Pharmaceutical Research

, 25:2786

Tumor Endothelial Cell Targeted Cyclic RGD-modified Heparin Derivative: Inhibition of Angiogenesis and Tumor Growth

  • Kyeongsoon Park
  • Yoo-Shin Kim
  • Gee Young Lee
  • Rang-Woon Park
  • In-San Kim
  • Sang Yoon Kim
  • Youngro Byun
Research Paper



We prepared tumor endothelium targeted cRGD-modified heparin derivative (cRGD-HL) by coupling heparin-lithocholic acid (HL) with cRGDyK, and evaluated inhibition effects of cRGD-HL on angiogenesis and tumor growth.


To evaluate antiangiogenic activity of cRGD-HL, we performed tests on endothelial cell adhesion and migration to vitronectin, tube formation, binding affinity to purified αvβ3 integrin, and in vivo Matrigel plug assay. The antitumor activity of cRGD-HL was also evaluated by monitoring tumor growth and microvessel formation in squamous cell carcinoma (SCC7) tumor.


The cRGD-HL significantly inhibited adhesion and migration of endothelial cells to vitronectin, and tubular structures of endothelial cells. Compared to cRGDyK and HL, cRGD-HL has high binding affinity to purified αvβ3 integrin. The enhanced antiangiogenic effect of cRGD-HL was confirmed in Matrigel assay by showing the significant inhibition of bFGF-driven angiogenesis and blood vessel formation. It was thought that potent antiangiogenic effect of cRGD-HL was probably due to the interference of αvβ3-mediated interaction, resulting in the enhanced antitumoral activity against SCC7 tumor.


These results demonstrated that cRGD-modified heparin derivative enhanced anti-angiotherapeutic effects against solid tumor, and therefore, it could be applied to treat various cancers and angiogenic diseases as a potent angiogenesis inhibitor.


