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Stefan Niewiarowski - One of the best experts on this subject based on the ideXlab platform.
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Extracellular signal-regulated kinase (ERK) activation is required for GP Ibalpha-dependent endothelial cell migration.
Thrombosis and haemostasis, 2001Co-Authors: Jie Lian, Cezary Marcinkiewicz, Stefan Niewiarowski, Dorothy A. BeachamAbstract:The GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibalpha, and Echistatin inhibited alphavbeta3 binding. Albo A, B and Echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibalpha monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to alphavbeta3, EC GP Ibalpha initiates ERK activation, and regulates ERK-induced EC migration on vWF.
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Extracellular Signal-regulated Kinase (ERK) Activation Is Required for GP Ibα-dependent Endothelial Cell Migration
Thrombosis and Haemostasis, 2001Co-Authors: Jie Lian, Cezary Marcinkiewicz, Stefan Niewiarowski, Dorothy A. BeachamAbstract:SummaryThe GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibα, and Echistatin inhibited αvβ3 binding. Albo A, B and Echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibα monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to αvβ3, EC GP Ibα initiates ERK activation, and regulates ERK-induced EC migration on vWF.
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ec3 a heterodimeric disintegrin from echis carinatus inhibits human and murine α4 integrin and attenuates lymphocyte infiltration of langerhans islets in pancreas and salivary glands in nonobese diabetic mice
Biochemical and Biophysical Research Communications, 2000Co-Authors: Clara Brando, Cezary Marcinkiewicz, M.a.r. Mclane, Bruce I Goldman, Stefan NiewiarowskiAbstract:Abstract The venom of Echis carinatus suchoreki contains a monomeric disintegrin Echistatin (Mr 5,500 Da) that strongly inhibits αIIbβ3, αvβ3, and α5β1 integrins and a heterodimeric disintegrin called EC3 (M r 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing α4β1 and α4β7 to immobilized VCAM-1; it has a lower inhibitory effect on α5β1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to Echistatin, inhibited binding of monoclonal anti-α4 and anti-α5 antibodies to cells expressing α4β7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas Echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas Echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking α4β1 and α4β7 on murine lymphocytes.
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Structural requirements of Echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.
The Journal of biological chemistry, 1999Co-Authors: Iwona Wierzbicka-patynowski, Cezary Marcinkiewicz, Stefan Niewiarowski, Juan J. Calvete, Mariola Marcinkiewicz, M.a.r. MclaneAbstract:There are key differences between the amino acid residues of the RGD loops and the C termini of Echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify Echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant Echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished Echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased Echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of Echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.
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Identification and characterization of endothelial glycoprotein Ib using viper venom proteins modulating cell adhesion.
Blood, 1999Co-Authors: Li Tan, M. Anna Kowalska, Gabriel M. Romo, José A. López, Zbigniew Darzynkiewicz, Stefan NiewiarowskiAbstract:The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and Echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks vβ3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with vβ3. We found the following: (1) Binding of monoclonal antibodies against GPIb to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and Echistatin, an vβ3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by Echistatin. (4) CHO cells expressing GPIbβ/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIb was required for this reaction. Echicetin, but not Echistatin, inhibited the adhesion of cells transfected with GPIbβ/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified vβ3 receptor did not interact with the alboaggregins, thereby excluding the contribution of vβ3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.
Cezary Marcinkiewicz - One of the best experts on this subject based on the ideXlab platform.
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Differences in Binding of 99mTc-Disintegrins to Integrin αvβ3 on Tumor and Vascular Cells
Nuclear medicine and biology, 2007Co-Authors: Linda C. Knight, Jan E. Romano, Stephen C. Cosenza, Nabisa M. Iqbal, Cezary MarcinkiewiczAbstract:Abstract Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as αvβ3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing αvβ3 and to cultured tumor cells which express αvβ3. Methods Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and Echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with 99m Tc and tested for binding to cells in vitro. Results 99m Tc-Kistrin, flavoridin and VLO4 had the highest binding, 99m Tc-Echistatin had moderate binding, and 99m Tc-bitistatin and 99m Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to αvβ3 to various extents: Echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, 99m Tc-kistrin was not displaced by competing peptide or plasma proteins. Conclusions These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting αvβ3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting αvβ3 on vasculature because they bind strongly to surface receptors and are not readily displaced.
