Two RGD-independent αvβ3 Integrin Binding Sites on Tumstatin Regulate Distinct Anti-tumor Properties

Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (α3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54–132) anti-angiogenic and a C-terminal (amino acids 185–203) anti-tumor cell activity (Maeshima, Y., et al.2000) J. Biol. Chem. 275, 21340–21348). Previous studies have identified the 185–203 amino acid sequence as a ligand for αvβ3integrin (Shahan, T. A., et al.(1999) Cancer Res. 59, 4584–4590). In the present study, we found distinct additional RGD-independent αvβ3 integrin binding site within 54–132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54–132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185–203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the αvβ3integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique αvβ3 integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (α3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54–132) anti-angiogenic and a C-terminal (amino acids 185–203) anti-tumor cell activity (Maeshima, Y., et al.2000) J. Biol. Chem. 275, 21340–21348). Previous studies have identified the 185–203 amino acid sequence as a ligand for αvβ3integrin (Shahan, T. A., et al.(1999) Cancer Res. 59, 4584–4590). In the present study, we found distinct additional RGD-independent αvβ3 integrin binding site within 54–132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54–132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185–203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the αvβ3integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique αvβ3 integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. noncollagenous 1 fetal calf serum phosphate-buffered saline bovine pulmonary arterial endothelial cells human umbilical vein endothelial cell(s) bovine serum albumin polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium vascular endothelial growth factor basic fibroblast growth factor Tumor growth and metastasis is associated with angiogenesis (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7153) Google Scholar,2Folkman J. Ann. Surg. 1972; 175: 409-416Crossref PubMed Scopus (1058) Google Scholar). Integrin αvβ3 is associated with angiogenic vascular cells and plays a critical role in angiogenesis promoting endothelial cell survival (3Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2704) Google Scholar, 4Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2161) Google Scholar). Vascular basement membrane is speculated to play an important role in regulating angiogenesis (5Madri J.A. Transplant. Immunol. 1997; 5: 179-183Crossref PubMed Scopus (58) Google Scholar,6Colorado P.C. Torre A. Kamphaus G. Maeshima Y. Hopfer H. Takahashi K. Volk R. Zamborsky E.D. Herman S. Ericksen M.B. Dhanabal M. Simons M. Post M. Kufe D.W. Weichselbaum R.R. Sukhatme V.P. Kalluri R. Cancer Res. 2000; 60: 2520-2526PubMed Google Scholar). Type IV collagen is expressed as six distinct α-chains, namely α1–α6 (7Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar), and assembled into triple helices and further forms a network to provide a scaffold for other macromolecules in basement membranes. These α-chains are composed of three domains, N-terminal 7 S domain, the middle triple-helical domain, and C-terminal globular noncollagenous domain (NC1)1(8Timpl R. Wiedemann H. van Delden V. Furthmayr H. Kuhn K. Eur. J. Biochem. 1981; 120: 203-211Crossref PubMed Scopus (643) Google Scholar). Type IV collagen is thought to play a crucial role in endothelial cell proliferation and behavior during the angiogenic process (5Madri J.A. Transplant. Immunol. 1997; 5: 179-183Crossref PubMed Scopus (58) Google Scholar, 6Colorado P.C. Torre A. Kamphaus G. Maeshima Y. Hopfer H. Takahashi K. Volk R. Zamborsky E.D. Herman S. Ericksen M.B. Dhanabal M. Simons M. Post M. Kufe D.W. Weichselbaum R.R. Sukhatme V.P. Kalluri R. Cancer Res. 2000; 60: 2520-2526PubMed Google Scholar,9Kamphaus G.D. Colorado P.C. Panka D.J. Hopfer H. Ramchandran R. Torre A. Maeshima Y. Mier J.W. Sukhatme V.P. Kalluri R. J. Biol. Chem. 2000; 275: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Synthetic peptides derived from NC1 domain of α3 chain of type IV collagen (α3(IV)NC1) has been shown to bind and inhibit the proliferation of melanoma, and other epithelial tumor cell lines,in vitro (10Han J. Ohno N. Pasco S. Monboisse J.C. Borel J.P. Kefalides N.A. J. Biol. Chem. 1997; 272: 20395-20401Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Shahan et al. (11Shahan T.A. Ziaie Z. Pasco