title: 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.affiliation: Institute of Pharmacology and Toxicology, Zhejiang University, Hangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ji
authors.initials: J
authors.lastname: Cao
authors.forename: Qinjie
authors.initials: Q
authors.lastname: Weng
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Hui
authors.initials: H
authors.lastname: Jing
authors.forename: Hong
authors.initials: H
authors.lastname: Zhu
authors.forename: Qiaojun
authors.initials: Q
authors.lastname: He
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases; D000077704:Tirapazamine; D014183:Transplantation, Heterologous; D014227:Triazines / Q000494:pharmacology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C087000:EIF4EBP1 protein, human; D015852:Eukaryotic Initiation Factor-2; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; D014227:Triazines; D000077704:Tirapazamine; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II