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. 2018 May;15(2):137-156.
doi: 10.20892/j.issn.2095-3941.2018.0012.

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-β-induced epithelial-mesenchymal transition

Aniket V Mali  1   2 , Asavari A Joshi  1 , Mahabaleshwar V Hegde  1 , Shivajirao S Kadam  2
Affiliations

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-β-induced epithelial-mesenchymal transition

Aniket V Mali et al. Cancer Biol Med. 2018 May.

Abstract

Objective: Triple-negative breast cancer (TNBC) is highly metastatic, and there is an urgent unmet need to develop novel therapeutic strategies leading to the new drug discoveries against metastasis. The transforming growth factor-β (TGF-β) is known to promote the invasive and migratory potential of breast cancer cells through induction of epithelial-mesenchymal transition (EMT) via the ERK/NF-κB/Snail signaling pathway, leading to breast cancer metastasis. Targeting this pathway to revert the EMT would be an attractive, novel therapeutic strategy to halt breast cancer metastasis.

Methods: Effects of enterolactone (EL) on the cell cycle and apoptosis were investigated using flow cytometry and a cleaved caspase-3 enzyme-linked immunosorbent assay (ELISA), respectively. Effects of TGF-β induction and EL treatment on the functional malignancy of MDA-MB-231 breast cancer cells were investigated using migration and chemo-invasion assays. The effects of EL on EMT markers and the ERK/NF-κB/Snail signaling pathway after TGF-β induction were studied using confocal microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and flow cytometry.

Results: Herein, we report that EL exhibits a significant antimetastatic effect on MDA-MB-231 cells by almost reverting the TGF-β-induced EMT in vitro. EL downregulates the mesenchymal markers N-cadherin and vimentin, and upregulates the epithelial markers E-cadherin and occludin. It represses actin stress fiber formation via inhibition of mitogen-activated protein kinase p-38 (MAPK-p38) and cluster of differentiation 44 (CD44). EL also suppresses ERK-1/2, NF-κB, and Snail at the mRNA and protein levels.

Conclusions: Briefly, EL was found to inhibit TGF-β-induced EMT by blocking the ERK/NF-κB/Snail signaling pathway, which is a promising target for breast cancer metastasis therapy.

Keywords: EMT; Enterolactone; breast cancer metastasis; invasion; migration.

