Suppression of hypoxia-inducible factor 1α (HIF-1α) by tirapazamine is dependent on eIF2α phosphorylation rather than the mTORC1/4E-BP1 pathway.

Abstract

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 IIα inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1α 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-1α 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-1α mRNA levels nor HIF-1α protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1α translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1α protein synthesis is dependent on the phosphorylation of translation initiation factor 2α (eIF2α) 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 IIα did not reverse the ability of TPZ to inhibit HIF-1α expression, suggesting that the HIF-1α inhibitory activity of TPZ is independent of its topoisomerase IIα inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1α and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1α expression.

• Authors: Jun Zhang, Ji Cao, Qinjie Weng, Rui Wu, Yan Yan, Hui Jing, Hong Zhu, Qiaojun He, Bo Yang
• Journal: PloS one (PLoS One) Vol. 5 Issue 11 Pg. e13910 (Nov 09 2010) ISSN: 1932-6203 [Electronic] United States
• PMID: 21085474 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• Antigens, Neoplasm
• Antineoplastic Agents
• Cell Cycle Proteins
• DNA-Binding Proteins
• EIF4EBP1 protein, human
• Eukaryotic Initiation Factor-2
• Hypoxia-Inducible Factor 1, alpha Subunit
• Multiprotein Complexes
• Phosphoproteins
• Proteins
• Triazines
• Tirapazamine
• Mechanistic Target of Rapamycin Complex 1
• TOR Serine-Threonine Kinases
• DNA Topoisomerases, Type II

Topics

• Adaptor Proteins, Signal Transducing (genetics, metabolism)
• Animals
• Antigens, Neoplasm (genetics, metabolism)
• Antineoplastic Agents (pharmacology)
• Blotting, Western
• Cell Cycle Proteins
• Cell Hypoxia
• Cell Line, Tumor
• DNA Topoisomerases, Type II (genetics, metabolism)
• DNA-Binding Proteins (genetics, metabolism)
• Eukaryotic Initiation Factor-2 (genetics, metabolism)
• Female
• HEK293 Cells
• HeLa Cells
• Hep G2 Cells
• Humans
• Hypoxia-Inducible Factor 1, alpha Subunit (genetics, metabolism)
• Liver Neoplasms, Experimental (metabolism, pathology)
• Mechanistic Target of Rapamycin Complex 1
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Multiprotein Complexes
• Phosphoproteins (genetics, metabolism)
• Phosphorylation (drug effects)
• Proteins (genetics, metabolism)
• RNA Interference
• Reverse Transcriptase Polymerase Chain Reaction
• Signal Transduction (drug effects)
• TOR Serine-Threonine Kinases
• Tirapazamine
• Transplantation, Heterologous
• Triazines (pharmacology)