S. Fawzi A. Bellon G. Monboisse J.C. Kefalides N.A. Cancer Res. 1999; 59: 4584-4590PubMed Google Scholar) localized the binding site for melanoma cells to amino acids 185–203 of α3(IV)NCI and also found this site bound to αvβ3integrin and CD47/IAP. Recently we identified that α3(IV)NC1 (termed “tumstatin” to include it in the family of endogenous inhibitor of angiogenesis derived from larger matrix protein with tumor “stasis” property) possessed a novel anti-angiogenic activity, and this activity was derived from amino acids 54–132, which does not contain the previously reported anti-tumor cell proliferation site (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Recently, Petitclerc et al. (13Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. J. Biol. Chem. 2000; 275: 8051-8061Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) reported similar results further supporting the notion that recombinant α3(IV)NCI inhibited angiogenesis and tumor growth. Although in these studies the issue of 185–203 αvβ3 binding site or RGD-independent sites within the NC1 domain were not addressed (13Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. J. Biol. Chem. 2000; 275: 8051-8061Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In the present study, we demonstrate that amino acids 54–132 binds to αvβ3 integrin in an RGD-independent manner, and this binding is essential for the anti-angiogenic property. The sequence encoding tumstatin or deletion mutants (tum-1–4) was amplified using polymerase chain reaction from the α3(IV)NCI/pDS vector (14Neilson E.G. Kalluri R. Sun M.J. Gunwar S. Danoff T. Mariyama M. Myers J.C. Reeders S.T. Hudson B.G. J. Biol. Chem. 1993; 268: 8402-8405Abstract Full Text PDF PubMed Google Scholar). The resulting cDNA fragment was ligated into pET22b(+) or pET28a(+) (Novagen, Madison, WI). Expression of recombinant protein in Escherichia coli and purification using nickel-nitrilotriacetic acid-agarose column (Qiagen) was performed as described previously (9Kamphaus G.D. Colorado P.C. Panka D.J. Hopfer H. Ramchandran R. Torre A. Maeshima Y. Mier J.W. Sukhatme V.P. Kalluri R. J. Biol. Chem. 2000; 275: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Recombinant mouse endostatin was expressed and purified as described previously (15Dhanabal M. Ramchandran R. Volk R. Stillman I.E. Lombardo M. Iruela-Arispe M.L. Simons M. Sukhatme V.P. Cancer Res. 1999; 59: 189-197PubMed Google Scholar). Only soluble protein was used in these experiments. Synthetic peptide CDCRGDCFC (RGD-4C), peptide CNGRC, and peptide PGLKGKRGDSGSPATWTTRG, which consists of the N-terminal 20 amino acids of tumstatin, were kindly provided by Ilex oncology, Inc. (San Antonio, TX) as gifts. These peptides were synthesized and characterized as described previously (16Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Crossref PubMed Scopus (1836) Google Scholar, 17Kalluri R. Gunwar S. Reeders S.T. Morrison K.C. Mariyama M. Ebner K.E. Noelken M.E. Hudson B.G. J. Biol. Chem. 1991; 266: 24018-24024Abstract Full Text PDF PubMed Google Scholar). Recombinant tumstatin and deletion mutants were analyzed by SDS-PAGE and immunoblotting as described previously (18Kalluri R. Sun M.J. Hudson B.G. Neilson E.G. J. Biol. Chem. 1996; 271: 9062-9068Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Rabbit antibody raised against C-terminal 36 amino acids of tumstatin (tum-4 antibody) was prepared as described previously (19Kalluri R. Shield C.F. Todd P. Hudson B.G. Neilson E.G. J. Clin. Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (258) Google Scholar). Goat anti-rabbit IgG antibody conjugated with horseradish peroxidase was purchased from Sigma. Bovine pulmonary arterial endothelial cells (C-PAE) and human umbilical vein endothelial cells (HUVECs) were all obtained from American Type Culture Collection. These cell lines were maintained in DMEM (C-PAE; Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS), 100 units/ml of penicillin, and 100 mg/ml streptomycin, or in EGM-2 (HUVEC; Clonetics, San Diego, CA). The melanoma cell line WM-164 was obtained from Dr. Meenhard Herlyn at the Wistar Institute (Philadelphia, PA) and maintained as described previously (20Herlyn D. Iliopoulos D. Jensen P.J. Parmiter A. Baird J. Hotta H. Adachi K. Ross A.H. Jambrosic J. Koprowski H. et al.Cancer Res. 1990; 50: 2296-2302PubMed Google Scholar). A suspension of C-PAE cells in DMEM containing was with medium was with DMEM containing and recombinant was performed as described previously (9Kamphaus G.D. Colorado P.C. Panka D.J. Hopfer H. Ramchandran R. Torre A. Maeshima Y. Mier J.W. Sukhatme V.P. Kalluri R. J. Biol. Chem. 2000; 275: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). was used to S. R. S. G. G. Clin. Biochem. 