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¹ Molecular Pharmacology Laboratory, ² Laboratory of Receptor Biology and Gene Expression, and ³ Cell and Cancer Biology Branch and Vascular Biology Faculty, National Cancer Institute, Bethesda, MD, and ⁴ DTP—Tumor Hypoxia Laboratory and ⁵ SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD

Requests for reprints: W.D. Figg, Building 10, Room 5A01, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402-3622; Fax: (301) 402-8606. E-mail: wdfigg{at}helix.nih.gov

Endostatin is a 20-kDa endogenous angiogenesis inhibitor that has recently been shown to inhibit the expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor that is up-regulated by hypoxia via the HIF-1 transcription factor complex. To determine if the anti-angiogenic activity of endostatin involves a modulation of the HIF-1/VEGF pathway in cancer cells, experiments were conducted to establish what effect endostatin has on HIF-1 activity, HIF-1 protein production, and cellular localization in prostate cancer cells and endothelial cells. Endothelial cell tube formation was inhibited by endostatin purchased from Calbiochem (San Diego, CA) but not endostatin obtained from EntreMed (Rockville, MD). Subsequent experiments using Calbiochem endostatin showed that it did not alter HIF-1 protein production or cellular localization in any of the cell lines tested, nor did it alter HIF-1 transactivational activity in hypoxia. Whether or not this is also true in vivo remains to be determined. Nevertheless, these data suggest that the anti-angiogenic activity of endostatin is independent of the HIF-1/VEGF pathway. Immunocytochemical staining results do not indicate a decreased production of VEGF in Calbiochem endostatin-treated LNCaP or human umbilical vein endothelial cells (HUVEC). Treatment of rat aortic cross sections with human endostatin from Calbiochem resulted in a dose-dependent inhibition of microvessel outgrowth. Importantly, inhibition of vessel outgrowth by Calbiochem endostatin in a human saphenous vein angiogenesis assay required early treatment. In view of this in vitro data, we suggest that clinical trials involving endostatin treatment of late-stage disease may not adequately represent the efficacy of this drug in early-stage cancer.

Key Words: endostatin • HIF-1 • HIF-1 • hypoxia • VEGF • angiogenesis • prostate cancer

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Grant Support: Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-C0-12400.

Note: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government.

¹ G. R. Macpherson, S. L. Forbes, and W. D. Figg, unpublished data.

Received 4/22/03; revised 6/ 6/03; accepted 6/20/03.