Clinical & Experimental Metastasis

, Volume 17, Issue 8, pp 687–694

Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line

  • Xin-Hua Liu
  • Alexander Kirschenbaum
  • Shen Yao
  • Mark E. Stearns
  • James F. Holland
  • Kevin Claffey
  • Alice C. Levine


Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE2 synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE2 production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis.

angiogenesis cyclooxygenase-2 cobalt chloride-simulated hypoxia metastasis NS398 prostaglandin E2 prostate cancer vascular endothelial growth factor 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990; 82: 4–6.PubMedGoogle Scholar
  2. 2.
    Bunn HF, Poyton RO. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 1996; 76: 839–85.PubMedGoogle Scholar
  3. 3.
    Wang GL, Semenza GL. Characterization of hypoxia-inducible factor-1 and regulation of DNA binding activity by hypoxia. J Biol Chem 1993; 268: 21513–8.PubMedGoogle Scholar
  4. 4.
    Guillemin K, Krasnow MA. The hypoxic response: Huffing and HIFing. Cell 1997; 89: 9–12.PubMedCrossRefGoogle Scholar
  5. 5.
    Damert A, Ikeda E, Risau W. Activator-protein-1 binding potentials the hypoxia-inducible factor-1 mediated hypoxia-induced transcriptional activation of vascular endothelial growth factor expression in C6 glioma. Biochem J 1997; 327: 419–23.PubMedGoogle Scholar
  6. 6.
    Taketo MM. Cyclooxygenase-2 inhibitor in tumorigenesis (Part II). J Natl Cancer Inst 1998; 90: 1609–20.PubMedCrossRefGoogle Scholar
  7. 7.
    Tjandrawinata RR, Dahiya R, Hughes-Fulford M. Induction of cyclooxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells. Br J Cancer 1997; 75: 1111–8.PubMedGoogle Scholar
  8. 8.
    Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase-2. Cell 1995; 83: 493–501.PubMedCrossRefGoogle Scholar
  9. 9.
    Liu XH, Rose DP. Differential expression and regulation of cyclooxygenase-1 and-2 in two human breast cancer cell lines. Cancer Res 1996; 56: 5125–7.PubMedGoogle Scholar
  10. 10.
    Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997; 94: 3336–40.PubMedCrossRefGoogle Scholar
  11. 11.
    Tsujii M, Kawano S, Tsuji S, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998; 93: 705–16.PubMedCrossRefGoogle Scholar
  12. 12.
    Schmedtji JF Jr., Ji YS, Liu WL et al. Hypoxia induces cyclooxygenase-2 via the NF-kB p65 transcription factor in human vascular endothelial cells. J Biol Chem 1997; 272: 601–8.CrossRefGoogle Scholar
  13. 13.
    Form DM, Auerbach R. Prostaglandin E2 and angiogenesis. Proc Soc Exp Biol Med 1983; 172: 214–8.PubMedGoogle Scholar
  14. 14.
    Brawer MK, Deering RE, Brown M et al. Predictors of pathologic stage in prostatic carcinomas: The role of neovascularity. Cancer 1994; 73: 678–87.PubMedCrossRefGoogle Scholar
  15. 15.
    Weidner N, Carroll PR, Flax J et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 1993; 143: 401–9.PubMedGoogle Scholar
  16. 16.
    Lissbrant IF, Stattin P, Damber JE, Bergh A. Vascular density is a predictor of cancer-specific survival in prostatic carcinoma. Prostate 1997; 33: 38–45.PubMedCrossRefGoogle Scholar
  17. 17.
    Toi M, Hoshina S, Takayanagi T, Tominaga T. Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res 1994; 85: 1045–52.PubMedGoogle Scholar
  18. 18.
    Mattern J, Koomagi R, Volm M. Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumor cell proliferation in human epidermoid lung carcinoma. Br J Cancer 1996; 73: 931–9.PubMedGoogle Scholar
  19. 19.
    Connolly JM, Rose DP. Angiogenesis in two human prostate cancer cell lines with differing metastatic potential when growing as solid tumors in nude mice. J Urol 1998; 160: 932–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Connolly JM, Rose DP. A comparison of the expression of the malignant phenotype in two androgen-independent human prostate cancer cell lines after orthotopic implantation in nude mice. Int J Oncol 1997; 11: 771–6.Google Scholar