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characterization of a monomeric disintegrin ocellatusin present in the venom of the nigerian carpet viper echis ocellatus
FEBS Letters, 2002Co-Authors: Bryan J Smith, Juan J. Calvete, David R G Theakston, Ana Lucia Coelho, Christina Barjafidalgo, Cezary MarcinkiewiczAbstract:Ocellatusin is a new RGD-containing short monomeric disintegrin. It is a better inhibitor of alpha(5)beta(1) integrin and a more potent inducer of the expression of a ligand-induced binding site epitope on beta(1) integrin subunit than Echistatin. In further contrast to Echistatin, ocellatusin has a direct chemotactic stimulus on human neutrophils in vitro. The distinct effects of these two close evolutionarily related disintegrins might be explained by the presence of methionine-22 and histidine-29 in the RGD loop of ocellatusin, which are arginine and aspartic acid, respectively, in Echistatin. These mutations may modulate the conformation and/or recognition properties of the integrin-binding loop of ocellatusin.
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Extracellular signal-regulated kinase (ERK) activation is required for GP Ibalpha-dependent endothelial cell migration.
Thrombosis and haemostasis, 2001Co-Authors: Jie Lian, Cezary Marcinkiewicz, Stefan Niewiarowski, Dorothy A. BeachamAbstract:The GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibalpha, and Echistatin inhibited alphavbeta3 binding. Albo A, B and Echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibalpha monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to alphavbeta3, EC GP Ibalpha initiates ERK activation, and regulates ERK-induced EC migration on vWF.
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Extracellular Signal-regulated Kinase (ERK) Activation Is Required for GP Ibα-dependent Endothelial Cell Migration
Thrombosis and Haemostasis, 2001Co-Authors: Jie Lian, Cezary Marcinkiewicz, Stefan Niewiarowski, Dorothy A. BeachamAbstract:SummaryThe GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibα, and Echistatin inhibited αvβ3 binding. Albo A, B and Echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibα monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to αvβ3, EC GP Ibα initiates ERK activation, and regulates ERK-induced EC migration on vWF.
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ec3 a heterodimeric disintegrin from echis carinatus inhibits human and murine α4 integrin and attenuates lymphocyte infiltration of langerhans islets in pancreas and salivary glands in nonobese diabetic mice
Biochemical and Biophysical Research Communications, 2000Co-Authors: Clara Brando, Cezary Marcinkiewicz, M.a.r. Mclane, Bruce I Goldman, Stefan NiewiarowskiAbstract:Abstract The venom of Echis carinatus suchoreki contains a monomeric disintegrin Echistatin (Mr 5,500 Da) that strongly inhibits αIIbβ3, αvβ3, and α5β1 integrins and a heterodimeric disintegrin called EC3 (M r 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing α4β1 and α4β7 to immobilized VCAM-1; it has a lower inhibitory effect on α5β1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to Echistatin, inhibited binding of monoclonal anti-α4 and anti-α5 antibodies to cells expressing α4β7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas Echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas Echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking α4β1 and α4β7 on murine lymphocytes.
Robert M Hoffman - One of the best experts on this subject based on the ideXlab platform.
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the disintegrin Echistatin in combination with doxorubicin targets high metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:// Yasunori Tome 1, 2, 3 , Hiroaki Kimura 1, 4 , Naotoshi Sugimoto 5 , Hiroyuki Tsuchiya 4 , Fuminori Kanaya 3 , Michael Bouvet 2 , Robert M. Hoffman 1, 2 1 AntiCancer, Inc., San Diego, CA 92111, USA 2 Department of Surgery, University of California, San Diego, CA 92103, USA 3 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0125 Japan 4 Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan 5 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan Correspondence to: Robert M. Hoffman, email: all@anticancer.com Keywords: α v β 3 integrin, Echistatin, green fluorescent protein, red fluorescent protein, osteosarcoma, nude mice, orthotopic Received: September 20, 2016 Accepted: October 27, 2016 Published: November 22, 2016 ABSTRACT Echistatin, a cyclic RGD peptide, which is an antagonist of α v β 3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing α v β 3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by Echistatin ( P <0.05) as was overall growth. A doxorubicin (DOX)-Echistatin combination inhibited orthotopic tumor growth compared to untreated control ( P <0.01) or DOX alone ( P <0.05) in nude mice. Tumor-bearing mice treated with the DOX-Echistatin combination survived longer than those treated with DOX alone or control PBS ( P <0.01 and P <0.01, respectively). Echistatin also inhibited experimental lung metastasis of 143B-LM4 cells in nude mice. These results suggest that DOX in combination with a disintegrin has potential to treat osteosarcoma and that α v β 3 integrin may be a target for osteosarcoma.
