Journal of Neuro-Oncology

, Volume 78, Issue 3, pp 233–247 | Cite as

Inhibition of hypoxia inducible factor-1α (HIF-1α) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas

  • Randy L. Jensen
  • Brian T. Ragel
  • Kum Whang
  • David Gillespie
Laboratory Investigation



Hypoxia inducible factor-1α (HIF-1α) regulates vascular endothelial growth factor (VEGF), the presumed principal mediator of angiogenesis in malignant gliomas, under normal physiologic conditions. We examined the effect of HIF-1α on VEGF secretion, tumor growth, and angiogenesis in malignant gliomas.


We examined 175 human gliomas for expression of HIF-1α and its downstream-regulated proteins. HIF-1α expression and VEGF secretion in glioma cell lines under normoxia and hypoxia were examined using␣ELISA and Western blot. Malignant glioma cell lines were transfected with dominant-negative HIF-1α (DN-HIF-1α) expression vector or siRNA constructs against the HIF-1α gene. Growth studies were conducted on cells with the highest VEGF/HIF-1α inhibition isolated from stable transfected cell lines. MIB-1-labeling index and microvascular density (MVD) measurements were performed on the in vivo tumors.


HIF-1 expression correlates with malignant glioma phenotype and was not confined to perinecrotic, pseudopalisading cells. VEGF and HIF-1 expression was high in glioma cell lines even under normoxia, and increased after exposure to hypoxia or growth factor stimulation. Cells transfected with DN-HIF-1α or HIF-1α siRNA demonstrated decreased HIF-1α and VEGF secretion. In vivo but not in vitro growth decreased in response to VEGF and HIF-1 inhibition. HIF-1 siRNA studies showed decreased VEGF secretion and in vitro and in vivo growth of glioma cell lines. MVD was unchanged but MIB-1 proliferation index decreased for both types of HIF-1 inhibition.


VEGF and HIF-1α are elevated in malignant gliomas. HIF-1α inhibition results in VEGF secretion inhibition. HIF-1α expression affects glioma tumor growth, suggesting clinical applications for malignant glioma treatment.


angiogenesis brain tumor glioblastoma multiforme hypoxia inducible factor siRNA vascular endothelial growth factor 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by a grant from the Western Institute of Biomedical Research. We wish to thank Elizabeth Jensen, M.D., for her help with the pathological interpretation, technical advice, and photography. We thank Kristin Kraus for her editorial guidance.


