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin alphavbeta3 is involved in tumor angiogenesis, we investigated whether integrin alphavbeta3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-alphavbeta3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-alpha2beta1 nor anti-alpha5beta1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-alphavbeta3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins alphavbeta3, alpha2beta1 and alpha5beta1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin alphavbeta3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin alphavbeta3.
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the recombinant Kringle Domain of urokinase plasminogen activator inhibits vegf165 induced angiogenesis of huvecs by suppressing vegfr2 dimerization and subsequent signal transduction
Iubmb Life, 2012Co-Authors: Hyun Jin Choi, Yongkil Hong, Su Jin Kang, Sung Hee HongAbstract:Summary The recombinant Kringle Domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGFinduced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGFinduced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals. 2012 IUBMB IUBMB Life, 64(3): 259–265, 2012
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abstract 1289 the recombinant Kringle Domain of urokinase plasminogen activator upa inhibits vegf165 induced proliferation of huvec cells
Cancer Research, 2010Co-Authors: Sung Hee HongAbstract:Angiogenesis is associated with tumor growth, invasion and metastasis. Many growth factors and cytokines are engaged in angiogenesis. Among them, vascular endothelial growth factor (VEGF) is one of the most potent and specific angiogenic factors and stimulates endothelial cell proliferation, protease expression, migration and subsequent differentiation to form new vessels. It has been previously revealed that the recombinant Kringle Domain of urokinase plasminogen activators (uPA) (UK1) exerts antiangiogenic activity against VEGF-induced angiogenesis using in vitro or in vivo model. In this study, we explored molecular signaling mechanisms involved in this activity. The antiangiogenic mechanism of UK1 was investigated in vitro by measuring phosphorylation levels of VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF/VEGFR2 signal molecules, and pre-treatment with UK1 blocked not only VEGF-induced phosphorylation of VEGFR2 but also phosphorylations of downstream signaling proteins, such as Erk and Akt. In addition, VEGF induced secretion of MMP2 in HUVEC, which efficiently suppressed by UK1 pre-treatment. However, mRNA levels of MMP2 as well as of MMP9, Timp1 and Timp2 remained unaffected by UK1. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation and migration of HUVEC by suppressing VEGF/VEGFR2-mediated angiogenic signals and MMP2 activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1289.
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enhancement of anti tumor activity by low dose combination of the recombinant urokinase Kringle Domain and celecoxib in a glioma model
Cancer Letters, 2010Co-Authors: O Eunju, Sung Hee Hong, Bae Jun Oh, Yongkil HongAbstract:Abstract The Kringle Domain of urokinase-type plasminogen activator (UK1) has anti-angiogenic and anti-tumor effects. Celecoxib, an inhibitor of cyclooxygenase type 2, also suppresses angiogenesis and tumor growth. To look for potential additive effects in their activities, we examined the anti-angiogenic and anti-tumor effects of the combination of UK1 and celecoxib for malignant gliomas. In vitro , the combination of UK1 and celecoxib enhanced inhibition of proliferation, migration, and tube formation of endothelial cells, although showing no enhancement of inhibition of U87 cell growth. However, in vivo models, combination treatment of intracerebral U87 malignant glioma xenografts in nude mice with UK1 (10 mg/kg/day) and celecoxib (10 mg/kg/day) at lower doses resulted in even more potent inhibition of tumor growth than each monotherapy (by 81% compared to untreated tumors), with drastic decrease of the expression of angiogenesis-related factors and increase of apoptosis in the tumor tissues. Interestingly, UK1 inhibited VEGF or bFGF-induced phosphorylation of ERK1/2 in ECs, whereas celecoxib showed no such effects. However, celecoxib inhibited U87 cell growth and directly suppressed their VEGF production. Therefore, our data suggest that combined use at low doses of UK1 and celecoxib with different anti-angiogenic mechanisms provides a desirable strategy for anti-glioma therapy.
Ermanno Gherardi - One of the best experts on this subject based on the ideXlab platform.
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exploring the chemical space of the lysine binding pocket of the first Kringle Domain of hepatocyte growth factor scatter factor hgf sf yields a new class of inhibitors of hgf sf met binding
Chemical Science, 2015Co-Authors: A G Sigurdardottir, Ermanno Gherardi, T L Blundell, Anja Winter, A Sobkowicz, Marco Fragai, Dimitri Y Chirgadze, David B AscherAbstract:The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the Kringle 1 Domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.
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Ciba Foundation Symposium 212 - Plasminogen-Related Growth Factors - Evolution of Plasminogen‐Related Growth Factors (HGF/SF and HGF1/MSP)
Ciba Foundation symposium, 2007Co-Authors: Ermanno Gherardi, Ramon Gonzalez Manzano, Amanda Cottage, Kelvin Hawker, Samuel AparicioAbstract:: HGF/SF and HGF1/MSP define a novel growth factor family whose members share the Domain structure and the proteolytic process of activation of the blood proteinase precursor plasminogen. The amino acid and RNA sequences of HGF/SF and HGF1/MSP, the intron-exon organization of their genes and the predicted 3D structure of individual Domains indicate that HGF/SF and HGF1/MSP evolved along with plasminogen and other members of the Kringle-serine proteinase (KSP) superfamily from an ancestral gene that contained a single copy of the Kringle Domain, a serine proteinase Domain and an activation peptide connecting the two Domains. A series of intragenic duplications of the Kringle Domain, gene duplications, exon shuffling and deletions is responsible for the genes currently present in mammals, avians and amphibians. Plasminogen, HGF/SF and HGF1/MSP represent paradigmatic examples of the modern, multi-Domain proteins typically associated with vertebrate organisms and illustrate a novel evolutionary pathway that led to the emergence of molecules with growth regulatory activity from proteolytic enzymes.
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structural basis of hepatocyte growth factor scatter factor and met signalling
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Ermanno Gherardi, George Vande F Woude, Mark Youles, Maxim V Petoukhov, Sara Sandin, J T Finch, Larsgoran Ofverstedt, Ricardo Nunez Miguel, T L Blundell, Ulf SkoglundAbstract:The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multiDomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectoDomain (MET928) with 1:1 stoichiometry in which the N-terminal and first Kringle Domain of HGF/SF contact the face of the seven-blade β-propeller Domain of MET harboring the loops connecting the β-strands b–c and d–a, whereas the C-terminal serine proteinase homology Domain binds the opposite “b” face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectoDomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.
