title: [Establishment of an aromatase inhibitor letrozole-resistant breast cancer cell model].
abstract: OBJECTIVE
  To establish a human breast cancer MCF-7 cell model stably overexpressing the aromatase gene (MCF-7-aromatase) and aromatase inhibitor letrozole-resistant MCF-7 cell model (MCF-7-LR).
  
  
  METHODS
  We utilized the lentivirus-mediated gene transfer approach to establish MCF-7-aromatase cell and MCF-7 cell model stably overexpressing green fluorescent protein (GFP) (MCF-7-GFP). The expression of aromatase in the MCF-7-aromatase and MCF-7-GFP cells was determined by reverse transcription polymerase chain reaction (RT-PCR), real time quantitative PCR (RT-qPCR), Western blot and immunoprecipitation (IP) assay. The proliferative ability in vitro of MCF-7-aromatase and MCF-7-GFP cells treated with testostorone and β-estradiol (E2) was determined by WST-1 cell proliferation assay. The proliferative ability of MCF-7-aromatase cells treated with letrozole was determined by WST-1 assay. The half maximal inhibitory concentration (IC50 value) for letrozole was calculated from the nonlinear regression line of the plot of cell viability (percentage of control) versus letrozole concentration using Graphpad Prism software. MCF-7-aromatase cells were continuously cultured in the presence of testosterone and letrozole, thus letrozole-resistant MCF-7-LR cells were obtained. WST-1 assay was performed to determine their chemoresistance to letrozole.
  
  
  RESULTS
  RT-PCR and RT-qPCR results revealed that the mRNA expression of aromatase was significantly increased in the MCF-7-aromatase cells compared with that in the MCF-7-GFP cells. Both Western blot and IP assays showed that the expression of aromatase protein was drastically increased in the MCF-7-aromatase cells, compared with that in the MCF-7-GFP cells. WST-1 assay showed that the cell proliferation rate of MCF-7-aromatase cells treated with 1 and 10 nmol/L testosterone was 1.43- and 1.53-fold higher than that of the control cells, respectively. The proliferation rate of MCF-7-aromatase cells treated with 1 and 10 nmol/L E2 was 1.41- and 1.55-fold higher than that of the control cells, respectively. In contrast, the proliferation rate of MCF-7-GFP cells treated with 10 nmol/L testosterone was 1.12-fold higher than that of the control cells, and the proliferation rate of MCF-7-GFP cells treated with 1 and 10 nmol/L E2 was 1.41- and 1.51-fold higher than that of the control cells, respectively. Letrozole treatment significantly inhibited the testosterone-induced proliferation ability of MCF-7-aromatase cells in a dose-dependent manner and the IC50 value was 5.3 nmol/L. In contrast, letrozole treatment showed no inhibitory effect on the proliferative ability of MCF-7-LR cells and the IC50 value was >1000 nmol/L.
  
  
  CONCLUSIONS
  MCF-7-aromatase and MCF-7-LR cells exhibit different response to letrozole treatment, which provides an important basis for further investigating the mechanism of letrozole resistance.
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Hong-yan
authors.initials: HY
authors.lastname: Chen
authors.forename: Zhi-hua
authors.initials: ZH
authors.lastname: Liu
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001141:Aromatase / Q000378:metabolism; D047072:Aromatase Inhibitors / Q000494:pharmacology*; D001943:Breast Neoplasms; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D006801:Humans; D000077289:Letrozole; D061986:MCF-7 Cells; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology*; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D047072:Aromatase Inhibitors; D009570:Nitriles; D014230:Triazoles; D000077289:Letrozole; D001141:Aromatase