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. MATERIALS AND 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.

译文

背景:多项研究表明,肺癌的进展受雌激素受体(ER)和表皮生长因子受体(EGFR)信号通路之间相互作用的支配。 EGFR和ER的联合靶向可能在肺癌治疗中具有协同作用。这项研究的目的是探讨在非小细胞肺癌(NSCLC)细胞系中使用阿那曲唑和吉非替尼联合抑制这两种途径的潜在效用。
材料与方法:实时检测肺癌细胞系(A549,H460,SPC-A-1,H1299)和正常支气管上皮细胞BEAS-2B中ER(ER-α和ER-β)的表达水平PCR和蛋白质印迹。免疫细胞化学法用于在具有最高ER表达水平的细胞系中定位ER-α和ER-β。单独或联合用阿那曲唑或吉非替尼处理细胞。 CCK8法检测细胞增殖抑制作用,流式细胞术检测细胞周期和凋亡效应。 Western blot检测磷酸化EGFR(p-EGFR),ERK,磷酸化ERK(p-ERK),AKT和磷酸化AKT(p-AKT)的表达水平。
结果:在这些细胞系中,ER在A549细胞中的表达水平最高。在A549细胞系中,ER-α主要位于细胞质中,而ER-β主要位于细胞质中,程度较小。两种药物的组合分别将24h,48h,72h的增殖抑制率分别提高到37.66±1.02%,63.41±2.02%,70.50±0.86%,这与G0 / G1相分数的升高密切相关( P <0.05)。单独或联合使用阿那曲唑,吉非替尼治疗的A549细胞凋亡率分别为10.72±1.12%,17.40±1.28%,23.02±2.32%(P <0.05)。与单独治疗相比,联合治疗的协同作用伴随着p-EGFR,p-ERK和p-AKT表达的降低。
结论:这项研究的结果表明,与单独使用这两种药物相比,阿那曲唑和吉非替尼的组合可以最大程度地抑制细胞增殖,诱导细胞凋亡并影响下游信号通路。我们的研究支持肺癌中ER和EGFR通路之间的功能相互作用,并为非小细胞肺癌患者提供了可临床利用的策略。

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