目的 探索槲皮素对 A549 肺癌干细胞活性及对 Wnt / β-连环蛋白(β-catenin)和音猬因子( sonic hedgehog,SHH)信 号通路的影响。 方法 使用不同浓度槲皮素(0、5、10、25、50、100 和 200 μmol / L)处理肺癌 A549 细胞 24、48 和 72 h,MTT 法检 测细胞存活率。 采用无血清培养法分离富集肺癌干细胞,观察槲皮素对 A549 悬浮细胞球体积、数量以及肺癌干细胞分子标 志物表达的影响;流式细胞术检测细胞凋亡;蛋白质印迹法检测增殖凋亡相关指标以及 Wnt / β-catenin 和 SHH 信号通路关键 分子的表达变化。 结果 槲皮素能够显著抑制 A549 细胞活力,且呈现时间和浓度依赖性;与空白对照组相比,槲皮素均显著抑 制 A549 成球能力和肺癌干细胞分子标志物[CD133、CD44 和醛脱氢酶 1A1(ALDH1A1)]表达;同时,肺癌干细胞增殖相关蛋 白增殖细胞核抗原(PCNA)、细胞周期蛋白 D1(Cyclin D1)和 B 细胞白血病/ 淋巴瘤-2(Bcl2)水平显著下调,凋亡相关指标 Bcl2 相关蛋白 X(Bax)和半胱氨酰天冬氨酸特异性蛋白酶-3(Cleaved Caspase 3)表达显著上调;槲皮素处理后,肺癌干细胞中 Wnt / β-catenin 和 SHH 信号通路关键分子磷酸化糖原合成酶激酶-3β(p-GSK3β)( Ser9)、β-catenin、原癌基因蛋白 c-Myc、胶 质瘤相关癌基因同源物 1(GLi1)、GLi2 和 SHH 的蛋白水平明显下降。 结论 槲皮素显著抑制肺癌干细胞活性,其机制可能与 调控 Wnt / β-catenin 和 SHH 通路有关。

Objective To explore the inhibitory effects of quercetin on the activity of A549 lung cancer stem cells. The role of quercetin on Wnt / β-catenin and Sonic Hedgehog SHH pathway was further illustrated. Method The lung cancer cell line A549 was cultured in vitro and treated with various concentrations of quercetin 0 5 10 25 50 100 and 200 μmol / L for 24 48 and 72 h cell vitality was detected by MTT assay. Lung cancer stem cells were isolated and enriched using sphere-forming assay. The effects of quercetin on the sphere formation and the expression of lung cancer stem cell specific markers were evaluated. The role of quercetin on the proliferation and apoptosis of sphere-forming cells was further determined by flow cytometry and western blot analysis. Finally the protein levels of key molecules of Wnt / β-catenin and SHH pathways were detected by western blot assay. Result Our results showed that quercetin significantly inhibited A549 cell viability in a time- dependent and concertation-dependent manner. Compared to the vehicle control group quercetin markedly suppressed the sphere formation ability of A549 cells and reduced the expression of lung cancer stem cell specific markers CD133 CD44 and aldehyde dehydrogenas 1A1 ALDH1A1 . Furthermore quercetin reduced proliferation and induced apoptosis of lung cancer stem cells. The levels of proliferating cell nuclear antigen PCNA Cyclin D1 and B-cell lymphoma- 2 Bcl2 were markedly downregulated while the expression of Bcl2-associated X Bax and cleaved cysteinyl aspartate specific proteinase Cleaved Caspase 3 was significantly upregulated upon quercetin treatment. The Wnt / β-catenin and SHH-related proteins phospho-glucogen synthase kinase-3β p-GSK3β Ser9 β-catenin c-Myc glioma-associated oncogene homolog 1 GLi1 GLi2 and SHH in lung cancer stem cells were also inhibited by quercetin. Conclusion Quercetin significantly suppressed the activity of lung cancer stem cells which might be associated with the downregulation of Wnt / β-catenin and SHH pathways.