title: Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1.
abstract: The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1alpha protein that acts as a dominant-negative HIF-1alpha (HIF-1alpha-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1alpha was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1alpha-no-TAD or nontoxic concentrations (0.1 microM; <IC(10)) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (<0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC(50) values in anoxia were 3-fold higher than those in air for HT1080 (2.2 +/- 0.3 versus 0.7 +/- 0.2 microM) and HCT116 (9 +/- 4 versus 3 +/- 2 microM) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC(50) in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1alpha-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC(50) values: HT1080, 0.7 +/- 0.04 microM; HCT116, 3 +/- 1 microM). HIF-1alpha-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure.
authors.affiliation: Experimental Oncology, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK.
authors.forename: Louisa M
authors.initials: LM
authors.lastname: Brown
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Cowen
authors.forename: Camille
authors.initials: C
authors.lastname: Debray
authors.forename: Amanda
authors.initials: A
authors.lastname: Eustace
authors.forename: Janine T
authors.initials: JT
authors.lastname: Erler
authors.forename: Freda C D
authors.initials: FC
authors.lastname: Sheppard
authors.forename: Catriona A
authors.initials: CA
authors.lastname: Parker
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Stratford
authors.forename: Kaye J
authors.initials: KJ
authors.lastname: Williams
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005047:Etoposide / Q000627:therapeutic use; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis / Q000235:genetics; D007546:Isoquinolines / Q000494:pharmacology; D009369:Neoplasms / Q000188:drug therapy*; D010100:Oxygen / Q000378:metabolism; D017384:Sequence Deletion; D019772:Topotecan / Q000494:pharmacology; D015533:Transcriptional Activation
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007546:Isoquinolines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C091971:DX 52-1; D005047:Etoposide; D019772:Topotecan; C038710:quinocarcin; D010100:Oxygen