Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway
Figure 1
TPZ decreases hypoxia-induced HIF-1α protein accumulation.
HeLa cells (A), and HEK-293, OVCAR8, HepG2, SMMC-7721 and HCT116 cells (B) were exposed to hypoxia and a gradient of concentrations of TPZ for 4 h. HIF-1α and β-actin protein levels were detected by western-blot analysis of whole-cell extracts, as described in the Materials and Methods. (C) Effect of TPZ treatment on the expression level of HIF-1α in HepG2 cell-derived tumors, as determined by immunoblot analysis. (D) Densitometry analyses of (C). (E) Hypoxia-dependent HIF-1α transcriptional activity was measured using HRE-dependent reporter assays, as described in the Materials and Methods. HeLa cells were transiently transfected with the HRE-Luc plasmid and then treated with TPZ for 8 h under hypoxic conditions. Luminescence was measured and fold stimulation was obtained by normalizing the relative luciferase activity to that of untreated cells under hypoxic conditions.