Advertisement

Molecular and Cellular Biochemistry

, Volume 339, Issue 1–2, pp 201–213 | Cite as

Angiostatic effects of K252a, a Trk inhibitor, in murine brain capillary endothelial cells

  • Shimon Lecht
  • Hadar Arien-Zakay
  • Martin Kohan
  • Peter I. Lelkes
  • Philip LazaroviciEmail author
Article

Abstract

Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCγ. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 ± 9%; (ii) reduced by 70 ± 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 ± 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 ± 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 ± 5% reduction of paracellular permeability and by 17 ± 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.

Keywords

K252a K252b Angiostatic Nerve growth factor TrkA receptor Signaling 

Notes

Acknowledgments

This work was supported in part by a grant-in-aid form the Stein Family Foundations (PL and PIL); PL is affiliated and partially supported by the David R. Bloom Center for Pharmacy; and the Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel; SL is supported by “Eshkol fellowship” from the Israeli Ministry of Science and Technology. The authors would like to acknowledge the help of Mrs. Zehava Cohen for graphics preparation and the constructive remarks of the referees.

References

  1. 1.
    Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6:273–286CrossRefPubMedGoogle Scholar
  2. 2.
    Herbst RS (2006) Therapeutic options to target angiogenesis in human malignancies. Expert Opin Emerg Drugs 11:635–650CrossRefPubMedGoogle Scholar
  3. 3.
    Calza L, Giardino L, Giuliani A, Aloe L, Levi-Montalcini R (2001) Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors. Proc Natl Acad Sci USA 98:4160–4165CrossRefPubMedGoogle Scholar
  4. 4.
    Nico B, Mangieri D, Benagiano V, Crivellato E, Ribatti D (2008) Nerve growth factor as an angiogenic factor. Microvasc Res 75:135–141CrossRefPubMedGoogle Scholar
  5. 5.
    Lecht S, Puxeddu I, Levi-Schaffer F, Reich R, Davidson B, Schaefer E, Marcinkiewicz C, Lelkes PI, Lazarovici P (2007) Nerve growth factor—a neurotrophin with angiogenic activity. In: Maragudakis M (ed) Angiogenesis: basic science and clinical applications. Transworld Research Network, Kerala, pp 99–113Google Scholar
  6. 6.
    Lazarovici P, Marcinkiewicz C, Lelkes PI (2006) Cross talk between the cardiovascular and nervous systems: neurotrophic effects of vascular endothelial growth factor (VEGF) and angiogenic effects of nerve growth factor (NGF)-implications in drug development. Curr Pharm Des 12:2609–2622CrossRefPubMedGoogle Scholar
  7. 7.
    Aloe L, Tirassa P, Bracci-Laudiero L (2001) Nerve growth factor in neurological and non-neurological diseases: basic findings and emerging pharmacological prospectives. Curr Pharm Des 7:113–123CrossRefPubMedGoogle Scholar
  8. 8.
    Kruttgen A, Schneider I, Weis J (2006) The dark side of the NGF family: neurotrophins in neoplasias. Brain Pathol 16:304–310CrossRefPubMedGoogle Scholar
  9. 9.
    Singer HS, Hansen B, Martinie D, Karp CL (1999) Mitogenesis in glioblastoma multiforme cell lines: a role for NGF and its TrkA receptors. J Neurooncol 45:1–8CrossRefPubMedGoogle Scholar
  10. 10.
    Menter DG, Herrmann JL, Marchetti D, Nicolson GL (1994) Involvement of neurotrophins and growth factors in brain metastasis formation. Invasion Metastasis 14:372–384PubMedGoogle Scholar
  11. 11.
    Ruggeri BA, Miknyoczki SJ, Singh J, Hudkins RL (1999) Role of neurotrophin-trk interactions in oncology: the anti-tumor efficacy of potent and selective trk tyrosine kinase inhibitors in pre-clinical tumor models. Curr Med Chem 6:845–857PubMedGoogle Scholar
  12. 12.
    Fiore M, Chaldakov GN, Aloe L (2009) Nerve growth factor as a signaling molecule for nerve cells and also for the neuroendocrine-immune systems. Rev Neurosci 20:133–145PubMedGoogle Scholar