1997; PubMed Scopus Google Scholar). In peptides were used for C-PAE cells were and as described and tumstatin or was with of human αvβ3 or for at the was and for were performed using In WM-164 cells were This was performed as described previously M. S. A. 1997; PubMed Scopus Google Scholar). were with recombinant mouse or human type IV collagen was at a of were with 100 mg/ml of bovine serum albumin for Synthetic peptides or were at a of and were with mg/ml of for an peptides were at a of and were as described or in were with of antibody for In cells were with peptides or 100 of the cell was and for at the of cells was with mouse and mouse antibody to the human integrin were purchased from Life Technologies, Inc. antibody to the human α1–α6 Integrin and integrin were purchased from WM-164 cells were in experiments. are expressed as of with a was used to significant in A of was Recombinant human tumstatin was in E. coli using an or as a protein with a C-terminal (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). deletion mutants of tumstatin were expressed in E. Tumstatin consists of amino amino acids from triple-helical in the N-terminal and amino acids derived from NC1 consists of amino acids and is N-terminal amino consists of amino acids in the N-terminal of tumstatin, and is the C-terminal amino consists of amino acids in the of tumstatin (amino acids The E. deletion mutants were as a soluble protein and SDS-PAGE a to the of protein as described previously (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). and were using antibody in 1 which does not contain the sequence and is was not by antibody effect of recombinant tumstatin and deletion tumstatin and deletion mutants were expressed in E. coli using or as described and were and with or in the presence of or of or of recombinant cell was by as described and of as to not in a Recombinant tumstatin and deletion mutants were expressed in E. coli using or as described and were and with or in the presence of or of or of recombinant cell was by as described and of as to not and inhibited the proliferation of in a or inhibited C-PAE proliferation by and Tumstatin at inhibited the proliferation of C-PAE cells by In and did not inhibit the proliferation of C-PAE cells. inhibited the proliferation of and or did not (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). inhibited the proliferation of WM-164 by In of did not inhibit the proliferation of C-PAE cells not the of and to in the presence of and αvβ3 integrin shown in αvβ3 antibody inhibited the of by and or antibody by as with IgG Although antibody inhibition antibody did not cell αvβ3 antibody and antibody were used cell binding was by inhibition was also using C-PAE cells of The binding of to tumstatin was not inhibited by antibody these results suggest that αvβ3 and play in the anti-angiogenic of tumstatin. with type IV and were used to demonstrate the activity of used in this The and bind M.J. M.E. S. A. PubMed Scopus Google Scholar, M.J. M. J.W. A. S. J. Biol. 1990; PubMed Scopus Google Scholar). binding type IV collagen was inhibited by and antibody as with cells with αvβ3 integrin is a for vitronectin Cell. Full Text PDF PubMed Scopus Google Scholar). binding was inhibited by αvβ3 antibody by Previous studies have shown that and bind J. Biol. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). or antibody the binding of endothelial cells by and These further the of these the sequence of tumstatin used in study, an RGD sequence at amino acids is and it is this RGD sequence binds to αvβ3 integrin on endothelial cells. this peptide peptides CNGRC, which were previously reported to bind to vascular endothelial cells (16Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Crossref PubMed Scopus (1836) Google Scholar), and which the N-terminal RGD containing sequence of tumstatin, were used in cell were with RGD cell of was by as with to with peptide was also by as was from the studies (16Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Crossref PubMed Scopus (1836) Google Scholar). were with and C-PAE cells were with or peptides and on RGD peptide inhibited cell vitronectin by peptides did not inhibit the binding of to vitronectin In of to was not inhibited by with peptide cells were on tumstatin in the presence of soluble αvβ3 integrin protein to with endothelial