Archives of Gynecology and Obstetrics

, Volume 282, Issue 6, pp 677–683

Reduction of hypoxia-induced angiogenesis in ovarian cancer cells by inhibition of HIF-1 alpha gene expression

  • Christopher S. Bryant
  • Adnan R. Munkarah
  • Sanjeev Kumar
  • Ramesh B. Batchu
  • Jay P. Shah
  • Jeremy Berman
  • Robert T. Morris
  • Zhong L. Jiang
  • Ghassan M. Saed
Gynecologic Oncology



The goal of this study was to investigate the effects of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF production and angiogenesis in epithelial ovarian cancer (EOC) cells.


Two EOC cell lines, MDAH-2774 and SKOV-3, were cultured under normoxic (20% O2) and hypoxic (2% O2) conditions using standard techniques. After EOC cells were transfected with siRNA, HIF-1 alpha and VEGF mRNA levels were measured by real-time RT–PCR. Angiogenesis was evaluated utilizing an in vitro assay model consisting of human umbilical vein endothelial cells (HUVEC) and polymerized ECM Matrix.


Both EOC cell lines evaluated constitutively expressed HIF-1 alpha and VEGF mRNA. HIF-1 alpha and VEGF mRNA levels were significantly increased in response to hypoxia (P < 0.05). Under hypoxic conditions, inhibition of HIF-1 alpha gene expression by a specific siRNA resulted in a significant reduction in HIF-1 alpha and VEGF mRNA levels (P < 0.05). In the in vitro angiogenesis model, supernatant from the hypoxic EOC cells induced the HUVEC to form a complex tubular network, a hallmark of angiogenesis. Semi-quantitative analysis of the angiogenesis assay revealed a significant reduction in tube formation when supernatant from HIF-1 alpha siRNA-treated hypoxic EOC cell was used (P < 0.05).


Inhibition of HIF-1 alpha expression by specific siRNA resulted in a significant decrease in VEGF production and angiogenesis. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.