  21. 21.
    Goldberg MA, Schneider TJ. Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 1994; 269: 4355–9.PubMedGoogle Scholar
  22. 22.
    Wang M, Stearns ME. Isolation and characterization of PC-3 human prostate tumor sublines which preferentially metastasize to select organs in SCID mice. Differentiation 1991; 48: 115–25.PubMedGoogle Scholar
  23. 23.
    Levine AC, Liu XH, Greenberg PD et al. Androgens induce the expression of vascular endothelial growth factor in human fetal prostatic fibroblasts. Endocrinology 1998; 139: 4672–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Nie DT, Hillman GG, Geddes T et al. Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth. Cancer Res 1998; 58: 4047–51.PubMedGoogle Scholar
  25. 25.
    Yang M, Sytkowski AJ. Cloning differentially expressed genes by linker capture subtraction. Anal Biochem 1996; 237: 109–14.PubMedCrossRefGoogle Scholar
  26. 26.
    Shweiki D, Itin A, Soffer D, Kesher E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-induced angiogenesis. Nature (London) 1992; 359: 843–5.CrossRefGoogle Scholar
  27. 27.
    Damert A, Machein M, Breier G et al. Upregulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms. Cancer Res 1997; 57: 3860–4.PubMedGoogle Scholar
  28. 28.
    Han JW, Sadowski H, Young DA, Macara IG. Persistent induction of cyclooxygenase in p60v–src –transformed 3T3 fibroblasts. Proc Natl Acad Sci USA 1990; 87: 3373–7.PubMedCrossRefGoogle Scholar
  29. 29.
    Rolland PH, Martin PM, Jacquemier J et al. Prostaglandins in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. J Natl Cancer Inst 1980; 64: 1061–70.PubMedGoogle Scholar
  30. 30.
    Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 1994; 12: 5447–54.Google Scholar
  31. 31.
    Appleby SB, Ristimaki A, Neilson K et al. Structure of the human cyclooxygenase-2 gene. Biochem J 1994; 302: 723–7.PubMedGoogle Scholar
  32. 32.
    Kraemer SA, Arthur KA, Dension MS et al. Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 1996; 330: 319–28.PubMedCrossRefGoogle Scholar
  33. 33.
    Harada S, Nagy JA, Sullivan KA et al. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J Clin Invest 1994; 93: 2490–6.PubMedCrossRefGoogle Scholar
  34. 34.
    Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: A potential mechanism for inflammatory angiogenesis. FEBS Lett 1995; 372: 83–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Warner TD, Giuliano F, Vojnovic I et al. Nonsteroid drug selectivities for cyclooxygenase-1 rather than cyclooxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci USA 1999; 96: 7563–8.PubMedCrossRefGoogle Scholar
  36. 36.
    Masferrer JL, Zweifel BS, Manning PT et al. Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic. Proc Natl Acad Sci USA 1994; 91: 3228–32.PubMedCrossRefGoogle Scholar
  37. 37.
    Futaki N, Yoshikawa K, Hamasaka Y et al. NS398, a novel nonsteroidal anti-inflammatory drug with potent analgestic and antipyretic effects, which causes minimal stomach lesions. Gen Pharmacol 1993; 24: 105–10.PubMedGoogle Scholar
  38. 38.
    Liu XH, Yao S, Kirschenbaum A, Levine AC. NS398, a selective cyclooxygen-ase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 1998; 58: 4245–9.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Xin-Hua Liu
    • 1
  • Alexander Kirschenbaum
    • 2
  • Shen Yao
    • 1
  • Mark E. Stearns
    • 3
  • James F. Holland
    • 1
  • Kevin Claffey
    • 4
  • Alice C. Levine
    • 1
  1. 1.Department of Medicine (Divisions of Endocrinology and Neoplastic Diseases)Mount Sinai School of MedicineNew YorkUSA
  2. 2.Department of UrologyMount Sinai School of MedicineNew YorkUSA
  3. 3.Department of PathologyMCP-Hahnemann UniversityPhiladelphiaUSA
  4. 4.Department of PathologyBeth Israel Deaconess Medical CenterBostonUSA

Personalised recommendations