Resveratrol and estradiol exert disparate effects on cell migration, cell surface actin structures, and focal adhesion assembly in MDA-MB-231 human breast cancer cells

Abstract

Resveratrol, a grape polyphenol, is thought to be a cancer preventive, yet its effects on metastatic breast cancer are relatively unknown. Since cancer cell invasion is dependent on cell migration, the chemotactic response of MDA-MB-231 metastatic human breast cancer cells to resveratrol, estradiol (E2), or epidermal growth factor (EGF) was investigated. Resveratrol decreased while E2 and EGF increased directed cell migration. Resveratrol may inhibit cell migration by altering the cytoskeleton. Resveratrol induced a rapid global array of filopodia and decreased focal adhesions and focal adhesion kinase (FAK) activity. E2 or EGF treatment did not affect filopodia extension but increased lamellipodia and associated focal adhesions that are integral for cell migration. Combined resveratrol and E2 treatment resulted in a filopodia and focal adhesion response similar to resveratrol alone. Combined resveratrol and EGF resulted in a lamellipodia and focal adhesion response similar to EGF alone. E2 and to a lesser extent resveratrol increased EGFR activity. The cytoskeletal changes and EGFR activity in response to E2 were blocked by EGFR1 inhibitor indicating that E2 may increase cell migration via crosstalk with EGFR signaling. These data suggest a promotional role for E2 in breast cancer cell migration but an antiestrogenic, preventative role for resveratrol.

Figure 1

Effects of E₂, EGF, and trans-resveratrol on directed cell migration of MDA-MB-231 cells. Cells were serum-starved in phenol red-free media for 24 hours and migration assays were conducted using the following as chemoattractants for 8 hours: DMSO as control (Un); 0.1 µM E₂ (E₂); 50 ng/ml EGF (EGF); 50 µM resveratrol (Res), pretreated with Res for 10 minutes followed by E₂ for 10 minutes (Res/E₂), or pretreated with Res for 10 minutes followed by EGF for 10 minutes (Res/EGF). The number of cells that migrated through the upper chamber of Costar wells was quantified and made relative to DMSO control. Data are expressed as mean cells migrated ± SEM of at least three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments at P < .05.

Figure 2

Effect of E₂ and trans-resveratrol on the actin cytoskeleton of ER (+) and (-) cells. (A) Micrographs of T47D, MDA-MB-231, and SKBR3 cells at x600 magnification. Cells were serum-starved in phenol red-free media for 24 hours and stimulated for 10 minutes with DMSO as control (Un), E₂ (0.1 µM), or 50 µM resveratrol (Res). Cells were stained with rhodamine phalloidin to visualize F-actin. Arrowheads (<) indicate a filopodium. (B) Western blot of whole cell lysates of T47D, MDA-MB-231, and SKBR3. Equal amounts of proteins from cell lysates were loaded on SDS-PAGE and Western-blotted using anti-ERα (65 kDa band) or anti-ERβ (53 kDa band). Equal amounts of cell lysates were also probed with anti-EGFR1 (185 kDa) or anti-phosphoEGFR (Y1173) (185 kDa).

Figure 3

Effect of E₂, EGF, or trans-resveratrol on the actin cytoskeleton of MDA-MB-231 cells. Cells were serum-starved in phenol red-free media for 24 hours and stimulated for 10 minutes with DMSO as control (Un); 0.1 µM E₂ (E₂); 50 ng/ml EGF (EGF); and 50 µM resveratrol (Res), pretreated with Res for 10 minutes followed by E₂ for 10 minutes (Res/E₂), or pretreated with Res for 10 minutes followed by EGF for 10 minutes (Res/EGF). Cells were stained with rhodamine phalloidin to visualize F-actin. (A) Micrographs at x600 magnification. Arrowheads (<) indicate a filopodium; arrows (←) indicate a lamellipodium. (B) Filopodia number was quantified for at least 10 microscopic fields per treatment per experiment and made relative to DMSO control (Un). Data expressed as mean filopodia ± SEM of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments at P < .05.

Figure 4

Effect of tyrphostin AG1478 on filopodia formation in MDA-MB-231 cells. (A) Cells were serum-starved in phenol red-free media for 24 hours and pretreated with vehicle (-AG1478) or tyrphostin AG1478 (+AG1478) for 15 minutes then treated with DMSO as control (Un), 0.1 µM E₂ (E₂), 50 ng/ml EGF (EGF), or 50 µM resveratrol (Res) for 10 minutes. Cells were stained with rhodamine phalloidin to visualize F-actin. Filopodia number was quantified for at least 10 microscopic fields per treatment per experiment and made relative to DMSO control (Un). Data expressed as mean filopodia ± SEM of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments at P < .05. (B) Quiescent cells were pretreated with DMSO (-AG1478) or tyrphostin AG1478 (+AG1478) for 15 minutes then treated with DMSO as control (Un), 0.1 µM E₂ (E₂), 50 ng/ml EGF (EGF), or 50 µM resveratrol (Res) for 10 minutes. Cells were immediately lysed and equal amounts of protein were separated on SDS-PAGE and Western-blotted for activated EGFR using an anti-phosphoEGFR (Y1173) antibody. The result is representative of two separate experiments.

Figure 5

Effect of E₂, EGF, or trans-resveratrol on focal adhesion assembly in MDA-MB-231 cells. Cells were serum-starved in phenol red-free media for 24 hours and stimulated for 10 minutes with DMSO as control (Un); 0.1 µM E₂ (E₂); 50 ng/ml EGF (EGF); 50 µM resveratrol (Res), pretreated with Res for 10 minutes followed by E₂ for 10 minutes (Res/E₂), or pretreated with Res for 10 minutes followed by EGF for 10 minutes (Res/EGF). Cells were probed with antiphosphotyrosine primary antibody and FITC-conjugated secondary antibody to visualize focal adhesions. (a) Micrographs at x600 magnification. (b) Focal adhesion number per cell was quantified for at least 10 microscopic fields per treatment per experiment and made relative to DMSO control (Un). Data expressed as mean focal adhesions ± SEM of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments at P < .05.

Figure 6

Effect of tyrphostin AG1478 on focal adhesion assembly in MDA-MB-231 cells. Cells were serum-starved in phenol red-free media for 24 hours and pretreated with vehicle (-AG1478) or tyrphostin AG1478 (+AG1478) for 15 minutes then treated with DMSO as control (Un), 0.1 µM E₂ (E₂), 50 ng/ml EGF (EGF), or 50 µM trans-resveratrol (Res) for 10 minutes. Cells were probed with an anti-phosphotyrosine antibody followed by FITC secondary antibody to visualize focal adhesions. Focal adhesion number was quantified for at least 10 microscopic fields per treatment per experiment and made relative to DMSO control (Un). Data expressed as mean focal adhesions ± SEM of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments at P < .05.

Figure 7

Effect of resveratrol on FAK activity in MDA-MB-231 cells. Cells were starved for 24 hours in phenol red- and serum-free media and treated with DMSO as vehicle (0) or resveratrol (1, 10, 25, 50, or 100 µM) for 10 minutes. (a) Equal amounts of protein were run on SDS-PAGE and Western-blotted using FAK (N-terminus) or phosphoFAK (tyr-397) antibodies. (b) The integrated density of phosphoFAK and FAK bands from Western blots was quantified. Relative activity is the difference between the ratio of phosphoFAK to total FAK with stimulation, and the ratio of phosphoFAK to FAK without stimulation. The result is representative of three separate experiments.