title: Estrogen receptor α and aryl hydrocarbon receptor independent growth inhibitory effects of aminoflavone in breast cancer cells.
abstract: BACKGROUND
  Numerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ERα) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF's effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines.
  
  
  METHODS
  AF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and β-galactosidase staining for senescence. Luciferase data was analyzed using Student's T test. Three-parameter nonlinear regression was performed for cell counting assays.
  
  
  RESULTS
  Here, we report that ERα-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ERα negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells.
  
  
  CONCLUSIONS
  Overall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ERα-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Brinkman
authors.forename: Jiacai
authors.initials: J
authors.lastname: Wu
authors.forename: Karen
authors.initials: K
authors.lastname: Ersland
authors.affiliation: Molecular and Environmental Toxicology Center, University of Wisconsin - Madison, Madison, WI, USA. wxu@oncology.wisc.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051792:Basic Helix-Loop-Helix Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D047628:Estrogen Receptor alpha / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D061986:MCF-7 Cells; D034622:RNA Interference; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D051792:Basic Helix-Loop-Helix Proteins; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; D047628:Estrogen Receptor alpha; D005419:Flavonoids; D018336:Receptors, Aryl Hydrocarbon; C111235:AHR protein, human; C506487:ESR1 protein, human; C413760:aminoflavone; C569463:CYP1A1 protein, human; C585496:CYP1B1 protein, human