Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway
Figure 2
TPZ does not affect HIF-1α mRNA expression or protein degradation, but decreases HIF-1α protein synthesis.
(A) HeLa cells were exposed to varying concentrations of TPZ for 4 h or a single concentration for the indicated times. Then, the total RNA was extracted and analyzed for HIF-1α mRNA expression by RT-PCR, using GAPDH as a control gene. (B) Cells exposed to hypoxia overnight were treated with cycloheximide (CHX) in the presence or absence of 20 µM TPZ for various periods, and HIF-1α protein levels were measured by western-blot analysis. HeLa cells were treated with TPZ, together with MG132 (C) or chloroquine diphosphate (CQ) (D), under the indicated conditions, followed by immunoblotting with anti-HIF-1α or anti-β-actin antibodies. Cells were pretreated for 30 min with MG132 and CQ to allow functional inhibition of the proteasome and lysosome. (E) HeLa cells were pre-incubated with CHX for 3 h in normoxic conditions and then placed in fresh medium and treated with or without 20 µM TPZ for the indicated times under hypoxic conditions. The cells were harvested and lysates were immunoblotted with an anti-HIF-1α antibody.