Increase in fatty acids and flotillins upon resveratrol treatment of human breast cancer cells

Figure 1. Effect of resveratrol on the cell viability of MCF-7 and MDA-MB-231 cells. (a) 4T1, (b) MCF-7 and (c) MDA-MB-231 cells (2 × 105) were incubated in the presence of different concentrations (10 to 300 μM) of resveratrol, or 0.1% DMSO (control), for 24 h at 37 °C. They were then washed and maintained with MTT (0.5 mg/mL) for 3 h at 37 °C for formazan crystals formation and the associated optical density was analyzed at 570 and 650 nm. The results were expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 significantly different (p < 0.05) from the values corresponding to the control, using One-way ANOVA, followed by multiple comparison test Dunnett.

Figure 2. Isolation of membrane microdomains and analysis of the sucrose gradient profile after resveratrol treatment. MCF-7 and MDA-MB-231 cells (1 × 108) after resveratrol treatment with 80 and 200 μM, respectively, or 0.1% DMSO (control) were sonicated (in TNE buffer) in the presence of different classes of proteases inhibitors. Afterwards, they were incubated with Triton X-100 detergent for 20 min at 4 °C. Subsequently, 80% sucrose solution was homogenized, followed by the addition of 30% sucrose and 5% sucrose solution, forming three distinct phases. The samples were centrifuged at 38,000 rpm for 22 h at 4 °C. Then divided into twelve 1 mL fractions. In each fraction, the refractive index was measured, which was converted to sucrose concentration. (a) A representative image of a tube with samples after centrifugation. The arrow indicates floating whitish material, visible in the median region of the tube, indicative of the presence of membrane lipid microdomains. Sucrose concentration profile on the gradient fractions of the (b) control, and (c) resveratrol-treated MCF-7 cells and, (d) control and (e) resveratrol-treated MDA-MB-231 cells.

Figure 3. Resveratrol alters membrane lipid microdomain distribution in sucrose gradient fractions. A volume of 500 μL of each fraction was applied to the nitrocellulose membrane and were submitted to Dot blot analysis. Membrane was then blocked with TBS-Tween-Milk for 2 h at room temperature. Subsequently, it was incubated with anti-flotillin-1 antibody (1:800) in TBS-Tween-Milk for 18 h at 4 °C. Membrane was then washed and incubated with peroxidase-conjugated secondary antibody (1:1000) for 2 h at room temperature. The detection of flotillin-1 was performed by chemiluminescence. (a) MCF-7 and (d) MDA-MB-231cells. Densitometry, in percentage, of the scanned image for the samples (b and e) control (0.1% DMSO), and (c and f) resveratrol-treated cells.

Figure 4. Immunofluorescence for flotillin-2 in MDA-MB-231 cells after resveratrol treatment. Cells (5 × 105) were treated with 200 μM resveratrol or 0.1% DMSO (control) for 24 h. Then were washed with 0.5% PBS-Triton X-100, and incubated with anti-flotillin-2 antibody (1:70) for 1 h at 37 °C. Samples were then washed, and incubated with the secondary antibody (1:100) conjugated to the fluorophore for 1 h at 37 °C. Subsequently, cells were washed, and incubated with DAPI (1: 2,000) for 3 min. Images show the labeling for flotillin-2 (green) in control cells (a) and treated cells (d). The nuclei in the control (b) and treated (e) samples were stained with DAPI (blue). (c,f) are the overlap of the images of the control samples (a,b) and treated (d.e), respectively. (g) representation in percentage of the area corresponding to flotillin-2. The results were expressed as mean ± SD, n = 5 for the control sample and n = 9 for the treated sample. Bar in (a) = 50 µm and bar in the inset = 10 µm. The arrow in the inset points to flotillin-2 dots.

Figure 5. Resveratrol effects on the distribution of flotillin-2 in MDA-MB-231 cells. Cells were treated with 200 μM resveratrol for 24 h, triple-labelled with an antibody against flotillin-2 (a,e), Bodipy-ceramide (a Golgi specific fluorescent probe, B and F) and DAPI (c,g) and analyzed under a Spinning Disk confocal microscope. Merged images are shown in (d,h). Arrow in (d) points to flotillin-2 in Golgi membranes and arrow in (h) points to flotillin-2 within the nuclei of cells. Bars in (a,e) = 10 µm.

Figure 6. Resveratrol effect on the mRNA expression of flotillin-1 and -2 in MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with 80 and 200 μM resveratrol, respectively, or 0.1% DMSO (control), for 24 h. Subsequently, the RNA was extracted using the Trizol reagent. Two micrograms of total RNA were used in cDNA synthesis. The qPCR was performed with flotillin-1 (a), and flotillin-2 (b) primers, and the results obtained after normalization as a function of actin expression, showed as mean ± S.D., n = 3, *p < 0.05.

Figure 7. Resveratrol effect on flotillin-1 (a) and -2 (b) in MCF-7 and MDA-MB-231 cells at the protein level. MCF-7 and MDA-MB-231 cells after resveratrol treatment with 80 and 200 μM, respectively, or 0.1% DMSO (control), were submitted to Western blot analysis (80 μg of total protein extract) under denaturing conditions. The values were expressed in arbitrary units, showed as mean ± S.E, n = 3, n.s.: non-significant.

Figure 8. Resveratrol alters the neutral lipid profile in MCF-7 and MDA-MB-231 cells. Cells (1 × 107) after resveratrol treatment, or 0.1% DMSO (control), for 24 h, were submitted to lipid extraction. Subsequently, the entire contents of total lipids extracted were subjected to TLC, in a solvent set consisting of n-hexane:diethylether:acetic acid (60:40:1 v/v). After evaporation of the solvents, (a) TLC was revealed with the Charring reagent and heating at 200 °C. CHOE (cholesterol ester), TG (triacylglycerol), FA (fatty acids), CHO (free cholesterol) content for (b) MCF-7 cells, and (c) MDA-MB-231 cells were expressed as μg/μL, after normalization with the respective standards.