cell binding to tumstatin, cell was inhibited by of cells with RGD peptides in to αvβ3 integrin protein did not the effect of αvβ3integrin protein on cell to tumstatin were on peptide RGD the cell was similar to that endothelial cells not to this tumstatin derived RGD similar results using not These results suggest that αvβ3 integrin bind to tumstatin RGD-independent the of to or in the presence of αvβ3 or shown in αvβ3 antibody inhibited the of to or by and of to and was not inhibited by the of WM-164 cells to tumstatin, or shown in melanoma cells bind to tumstatin, or and as with Tumstatin was with αvβ3 integrin protein for and to which were on and effect of tumstatin was with of protein The protein at of effect by with αvβ3 protein tumstatin did not inhibit endothelial cell proliferation. integrin protein was with tumstatin of αvβ3 effect of tumstatin was not was with αvβ3 protein for and to as described the effect of was also with of protein The αvβ3 protein at effect by This of activity, as with the of by the binding of αvβ3 integrin protein to with tumstatin, to and to for binding to αvβ3 In we reported that tumstatin, and that inhibition of endothelial cell proliferation (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). These did not inhibition of melanoma cell proliferation. In and protein did not inhibition of endothelial cell but inhibition of melanoma cell proliferation. In this study, was with αvβ3 protein for and to WM-164 cells. effect of was with of protein The αvβ3 protein at effect by with αvβ3 protein did not inhibit melanoma cell proliferation. which N-terminal RGD sequence of tumstatin, was for effect on endothelial cell proliferation. peptides at and did not inhibit proliferation of C-PAE cells This further that activity to inhibit endothelial cell proliferation is the cell of tumstatin and integrin an of tumstatin and integrin antibody was to C-PAE cells to on endothelial cell proliferation. integrin antibody did not inhibit proliferation at of the used in this In both tumstatin and endostatin inhibition of endothelial cell proliferation IgG did not inhibit proliferation of these cells. have that a to an angiogenic both of angiogenic and of angiogenesis (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7153) Google Scholar, D. J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In this vascular endothelial growth factor and basic fibroblast growth factor are of A of angiogenesis have been and such as Y. R.A. M. Y. J. Cell. 1994; 79: Full Text PDF PubMed Scopus Google Scholar), endostatin T. Y. N. G. E. J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), (9Kamphaus G.D. Colorado P.C. Panka D.J. Hopfer H. Ramchandran R. Torre A. Maeshima Y. Mier J.W. Sukhatme V.P. Kalluri R. J. Biol. Chem. 2000; 275: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), and P.C. Torre A. Kamphaus G. Maeshima Y. Hopfer H. Takahashi K. Volk R. Zamborsky E.D. Herman S. Ericksen M.B. Dhanabal M. Simons M. Post M. Kufe D.W. Weichselbaum R.R. Sukhatme V.P. Kalluri R. Cancer Res. 2000; 60: 2520-2526PubMed Google Scholar), are angiogenesis that are in Recently, tumstatin, recombinant NC1 domain of the α3 chain of human type IV collagen R. Wiedemann H. van Delden V. Furthmayr H. Kuhn K. Eur. J. Biochem. 1981; 120: 203-211Crossref PubMed Scopus (643) Google Scholar, N. P.J. R. M. S. A. J. Clin. Invest. PubMed Scopus Google Scholar), was identified as a protein with the anti-tumor Tumstatin was identified as an inhibitor of vascular endothelial cell proliferation and of using in vitro and in of angiogenesis and tumor growth (10Han J. Ohno N. Pasco S. Monboisse J.C. Borel J.P. Kefalides N.A. J. Biol. Chem. 1997; 272: 20395-20401Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Petitclerc et al. (13Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. J. Biol. Chem. 2000; 275: 8051-8061Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) that NC1 domain of and anti-angiogenic activity binding to αvβ3 or Although in these were not performed (13Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. J. Biol. Chem. 2000; 275: 8051-8061Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). to cell α3(IV)NC1 was a ligand for and not for integrin (13Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. J. Biol. Chem. 2000; 275: 8051-8061Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). results using cell with two endothelial cell suggest that αvβ3 and bind to tumstatin and that the αvβ3 binding is Recently, peptides amino to the C-terminal of tumstatin (amino acids 