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Research Articles: Therapeutics, Targets, and Development

Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1 and -2

¹ Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy; ² Department of Human Anatomy and Histology, University of Bari, Bari, Italy; and ³ Italian Neuroblastoma Foundation, Genca, Italy

Requests for reprints: Maria Carla Bosco, Laboratory of Molecular Biology, G. Gaslini Institute, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. Phone: 39-10-5636633; Fax: 39-10-3733346. E-mail: mcbosco1{at}virgilio.it

Abstract

Neuroblastoma produce angiogenic peptides, and the extent of angiogenesis correlates with tumor progression and poor clinical outcome. Hence, angiogenic factor inhibition represents an important therapeutic option. One of the major drives to tumor angiogenesis is hypoxia, a decrease in oxygen tension that characterizes the tumor microenvironment. We investigated the effects of the topoisomerase I inhibitor, topotecan, on vascular endothelial growth factor (VEGF) induction by hypoxia in advanced-stage human neuroblastoma cells. Topotecan counteracted hypoxic induction of VEGF and decreased angiogenic activity of conditioned medium from hypoxic cultures in vivo in the chick chorioallantoic membrane. Promoter-driven reporter studies showed the role of both hypoxia-inducible factor (HIF)-1 and -2 in VEGF transcription activation by hypoxia, because (a) overexpression of either protein by cotransfection with expression vectors resulted in VEGF promoter transactivation, which was abrogated by mutation in the HIF-binding site, and (b) targeted knockdown of HIF-1/2 by RNA interference inhibited hypoxia-stimulated VEGF transcriptional activity and protein secretion. Topotecan-inhibitory effects on VEGF induction by hypoxia were mediated through suppression of both HIF-1 and HIF-2 protein accumulation and transactivation properties, which was specific and required ongoing RNA transcription. A similar pattern of results was obtained in cells treated with the hypoxia-mimetic agent, desferrioxamine. These data provide the first evidence that topotecan is a potent inhibitor of HIF-1 and HIF-2 subunits in hypoxic neuroblastoma cells, leading to decreased VEGF expression and angiogenic activity. An important clinical implication of these findings is that therapies targeted to the HIF pathway have the potential to inhibit neuroblastoma angiogenesis and growth. [Mol Cancer Ther 2008;7(7):1974–84]

Grant support: Fondazione Italiana per la Lotta al Neuroblastoma, Italian Association for Cancer Research, Italian Health Ministry, and Ministero Istruzione Universita e Ricerca.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/10/07; revised 4/24/08; accepted 4/30/08.