angiogenesis heparin derivative lithocholic acid RGD SCC7 



basic fibroblast growth factor


extracellular matrix


extracellular signal-regulated kinase


fibroblast growth factor receptor


heparin-lithocholic acid


human umbilical vein endothelial cells


mitogen-activated protein kinase


squamous cell carcinoma


  1. 1.
    J. Folkman. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285:1182–1186 (1971).PubMedGoogle Scholar
  2. 2.
    J. Folkman. What is the evidence that tumors are angiogenesis dependent? J. Natl. Cancer Inst. 82:4–6 (1990) doi:10.1093/jnci/82.1.4.PubMedCrossRefGoogle Scholar
  3. 3.
    J. Folkman. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1:27–31 (1995) doi:10.1038/nm0195-27.PubMedCrossRefGoogle Scholar
  4. 4.
    A. W. Griffioen, and G. Molema. Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases and chronic inflammation. Pharmacol. Rev. 53:237–268 (2000).Google Scholar
  5. 5.
    P. Carmeliet, and R. K. Jain. Angiogenesis in cancer and other diseases. Nature. 407:249–257 (2000) doi:10.1038/35025220.PubMedCrossRefGoogle Scholar
  6. 6.
    D. Srivastava, P. Cserjesi, and E. N. Olson. A subclass of bHLH proteins required for cardiac morphogenesis. Science. 270:1995–1999 (1995) doi:10.1126/science.270.5244.1995.PubMedCrossRefGoogle Scholar
  7. 7.
    H. P. Hammes, M. Brownlee, A. Jonczyk, A. Sutter, and K. T. Preissner. Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nat. Med. 2:529–533 (1996) doi:10.1038/nm0596-529.PubMedCrossRefGoogle Scholar
  8. 8.
    R. O. Schlingemann, F. J. Rietveld, R. M. de Waal, S. Ferrone, and D. J. Ruiter. Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol. 136:1393–1405 (1990).PubMedGoogle Scholar
  9. 9.
    M. A. Burg, R. Pasqualini, W. Arap, E. Ruoslahti, and W. B. Stallcup. NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res. 59:2869–2874 (1999).PubMedGoogle Scholar
  10. 10.
    S. Zitxmann, V. Ehemann, and M. Schwab. Arginine–glycine–aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo. Cancer Res. 62:5139–5143 (2000).Google Scholar
  11. 11.
    X. Chen, P. S. Conti, and R. A. Moats. In vivo near-infrared fluorescence imaging of integrin αvβ3 in brain tumor xenografts. Cancer Res. 64:8009–8014 (2004) doi:10.1158/0008-5472.CAN-04-1956.PubMedCrossRefGoogle Scholar
  12. 12.
    Z. Cheng, W. Yun, X. Zhengming, S. S. Gambhir, and X. Chen. Near-infrared fluorescent RGD peptides for optical imaging of integrin αvβ3 expression in living mice. Bioconj. Chem. 16:1433–1441 (2005) doi:10.1021/bc0501698.CrossRefGoogle Scholar
  13. 13.
    B. Haubner, H. J. Wester, W. A. Weber, and M. Schwaiger. Radiotracer-based strategies to image angiogenesis. Q. J. Nucl. Med. 47:189–199 (2003).PubMedGoogle Scholar
  14. 14.
    W. Arap, R. Pasqualini, and E. Ruoslahti. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 279:377–380 (1998) doi:10.1126/science.279.5349.377.PubMedCrossRefGoogle Scholar
  15. 15.
    H. M. Ellerby, W. Arap, L. M. Ellerby, R. Kain, R. Andrusiak, G. D. Rio, S. Krajewski, C. R. Lombardo, R. Rao, E. Ruoslahti, D. E. Bredesen, and R. Pasqualini. Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5:1032–1038 (1995).Google Scholar
  16. 16.
    W. Suh, S. O. Han, L. Yu, and S. W. Kim. An angiogenic, endothelial-cell-targeted polymeric gene carrier. Mol. Ther. 6:664–672 (2002) doi:10.1016/S1525-0016(02)90721-5.PubMedCrossRefGoogle Scholar
  17. 17.
    W. J. Kim, J. W. Yockman, M. Lee, J. H. Jeong, Y. H. Kim, and S. W. Kim. Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis. J. Control. Release. 106:224–234 (2005) doi:10.1016/j.jconrel.2005.04.016.PubMedCrossRefGoogle Scholar
  18. 18.
    W. J. Kim, J. W. Yockman, J. H. Jeong, L. V. Christensen, M. Lee, Y. H. Kim, and S. W. Kim. Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice. J. Control. Release. 114:381–388 (2006) doi:10.1016/j.jconrel.2006.05.029.PubMedCrossRefGoogle Scholar
  19. 19.
