Functional Role of Resveratrol in Inducing Apoptosis in Breast Cancer Subtypes via Inhibition of Intracellular Fatty Acid Synthase

Abstract

Fatty acid synthase (FASN) is frequently overexpressed in human breast cancer and has emerged as a potential therapeutic target. Resveratrol has been shown to inhibit FASN activity in vitro through both fast-reversible and slow-irreversible mechanisms. In this study, resveratrol reduced intracellular fatty acid levels by inhibiting FASN activity and downregulating its expression across various breast cancer subtypes, including SK-BR-3, MCF-7, and MDA-MB-231 cells. Knockdown of FASN via small interfering RNA (siRNA) further enhanced resveratrol-induced cytotoxicity. Resveratrol significantly suppressed cell viability and triggered apoptosis, as evidenced by increased cleavage of poly(ADP-ribose) polymerase (PARP) and disruption of Bcl-2 family protein balance. Furthermore, resveratrol inhibited key signaling pathways involved in cell proliferation and survival, notably FAK, AKT, and ERK1/2. FASN silencing by siRNA also modulated the activation states of these signaling proteins. Collectively, these findings support resveratrol as a promising anti-cancer candidate that induces apoptosis in diverse breast cancer subtypes via FASN inhibition.

Keywords: breast cancer cells; cell apoptosis; fatty acid synthase; inhibitor; resveratrol.

Figure 1

Effect of resveratrol on the viability of breast cancer cells. (A) Chemical structure of resveratrol. (B) The viability of SK-BR-3, MCF-7, and MDA-MB-231 cells treated with various concentrations of resveratrol (0, 12.5, 25, 50, 100, 150, 200 μM) for 24 h and 48 h as measured by a CCK-8 assay. The percentage of cell viability was calculated as the ratio of resveratrol-treated cells to control cells. Data represent the mean ± SD of three independent experiments.

Figure 2

Resveratrol-induced apoptosis in breast cancer cells. (A) SK-BR-3, MCF-7, and MDA-MB-231 cells were treated with 0, 50, 100, 150 μM resveratrol for 24 h, then stained with Annexin V/PI and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated (bottom left: viable; top left: necrotic; bottom right: early apoptotic; top right: late apoptotic). The bar graphs below show the quantification of PI-positive (necrotic and late apoptotic) cells at each resveratrol concentration. (B) After treatment as described above, the expression levels of PARP, cleaved PARP, Bax, and Bcl-2 were analyzed by Western blotting with GAPDH as a loading control. Blots shown are representative of at least two independent experiments. Densitometric analysis of protein bands was performed using ImageJ version 1.53c (National Institutes of Health, USA), and quantification is presented in the bar graphs below each blot. Data are shown as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

Resveratrol down-regulated FASN expression and inhibited intracellular FASN activity. (A) The expression levels of FASN in SK-BR-3, MCF-7, and MDA-MB-231 cells were examined by Western blotting. Representative blots and densitometric quantification (FASN/GAPDH ratio) from three independent experiments are shown. (B) Intracellular FASN activity in breast cancer cells was measured by spectrophotometrically monitoring the oxidation of NADPH at 340 nm. (C) Treatment with various concentrations of resveratrol for 24 h in breast cancer cells inhibited FASN expression, as determined by Western blotting. Densitometric analysis of protein bands was performed using ImageJ software (version 1.53c, National Institutes of Health, USA). (D) Resveratrol-induced inhibition of intracellular FASN activity in breast cancer cells; FASN activity was measured by monitoring the decrease in NADPH at 340 nm. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. control (0 μM); ** p < 0.01 vs. control; *** p < 0.001 vs. control.

Figure 4

Resveratrol showed more potent cytotoxicity in FASN knockdown breast cancer cells. (A) SK-BR-3, MCF-7, and MDA-MB-231 cells were transfected with scrambled siRNA or siRNA targeting FASN for 72 h; the expression levels of FASN were analyzed by Western blotting, with GAPDH as a loading control. Densitometric analysis of FASN bands was quantified and normalized to GAPDH. (B) After transfection with FASN-targeting siRNA or control siRNA for 72 h, cells were treated with resveratrol for 24 h, and cell viability was determined by CCK-8 assay. The percentage of cell viability was calculated as the ratio of resveratrol-treated cells to control cells. Data are presented as the mean ± SD from three independent biological replicates (n = 3). Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s post hoc test followed for multiple comparisons. ** p < 0.01, and *** p < 0.001 versus the indicated controls.

Figure 5

Resveratrol or siRNA-targeted FASN reduced intracellular fatty acid levels in breast cancer cells. (A) SK-BR-3, MCF-7, and MDA-MB-231 cells were treated with increasing concentrations of resveratrol (0, 50, 100, and 150 μM) for 24 h. Intracellular fatty acid content was measured using a Free Fatty Acid Quantification Kit. (B) Cells were transfected with siRNA targeting FASN or control siRNA for 72 h, followed by treatment with resveratrol (0, 50, 100, or 150 μM) for 24 h. Intracellular fatty acid levels were subsequently determined. Data are presented as the mean ± SD from three independent biological replicates (n = 3). Statistical analysis was performed using one-way ANOVA by Tukey’s post hoc test or two-way ANOVA by Bonferroni’s post hoc test followed for multiple comparisons. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistical significance compared to the indicated controls.

Figure 6

Resveratrol or siRNA-mediated FASN attenuation of signaling pathways associated with breast cancer cell proliferation and survival. (A) SK-BR-3, MCF-7, and MDA-MB-231 cells were treated with various concentrations of resveratrol (0, 50, 100, and 150 μM) for 24 h, and phosphorylation of FAK was analyzed by Western blotting. (B) Phosphorylation of AKT and ERK1/2 was assessed in cells treated with increasing concentrations of resveratrol for 24 h, as determined by Western blotting. (C) Breast cancer cells were transfected with scrambled control siRNA or siRNA targeting FASN for 72 h. The phosphorylation levels of FAK, AKT, and ERK1/2 were determined by Western blotting to confirm the regulatory effect of FASN knockdown. GAPDH served as a loading control. Densitometric analysis was performed and data are shown as the mean ± SD from three independent biological replicates (n = 3). Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 7

The proposed mechanism of resveratrol-induced apoptosis via inhibition of FASN.