Ovarian cancer Angiogenesis siRNA Gene silencing HIF-1 alpha VEGF 


  1. 1.
    Folkman J (2000) Tumor angiogenesis. In: Holland JF (ed) Cancer medicine, 5th edn. BC Decker, Ontario, pp 132–152Google Scholar
  2. 2.
    Liotta LA, Stetler-Stevenson WG (1991) Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res 15(18):5054–5059Google Scholar
  3. 3.
    Yamamoto S, Konishi I, Mandai M, JKuroda H et al (1997) Expression of vascular endothelial growth factor (VEGF) in epithelial ovarian neoplasms: correlation with clinicopatholgy and patient survivial, and analysis of serum VEGF levels. Br J Cancer 76:1221–1227PubMedGoogle Scholar
  4. 4.
    Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407:249–257CrossRefPubMedGoogle Scholar
  5. 5.
    Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M et al (1998) Role of HIF-1alpha in hypoxia-mediated apopotosis, cell proliferation and tumour angiogenesis. Nature 394:485–490CrossRefPubMedGoogle Scholar
  6. 6.
    Marxsen JH, Schmitt O, Metzen E, Jelkmann W, Hellwig-Burgel T (2001) Vascular endothelial growth factor gene expression in the human breast cancer cell line MX-1 is controlled by O2 availability in vitro and in vivo. Ann Anat 183:243–249CrossRefPubMedGoogle Scholar
  7. 7.
    Semenza GL (2000) HIF-1: using two hands to flip the angiogenic switch. Cancer Metastasis Rev 19:59–65CrossRefPubMedGoogle Scholar
  8. 8.
    Wang GL, Jiang B, Rue EA, Semenza GL (1995) Hypoxia-inducible factor 1 is a basic-helix-loop-helix-pas heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 92:5510–5514CrossRefPubMedGoogle Scholar
  9. 9.
    Semenza GL (2002) Signal transduction to hypoxia-inducible factor 1. Biochem Pharm 64:993–998CrossRefPubMedGoogle Scholar
  10. 10.
    Zhu G, Saed GM, Deppe G, Diamond M, Munkarah AR (2004) Hypoxia up-regulates the effects of prostaglandin E2 on tumor angiogenesis in ovarian cancer cells. Gynecol Oncol 94:422–426CrossRefPubMedGoogle Scholar
  11. 11.
    Munkarah AR, Ali-Fehmi R, Malone J Jr, Deppe G, Saed GM (2006) Knockout of inducible nitric oxide synthase reduces VEGF production and angiogenesis in epithelial ovarian cancer cell lines. Gynecol Oncol 101(Suppl 1):S96Google Scholar
  12. 12.
    Saed GM, Zhang W, Chegini N, Holmadhal L, Diamond MP (1999) Alteration of type I and III collagen expression in human peritoneal mesothelial cells in response to hypoxia and transforming growth factor-beta. Wound Repair Regen 7:504–510CrossRefPubMedGoogle Scholar
  13. 13.
    Saed GM, Diamond M (2002) Hypoxia-induced irreversible up-regulation of type I collagen and transforming growth factor-beta 1 in human peritoneal fibroblasts. Fertil Steril 78:144–147CrossRefPubMedGoogle Scholar
  14. 14.
    Wang Y, Minko T (2004) A novel cancer therapy: combined liposomal hypoxia inducible factor 1 alpha antisense oligonucleotides and an anticancer drug. Biochem Pharmacol 68:2031–2042CrossRefPubMedGoogle Scholar
  15. 15.
    Jensen RL, Ragel BT, Whang K, Gillespie D (2006) Inhibition of hypoxia inducible factor-1 alpha (HIF-1 alpha) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas. J Neuronocol 78:233–247CrossRefGoogle Scholar
  16. 16.
    Yang G, Cai KQ, Thompson-Lansa JA, Bast RC Jr, Liu J (2004) Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNa against Her-2/neu gene expression. J Biol Chem 279:4339–4345CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang L, Yang N, Park JW, Katsaros D, Fracchioli S, Cao G et al (2003) Tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature, supporting angiogenesis in ovarian cancer. Cancer Res 63:3403–3412PubMedGoogle Scholar
  18. 18.
    Milkiewicz M, Ispanovic E, Doyle JL, Haas TL (2006) Regulators of angiogenesis and strategies for their therapeutic manipulation. Int J Biochem Cell Biol 38:333–357CrossRefPubMedGoogle Scholar
  19. 19.
    Malone J Jr, Saed GM, Diamond M, Sokol RJ, Munkarah AR (2006) The effects of the inhibition of inducible nitric oxide synthase on angiogenesis of epithelial ovarian cancer. Am J Obstet Gynecol 194:1110–1118CrossRefPubMedGoogle Scholar
  20. 20.
    Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D et al (1999) Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases. Cancer Res 59:5830–5835PubMedGoogle Scholar
  21. 21.
    Parnsare V, Munkarah AR, Schimp Vl, Arabi HM, Saed GM, Morris RT et al (2007) Increased expression of hypoxia-inducible factor 1 α in type I and type II endometrial carcinomas. Mod Pathol 20:35–43CrossRefGoogle Scholar
  22. 22.
    Wang Y, Pakunlu RI, Tsao W, Pozharov V, Minko T (2004) Bimodal effect of hypoxia in cancer: the role of hypoxia inducible factor in apoptosis. Mol Pharm 1:156–165CrossRefPubMedGoogle Scholar
  23. 23.
    Salceda S, Caro J (1997) Hypoxia-inducible factor 1 alpha protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced chagnes. J Biol Chem 272:22642–22647CrossRefPubMedGoogle Scholar
  24. 24.
    Gartel AL (2006) RNA interference in cancer. Biomol Eng 23:17–34CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Christopher S. Bryant
    • 1
  • Adnan R. Munkarah
    • 2
  • Sanjeev Kumar
    • 1
  • Ramesh B. Batchu
    • 3
  • Jay P. Shah
    • 1
  • Jeremy Berman
    • 1
  • Robert T. Morris
    • 1
  • Zhong L. Jiang
    • 4
  • Ghassan M. Saed
    • 4
  1. 1.Division of Gynecologic Oncology, Barbara Ann Karmanos Cancer InstituteWayne State UniversityDetroitUSA
  2. 2.Division of Gynecologic OncologyHenry Ford Health SystemDetroitUSA
  3. 3.Department of Surgery, Barbara Ann Karmanos Cancer InstituteWayne State UniversityDetroitUSA
  4. 4.Department of Obstetrics and GynecologyWayne State UniversityDetroitUSA

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