    J. D. Hood, M. Bednarski, R. Frausto, S. Guccione, R. A. Reisfeld, R. Xiang, and D. A. Cheresh. Tumor regression by targeted gene delivery to the neovasculature. Science. 296:2404–2407 (2002) doi:10.1126/science.1070200.PubMedCrossRefGoogle Scholar
  20. 20.
    N. Nasongkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J. S. Guthi, S. F. Chin, A. D. Sherry, D. A. Boothman, and J. Gao. Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems. Nano Lett. 6:2427–2430 (2006) doi:10.1021/nl061412u.PubMedCrossRefGoogle Scholar
  21. 21.
    K. Temming, D. L. Meyer, R. Zabinski, E. C. Dijkers, K. Poelstra, G. Molema, and R. J. Kok. Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels. Bioconjug. Chem. 17:1385–1394 (2006) doi:10.1021/bc060087z.PubMedCrossRefGoogle Scholar
  22. 22.
    K. Temming, D. L. Meyer, R. Zabinski, P. D. Senter, K. Poelstra, G. Molema, and R. J. Kok. Improved efficacy of alphavbeta3-targeted albumin conjugates by conjugation of a novel auristatin derivative. Mol. Pharm. 4:686–694 (2007) doi:10.1021/mp0700312.PubMedCrossRefGoogle Scholar
  23. 23.
    J. A. Varner, and D. A. Cheresh. Integrins and cancer. Curr. Opin. Cell Biol. 8:724–730 (1996) doi:10.1016/S0955-0674(96)80115-3.PubMedCrossRefGoogle Scholar
  24. 24.
    K. Park, K. Kim, I. C. Kwon, S. K. Kim, S. Lee, D. Y. Lee, and Y. Byun. Preparation and characterization of self-assembled nanoparticles of heparin-deoxycholic acid conjugates. Langmuir. 20:11726–11731 (2004) doi:10.1021/la048646i.PubMedCrossRefGoogle Scholar
  25. 25.
    K. Park, G. Y. Lee, Y. S. Kim, M. Yu, R. W. Park, I. S. Kim, S. Y. Kim, and Y. Byun. Heparin-deoxycholic acid chemical conjugate as an anticancer drug carrier and its antitumor activity. J. Control. Release. 114:300–306 (2006) doi:10.1016/j.jconrel.2006.05.017.PubMedCrossRefGoogle Scholar
  26. 26.
    K. Park, Y. S. Kim, G. Y. Lee, J. O. Nam, S. K. Lee, R. W. Park, S. Y. Kim, I. S. Kim, and Y. Byun. Antiangiogenic effect of bile acid acylated heparin derivative. Pharm. Res. 24:176–185 (2007) doi:10.1007/s11095-006-9139-6.PubMedCrossRefGoogle Scholar
  27. 27.
    M. K. Yu, D. Y. Lee, Y. S. Kim, K. Park, S. A. Park, D. H. Son, G. Y. Lee, J. H. Nam, S. Y. Kim, I. S. Kim, R. W. Park, and Y. Byun. Antiangiogenic and apoptotic properties of a novel amphiphilic folate-heparin-lithocholate derivative having cellular internality for cancer therapy. Pharm. Res. 24:705–714 (2007) doi:10.1007/s11095-006-9190-3.PubMedCrossRefGoogle Scholar
  28. 28.
    M. N. Levin, J. Hirsh, and J. G. Kelton. Heparin-induced bleeding. In D. A. Lane, and E. Lindhal (eds.), In heparin: chemical and biological properties, clinical applications, CRC Press, Boca Raton, 1989.Google Scholar
  29. 29.
    J. D. Douketis, J. S. Ginsberg, R. F. Burrows, E. K. Duku, C. E. Webber, and P. Brill-Edwards. The effects of long-term heparin therapy during pregnancy on bone density. A prospective matched cohort study. Thromb. Haemost. 75:254–257 (1996) doi:10.1159/000134495.PubMedCrossRefGoogle Scholar
  30. 30.
    T. Irimura, M. Nakajima, and G. L. Nicolson. Chemically modified heparins as inhibitors of heparan sulfate specific endo-beta-glucuronidase (heparanase) of metastatic melanoma cells. Biochemistry. 25:5322–5328 (1986) doi:10.1021/bi00366a050.PubMedCrossRefGoogle Scholar
  31. 31.
    P. E. Thorpe, E. J. Derbyshire, S. P. Andrade, N. Press, P. P. Knowles, S. King, G. J. Watson, Y. C. Yang, and M. Rao-Bette. Heparin-steroid conjugates: new angiogenesis inhibitors with antitumor activity in mice. Cancer Res. 53:3000–3007 (1993).PubMedGoogle Scholar
  32. 32.
    K. Ono, M. Ishihara, K. Ishikawa, Y. Ozeki, H. Deguchi, M. Sato, H. Hashimoto, Y. Saito, H. Yura, A. Kurita, and T. Maehara. Periodate-treated, non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung. Br. J. Cancer. 86:1803–1812 (2002) doi:10.1038/sj.bjc.6600307.PubMedCrossRefGoogle Scholar
  33. 33.
    C. Y. Pumphrey, A. M. Theus, S. Li, R. S. Parrish, and R. D. Sanderson. Neoglycans, carbodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis. Cancer Res. 62:3722–3728 (2002).PubMedGoogle Scholar