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The disintegrin Echistatin in combination with doxorubicin targets high-metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models.
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:// Yasunori Tome 1, 2, 3 , Hiroaki Kimura 1, 4 , Naotoshi Sugimoto 5 , Hiroyuki Tsuchiya 4 , Fuminori Kanaya 3 , Michael Bouvet 2 , Robert M. Hoffman 1, 2 1 AntiCancer, Inc., San Diego, CA 92111, USA 2 Department of Surgery, University of California, San Diego, CA 92103, USA 3 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0125 Japan 4 Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan 5 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan Correspondence to: Robert M. Hoffman, email: all@anticancer.com Keywords: α v β 3 integrin, Echistatin, green fluorescent protein, red fluorescent protein, osteosarcoma, nude mice, orthotopic Received: September 20, 2016 Accepted: October 27, 2016 Published: November 22, 2016 ABSTRACT Echistatin, a cyclic RGD peptide, which is an antagonist of α v β 3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing α v β 3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by Echistatin ( P
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disintegrin targeting of an αvβ3 integrin over expressing high metastatic human osteosarcoma with Echistatin inhibits cell proliferation migration invasion and adhesion in vitro
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Tasuku Kiyuna, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:The in vitro efficacy of the disintegrin Echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, Echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with Echistatin in a time-dependent and dose-dependent manner (P <0.01). In vitro migration and invasion of 143B-LM4 cells were also inhibited by Echistatin in a dose-dependent manner (P <0.01, respectively). Cell adhesion to vitronectin of 143B-LM4 cells was also inhibited by Echistatin in a dose-dependent manner (P <0.01). These results suggest that αvβ3 integrin may be an effective target for osteosarcoma.
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Disintegrin targeting of an αvβ3 integrin-over-expressing high-metastatic human osteosarcoma with Echistatin inhibits cell proliferation, migration, invasion and adhesion in vitro.
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Tasuku Kiyuna, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:The in vitro efficacy of the disintegrin Echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, Echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with Echistatin in a time-dependent and dose-dependent manner (P
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abstract 5177 molecular targeting of αvβ3integrin by Echistatin inhibits tumor progression and metastasis in osteosarcoma
Cancer Research, 2012Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Hiroki Maehara, Katsuro Tomita, Robert M HoffmanAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Expression of αvβ3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. We tested Echistatin, an antagonist of αvβ3 integrin, as a molecular targeting drug, in an imageable orthotopic mouse model of human metastatic osteosarcoma. αvβ3 integrin expression was measured in a series of human osteosarcoma cell lines (143B parental and LM1-LM4) with increasing metastatic potential, using RT-PCR and immunoprecipitation. The cell lines expressed GFP in the nucleus and RFP in the cytoplasm and therefore could be imaged down to the subcellular level in vitro and in vivo. αvβ3 integrin expression correlated with the metastatic potential of the cells, with the LM4 cells showing the highest expression and highest metastatic capability, 6-fold and 18-fold, respectively, compared to parental 143B cells. In vitro cell proliferation of LM4 was inhibited by Echistatin (p<0.01). In vitro migration, invasion, and adhesion of LM4 were also inhibited by Echistatin (p<0.01). Tumor-induced angiogenesis by LM4 in the chick CAM model was also inhibited by Echistatin (p<0.05). Mice treated with an Echistatin-doxorubicin conjugate had significantly smaller primary tumors and longer survival compared to control untreated tumor-bearing mice (p<0.05 and p<0.01, respectively). Treatment with Echistatin resulted in decreased experimental pulmonary metastases, imaged at the cellular level, in a nude mouse model compared to the untreated control (p<0.01). The results of this study indicate that αvβ3 integrin plays an essential role in osteosarcoma metastasis and that Echistatin has potential to target αvβ3 integrin and inhibit tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5177. doi:1538-7445.AM2012-5177
Juan J. Calvete - One of the best experts on this subject based on the ideXlab platform.
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Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of Echistatin revealed by homonuclear NMR.
The Biochemical journal, 2005Co-Authors: Daniel Monleón, Juan J. Calvete, Vicent Esteve, Helena Kovacs, Bernardo CeldaAbstract:Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of Echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of Echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length Echistatin-alpha(v)beta3 docking model suggests that Echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.