  1. 1.
    Lantos PL, Vandenberg SR, Kleihues P, (2002). Tumours of the nervous system In: Graham DI, Lantos PL (eds). Greenfield's Neuropathology. Arnold, London, pp 583795Google Scholar
  2. 2.
    Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, Cavenee WK The WHO classification of tumors of the nervous system J Neuropathol Exp Neurol 61:215--225; 2002 discussion 226--229PubMedGoogle Scholar
  3. 3.
    Chan AS, Leung SY, Wong MP, Yuen ST, Cheung N, Fan YW, Chung LP Expression of vascular endothelial growth factor and its receptors in the anaplastic progression of astrocytoma, oligodendroglioma, and ependymoma Am J Surg Pathol 22:816--826, 1998CrossRefPubMedGoogle Scholar
  4. 4.
    Ke LD, Shi YX, Im SA, Chen X, Yung WK The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines Clin Cancer Res 6:2562--2572, 2000PubMedGoogle Scholar
  5. 5.
    Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 Mol Cell Biol 16:4604--4613, 1996PubMedGoogle Scholar
  6. 6.
    Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, Buechler P, Isaacs WB, Semenza GL, Simons JW Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases Cancer Res 59:5830--5835, 1999PubMedGoogle Scholar
  7. 7.
    Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E, Keshet E Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis Nature 394:485--490, 1998CrossRefPubMedGoogle Scholar
  8. 8.
    Maxwell PH, Dachs GU, Gleadle JM, Nicholls LG, Harris AL, Stratford IJ, Hankinson O, Pugh CW, Ratcliffe PJ Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth Proc Natl Acad Sci USA 94:8104--8109, 1997CrossRefPubMedGoogle Scholar
  9. 9.
    Ryan HE, Lo J, Johnson RS HIF-1 alpha is required for solid tumor formation and embryonic vascularization EMBO J 17:3005--3015, 1998CrossRefPubMedGoogle Scholar
  10. 10.
    Ryan HE, Poloni M, McNulty W, Elson D, Gassmann M, Arbeit JM, Johnson RS Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth Cancer Res 60:4010--4015, 2000PubMedGoogle Scholar
  11. 11.
    Zagzag D, Zhong H, Scalzitti JM, Laughner E, Simons JW, Semenza GL Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression Cancer 88:2606--2618, 2000CrossRefPubMedGoogle Scholar
  12. 12.
    Weidner N, Semple JP, Welch WR, Folkman J Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma N Engl J Med 324:1--8, 1991PubMedCrossRefGoogle Scholar
  13. 13.
    Jensen RL, Lee YS, Guijrati M, Origitano TC, Wurster RD, Reichman OH Inhibition of in vitro meningioma proliferation after growth factor stimulation by calcium channel antagonists: Part II -- Additional growth factors, growth factor receptor immunohistochemistry, and intracellular calcium measurements Neurosurgery 37:937--946, 1995 discussion 946--947PubMedCrossRefGoogle Scholar
  14. 14.
    An WG, Kanekal M, Simon MC, Maltepe E, Blagosklonny MV, Neckers LM Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha Nature 392:405--408, 1998CrossRefPubMedGoogle Scholar
  15. 15.
    Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway Cancer Res 62:4316--4324, 2002PubMedGoogle Scholar
  16. 16.
    Semenza GL Expression of hypoxia-inducible factor 1: mechanisms and consequences Biochem Pharmacol 59:47--53, 2000CrossRefPubMedGoogle Scholar
  17. 17.
    Jiang BH, Rue E, Wang GL, Roe R, Semenza GL Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1 J Biol Chem 271:17771--17778, 1996CrossRefPubMedGoogle Scholar
  18. 18.
    Semenza GL HIF-1: mediator of physiological and pathophysiological responses to hypoxia J Appl Physiol 88:1474--1480, 2000PubMedGoogle Scholar
  19. 19.
    Cho S, Choi YJ, Kim JM, Jeong ST, Kim JH, Kim SH, Ryu SE Binding and regulation of HIF-1alpha by a subunit of the proteasome complex, PSMA7 FEBS Lett 498:62--66, 2001CrossRefPubMedGoogle Scholar
  20. 20.
    Jensen RL, Leppla D, Rokosz N, Wurster RD Matrigel augments xenograft transplantation of meningioma cells into athymic mice Neurosurgery 42:130--135, 1998 discussion 135--136CrossRefPubMedGoogle Scholar
  21. 21.
    Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer Cancer Res 60:4693--4696, 2000PubMedGoogle Scholar
  22. 22.
    Lund EL, Hog A, Olsen MW, Hansen LT, Engelholm SA, Kristjansen PE Differential regulation of VEGF, HIF1alpha and angiopoietin-1, -2 and -4 by hypoxia and ionizing radiation in human glioblastoma Int J Cancer 108:833--838, 2004CrossRefPubMedGoogle Scholar
  23. 23.
    Nishikawa R, Cheng SY, Nagashima R, Huang HJ, Cavenee WK, Matsutani M Expression of vascular endothelial growth factor in human brain tumors Acta Neuropathol (Berl) 96:453--462, 1998CrossRefGoogle Scholar