  34. 34.
    B. Gimelius, C. Busch, and M. Hook. Binding of heparin on the surface of cultured human endothelial cells. Thromb. Res. 12:773–782 (1978) doi:10.1016/0049-3848(78)90271-2.CrossRefGoogle Scholar
  35. 35.
    L. M. Hiebert, and L. B. Jaques. The observation of heparin on endothelium after injection. Thromb. Res. 8:195–204 (1976) doi:10.1016/0049-3848(76)90262-0.PubMedCrossRefGoogle Scholar
  36. 36.
    N. Sakamoto, and N. G. Tanaka. Mechanism of the synergistic effect of heparin and cortisone against angiogenesis and tumor growth. Cancer J. 2:9–16 (1988).Google Scholar
  37. 37.
    Y. Lee, H. T. Moon, and Y. Byun. Preparation of slightly hydrophobic heparin derivatives which can be used for solvent casting in polymeric formulation. Thromb. Res. 92:149–156 (1998) doi:10.1016/S0049-3848(98)00124-8.PubMedCrossRefGoogle Scholar
  38. 38.
    D. A. Jaffe, R. L. Nachman, C. G. Becker, and C. R. Minick. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745–2756 (1973) doi:10.1172/JCI107470.PubMedCrossRefGoogle Scholar
  39. 39.
    M. Aumailley, M. Gurrath, G. Muller, J. Calvete, R. Timpl, and H. Kessler. Arg–Gly–Asp, constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. FEBS Lett. 291:50–54 (1991) doi:10.1016/0014-5793(91)81101-D.PubMedCrossRefGoogle Scholar
  40. 40.
    S. J. Bogdanowich-Knipp, S. Chakrabarti, T. D. Williams, R. K. Dillman, and T. J. Siahaan. Solution stability of linear vs. cyclic RGD peptides. J. Pept. Res. 53:530–541 (1999).PubMedCrossRefGoogle Scholar
  41. 41.
    K. M. Yamada. Adhesive recognition sequences. J. Biol. Chem. 266:12809–12812 (1991).PubMedGoogle Scholar
  42. 42.
    J. Denekamp. Review article: angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Br. J. Radiol. 66:181–196 (1993).PubMedCrossRefGoogle Scholar
  43. 43.
    F. J. Burrows, and P. E. Thorpe. Vascular targeting: a new approach to the therapy of solid tumors. Pharmacol. Ther. 64:155–174 (1994) doi:10.1016/0163-7258(94)90037-X.PubMedCrossRefGoogle Scholar
  44. 44.
    J. P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S. L. Goodman, and M. A. Arnaout. Crystal structure of the extracellular segment of integrin alpha v beta3 in complex with an Arg-Gly-Asp ligand. Science. 296:151–155 (2002) doi:10.1126/science.1069040.PubMedCrossRefGoogle Scholar
  45. 45.
    P. A. Raj, E. Marcus, and R. Rein. Conformational requirements of suramin to target angiogenic growth factors. Angiogenesis. 2:183–199 (1998) doi:10.1023/A:1009244623717.PubMedGoogle Scholar
  46. 46.
    D. I. Leavesley, G. D. Ferguson, E. A. Wayner, and D. A. Cheresh. Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. J. Cell Biol. 117:1101–1107 (1991) doi:10.1083/jcb.117.5.1101.CrossRefGoogle Scholar
  47. 47.
    F. Hunter, J. Xie, C. Trimble, M. Bur, and K. C. Li. Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model. Mol. Cancer. 5:5 (2006) doi:10.1186/1476-4598-5-5.PubMedCrossRefGoogle Scholar
  48. 48.
    Y. Matsumura, and H. Maeda. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and antitumor agent smancs. Cancer Res. 46:6387–6392 (1986).PubMedGoogle Scholar
  49. 49.
    G. Y. Lee, S. K. Kim, and Y. Byun. Glucosylated heparin derivatives as non-toxic anti-cancer drugs. J. Control. Release. 123:46–55 (2007) doi:10.1016/j.jconrel.2007.07.017.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Kyeongsoon Park
    • 1
  • Yoo-Shin Kim
    • 2
  • Gee Young Lee
    • 3
  • Rang-Woon Park
    • 2
  • In-San Kim
    • 2
  • Sang Yoon Kim
    • 4
  • Youngro Byun
    • 3
  1. 1.Biomedical Research CenterKorea Institute of Science and TechnologySeoulSouth Korea
  2. 2.Department of Biochemistry and Cell Biology and Cell and Matrix Research Institute, School of MedicineKyungpook National UniversityDaeguSouth Korea
  3. 3.College of PharmacySeoul National UniversitySeoulSouth Korea
  4. 4.Department of Otolaryngology–Head and Neck Surgery, Asan Medical Center, College of MedicineUniversity of UlsanSeoulKorea

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