PX-478 inhibits hypoxic HIF-1 protein expression and signaling activity in a dose-dependent manner. PX-478 inhibits hypoxic HIF-1 protein expression and.

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PX-478 inhibits hypoxic HIF-1 protein expression and signaling activity in a dose-dependent manner. PX-478 inhibits hypoxic HIF-1 protein expression and signaling activity in a dose-dependent manner. A, HN5 and UMSCCA10 cells were treated with PX-478 at indicated concentrations for 24 h under normoxic (air, 5% CO2) or hypoxic (1% O2, 5% CO2, 94% N2) conditions. Western blots were quantified by densitometry relative to actin. Columns, normalized mean absorbance values plotted from triplicate experiments; bars, SE. B, C6 HIF-1 reporter cells were created via stable transfection with a bifunctional genetic reporter consisting of HSV-tk and GFP under transcriptional control of the hypoxia response element (HRE-tk/eGFP-cmvRed 2XPRT). Cells were treated with PX-478 at concentrations ranging from 0.1 to 200 μmol/L, with or without concurrent 150 μmol/L CoCl2 chemical stimulation of HIF-1 signaling for 24 h. Experiments were repeated in hypoxic conditions, with cells incubated at 1% O2 (hypoxia, via chamber) for 24 h and then treated with PX-478 at equivalent concentrations for 24 h. HIF-1–dependent GFP signal normalized to beacon RFP signal was measured in quadruplicate. Points, mean; bars, SE. C, VEGF protein per milligram of total secreted protein measured by ELISA in supernatant medium from HN5 and UMSCCa10 cells cultured in normoxic or 1% O2 (24 h, via chamber) conditions, with or without 24-h treatment with indicated concentrations of PX-478. Points, mean from three separate experiments; bars, SD. ***, P < 0.001. David L. Schwartz et al. Mol Cancer Ther 2009;8:947-958 ©2009 by American Association for Cancer Research