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Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of Echistatin revealed by homonuclear NMR
Biochemical Journal, 2005Co-Authors: Daniel Monleón, Juan J. Calvete, Vicent Esteve, Helena Kovacs, Bernardo CeldaAbstract:Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-GlyAsp) motif expressed atthe tip ofthe integrin-binding loop and on its C-terminal tail. Previous NMR structures of Echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of Echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frameOverhauserenhancementspectroscopy).Thefulllength C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro 43 , His 44 and Lys 45 . The side chains of the amino acids of the RGD motifhavewell-definedconformations.Theintegrin-bindingloop displays an overall movement with maximal amplitude of 30 ◦ . Internal angular motions in the 100‐300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala 23 (flanking the R 24 GD 26 tripeptide) and Asp 26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length Echistatin‐ αvβ3 docking model suggests that Echistatin’s C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure‐function correlations.
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characterization of a monomeric disintegrin ocellatusin present in the venom of the nigerian carpet viper echis ocellatus
FEBS Letters, 2002Co-Authors: Bryan J Smith, Juan J. Calvete, David R G Theakston, Ana Lucia Coelho, Christina Barjafidalgo, Cezary MarcinkiewiczAbstract:Ocellatusin is a new RGD-containing short monomeric disintegrin. It is a better inhibitor of alpha(5)beta(1) integrin and a more potent inducer of the expression of a ligand-induced binding site epitope on beta(1) integrin subunit than Echistatin. In further contrast to Echistatin, ocellatusin has a direct chemotactic stimulus on human neutrophils in vitro. The distinct effects of these two close evolutionarily related disintegrins might be explained by the presence of methionine-22 and histidine-29 in the RGD loop of ocellatusin, which are arginine and aspartic acid, respectively, in Echistatin. These mutations may modulate the conformation and/or recognition properties of the integrin-binding loop of ocellatusin.
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Structural requirements of Echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.
The Journal of biological chemistry, 1999Co-Authors: Iwona Wierzbicka-patynowski, Cezary Marcinkiewicz, Stefan Niewiarowski, Juan J. Calvete, Mariola Marcinkiewicz, M.a.r. MclaneAbstract:There are key differences between the amino acid residues of the RGD loops and the C termini of Echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify Echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant Echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished Echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased Echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of Echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.
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Importance of the structure of the RGD-containing loop in the disintegrins Echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins.
FEBS letters, 1996Co-Authors: M.a.r. Mclane, Cezary Marcinkiewicz, S Vijay-kumar, Juan J. Calvete, Stefan NiewiarowskiAbstract:Echistatin and eristostatin are structurally homologous distintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 AND 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of S-S bridges as Echistatin. Synthetic Echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to alpha IIb beta 3, and decreased potency to block vitronectin binding to alpha v beta 3 as compared to wild-type Echistatin. Since eristostatin and Echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on Echistatin NMR coordinates. The RGD loops of eristostatin and Echistatin D27W were wider than Echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of alpha IIb beta 3 and alpha v beta 3.
Yasunori Tome - One of the best experts on this subject based on the ideXlab platform.
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the disintegrin Echistatin in combination with doxorubicin targets high metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:// Yasunori Tome 1, 2, 3 , Hiroaki Kimura 1, 4 , Naotoshi Sugimoto 5 , Hiroyuki Tsuchiya 4 , Fuminori Kanaya 3 , Michael Bouvet 2 , Robert M. Hoffman 1, 2 1 AntiCancer, Inc., San Diego, CA 92111, USA 2 Department of Surgery, University of California, San Diego, CA 92103, USA 3 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0125 Japan 4 Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan 5 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan Correspondence to: Robert M. Hoffman, email: all@anticancer.com Keywords: α v β 3 integrin, Echistatin, green fluorescent protein, red fluorescent protein, osteosarcoma, nude mice, orthotopic Received: September 20, 2016 Accepted: October 27, 2016 Published: November 22, 2016 ABSTRACT Echistatin, a cyclic RGD peptide, which is an antagonist of α v β 3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing α v β 3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by Echistatin ( P <0.05) as was overall growth. A doxorubicin (DOX)-Echistatin combination inhibited orthotopic tumor growth compared to untreated control ( P <0.01) or DOX alone ( P <0.05) in nude mice. Tumor-bearing mice treated with the DOX-Echistatin combination survived longer than those treated with DOX alone or control PBS ( P <0.01 and P <0.01, respectively). Echistatin also inhibited experimental lung metastasis of 143B-LM4 cells in nude mice. These results suggest that DOX in combination with a disintegrin has potential to treat osteosarcoma and that α v β 3 integrin may be a target for osteosarcoma.
