Quercetin inhibits epithelial-mesenchymal transition, decreases invasiveness and metastasis, and reverses IL-6 induced epithelial-mesenchymal transition, expression of MMP by inhibiting STAT3 signaling in pancreatic cancer cells

Abstract

Quercetin, a flavone, is multifaceted, having anti-oxidative, anti-inflammatory, and anticancer properties. In the present study, we explored the effects of quercetin on the epithelial-mesenchymal transition (EMT) and invasion of pancreatic cancer cells and the underlying mechanisms. We noted that quercetin exerted pronounced inhibitory effects in PANC-1 and PATU-8988 cells. Moreover, quercetin inhibited EMT and decreased the secretion of matrix metalloproteinase (MMP). Meanwhile, we determined the activity of STAT3 after quercetin treatment. STAT3 phosphorylation decreased following treatment with quercetin. We also used activating agent of STAT3, IL-6, to induce an increase in cell malignancy and to observe the effects of treatment with quercetin. As expected, the EMT and MMP secretion increased with activation of the STAT3 signaling pathway, and quercetin reversed IL-6-induced EMT, invasion, and migration. Therefore, our results demonstrate that quercetin triggers inhibition of EMT, invasion, and metastasis by blocking the STAT3 signaling pathway, and thus, quercetin merits further investigation.

Keywords: EMT; MMPs; STAT3; pancreatic cancer; quercetin.

Figure 1

Dose-dependent inhibition of cell viability in PANC-1 and PATU-8988 cells, and of epithelial–mesenchymal transition in PATU-8988 cells. Notes: PANC-1 and PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h in the cell viability experiments, and PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h in the rest of the experiments. (A, B) Fold changes in cell viability of PANC-1 and PATU-8988 cells. (C) Fold changes in mRNA expression of E-cadherin, N-cadherin, and Vimentin in PATU-8988 cells. PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h. (D) The protein expression of E-cadherin, N-cadherin, and Vimentin was measured with a Western blotting assay. β-actin was used as an internal control. (E) Fold changes in mRNA expression of Zeb1, Twist, Slug, and Snail in PATU-8988 cells. (F) The protein expression of Zeb1, Twist, Slug, and Snail was measured with a Western blotting assay in PATU-8988 cells. β-actin was used as an internal control. (G) The immunofluorescence of N-cadherin, Vimentin (green), and the cell nuclei (blue). The data are presented as the mean ± standard error of the mean (SEM); n=3; *P<0.05 versus the 0 μM group.

Figure 1

Dose-dependent inhibition of cell viability in PANC-1 and PATU-8988 cells, and of epithelial–mesenchymal transition in PATU-8988 cells. Notes: PANC-1 and PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h in the cell viability experiments, and PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h in the rest of the experiments. (A, B) Fold changes in cell viability of PANC-1 and PATU-8988 cells. (C) Fold changes in mRNA expression of E-cadherin, N-cadherin, and Vimentin in PATU-8988 cells. PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h. (D) The protein expression of E-cadherin, N-cadherin, and Vimentin was measured with a Western blotting assay. β-actin was used as an internal control. (E) Fold changes in mRNA expression of Zeb1, Twist, Slug, and Snail in PATU-8988 cells. (F) The protein expression of Zeb1, Twist, Slug, and Snail was measured with a Western blotting assay in PATU-8988 cells. β-actin was used as an internal control. (G) The immunofluorescence of N-cadherin, Vimentin (green), and the cell nuclei (blue). The data are presented as the mean ± standard error of the mean (SEM); n=3; *P<0.05 versus the 0 μM group.

Figure 2

Quercetin inhibits invasion and metastasis of PATU-8988 cells in a dose-dependent manner. Notes: PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h. (A) Fold changes in mRNA expression of MMP-2 and MMP-7 in PATU-8988 cells. (B) The protein expression of MMP-2 and MMP-7 was measured with a Western blotting assay. β-actin was used as an internal control. (C) Quercetin inhibited the invasion and migration capacity of PATU-8988 cells. (D) Image of the wound healing assay and the average width of the wound area. The data are presented as the mean ± standard error of the mean (SEM); n=3; *P<0.05 versus the 0 μM group.

Figure 2

Quercetin inhibits invasion and metastasis of PATU-8988 cells in a dose-dependent manner. Notes: PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h. (A) Fold changes in mRNA expression of MMP-2 and MMP-7 in PATU-8988 cells. (B) The protein expression of MMP-2 and MMP-7 was measured with a Western blotting assay. β-actin was used as an internal control. (C) Quercetin inhibited the invasion and migration capacity of PATU-8988 cells. (D) Image of the wound healing assay and the average width of the wound area. The data are presented as the mean ± standard error of the mean (SEM); n=3; *P<0.05 versus the 0 μM group.

Figure 3

Quercetin inhibits the STAT3 signaling pathway and IL-6-induced STAT3 signaling. Notes: (A) The protein expression of STAT3 and p-STAT3 was measured with a Western blotting assay. β-actin was used as an internal control. PATU-8988 cells were treated with different concentrations of quercetin (0, 20, 40, and 80 μM) for 24 h. (B) Morphological changes after IL-6 (100 ng/mL) treatment in PATU-8988 cells. (C) The protein expression of IL-6-induced STAT3 and p-STAT3 was measured with a Western blotting assay. β-actin was used as an internal control. PATU-8988 cells were treated with human recombinant IL-6 (100 ng/mL) for 72 h, and in the last 24 h, quercetin (80 μM) was added. (D) The protein expression of IL-6-induced p-STAT3 in cells treated with quercetin and AG490. β-actin was used as an internal control. Before being treated with quercetin for 24 h, PANC-1 cells were pretreated with AG490 for 30 min.

Figure 4

Quercetin inhibits IL-6-induced epithelial–mesenchymal transition and MMP secretion in PATU-8988 cells. Notes: PATU-8988 cells were treated with human recombinant IL-6 (100 ng/mL) for 72 h, and in the last 24 h, quercetin (80 μM) was added. (A) Fold changes in mRNA expression of E-cadherin, N-cadherin, and Vimentin in PATU-8988 cells. (B) The protein expression of E-cadherin, N-cadherin, and Vimentin was measured with a Western blotting assay. β-actin was used as an internal control. (C) Fold changes in the mRNA expression of Zeb1, Twist, Slug, and Snail in PATU-8988 cells. (D) The protein expression of Zeb1, Twist, Slug, and Snail was measured with a Western blotting assay in PATU-8988 cells. β-actin was used as an internal control. (E) Fold changes in the mRNA expression of MMP-2 and MMP-7 in PATU-8988 cells. (F) The protein expression of MMP-2 and MMP-7 was measured with a Western blotting assay. β-actin was used as an internal control. The data are presented as the mean ± standard error of the mean (SEM); n=3; *P<0.05 versus the 0 μM group; ^#P<0.05 versus the 20 μM group.