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Figures

S1
S1
Cell cycle arrest in ‘S’ phase by increasing concentrations of EL. Effects of EL, TAM and DOXO on cell cycle of MDA-MB-231 cells representing % of cell population in each phase upon 48 h of treatment.
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Confocal microscopy images of immunofluorescence staining showing the effects of EL. (A) Induction of EMT by TGF-β (10 ng/mL) characterized by fibroblast-like mesenchymal morphology, which was reverted in a dose-dependent manner by EL by retaining a more epithelial-like appearance. (B) Decreased expression of E-cadherin after TGF-β stimulation (10 ng/mL), and increased expression of E-cadherin when concentrations of 25, 50, and 75 μM EL were used. (C) Increased expression of vimentin after TGF-β stimulation (10 ng/mL), and decreased expression of vimentin when concentrations of 25, 50, and 75 μM EL were used. Values are represented as mean ± SEM of three independent experiments (n = 3), with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 indicating statistical significance.
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Effects of EL on epithelial markers E-cadherin and occludin, and mesenchymal markers vimentin and N-cadherin at the mRNA and protein levels. (A) Dose-dependent reversal of decreased expression of E-cadherin after TGF-β induction when concentrations of 25, 50, and 75 μM EL were used. (B) Dose-dependent reversal of increased expression of vimentin after TGF-β induction when concentrations of 25, 50, and 75 μM EL were used. (C) Decreased occludin gene expression after TGF-β stimulation, and increased occludin gene expression when concentrations of 25, 50, and 75 μM EL were used. (D) Increased gene expression of N-cadherin after TGF-β stimulation, and decreased gene expression of occludin when concentrations of 25, 50, and 75 μM EL were used. (E) Representative Western blot results of the indicated EMT-related proteins derived from cells treated with TGF-β (10 ng/mL) alone or combined with EL (25, 50, and 75 μM). Images of the full-length Western blot gels are shown in supplementary files. Values are represented as mean ± SEM of three independent experiments (n = 3), with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 indicating statistical significance.
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Effects of EL on the formation of actin stress fibers. TGF-β stimulation (10 ng/mL for 48 h) resulted in increased actin stress fiber formation, while EL treatment with 25, 50 and 75 μM for 48 h, actin stress fiber formation decreased in a dose-dependent manner.
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Effects of EL on CD44 and MAPK-p38 expression at the mRNA and protein levels. (A) Increased protein expression of CD44 after transforming growth factor beta (TGF-β) stimulation (10 ng/mL), and decreased CD44 expression with 25, 50, and 75 μM of EL treatments, indicated by median fluorescence intensity and mean fluorescence intensity (MFI). (B) Representative histograms of fluorescence-activated cell sorting (FACS) analysis indicating changes in CD44 expression in terms of MFI and median fluorescence intensity after EL treatment. (C) Increased gene expression of CD44 after TGF-β stimulation, and dose-dependent reduction in CD44 gene expression when concentrations of 25, 50, and 75 μM EL were used. (D) Increased gene expression of MAPK-p38 after TGF-β stimulation, and decreased MAPK-p38 gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (E) Increased protein expression of MAPK-p38 after TGF-β stimulation, and decreased MAPK-p38 protein expression with 25, 50, and 75 μM of EL treatments. (F) Representative Western blot results for the MAPK-p38 protein derived from cells treated with TGF-β (10 ng/mL) alone or combined with EL (25, 50, 75 μM). The full-length Western blot gel images are shown in supplementary files. Values are represented as mean ± SEM of three independent experiments (n = 3), with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 indicating statistical significance.
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EL on the levels of ERK-1/2, NF-κB/p65, IκB-α and Snail gene expression. (A) Increased gene expression of ERK-1 after TGF-β stimulation, and decreased ERK-1 gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (B) Increased gene expression of ERK-2 after TGF-β stimulation, and decreased ERK-2 gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (C) Increased gene expression of NF-κB/p65 after TGF-β stimulation and decreased NF-κB/p65 gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (D) Decreased gene expression of IκB-α after TGF-β stimulation, and increased IκB-α gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (E) Increased gene expression of Snail after TGF-β stimulation, and decreased Snail gene expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. Values are represented as mean ± SEM of three independent experiments (n = 3), with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 indicating statistical significance.
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Effects of EL on the levels of ERK-1/2, NF-κB/p65, and Snail protein expression. (A) Increased protein expression of tERK-1/2 after TGF-β stimulation, and decreased tERK-1/2 protein expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (B) Increased protein expression of pERK-1/2 after TGF-β stimulation, and decreased pERK-1/2 protein expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (C) Increased protein expression of NF-κB/p65 after TGF-β stimulation, and decreased NF-κB/p65 protein expression with 25, 50, and 75 μM of EL treatments, in a dose dependent manner. (D) Increased protein expression of Snail after TGF-β stimulation, and decreased Snail protein expression with 25, 50, and 75 μM of EL treatments, in a dose-dependent manner. (E) Representative Western blot results for the tERK-1/2, pERK-1/2, NF-κB/p65, and Snail proteins derived from cells treated with TGF-β (10 ng/mL) alone or combined with EL (25, 50, 75 μM). The full-length Western blot gel images are shown in supplementary files. Values are represented as mean ± SEM of three independent experiments (n =3), with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 indicating statistical significance.
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Inhibition of TGF-β-induced EMT in MDA-MB-231 breast cancer cells by blocking the ERK/NF-κB/Snail signaling pathway in vitro.
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