  24. 24.
    Berkman RA, Merrill MJ, Reinhold WC, Monacci WT, Saxena A, Clark WC, Robertson JT, Ali IU, Oldfield EH Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms J Clin Invest 91:153--159, 1993PubMedCrossRefGoogle Scholar
  25. 25.
    Sondergaard KL, Hilton DA, Penney M, Ollerenshaw M, Demaine AG Expression of hypoxia-inducible factor 1alpha in tumours of patients with glioblastoma Neuropathol Appl Neurobiol 28:210--217, 2002CrossRefPubMedGoogle Scholar
  26. 26.
    Kaur B, Khwaja FW, Severson EA, Matheny SL, Brat DJ, Van Meir EG Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis Neuro-oncol 7:134--153, 2005CrossRefPubMedGoogle Scholar
  27. 27.
    Korkolopoulou P, Patsouris E, Konstantinidou AE, Pavlopoulos PM, Kavantzas N, Boviatsis E, Thymara I, Perdiki M, Thomas-Tsagli E, Angelidakis D, Rologis D, Sakkas D Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis Neuropathol Appl Neurobiol 30:267--278, 2004CrossRefPubMedGoogle Scholar
  28. 28.
    Brat DJ, Castellano-Sanchez AA, Hunter SB, Pecot M, Cohen C, Hammond EH, Devi SN, Kaur B, Van Meir EG Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population Cancer Res 64:920--927, 2004CrossRefPubMedGoogle Scholar
  29. 29.
    Plate KH Mechanisms of angiogenesis in the brain. J Neuropathol Exp Neurol 58:313--320, 1999PubMedCrossRefGoogle Scholar
  30. 30.
    Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant Nature 367:576--579, 1994CrossRefPubMedGoogle Scholar
  31. 31.
    Bernsen HJ, Rijken PF, Peters JP, Bakker H, van der Kogel AJ Delayed vascular changes after antiangiogenic therapy with antivascular endothelial growth factor antibodies in human glioma xenografts in nude mice Neurosurgery 43:570--575; 1998 discussion 575--576CrossRefPubMedGoogle Scholar
  32. 32.
    Clerici C, Matthay MA Hypoxia regulates gene expression of alveolar epithelial transport proteins J Appl Physiol 88:1890--1896, 2000PubMedGoogle Scholar
  33. 33.
    Semenza GL Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1 Annu Rev Cell Dev Biol 15:551--578, 1999CrossRefPubMedGoogle Scholar
  34. 34.
    Richard DE, Berra E, Pouyssegur J Angiogenesis: how a tumor adapts to hypoxia Biochem Biophys Res Commun 266:718--722, 1999CrossRefPubMedGoogle Scholar
  35. 35.
    Li J, Post M, Volk R, Gao Y, Li M, Metais C, Sato K, Tsai J, Aird W, Rosenberg RD, Hampton TG, Sellke F, Carmeliet P, Simons M PR39, a peptide regulator of angiogenesis Nat Med 6:49--55, 2000CrossRefPubMedGoogle Scholar
  36. 36.
    Richard DE, Berra E, Pouyssegur J Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells J Biol Chem 275:26765--26771, 2000PubMedCrossRefGoogle Scholar
  37. 37.
    Bernsen HJ, Rijken PF, Peters H, Raleigh JA, Jeuken JW, Wesseling P, van der Kogel AJ Hypoxia in a human intracerebral glioma model J Neurosurg 93:449--454, 2000PubMedCrossRefGoogle Scholar
  38. 38.
    Evans SM, Judy KD, Dunphy I, Jenkins WT, Hwang WT, Nelson PT, Lustig RA, Jenkins K, Magarelli DP, Hahn SM, Collins RA, Grady MS, Koch CJ Hypoxia is important in the biology and aggression of human glial brain tumors Clin Cancer Res 10:8177--8184, 2004CrossRefPubMedGoogle Scholar
  39. 39.
    Evans SM, Judy KD, Dunphy I, Jenkins WT, Nelson PT, Collins R, Wileyto EP, Jenkins K, Hahn SM, Stevens CW, Judkins AR, Phillips P, Geoerger B, Koch CJ Comparative measurements of hypoxia in human brain tumors using needle electrodes and EF5 binding Cancer Res 64:1886--1892, 2004CrossRefPubMedGoogle Scholar
  40. 40.
    Jiang BH, Agani F, Passaniti A, Semenza GL V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression Cancer Res 57:5328--5335, 1997PubMedGoogle Scholar
  41. 41.
    Stratman NC, Carter DB, Sethy VH Ibuprofen: effect on inducible nitric oxide synthase Brain Res Mol Brain Res 50:107--112, 1997CrossRefPubMedGoogle Scholar
  42. 42.
    Kimura H, Weisz A, Kurashima Y, Hashimoto K, Ogura T, D'Acquisto F, Addeo R, Makuuchi M, Esumi H Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide Blood 95:189--197, 2000PubMedGoogle Scholar
  43. 43.
    Zundel W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, Gottschalk AR, Ryan HE, Johnson RS, Jefferson AB, Stokoe D, Giaccia AJ Loss of PTEN facilitates HIF-1-mediated gene expression Genes Dev 14:391--396, 2000PubMedGoogle Scholar
  44. 44.
    Newcomb EW, Ali MA, Schnee T, Lan L, Lukyanov Y, Fowkes M, Miller DC, Zagzag D Flavopiridol downregulates hypoxia-mediated hypoxia-inducible factor-1alpha expression in human glioma cells by a proteasome-independent pathway: implications for in vivo therapy Neuro-oncol 7:225--235, 2005CrossRefPubMedGoogle Scholar