  13. 13.
    Tuszynski MH, Thal L, Pay M, Salmon DP, Hoi Sang U, Bakay R, Patel P, Blesch A, Vahlsing HL, Ho G, Tong G, Potkin SG, Fallon J, Hansen L, Mufson EJ, Kordower JH, Gall C, Conner J (2005) A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 11:551–555CrossRefPubMedGoogle Scholar
  14. 14.
    Pittenger G, Vinik A (2003) Nerve growth factor and diabetic neuropathy. Exp Diabesity Res 4:271–285PubMedGoogle Scholar
  15. 15.
    Aloe L, Tirassa P, Lambiase A (2008) The topical application of nerve growth factor as a pharmacological tool for human corneal and skin ulcers. Pharmacol Res 57:253–258CrossRefPubMedGoogle Scholar
  16. 16.
    Dolle L, El Yazidi-Belkoura I, Adriaenssens E, Nurcombe V, Hondermarck H (2003) Nerve growth factor overexpression and autocrine loop in breast cancer cells. Oncogene 22:5592–5601CrossRefPubMedGoogle Scholar
  17. 17.
    Davidson B, Reich R, Lazarovici P, Nesland JM, Skrede M, Risberg B, Trope CG, Florenes VA (2003) Expression and activation of the nerve growth factor receptor TrkA in serous ovarian carcinoma. Clin Cancer Res 9:2248–2259PubMedGoogle Scholar
  18. 18.
    Cantarella G, Lempereur L, Presta M, Ribatti D, Lombardo G, Lazarovici P, Zappala G, Pafumi C, Bernardini R (2002) Nerve growth factor-endothelial cell interaction leads to angiogenesis in vitro and in vivo. FASEB J 16:1307–1309PubMedGoogle Scholar
  19. 19.
    Lazarovici P, Matsuda Y, Kaplan D, Guroff G (1997) The protein kinase inhibitors K252a and Staurosporine as modifiers of neurotrophin receptor signal transduction. In: Gutman Y, Lazarovici P (eds) Cellular and molecular mechanisms of toxin action: toxins and signal transduction. Harwood Academic Publishers, Amsterdam, pp 69–93Google Scholar
  20. 20.
    Tapley P, Lamballe F, Barbacid M (1992) K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene 7:371–381PubMedGoogle Scholar
  21. 21.
    Roux PP, Dorval G, Boudreau M, Angers-Loustau A, Morris SJ, Makkerh J, Barker PA (2002) K252a and CEP1347 are neuroprotective compounds that inhibit mixed-lineage kinase-3 and induce activation of Akt and ERK. J Biol Chem 277:49473–49480CrossRefPubMedGoogle Scholar
  22. 22.
    Perez-Pinera P, Hernandez T, Garcia-Suarez O, de Carlos F, Germana A, Del Valle M, Astudillo A, Vega JA (2007) The Trk tyrosine kinase inhibitor K252a regulates growth of lung adenocarcinomas. Mol Cell Biochem 295:19–26CrossRefPubMedGoogle Scholar
  23. 23.
    Morotti A, Mila S, Accornero P, Tagliabue E, Ponzetto C (2002) K252a inhibits the oncogenic properties of Met, the HGF receptor. Oncogene 21:4885–4893CrossRefPubMedGoogle Scholar
  24. 24.
    Takai N, Ueda T, Nishida M, Nasu K, Narahara H (2008) K252a is highly effective in suppressing the growth of human endometrial cancer cells, but has little effect on normal human endometrial epithelial cells. Oncol Rep 19:749–753PubMedGoogle Scholar
  25. 25.
    Takai N, Ueda T, Nishida M, Nasu K, Fukuda J, Miyakawa I (2005) K252a inhibits proliferation of ovarian cancer cells by upregulating p21WAF1. Oncol Rep 14:141–143PubMedGoogle Scholar
  26. 26.
    Illmer T, Ehninger G (2007) FLT3 kinase inhibitors in the management of acute myeloid leukemia. Clin Lymphoma Myeloma 8(Suppl 1):S24–S34CrossRefPubMedGoogle Scholar
  27. 27.
    Zacchigna S, Lambrechts D, Carmeliet P (2008) Neurovascular signalling defects in neurodegeneration. Nat Rev Neurosci 9:169–181CrossRefPubMedGoogle Scholar
  28. 28.
    Lecht S, Arien-Zakay H, Marcinkiewicz C, Lelkes PI, Lazarovici P (2009) Nerve growth factor-induced protection of brain capillary endothelial cells exposed to oxygen-glucose deprivation involves attenuation of Erk phosphorylation. J Mol Neurosci doi: 10.1007/s12031-12009-19318-12030
  29. 29.
