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. 2019 Mar 5;8(3):218.
doi: 10.3390/cells8030218.

Licochalcone A Inhibits Cellular Motility by Suppressing E-cadherin and MAPK Signaling in Breast Cancer

Wen-Chung Huang  1   2 , Haiso-Han Su  3 , Li-Wen Fang  4 , Shu-Ju Wu  5   6 , Chian-Jiun Liou  7   8
Affiliations

Licochalcone A Inhibits Cellular Motility by Suppressing E-cadherin and MAPK Signaling in Breast Cancer

Wen-Chung Huang et al. Cells. .

Abstract

A compound isolated from Glycyrrhizauralensis, licochalcone A (LA) exhibits anti-inflammatory and anti-tumor properties in various cell lines. LA has been found to promote autophagy and suppress specificity protein 1, inducing apoptosis in breast cancer cells. However, the regulation of breast cancer cell invasion and migration by LA is elusive. Thus, the present study investigated whether LA induces apoptosis and cellular motility in MDA-MB-231 breast cells, and investigated the underlying molecular mechanisms. MDA-MB-231 cells treated with LA and cell viability measured by cell counting kit-8 assay. Apoptotic signal proteins checked by flow cytometry, fluorescent staining, and Western blot. LA effectively suppressed cell migration, and modulated E-cadherin and vimentin expression by blocking MAPK and AKT signaling. LA inhibited cell proliferation and cell cycle, modulated mitochondrial membrane potential and DNA damage, and reduced oxidative stress in MDA-MB-231 cells. LA also activated cleaved-caspase 3 and 9, significantly decreased Bcl-2 expression, ultimately causing the release of cytochrome c from the mitochondria into the cytoplasm. Overall, our findings suggest that LA decreases cell proliferation and increases reactive oxygen species production for induced apoptosis, and regulates E-cadherin and vimentin by reducing MAPK and AKT signaling, resulting in suppressed MDA-MB-231 cell migration and invasion.

Keywords: MDA-MB-231 cells; apoptosis; caspase-3; cellular motility; licochalcone A.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Effects of licochalcone A (LA) on MDA-MB-231 cell viability. (A) The chemical structure of licochalcone A (LA). (B) Cell viability of MDA-MB-231 cells and BEAS-2B cells treated with the indicated LA concentrations (0–100 μM) for 24 h. (C) Morphological changes in MDA-MB-231 cells treated with LA for 24 h and stained with DAPI (arrows represent apoptotic cells). (D) Quantification of apoptotic cells. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 2
Figure 2
Effects of licochalcone A (LA) on clonogenic survival in MDA-MB-231 cells. (A) Cells were seeded on a culture plate and treated with LA for 24 h. Cells were fixed with 1% formalin-containing 1% crystal violet and colony formation inspected using an inverted microscope. (B) Colony cells were measured in culture plate. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 3
Figure 3
Effects of licochalcone A (LA) on cell cycle analysis. (A) Cells were seeded on a culture plate and treated with LA for 24 h. Cells were treated with MuseTM Cell Cycle reagent and the cell cycle status detected by flow cytometry. (B) Percentages of cells in each cell-cycle phase. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA). (C) p21 and cyclin D1 were assessed by Western blot, with β-actin as an internal control.
Figure 4
Figure 4
Licochalcone A (LA) treatment inhibited migration and invasion in wound healing assay (A,B) and transwell invasion assay (C,D). (A) Cell migration with and without LA treatment at different time points. (B) Measured width of the cell-free gap. A greater width indicates reduced cell migration. (C) Crystal violent-stained invasive cells, indicating that LA inhibited invasion. (D) Invasive cell numbers in the transwell assay. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 5
Figure 5
Effects of licochalcone A (LA) on AKT and the MAPK signal pathways. (A) Effect of varying concentrations (0–40 μM) of licochalcone A (LA) on the phosphorylation of PI3K, AKT, p38, JNK, and NF-κB. (B) E-cadherin and vimentin were also assessed by Western blot, with β-actin as an internal control.
Figure 6
Figure 6
Licochalcone A (LA) regulated apoptosis in MDA-MB-231 cells. (A) Flow cytometry results showing that 24-h treatment with licochalcone A (LA) induced apoptosis in MDA-MB-231cells. (B) Percentage of apoptotic cells in each group. (C) Western blot of cleaved (cl) caspase-3, cleaved caspase-9, and cleaved PAPR-1, cytochrome c, Bal2, and Bax expression. β-actin levels were used as internal controls. (D) LA increased caspase-3 activity and (E) depolarized cells (i.e., decreased mitochondrial membrane potential) in MDA-MB-231 cells. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 7
Figure 7
Licochalcone A (LA) regulated PPAR and cytochrome c expression. (A) Licochalcone A (LA) induced PPAR translocation from the cytoplasm to nucleus in MDA-MB-231 cells as detected by fluorescence microscopy. The cell nucleus was stained with DAPI solution. (B) 40 μM LA increased cytochrome c translocation from mitochondria to the cytoplasm of MDA-MB-231 cells at the indicated time points (0 to 24 h). The cell nucleus was stained with DAPI solution and mitochondria with Mito Trac solution.
Figure 8
Figure 8
Effect of licochalcone A (LA) on DNA damage in MDA-MB-231 cells. (A) Cells were seeded on a culture plate and treated with LA, incubated with γ-H2AX antibodies, and detected by fluorescence microscopy. The cell nucleus was stained with DAPI solution. (B) The fold expression of γ-H2AX relative to cells not treated with LA. (C) DNA damage assay using the Multi-Color DNA Damage Kit, and cellular DNA damage detected by flow cytometry. (D) Percentage of DNA damaged cells in each group. (E) γ-H2AX protein expression assessed by Western blot, with β-actin as an internal control. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 9
Figure 9
Effects of licochalcone A (LA) on reactive oxygen species (ROS) production in MDA-MB-231 cells. (A) ROS levels detected in MDA-MB-231 cells treated with 0-40 μM LA. (B) ROS levels detected in MDA-MB-231 cells treated with 40 μM LA for the indicated times. (C) Fluorescence microscope images of intracellular ROS. (D) Percentage of ROS detected in cells with the indicated LA concentrations compared to untreated cells. (E) ROS measured using the Muse™ Oxidative Stress Kit and detected by flow cytometry. (F) Quantification of ROS as the fold expression relative to untreated cells (0 μM LA). (G) Mitochondrial ROS detected by MitoSOX, with MitoTracker Green FM as the mitochondrial control. Mitochondrial ROS were observed using a fluorescence microscope. (H) Quantification of ROS as the fold expression relative to untreated cells (0 μM LA). The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 10
Figure 10
Licochalcone A (LA) regulated autophagy in MDA-MB-231 cells. (A) Fluorescence microscopy showed LC3B expression and the nucleus was stained with DAPI solution. (B) Quantification as the fold expression relative to the expression of untreated cells (0 μM LA). (C) Western blot assay of the expression of Beclin 1, ATG5, LC3 I, LC3II, and P62 in MDA-MB-231 cells. The data are presented as mean ± SD of three independent experiments (n = 6). * p < 0.05, ** p < 0.01 compared to untreated cells (0 μM LA).
Figure 11
Figure 11
Proposed model mechanism for licochalcone A (LA) suppression of cellular motility and induced apoptosis and autophagy in MDA-MB-231 breast cancer cells.
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