  45. 45.
    Parliament MB, Franko AJ, Allalunis-Turner MJ, Mielke BW, Santos CL, Wolokoff BG, Mercer JR Anomalous patterns of nitroimidazole binding adjacent to necrosis in human glioma xenografts: possible role of decreased oxygen consumption Br J Cancer 75:311--318, 1997PubMedGoogle Scholar
  46. 46.
    Mizukami Y, Li J, Zhang X, Zimmer MA, Iliopoulos O, Chung DC Hypoxia-inducible factor-1-independent regulation of vascular endothelial growth factor by hypoxia in colon cancer Cancer Res 64:1765--1772, 2004CrossRefPubMedGoogle Scholar
  47. 47.
    Maity A, Pore N, Lee J, Solomon D, O'Rourke DM Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3′-kinase and distinct from that induced by hypoxia Cancer Res 60:5879--5886, 2000PubMedGoogle Scholar
  48. 48.
    Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, Georgescu MM, Simons JW, Semenza GL Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics Cancer Res 60:1541--1545, 2000PubMedGoogle Scholar
  49. 49.
    Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG Jr Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1:237--246, 2002CrossRefPubMedGoogle Scholar
  50. 50.
    Blagosklonny MV, An WG, Romanova LY, Trepel J, Fojo T, Neckers L p53 inhibits hypoxia-inducible factor-stimulated transcription J Biol Chem 273:11995--11998, 1998CrossRefPubMedGoogle Scholar
  51. 51.
    Fults D, Pedone C Immunocytochemical mapping of the phosphatase and tensin homolog (PTEN/MMAC1) tumor suppressor protein in human gliomas Neuro-oncol 2:71--79, 2000CrossRefPubMedGoogle Scholar
  52. 52.
    Sano T, Lin H, Chen X, Langford LA, Koul D, Bondy ML, Hess KR, Myers JN, Hong YK, Yung WK, Steck PA Differential expression of MMAC/PTEN in glioblastoma multiforme: relationship to localization and prognosis Cancer Res 59:1820--1824, 1999PubMedGoogle Scholar
  53. 53.
    Blum R, Jacob-Hirsch J, Amariglio N, Rechavi G, Kloog Y Ras inhibition in glioblastoma down-regulates hypoxia-inducible factor-1alpha, causing glycolysis shutdown and cell death Cancer Res 65:999--1006, 2005PubMedGoogle Scholar
  54. 54.
    Arbiser JL, Moses MA, Fernandez CA, Ghiso N, Cao Y, Klauber N, Frank D, Brownlee M, Flynn E, Parangi S, Byers HR, Folkman J Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways Proc Natl Acad Sci USA 94:861--866, 1997CrossRefPubMedGoogle Scholar
  55. 55.
    Gaddipati JP, Madhavan S, Sidhu GS, Singh AK, Seth P, Maheshwari RK Picroliv -- a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation Mol Cell Biochem 194:271--281, 1999CrossRefPubMedGoogle Scholar
  56. 56.
    Zelzer E, Levy Y, Kahana C, Shilo BZ, Rubinstein M, Cohen B Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT Embo J 17:5085--5094, 1998CrossRefPubMedGoogle Scholar
  57. 57.
    Kanno H, Shuin T, Kondo K, Yamamoto I, Ito S, Shinonaga M, Yoshida M, Yao M Somatic mutations of the von Hippel-Lindau tumor suppressor gene and loss of heterozygosity on chromosome 3p in human glial tumors Cancer Res 57:1035--1038, 1997PubMedGoogle Scholar
  58. 58.
    Feldser D, Agani F, Iyer NV, Pak B, Ferreira G, Semenza GL Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2 Cancer Res 59:3915--3918, 1999PubMedGoogle Scholar
  59. 59.
    Blouw B, Song H, Tihan T, Bosze J, Ferrara N, Gerber HP, Johnson RS, Bergers G The hypoxic response of tumors is dependent on their microenvironment Cancer Cell 4:133--146, 2003CrossRefPubMedGoogle Scholar
  60. 60.
    Zhang X, Kon T, Wang H, Li F, Huang Q, Rabbani ZN, Kirkpatrick JP, Vujaskovic Z, Dewhirst MW, Li CY Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1alpha Cancer Res 64:8139--8142, 2004CrossRefPubMedGoogle Scholar
  61. 61.
    Tan C, de Noronha RG, Roecker AJ, Pyrzynska B, Khwaja F, Zhang Z, Zhang H, Teng Q, Nicholson AC, Giannakakou P, Zhou W, Olson JJ, Pereira MM, Nicolaou KC, Van Meir EG Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway Cancer Res 65:605--612, 2005PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Randy L. Jensen
    • 1
    • 2
    • 4
  • Brian T. Ragel
    • 1
  • Kum Whang
    • 2
    • 3
  • David Gillespie
    • 1
    • 2
  1. 1.Department of NeurosurgeryUniversity of UtahSalt Lake CityUSA
  2. 2.Huntsman Cancer Institute,University of UtahSalt Lake CityUSA
  3. 3.Yonsei University, Wonju College of MedicineWonjuKorea
  4. 4.Department of NeurosurgeryUniversity of Utah Salt Lake CityUSA

Personalised recommendations