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Novel Thioredoxin Inhibitors Paradoxically Increase Hypoxia-Inducible Factor- Expression but Decrease Functional Transcriptional Activity, DNA Binding, and Degradation

Authors' Affiliations: ¹ Cancer Research UK Growth Factor Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford; ² The Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom; and ³ Cancer Research UK Experimental Cancer Chemotherapy Research Group, University of Nottingham, Nottingham, United Kingdom

Requests for reprints: Adrian L. Harris, Cancer Research UK Growth Factor Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DS, United Kingdom. Phone: 44-1865226184; Fax: 44-1865226179; E-mail: aharris.lab{at}cancer.org.uk.

Purpose: Hypoxia-inducible factor- (HIF-) is a transcription factor that regulates the response to hypoxia. HIF- protein is found at high levels in many cancers, and the redox protein thioredoxin-1 (Trx-1) increases both aerobic and hypoxia-induced HIF-. Therefore, Trx-1 and HIF- are attractive molecular targets for novel cancer therapeutics.

Experimental Design: We investigated whether two novel anticancer drugs AJM290 and AW464 (quinols), which inhibit Trx-1 function, can inhibit the HIF pathway.

Results: Treatment of several cancer cell lines with AJM290 or AW464 prevented the hypoxia-induced increase of vascular endothelial growth factor (VEGF) at subtoxic concentrations. AJM290 and AW464 also decreased VEGF in pVHL mutant renal cell carcinoma cells that constitutively overexpress HIF- protein. They surprisingly up-regulated HIF- expression in breast cancer cell lines in normoxia and hypoxia as well as in pVHL mutant cells. In the MDA-MB-468 breast cancer cell line, the compounds inhibited RNA and protein expression of the HIF- target genes, carbonic anhydrase IX, VEGF, and BNIP3, concordantly with HIF- up-regulation. Both compounds specifically inhibited HIF--dependent induction of hypoxia regulatory element-luciferase and HIF-1 hypoxia regulatory element-DNA binding. To analyze the HIF-1 domain inhibited by AJM290, we transfected cells with plasmids expressing a fusion protein of Gal linked to HIF-1 or HIF-1 COOH-terminal transactivation domain (CAD) with a Gal4-responsive luciferase reporter gene. AJM290 inhibited both the full-length HIF-1 and HIF-1 CAD transcriptional activity.

Conclusions: AJM290 and AW464 are inhibitors of HIF-1 CAD transcription activity and DNA binding, but they also inhibit degradation of HIF, in contrast to other Trx inhibitors.