185–203) was reported to bind to αvβ3 integrin and inhibit tumor cell proliferation melanoma cell lines (11Shahan T.A. Ziaie Z. Pasco S. Fawzi A. Bellon G. Monboisse J.C. Kefalides N.A. Cancer Res. 1999; 59: 4584-4590PubMed Google Scholar). In study, we identified that anti-angiogenic activity of tumstatin is within amino acids by deletion and that the C-terminal fragment of tumstatin, amino acids did not possess anti-angiogenic activity (12Maeshima Y. Colorado P.C. Torre A. Holthaus K.A. Grunkemeyer J.A. Ericksen M.D. Hopfer H. Xiao Y. Stillman I.E. Kalluri R. J. Biol. Chem. 2000; 275: 21340-21348Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In this study, we used tumstatin and deletion mutants of tumstatin in cell to for integrin binding the N-terminal of tumstatin used in this study, is a RGD sequence (amino acids derived from triple-helical noncollagenous The RGD sequence is as an important binding site for αvβ3 integrin In this study, we that N-terminal amino acids this RGD binds to αvβ3 a peptide from amino acids of tumstatin containing the RGD sequence bound to endothelial cells inhibited endothelial cell proliferation. These results suggest that RGD sequence in the N-terminal of tumstatin is not a αvβ3 integrin binding the binding of tumstatin to endothelial cells is RGD-independent as was previously shown for the 185–203 amino acid sequence of tumstatin (11Shahan T.A. Ziaie Z. Pasco S. Fawzi A. Bellon G. Monboisse J.C. Kefalides N.A. Cancer Res. 1999; 59: 4584-4590PubMed Google Scholar). (amino acids which does not contain the C-terminal site (amino acids was shown to bind to αvβ3 in cell and inhibit endothelial cell proliferation. tumstatin or were with αvβ3 integrin protein to binding effect of tumstatin was This that the of soluble αvβ3 protein for tumstatin and the binding as with αvβ3 This that the αvβ3 binding site is within amino acids soluble integrin protein was used of αvβ3 the effect was not that the effect was on the of tumstatin binding to αvβ3 endothelial cells were with cell to was not the peptide binds to αvβ3 integrin and vitronectin binding to endothelial cells, we that tumstatin bind to a site on the αvβ3 integrin which is from RGD binding αvβ3 integrin was to inhibit endothelial cell to tumstatin, by binding to tumstatin and cell binding These results demonstrate the of tumstatin with αvβ3 integrin the αvβ3 integrin site by cyclic RGD peptide did not inhibit tumstatin binding to αvβ3 integrin on endothelial cells, that tumstatin binds to a RGD-independent and distinct site on αvβ3integrin the of an additional for tumstatin to the used in the results of αvβ3 integrin for anti-angiogenic activity is with the notion that the of αvβ3 on endothelial cells A. M. J. 1996; Google Scholar, K. H. A. Y. 1998; Google Scholar). angiogenesis on endothelial cell by αvβ3 integrin (3Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2704) Google Scholar, 4Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2161) Google Scholar), it is that the anti-angiogenic effect of tumstatin is by the of endothelial cells to the matrix such as vitronectin and as important J. Cell. 1998; 175: PubMed Scopus Google Scholar). tumstatin by the activity of to results two RGD-independent sites on tumstatin. The previously identified site (11Shahan T.A. Ziaie Z. Pasco S. Fawzi A. Bellon G. Monboisse J.C. Kefalides N.A. Cancer Res. 1999; 59: 4584-4590PubMed Google Scholar) and of a site no sequence and bind to distinct sites on αvβ3 integrin or bind to the site by protein for although the 185–203 sequence containing binds to endothelial cells, and this binding inhibited by integrin this is not to an anti-angiogenic to this these results suggest that although both tumstatin sites have the to bind to αvβ3 integrin on the cell of both cell additional necessary for the on endothelial or melanoma cells E. S. J. Montgomery A.M. Cheresh D.A. Brooks P.C. Cancer Res. 1999; 59: Google Scholar). In this many studies suggest that other cell to into the cell P. M. E. M. H. D. R. G. M. J. Biol. 1999; PubMed Scopus Google Scholar). The of αvβ3 integrin by as the two sequence of by additional binding protein on the cell such as P. M. E. M. H. D. R. G. M. J. Biol. 1999; PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google Scholar, M.B. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of two novel RGD-independent αvβ3 sites on tumstatin, two unique anti-tumor it a for inhibition of tumor growth. CNGRC, and peptides were from Ilex oncology, Inc. we Dr. Meenhard Herlyn for with WM-164 melanoma cells.