Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Tirapazamine (TPZ), a well-characterized bioreductive anticancer agent, is currently in Phase II and III clinical trials. A major aspect of the anticancer activity of TPZ is its identity as a tumor-specific topoisomerase II alpha inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1 alpha accumulation driven by hypoxia or growth factors in human cancer cells and in HepG2 cell-derived tumors in athymic nude mice. We investigated the mechanism of TPZ on HIF-1 alpha in HeLa human cervical cancer cells by western blot analysis, reverse transcription-PCR assay, luciferase reporter assay and small interfering RNA (siRNA) assay. Mechanistic studies demonstrated that neither HIF-1 alpha mRNA levels nor HIF-1 alpha protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1 alpha translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1 alpha protein synthesis is dependent on the phosphorylation of translation initiation factor 2 alpha (eIF2 alpha) rather than the mTOR complex 1/eukaryotic initiation factor 4E-binding protein-1 (mTORC1/4E-BP1) pathway. Immunofluorescence analysis of tumor sections provide the in vivo evidences to support our hypothesis. Additionally, siRNA specifically targeting topoisomerase II alpha did not reverse the ability of TPZ to inhibit HIF-1 alpha expression, suggesting that the HIF-1 alpha inhibitory activity of TPZ is independent of its topoisomerase II alpha inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1 alpha and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1 alpha expression.