Methylalpinumisoflavone Inhibits Hypoxia-inducible Factor-1 (HIF-1) Activation by Simultaneously Targeting Multiple Pathways

Yang Liu,Coothan K Veena,J Brian Morgan,Kaleem A Mohammed,Mika B Jekabsons,Dale G Nagle,Yu-Dong Zhou

doi:10.1074/jbc.m806744200

Yang Liu, Coothan K Veena + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m806744200

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Abstract

Hypoxia is a common feature of solid tumors, and the extent of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective molecular target for anticancer drug discovery directed at tumor hypoxia. A natural product chemistry-based approach was employed to discover small molecule inhibitors of HIF-1. Bioassay-guided isolation of an active lipid extract of the tropical legumaceous plant Lonchocarpus glabrescens and structure elucidation afforded two new HIF-1 inhibitors: alpinumisoflavone (compound 1) and 4'-O-methylalpinumisoflavone (compound 2). In human breast tumor T47D cells, compounds 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC(50) values of 5 and 0.6 mum, respectively. At the concentrations that in hibited HIF-1 activation, compound 2 inhibited hypoxic induction of HIF-1 target genes (CDKN1A, GLUT-1, and VEGF), tumor angiogenesis in vitro, cell migration, and chemotaxis. Compound 2 inhibits HIF-1 activation by blocking the induction of nuclear HIF-1alpha protein, the oxygen-regulated subunit that controls HIF-1 activity. Mechanistic studies indicate that, unlike rotenone and other mitochondrial inhibitors, compound 2 represents the first small molecule that inhibits HIF-1 activation by simultaneously suppressing mitochondrial respiration and disrupting protein translation in vitro. This unique mechanism distinguishes compound 2 from other small molecule HIF-1 inhibitors that are simple mitochondrial inhibitors or flavanoid-based protein kinase inhibitors.

Highlights

Oxygen supply, hypoxic tumor cells activate the expression of genes that range in function from those that promote anaerobic metabolism to those that initiate tumor angiogenesis [1,2,3]
Alpinumisoflavone [1] and 4Ј-O-Methylalpinumisoflavone [2] Inhibit hypoxia-inducible factor-1 (HIF-1) Activation in T47D Cells—Over 15,000 natural product-rich extracts from plants, marine invertebrates, algae, and microorganisms were evaluated in a T47D human breast tumor cell-based reporter assay for HIF-1 inhibitory activity [29]
At the minimum To investigate if the inhibition of VEGF protein production inhibitory concentration (MIC) value determined in the T47D correlates with the inhibition of tumor angiogenesis, a cell-based reporter assay, compound 2 (10 ␮M) blocked the HUVEC-based in vitro angiogenesis assay was utilized for the hypoxic (1% O2) induction of all three HIF-1 target genes at the follow-up study

Summary

EXPERIMENTAL PROCEDURES

Plant Material, Extraction, and Isolation—The plant material (L. glabrescens) was collected by Dr Sidney McDaniel from Punchana, Peru, on August 8, 1991. The cells were washed twice with a serum-free medium, and a fresh DMEM/F-12 medium supplemented with 5% (v/v) FBS (Hyclone), penicillin/streptomycin, and each specific test compound was added. Nuclear Extract Preparation and Western Blot Analysis— Plating of T47D cells, compound treatment, and exposure to hypoxic conditions were described [29]. The plasma membrane was selectively permeabilized with digitonin (30 ␮M) so that the substrates available to the mitochondria could be manipulated This required the use of a mitochondrial buffer containing 20 mM HEPES, pH 7.3, 120 mM KCl, 2 mM KH2PO4, 2 mM MgCl2, 1 mM EGTA, and 0.3% bovine serum albumin (fat-free) in place of the DMEM/F-12 medium.

RESULTS

Induction of VEGF Protein and

DISCUSSION