A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth

Abstract

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.

Keywords: anti-inflammatory activity; antiproliferative activity; breast cancer cell lines; cell cycle arrest; cell death; phenylacetamide RSV derivatives; resveratrol.

Figure 1

Molecular structure of RSV and the tested RSV derivatives (1–6).

Figure 2

Effects of phenylacetamide RSV derivatives on cancer cell viability. MCF7, MDA-MB231, H295R, R2C, U937, and 3T3L1 cells were treated with vehicle DMSO (0) or the following phenylacetamide RSV derivatives: 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), or 6 (F) at the indicated concentrations (2.5, 5, 10, 20, and 40 µM). Cell viability was assessed by MTT assay after 72 h exposure. Results are expressed as mean ± SE of three separate experiments (* p < 0.05 with respect to control (0)).

Figure 3

Effects of low doses of phenylacetamide RSV derivatives on U937 cell viability, NO and ROS levels, and PGE2 production. (A) U937 cells were treated with vehicle DMSO (0) or the phenylacetamide RSV derivatives (1–6) at the indicated concentrations (0.01, 0.1, or 1 µM). Cell viability was assessed by MTT assay after 72 h exposure. Results are expressed as mean ± SE of three separate experiments (* p < 0.05 with respect to control (0)). (B–D) In PMA-treated U937 cells, unstimulated (C) or activated with LPS alone (LPS), or in the presence of 0.1 µM of the phenylacetamide RSV derivatives (1–6), NO (B), ROS (C) and PGE2 (D) levels were quantified. Means ± SE of four independent experiments are shown. In (B–D), different letters indicate significant differences between treatments at p < 0.05 (Tukey’s post hoc test).

Figure 4

Effects of derivative 2 on the MCF7 and MDA-MB231 cell cycle distribution. (A,C) MCF7 (A) and MDA-MB231 (C) cells were synchronized in serum-free media for 12 h and then exposed to vehicle (0) or derivative 2 for 24 h at different concentrations (20 and 40 µM). The distribution of MCF7 and MDA-MB231 in the cell cycle was determined by flow cytometry using propidium iodide stained nuclei. (B,D) Western blot analysis of CCND1 and CDK4 was performed on equal amounts of total proteins extracted from MCF7 (B) and MDA-MB231 (D) cells treated with derivative 2 (20 and 40 µM) for 24 h. Blots are representative of three independent experiments with similar results. GAPDH was used as a loading control.

Figure 5

Effects of derivative 2 on MCF7 and MDA-MB231 cell morphology and apoptosis. MCF7 (A) and MDA-MB231 (D) cells were untreated (0) or treated with derivative 2 (20 and 40 μM) for 24 h; after treatment, cells were examined with a phase-contrast microscope (×10 objective). Images are from a representative experiment. Scale bar: 100 µm. (B,E) MCF7 (B) and MDA-MB231 (E) cells were untreated (0) or treated with derivative 2 (10 μM) for 72 h; after treatment, cells were fixed with paraformaldehyde and processed for TUNEL staining. Nuclei counterstaining was performed using DAPI. Fluorescent signals were observed under a fluorescent microscope (×20 objective). Images are from a representative experiment. Scale bar: 50µm. (C,F) MCF7 (C) and MDA-MB231 (F) cells were untreated (0) or treated with derivative 2 (20 and 40 μM) for 24 h. Western blot analyses of bax, bcl-2, and parp1 were performed on equal amounts of total proteins. Blots are representative of three independent experiments with similar results. GAPDH was used as a loading control.