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The disintegrin Echistatin in combination with doxorubicin targets high-metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models.
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:// Yasunori Tome 1, 2, 3 , Hiroaki Kimura 1, 4 , Naotoshi Sugimoto 5 , Hiroyuki Tsuchiya 4 , Fuminori Kanaya 3 , Michael Bouvet 2 , Robert M. Hoffman 1, 2 1 AntiCancer, Inc., San Diego, CA 92111, USA 2 Department of Surgery, University of California, San Diego, CA 92103, USA 3 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0125 Japan 4 Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan 5 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-8641 Japan Correspondence to: Robert M. Hoffman, email: all@anticancer.com Keywords: α v β 3 integrin, Echistatin, green fluorescent protein, red fluorescent protein, osteosarcoma, nude mice, orthotopic Received: September 20, 2016 Accepted: October 27, 2016 Published: November 22, 2016 ABSTRACT Echistatin, a cyclic RGD peptide, which is an antagonist of α v β 3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing α v β 3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by Echistatin ( P
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disintegrin targeting of an αvβ3 integrin over expressing high metastatic human osteosarcoma with Echistatin inhibits cell proliferation migration invasion and adhesion in vitro
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Tasuku Kiyuna, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:The in vitro efficacy of the disintegrin Echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, Echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with Echistatin in a time-dependent and dose-dependent manner (P <0.01). In vitro migration and invasion of 143B-LM4 cells were also inhibited by Echistatin in a dose-dependent manner (P <0.01, respectively). Cell adhesion to vitronectin of 143B-LM4 cells was also inhibited by Echistatin in a dose-dependent manner (P <0.01). These results suggest that αvβ3 integrin may be an effective target for osteosarcoma.
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Disintegrin targeting of an αvβ3 integrin-over-expressing high-metastatic human osteosarcoma with Echistatin inhibits cell proliferation, migration, invasion and adhesion in vitro.
Oncotarget, 2016Co-Authors: Yasunori Tome, Hiroaki Kimura, Tasuku Kiyuna, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Robert M HoffmanAbstract:The in vitro efficacy of the disintegrin Echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, Echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with Echistatin in a time-dependent and dose-dependent manner (P
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abstract 5177 molecular targeting of αvβ3integrin by Echistatin inhibits tumor progression and metastasis in osteosarcoma
Cancer Research, 2012Co-Authors: Yasunori Tome, Hiroaki Kimura, Naotoshi Sugimoto, Hiroyuki Tsuchiya, Fuminori Kanaya, Michael Bouvet, Hiroki Maehara, Katsuro Tomita, Robert M HoffmanAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Expression of αvβ3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. We tested Echistatin, an antagonist of αvβ3 integrin, as a molecular targeting drug, in an imageable orthotopic mouse model of human metastatic osteosarcoma. αvβ3 integrin expression was measured in a series of human osteosarcoma cell lines (143B parental and LM1-LM4) with increasing metastatic potential, using RT-PCR and immunoprecipitation. The cell lines expressed GFP in the nucleus and RFP in the cytoplasm and therefore could be imaged down to the subcellular level in vitro and in vivo. αvβ3 integrin expression correlated with the metastatic potential of the cells, with the LM4 cells showing the highest expression and highest metastatic capability, 6-fold and 18-fold, respectively, compared to parental 143B cells. In vitro cell proliferation of LM4 was inhibited by Echistatin (p<0.01). In vitro migration, invasion, and adhesion of LM4 were also inhibited by Echistatin (p<0.01). Tumor-induced angiogenesis by LM4 in the chick CAM model was also inhibited by Echistatin (p<0.05). Mice treated with an Echistatin-doxorubicin conjugate had significantly smaller primary tumors and longer survival compared to control untreated tumor-bearing mice (p<0.05 and p<0.01, respectively). Treatment with Echistatin resulted in decreased experimental pulmonary metastases, imaged at the cellular level, in a nude mouse model compared to the untreated control (p<0.01). The results of this study indicate that αvβ3 integrin plays an essential role in osteosarcoma metastasis and that Echistatin has potential to target αvβ3 integrin and inhibit tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5177. doi:1538-7445.AM2012-5177
