Quercetin Suppresses the Migration and Invasion in Human Colon Cancer Caco-2 Cells Through Regulating Toll-like Receptor 4/Nuclear Factor-kappa B Pathway.
Quercetin Suppresses the Migration and Invasion in Human Colon Cancer Caco-2 Cells Through Regulating Toll-like Receptor 4/Nuclear Factor-kappa B Pathway.
复制标题
槲皮素通过调节 Toll-like Receptor 4/Nuclear Factor-kappa B 通路抑制人结肠癌 Caco-2 细胞的迁移和侵袭
DOI：
10.4103/0973-1296.182154
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发表时间：
2016-05
影响因子：
0.7
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Han M;Song Y;Zhang X
与炎症反应相关的迁移和侵袭特征在结肠癌的发生发展中起着至关重要的作用。槲皮素是一种生物类黄酮化合物，广泛存在于蔬菜和水果中。尽管槲皮素具有抗氧化和抗癌活性，但人类结肠癌细胞中的分子信号传导途径仍不清楚。因此，本研究旨在探讨槲皮素对结肠癌迁移和侵袭活性的抑制及其潜在机制。分别通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物测定、伤口愈合测定和Transwell小室测定分析槲皮素对Caco-2细胞的细胞活力、迁移和侵袭的影响。采用Western blot法检测Toll样受体4（TLR4）、核因子κB（NF-κB）p65、线粒体膜电位2（MMP-2）、MMP-9蛋白表达。采用酶联免疫吸附法检测细胞上清液中肿瘤坏死因子-α（TNF-α）、环氧合酶-2（Cox-2）、白细胞介素-6（IL-6）等炎症因子。选择<20μM的槲皮素浓度用于进一步的实验。槲皮素 (5 μM) 可以显着抑制 Caco-2 细胞的迁移和侵袭能力。槲皮素可降低转移相关蛋白MMP-2、MMP-9的表达，而增加E-cadherin蛋白的表达，且呈剂量依赖性。有趣的是，抗TLR4（2 μg）抗体或吡咯烷二硫代氨基甲酸酯（PDTC；1 μM）可以影响槲皮素对细胞迁移和侵袭的抑制，以及MMP-2、MMP-9、E-cadherin、TLR4和NF-κB p65的蛋白表达。此外，槲皮素还可减少炎症因子TNF-α、Cox-2和IL-6的产生。研究结果首次表明槲皮素可能通过 TLR4 和/或 NF-κB 介导的信号通路发挥其抗结肠癌活性。槲皮素可显着抑制Caco-2细胞的迁移和侵袭能力，槲皮素可降低线粒体膜电位2（MMP-2）、MMP-9转移相关蛋白的表达，而上皮细胞钙粘蛋白（E-cadherin）的表达则呈剂量依赖性增加。槲皮素可减少肿瘤坏死因子-α、环氧合酶-2、白细胞介素-6等炎症因子的产生。 使用的缩写：MTT：3-(4,5-二甲基噻唑-2-基)- 2,5-联苯四唑溴化物、TLR4：Toll 样受体 4、NF-κB：核因子-κ B、MMP-2：线粒体膜电位-2、MMP-9：线粒体膜电位-9、TNF-α：肿瘤坏死因子-α、 Cox-2：环氧合酶-2、IL-6：白细胞介素-6、ELISA：酶联免疫吸附测定、PDTC：吡咯烷二硫代氨基甲酸酯、ROS：活性氧、DMSO：二甲亚砜、FBS：胎牛血清、DMEM：杜尔贝科改良 Eagle 培养基、OD：光密度、IPP：Image Pro-plus、PBS：磷酸盐缓冲盐水、SD：标准差、 ANOVA：单向方差分析，SPSS：社会科学统计软件包，ECM：细胞外基质，TLR：Toll 样受体，LPS：脂多糖。
The migration and invasion features, which were associated with inflammatory response, acted as vital roles in the development of colon cancer. Quercetin, a bioflavonoid compound, was widely spread in vegetables and fruits. Although quercetin exerts antioxidant and anticancer activities, the molecular signaling pathways in human colon cancer cells remain unclear. Hence, the present study was conducted to investigate the suppression of quercetin on migratory and invasive activity of colon cancer and the underlying mechanism. The effect of quercetin on cell viability, migration, and invasion of Caco-2 cells was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound-healing assay, and transwell chambers assay, respectively. The protein expressions of toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB) p65, mitochondrial membrane potential-2 (MMP-2), and MMP-9 were detected by Western blot assay. The inflammatory factors, such as tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (Cox-2), and interleukin-6 (IL-6), in cell supernatant were detected by enzyme-linked immunosorbent assay. The concentration of quercetin <20 μM was chosen for further experiments. Quercetin (5 μM) could remarkably suppress the migratory and invasive capacity of Caco-2 cells. The expressions of metastasis-related proteins of MMP-2, MMP-9 were decreased, whereas the expression of E-cadherin protein was increased by quercetin in a dose-dependent manner. Interestingly, the anti-TLR4 (2 μg) antibody or pyrrolidine dithiocarbamate (PDTC; 1 μM) could affect the inhibition of quercetin on cell migration and invasion, as well as the protein expressions of MMP-2, MMP-9, E-cadherin, TLR4, and NF-κB p65. In addition, quercetin could reduce the inflammation factors production of TNF-α, Cox-2, and IL-6. The findings suggested for the 1st time that quercetin might exert its anticolon cancer activity via the TLR4- and/or NF-κB-mediated signaling pathway. Quercetin could remarkably suppress the migratory and invasive capacity of Caco-2 cells The expressions of metastasis-related proteins of mitochondrial membrane potential-2 (MMP-2), MMP-9 were decreased, whereas the expression of E-cadherin protein was increased by quercetin in a dose-dependent manner The anti-toll-like receptor 4 (TLR4) antibody or pyrrolidine dithiocarbamate affected the inhibition of quercetin on cell migration and invasion, as well as the protein expressions of MMP-2, MMP-9, E-cadherin, TLR4, and nuclear factor-kappa B p65 Quercetin could reduce the inflammation factors production of tumor necrosis factors-α, cyclooxygenase-2, and interleukin-6. Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphen yltetrazolium bromide, TLR4: Toll-like receptor 4, NF-κB: Nuclear factor-kappa B, MMP-2: Mitochondrial membrane potential-2, MMP-9: Mitochondrial membrane potential-9, TNF-α: Tumor necrosis factor-α, Cox-2: Cyclooxygenase-2, IL-6: Interleukin-6, ELISA: Enzyme-linked immunosorbent assay, PDTC: Pyrrolidine dithiocarbamate, ROS: Reactive oxygen species, DMSO: Dimethyl sulfoxide, FBS: Fetal bovine serum, DMEM: Dulbecco modified Eagle medium, OD: Optical density, IPP: Image Pro-plus, PBS: Phosphate buffered saline, SD: Standard deviation, ANOVA: One-way analysis of variance, SPSS: Statistical Package for the Social Sciences, ECM: Extracellular matrix, TLRs: Toll-like receptors, LPS: Lipopolysaccharide.