Acacetin and Pinostrobin Inhibit Malignant Breast Epithelial Cell Adhesion and Focal Adhesion Formation to Attenuate Cell Migration

Abstract

Naturally occurring flavonoids, such as acacetin and pinostrobin, disrupt a wide range of processes during tumor progression, such as cell proliferation, apoptosis, and angiogenesis. Although the antiproliferative and antiapoptotic effects of acacetin and pinostrobin have been studied using various cell lines, relatively little is known about the effects of acacetin and pinostrobin on cancer cell migration and metastasis. For instance, it is unclear whether acacetin or pinostrobin have any effect on breast cancer cell migration or adhesion. In this study, we assessed the effects of acacetin and pinostrobin on malignant MDA-MB-231 and T47D breast epithelial cells and non-tumorigenic MCF10A breast epithelial cells. Our results demonstrate that both acacetin and pinostrobin selectively inhibit the migration of both MDA-MB-231 and T47D cells in a dose-dependent manner while exhibiting blunted effects on MCF10A cells. Interestingly, neither compound had an effect on cell proliferation in any of the 3 cell lines. Furthermore, both acacetin and pinostrobin inhibit MDA-MB-231 and T47D cell adhesion, cell spreading, and focal adhesion formation, but have no significant effect on MCF10A cells. Collectively, these results suggest that both acacetin and pinostrobin selectively inhibit malignant breast epithelial cell migration through attenuation of cell adhesion and focal adhesion formation. These findings indicate that both acacetin and pinostrobin may serve as potential therapeutic options to target breast tumor cell migration during late-stage tumor progression.

Keywords: acacetin; adhesion; flavonoids; focal adhesion; migration; pinostrobin.

Figure 1.

Chemical structure of acacetin (A) and (±)-pinostrobin (B).

Figure 2.

Both acacetin and pinostrobin have no effect on cell proliferation. MDA-MB-231 (A), T47D (B), and MCF10A cells (C) were cultured for 24 hours in the presence of either pinostrobin or acacetin. Cell proliferation was determined using CellTiter 96 AQueous One Solution Reagent and absorbance was measured at 490 nm. Data are presented as average absorbance ± SEM (standard error of mean) from a minimum of 8 wells. There were no statistically significant differences between DMSO control and treatments for all cell lines.

Figure 3.

Acacetin and pinostobin inhibit malignant breast epithelial cell migration. Example images of MDA-MB-231, T47D, and MCF10A cells (stained with crystal violet) in a transwell migration assay in response to increasing concentrations of acacetin (A) and pinostrobin (B). Scale bar = 100 µm. (C and D) 20 µM acacetin or pinostrobin inhibited MDA-MB-231 transwell migration by approximately 45% and 70%, while T47D transwell migration was reduced by approximately 46% and 77%, respectively. Only 20 µM acacetin produced a significant inhibition of 13% on non-tumorigenic MCF10A cells. Data in C and D represent the mean ± SEM (standard error of mean) from a minimum of 3 independent experiments performed in duplicate. *P < .05, **P < .01, ***P < .001 indicate statistical significance relative to DMSO control; 2-sample t test.

Figure 4.

Malignant breast epithelial cells are more sensitive, compared with non-tumorigenic cells, to the inhibitory effects of acacetin and pinostrobin on cell migration. (A and B) Example phase-contrast images of cells immediately following scratch formation (0 hour) and following migration for 24 hours (MDA-MB-231), 30 hours (T47D), or 18 hours (MCF10A) in the absence or presence of acacetin (A) or pinostrobin (B). Scale bar = 100 µm. (C and D) Both acacetin and pinostrobin produced a dose-dependent inhibition of malignant cell migration, while only 20 µM acacetin or pinostrobin produced a significant inhibition on MCF10A cells. Data in C and D are presented as mean ± SEM (standard error of mean) from a minimum of 4 independent experiments performed in triplicate. *P < .05, **P < .01, ***P < .001 indicate statistical significance relative to DMSO control; 2-sample t test.

Figure 5.

Acacetin and pinostrobin produced a dose-dependent inhibition of malignant cell adhesion. Both acacetin (A) and pinostrobin (B) selectively inhibited cell adhesion of MDA-MB-231 and T47D cells but had no measurable effect on MCF10A cells. The data are presented as mean ± SEM (standard error of mean) from a minimum of 3 independent experiments performed in quadruplicate. *P < .05, **P < .01, ***P < .001 indicate statistical significance relative to DMSO control; 2-sample t test.

Figure 6.

Acacetin and pinostrobin reduced focal adhesion formation in malignant breast epithelial cells. (A-C) Representative fluorescence images of MDA-MB-231 (A), T47D (B), and MCF10A (C) cells treated with DMSO control, 20 µM acacetin, or pinostrobin. Indirect immunofluorescence of focal adhesions was assessed with a vinculin antibody and counterstained with TRITC-phalloidin. Scale bar = 10 µm. (D and E) Both acacetin and pinostrobin produced a dose-dependent reduction in average vinculin area in both MDA-MB-231 cells and T47D cells. Treatment with 20 µM acacetin reduced average vinculin area by 57% and 76% in MDA-MB-231 cells and T47D cells, respectively, while 20 µM pinostrobin decreased average vinculin area by 59% and 73% in MDA-MB-231 and T47D cells, respectively. Acacetin and pinostrobin produced no statistically significant effect in average vinculin area in MCF10A cells. (F and G) Relative vinculin area was examined by normalizing the average total surface area containing vinculin to the total cell area as assessed by TRITC-phalloidin. Data are presented as average ± SEM (standard error of mean) from a minimum of 72 cells for each condition. *P < .05, **P < .01, ***P < .001 indicate statistical significance relative to DMSO control; 2-sample t test.