    Arien-Zakay H, Lecht S, Perets A, Roszell B, Lelkes PI, Lazarovici P (2009) Quantitative assessment of neuronal differentiation in three-dimensional collagen gels using enhanced green fluorescence protein expressing PC12 pheochromocytoma cells. J Mol Neurosci 37:225–237CrossRefPubMedGoogle Scholar
  30. 30.
    Arien-Zakay H, Lecht S, Bercu MM, Tabakman R, Kohen R, Galski H, Nagler A, Lazarovici P (2008) Neuroprotection by cord blood neural progenitors involves antioxidants, neurotrophic and angiogenic factors. Exp Neurol 216:83–94CrossRefPubMedGoogle Scholar
  31. 31.
    Lelkes PI, Hahn KA, Karmiol S, Schmidt DH (1998) Hypoxia/reoxygenation enhances tube formation of cultured human microvascular endothelial cells: role of reactive oxygen species. In: Maragoudakis ME (ed) Angiogenesis. Plenum Press, New York and LondonGoogle Scholar
  32. 32.
    Papadimitriou E, Unsworth BR, Maragoudakis ME, Lelkes PI (1993) Time course and quantitation of extracellular matrix maturation in the chick chorioallantoic membrane and in cultured endothelial cells. Endothelium 1:207–219CrossRefGoogle Scholar
  33. 33.
    Mizuguchi H, Hashioka Y, Fujii A, Utoguchi N, Kubo K, Nakagawa S, Baba A, Mayumi T (1994) Glial extracellular matrix modulates gamma-glutamyl transpeptidase activity in cultured bovine brain capillary and bovine aortic endothelial cells. Brain Res 651:155–159CrossRefPubMedGoogle Scholar
  34. 34.
    Kohan M, Bader R, Puxeddu I, Levi-Schaffer F, Breuer R, Berkman N (2007) Enhanced osteopontin expression in a murine model of allergen-induced airway remodelling. Clin Exp Allergy 37:1444–1454PubMedGoogle Scholar
  35. 35.
    Omidi Y, Campbell L, Barar J, Connell D, Akhtar S, Gumbleton M (2003) Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. Brain Res 990:95–112CrossRefPubMedGoogle Scholar
  36. 36.
    Katzir I, Shani J, Regev K, Shabashov D, Lazarovici P (2002) A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors. J Mol Neurosci 18:251–264CrossRefPubMedGoogle Scholar
  37. 37.
    Lecht S, Foerster C, Arien-Zakay H, Marcinkiewicz C, Lazarovici P, Lelkes PI (2009) Cardiac microvascular endothelial cells express and release nerve growth factor but not fibroblast growth factor-2. In Vitro Cell Dev Biol Anim doi: 10.1007/s11626-11009-19267-11625
  38. 38.
    Kim H, Li Q, Hempstead BL, Madri JA (2004) Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells. J Biol Chem 279:33538–33546CrossRefPubMedGoogle Scholar
  39. 39.
    Kaplan DR, Miller FD (2000) Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10:381–391CrossRefPubMedGoogle Scholar
  40. 40.
    Ohmi K, Yamashita S, Hashimoto Y, Nonomura Y (1993) Induction of giant endothelial cells in culture by K-252a, a protein kinase inhibitor. Jpn J Pharmacol 63:195–202CrossRefPubMedGoogle Scholar
  41. 41.
    Vaudry D, Stork PJ, Lazarovici P, Eiden LE (2002) Signaling pathways for PC12 cell differentiation: making the right connections. Science 296:1648–1649CrossRefPubMedGoogle Scholar
  42. 42.
    Hartmann C, Zozulya A, Wegener J, Galla HJ (2007) The impact of glia-derived extracellular matrices on the barrier function of cerebral endothelial cells: an in vitro study. Exp Cell Res 313:1318–1325CrossRefPubMedGoogle Scholar
  43. 43.
    Grant DS, Lelkes PI, Fukuda K, Kleinman HK (1991) Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell Dev Biol 27A:327–336CrossRefPubMedGoogle Scholar
  44. 44.
    Papadimitriou E, Waters CR, Manolopoulos VG, Unsworth BR, Maragoudakis ME, Lelkes PL (2001) Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells. Endothelium 8:181–194CrossRefPubMedGoogle Scholar
  45. 45.
    Utoguchi N, Ikeda K, Saeki K, Oka N, Mizuguchi H, Kubo K, Nakagawa S, Mayumi T (1995) Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer. J Cell Physiol 163:393–399CrossRefPubMedGoogle Scholar
  46. 46.
