Quercetin Suppresses the Migration and Invasion in Human Colon Cancer Caco-2 Cells Through Regulating Toll-like Receptor 4/Nuclear Factor-kappa B Pathway

Abstract

Objective: 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.

Materials and methods: 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.

Results: 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.

Conclusion: The findings suggested for the 1(st) time that quercetin might exert its anticolon cancer activity via the TLR4- and/or NF-κB-mediated signaling pathway.

Summary: Quercetin could remarkably suppress the migratory and invasive capacity of Caco-2 cellsThe 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 mannerThe 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 p65Quercetin 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.

Keywords: Colon cancer; invasion; migration; quercetin; toll-like receptor 4.

Figure 1

Effect of quercetin on cell viability of Caco-2 cells. The data were obtained from triplicate experiments and expressed as the means ± standard deviation (n = 3)

Figure 2

Effect of quercetin on migration of Caco-2 cells. (a) Cells were incubated in 24 well plates and treated with quercetin (5 μM) for 24 h in the presence or absence of anti-toll-like receptor 4 antibody (2 μg) or pyrrolidine dithiocarbamate (1 μM). a’: Blank control; b’: Quercetin of 5 μM; c’: Anti-toll-like receptor 4 antibody (2 μg); d’: Pyrrolidine dithiocarbamate (1 μM); e’: Quercetin (5 μM) + anti-toll-like receptor 4 antibody (2 μg); f’: Quercetin (5 μM) + pyrrolidine dithiocarbamate (1 μM). (b) The closure rate of cells was recorded, and the data were obtained from triplicate experiments and expressed as the means ± standard deviation (n = 3). *P < 0.05, **P < 0.01, versus blank control; #P < 0.05, quercetin (5 μM) + anti-toll-like receptor 4 versus anti-toll-like receptor 4 alone; ^&P < 0.05, quercetin (5 μM) + pyrrolidine dithiocarbamate versus pyrrolidine dithiocarbamate alone

Figure 3

Effect of quercetin on invasion of Caco-2 cells. (a) The invasion of Caco-2 cells was evaluated by cell invasion assay, which was performed on 8 μm polycarbonate Matrigel-coated transwell cell culture chambers. Cells were incubated in 24 well plates and treated with quercetin (5 μM) for 24 h in the presence or absence of anti-toll-like receptor 4 antibody (2 μg) or pyrrolidine dithiocarbamate (1 μM). a’: Blank control; b’: Quercetin of 5 μM; c’: Anti-toll-like receptor 4 antibody (2 μg); d’: Pyrrolidine dithiocarbamate (1 μM); e’: Quercetin (5 μM) + anti-toll-like receptor 4 antibody (2 μg); f’: Quercetin (5 μM) + pyrrolidine dithiocarbamate (1 μM). (b) Optical density for the permeated cells. Data were obtained from triplicate experiments and expressed as the means ± standard deviation (n = 3). **P < 0.01, versus blank control; #P < 0.05, quercetin (5 μM) + anti-toll-like receptor 4 versus. anti-toll-like receptor 4 alone; ^&&P < 0.05, quercetin (5 μM) + pyrrolidine dithiocarbamate versus pyrrolidine dithiocarbamate alone

Figure 4

Effect of quercetin on the expressions of metastasis-related protein with Western blot assay. (a) Western blot for the expressions of E-cadherin, mitochondrial membrane potential-2 and mitochondrial membrane potential-9 in Caco-2 cells. (b) E-cadherin protein level; (c) mitochondrial membrane potential-2 protein level. (d) Mitochondrial membrane potential-9 protein level. Data were expressed as the means ± standard deviation (n = 3). *P < 0.05, **P < 0.01 versus control blank group

Figure 5

Effect of quercetin on the toll-like receptor 4/nuclear factor-kappa B pathway in Caco-2 cells. (a) Western blot for the expressions of toll-like receptor 4, nuclear factor-kappa B p65, mitochondrial membrane potential-2, mitochondrial membrane potential-9, and E-cadherin; (b) toll-like receptor 4 protein level; (c) nuclear factor-kappa B p65 protein level; (d) E-cadherin protein level; (e) mitochondrial membrane potential-2 protein level; (f) mitochondrial membrane potential-9 protein level. Data were expressed as the means ± standard deviation (n = 3). *P < 0.05, **P < 0.01, versus blank control; #P < 0.05, quercetin (5 μM) + anti-toll-like receptor 4 versus anti-toll-like receptor 4 alone; ^&P < 0.05, ^&&P < 0.01, quercetin (5 μM) + pyrrolidine dithiocarbamate versus pyrrolidine dithiocarbamate alone

Figure 6

Effect of quercetin on the expression of inflammation factors with enzyme-linked immunosorbent assay. (a) Tumor necrosis factor-α content in the cell supernatant; (b) cyclooxygenase-2 content in the cell supernatant; (c) interleukin-6 content in the cell supernatant. Data were expressed as the means ± standard deviation (n = 3). *P < 0.05, **P < 0.01 versus control blank group

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Yucheng Song