title: The synergistic effect of EGFR tyrosine kinase inhibitor gefitinib in combination with aromatase inhibitor anastrozole in non-small cell lung cancer cell lines.
abstract: BACKGROUND
  Several studies implicated that lung cancer progression was governed by the interaction between estrogen receptor (ER) and epidermal growth factor receptor (EGFR) signaling pathways. Combined targeting of EGFR and ER may have the synergistic effect in lung cancer treatment. The aim of this study was to explore the potential utility of inhibiting these two pathways with combination of anastrozole and gefitinib in non-small cell lung cancer (NSCLC) cell lines.
  
  
  METHODS
  The expression levels of ER (ER-α and ER-β) in lung cancer cell lines (A549, H460, SPC-A-1, H1299) and normal bronchus epithelial cell BEAS-2B were detected using real-time PCR and Western blot. Immunocytochemistry was used to locate ER-α and ER-β in cell line with highest ER expression levels. The cells were treated with anastrozole or gefitinib alone or in combination. The cell proliferation inhibition was detected by the CCK8 assay, cell cycle and apoptosis effects were detected by flow cytometry; the expression levels of phosphorylated-EGFR (p-EGFR), ERK, phosphorylated-ERK (p-ERK), AKT and phosphorylated-AKT (p-AKT) were detected by Western blot.
  
  
  RESULTS
  Among these cell lines the expression levels of ER in A549 cells were highest. In A549 cell line, ER-α was mainly localized in the cytoplasm, whereas ER-β was mainly localized in the cytoplasm and to a lesser degree in the nucleus. The combination of two drugs increased the proliferation inhibition rates for 24h, 48 h, 72 h to 37.66 ± 1.02%, 63.41 ± 2.02%, 70.50 ± 0.86%, respectively, which was closely associated with elevation of the G0/G1 phase fraction (P<0.05). Apoptosis rates of A549 cells treated with anastrozole, gefitinib alone or in combination were 10.72 ± 1.12%, 17.40±1.28%, 23.02 ± 2.32%, respectively (P<0.05). The synergistic effects of the combination therapy were accompanied by reduction of p-EGFR, p-ERK and p-AKT expression compared with individual treatment.
  
  
  CONCLUSIONS
  The results of this study suggest that the combination of anastrozole and gefitinib compared with either drug alone can maximally inhibit cell proliferation, induce apoptosis, and affect downstream signaling pathways. Our study supports functional interaction between the ER and the EGFR pathways in lung cancer and provides a clinically exploitable strategy for non-small cell lung cancer patients.
authors.affiliation: Shanghai Lung Cancer Center, Shanghai Chest Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200030, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Shen
authors.forename: Ziming
authors.initials: Z
authors.lastname: Li
authors.forename: Shengping
authors.initials: S
authors.lastname: Shen
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Niu
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Yu
authors.forename: Zonghai
authors.initials: Z
authors.lastname: Li
authors.forename: Meilin
authors.initials: M
authors.lastname: Liao
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Chen
authors.forename: Shun
authors.initials: S
authors.lastname: Lu
mesh_terms: D000077384:Anastrozole; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D047072:Aromatase Inhibitors / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004357:Drug Synergism; D066246:ErbB Receptors / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000077156:Gefitinib; D015870:Gene Expression; D006801:Humans; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000188:drug therapy*; D020935:MAP Kinase Signaling System; D009570:Nitriles / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D018931:Antineoplastic Agents, Hormonal; D047072:Aromatase Inhibitors; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D014230:Triazoles; D000077384:Anastrozole; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D000077156:Gefitinib