    Ashino H, Shimamura M, Nakajima H, Dombou M, Kawanaka S, Oikawa T, Iwaguchi T, Kawashima S (2003) Novel function of ascorbic acid as an angiostatic factor. Angiogenesis 6:259–269CrossRefPubMedGoogle Scholar
  47. 47.
    Moser KV, Reindl M, Blasig I, Humpel C (2004) Brain capillary endothelial cells proliferate in response to NGF, express NGF receptors and secrete NGF after inflammation. Brain Res 1017:53–60CrossRefPubMedGoogle Scholar
  48. 48.
    Andjelkovic AV, Stamatovic SM, Keep RF (2003) The protective effects of preconditioning on cerebral endothelial cells in vitro. J Cereb Blood Flow Metab 23:1348–1355CrossRefPubMedGoogle Scholar
  49. 49.
    Thijssen VL, van Beijnum JR, Mayo KH, Griffioen AW (2007) Identification of novel drug targets for angiostatic cancer therapy; it takes two to tango. Curr Pharm Des 13:3576–3583CrossRefPubMedGoogle Scholar
  50. 50.
    Satoh M, Kokubu N, Matsuo K, Takayanagi I (1995) Alpha 1A-adrenoceptor subtype effectively increases Ca(2+)-sensitivity for contraction in rabbit thoracic aorta. Gen Pharmacol 26:357–362CrossRefPubMedGoogle Scholar
  51. 51.
    Ohmi K, Yamashita S, Nonomura Y (1990) Effect of K252a, a protein kinase inhibitor, on the proliferation of vascular smooth muscle cells. Biochem Biophys Res Commun 173:976–981CrossRefPubMedGoogle Scholar
  52. 52.
    Miura S, Matsuo Y, Saku K (2004) Simvastatin suppresses coronary artery endothelial tube formation by disrupting Ras/Raf/ERK signaling. Atherosclerosis 175:235–243CrossRefPubMedGoogle Scholar
  53. 53.
    Yang YH, Wang Y, Lam KS, Yau MH, Cheng KK, Zhang J, Zhu W, Wu D, Xu A (2008) Suppression of the Raf/MEK/ERK signaling cascade and inhibition of angiogenesis by the carboxyl terminus of angiopoietin-like protein 4. Arterioscler Thromb Vasc Biol 28:835–840CrossRefPubMedGoogle Scholar
  54. 54.
    Tabruyn SP, Griffioen AW (2007) Molecular pathways of angiogenesis inhibition. Biochem Biophys Res Commun 355:1–5CrossRefPubMedGoogle Scholar
  55. 55.
    Ali AS, Ali S, El-Rayes BF, Philip PA, Sarkar FH (2009) Exploitation of protein kinase C: a useful target for cancer therapy. Cancer Treat Rev 35:1–8CrossRefPubMedGoogle Scholar
  56. 56.
    Grothey A, Galanis E (2009) Targeting angiogenesis: progress with anti-VEGF treatment with large molecules. Nat Rev Clin Oncol 6:507–518CrossRefPubMedGoogle Scholar
  57. 57.
    Abdiche YN, Malashock DS, Pons J (2008) Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors. Protein Sci 17:1326–1335CrossRefPubMedGoogle Scholar
  58. 58.
    Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, Schwartz B, Simantov R, Kelley S (2006) Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 5:835–844CrossRefPubMedGoogle Scholar
  59. 59.
    Faivre S, Demetri G, Sargent W, Raymond E (2007) Molecular basis for sunitinib efficacy and future clinical development. Nat Rev Drug Discov 6:734–745CrossRefPubMedGoogle Scholar
  60. 60.
    Quintas-Cardama A, Cortes J (2008) Nilotinib: a phenylamino-pyrimidine derivative with activity against BCR-ABL, KIT and PDGFR kinases. Future Oncol 4:611–621CrossRefPubMedGoogle Scholar
  61. 61.
    Festuccia C, Muzi P, Gravina GL, Millimaggi D, Speca S, Dolo V, Ricevuto E, Vicentini C, Bologna M (2007) Tyrosine kinase inhibitor CEP-701 blocks the NTRK1/NGF receptor and limits the invasive capability of prostate cancer cells in vitro. Int J Oncol 30:193–200PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Shimon Lecht
    • 1
  • Hadar Arien-Zakay
    • 1
  • Martin Kohan
    • 1
  • Peter I. Lelkes
    • 2
  • Philip Lazarovici
    • 1
    Email author
  1. 1.School of Pharmacy-Institute for Drug Research, Faculty of MedicineThe Hebrew University of JerusalemJerusalemIsrael
  2. 2.Laboratory of Cellular Tissue Engineering, School of Biomedical Engineering, Science and Health SystemsDrexel UniversityPhiladelphiaUSA

Personalised recommendations