MCF-7 and MDA-MB-231 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM), supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO₂.

Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA), and dissolved at a concentration of 80 mmol/l in ethanol, stored at −20°C and diluted with DMEM to a 100-µM working concentration.

Total RNA was processed and hybridized into the GeneChip Human Gene 1.0 ST (Affymetrix Inc., Santa Clara, CA, USA), following the manufacturer's recommendations. Microarray quality assessment, condensing of the probe sets, data normalization, and filtering were conducted using the Transcriptome Analysis Console version 2.0 (Affymetrix). To identify those biological processes that show differentially expressed genes, we used KEGG and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 for functional annotation. To validate the microarray data, real-time quantitative PCR (RT-qPCR) was performed for 13 selected genes using specific primers (Table I). The SYBR-Green reaction was carried out using a QuantiTect SYBR Green PCR reagents kit (Qiagen) following the manufacturer's recommendations. GADPH expression was used as control. Samples were tested in triplicate and data were analyzed using the 2^−∆∆Ct method (11).

MDA-MB-231 and MCF-7 cells were plated at a density of 5×10⁴ cells/ml for 24 h, and then treated with resveratrol (100 µM) for 24 and 48 h. Cells were harvested by trypsinization and fixed in ice-cold 70% methanol overnight at −20°C. Cells were centrifuged at 1,200 rpm for 5 min and incubated with a propidium iodide (PI) working solution (100 µg/ml PI and 100 µg/ml RNase A) for 30 min at 37°C. Cell cycle distribution was analyzed using a FACScan flow cytometer (FACSCalibur; Becton-Dickinson).

Protein extracts (50 µg) were separated by 12% SDS-PAGE and electrotransferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween-20 and then probed with primary antibodies as follows: anti-AURKA (1:1,000; Cell Signaling Technology), anti-PLK1 (1:500), anti-cyclin D1 (1:1,000), anti-cyclin B1 (1:1,000) and anti-BRCA1 (1:1,000) (all from Santa Cruz Biotechnology), followed by secondary antibodies, anti-rabbit (1:2,500) or anti-mouse (1:2,500) (both from Santa Cruz Biotechnology). The membranes were probed with anti-α-tubulin (1:1,000; Sigma-Aldrich) or actin (1:1,000; Abcam) as a loading control. Immunoreactive bands were developed using the ECL chemiluminescence system (Amersham Pharmacia Biotech).

A two-way ANOVA was performed to identify differentially expressed genes of the microarray data. Only genes with statistically significant differences in expression levels (P-value <0.05) and a fold change criteria of ≥1.5 were included in the final set of differentially expressed genes. RT-qPCR results were analyzed using the Student's test. Differences of P<0.05 were considered statistically significant. Values are presented as the mean ± standard deviation of three independent experiments.

Resveratrol is able to inhibit cell viability through apoptosis activation in diverse types of cancer cells (12,13). Here, the effects of resveratrol (100 µM) on cell viability and apoptosis of triple-negative MDA-MB-231 breast cancer cells were evaluated. Results of the MTT, Annexin V and TUNEL assays confirmed that resveratrol treatment was able to significantly (P<0.05) inhibit cell viability and induce apoptosis after 24 and 48 h treatments (data not shown). These results concur with previous reports on diverse types of cancer cells (14–17). Then, in order to obtain insight concerning the cellular transcripts modulated by resveratrol that may explain its anticancer effects, we performed a genome-wide analysis of the transcriptome of MDA-MB-231 breast cancer cells after treatment with resveratrol (100 µM) for 24 and 48 h using DNA microarrays. Data from two biological replicates were analyzed, normalized, and raw P-values adjusted. Only genes with a significant fold change (FC>1.5; P<0.05) were included in this analysis. Transcriptional profiling showed that 375 genes (206 upregulated and 169 downregulated) were significantly modulated at 24 h (Table II). After a 48-h treatment with resveratrol, changes in the expression of 579 genes were detected (338 upregulated and 241 downregulated). Of these, 290 genes (30%) were modulated in common at 24 and 48 h (Fig. 1A).

To evaluate the impact of resveratrol on cellular processes and pathways of MDA-MB-231 cells, a bioinformatic analysis of the DNA microarray data set was performed. We used the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Reactome Database for functional annotation and visualization of the expression data in the KEGG biological pathway context. Gene ontology category analysis indicated that a number of genes involved in cancer-related processes were modulated by resveratrol at both times (Fig. 1B). Of these, 47 suppressed genes are well-known oncogenes, which highlight the potential effects of resveratrol on the suppression of cancer-related processes. Notably, a significant decrease in the expression of genes involved in the cell cycle, DNA metabolic processes, cytoskeleton organization, metabolism of xenobiotics by cytochrome P450, and angiogenesis was detected at 24 h after resveratrol treatment (Table II). Several of these genes, including SLC6A6, HK2, GPR110, IGHG1, ALOX5AP, CCNB1, CYP1B1, JUN, VEGFA, ANGPTL4 and histones, are clinical therapeutic targets in several types of cancers (18–20). Remarkably, after a 24-h resveratrol treatment we identified the overexpression of a set of DNA repair genes including BRIP1, UNG, BRCA2, MLH1, FANCL, BRCA1, EXO1, CLSPN, BLM, ESCO2, DNA2, POLN (Table II).

In contrast, at 48 h after resveratrol treatment we detected the upregulation of genes involved in cellular response to stress, regulation of cell death, glycolytic process, chromatin organization and cellular senescence. Notably, DNA repair genes involved in homologous recombinational repair (RAD54L, EXO1, RECQL, BRCA1, BRCA2, BLM, DNA2, RBBP8, POLN), mismatch repair (MLH1, MSH2), Fanconi anemia pathway (FANCB, FANCD2, FANCL), base excision repair (UNG), DNA double-strand break repair (BRIP1, FAM175A) and repair of DNA inter-strand crosslinks (EME1) were upregulated at 48 h (Table III). Moreover, several genes involved in cell death, such as TP53I3, CCNE1 and SESN3, were overexpressed at 48 h. Remarkably, 22 and 63 genes involved in the regulation of cell cycle progression were modulated at 24 and 48 h, respectively (Tables II and III). Repressed cell cycle genes at 24 h included AURKA, PLK1, TXNIP, CCNB1, FAM83D, CDCA8, HJURP, CENPA, DLGAP5, BUB1, ASPM, and WEE1; whereas at 48 h of treatment, AURKA, PLK1, JUN, ARHGAP18, KIF20A, SH3KBP, EDN1 and PSMD11 were suppressed. To validate the DNA microarray data, we analyzed using RT-qPCR the expression of 13 modulated genes by resveratrol including six oncogenes at 24 and 48 h. In all cases, the RT-qPCR data were similar to those obtained by the DNA microarray analyses (Fig. 2).

In our genome-wide analysis of gene expression in MDA-MB-231 cells treated with resveratrol at 24 and 48 h, we identified 63 modulated genes involved in the cell cycle (Tables II and III). Thus, we decided to confirm the effects of resveratrol (100 µM) on cell cycle progression in the MDA-MB-231 and MCF-7 breast cancer cell lines. The cells were treated with resveratrol and the distribution of cell cycle phases was determined by flow cytometric analysis as described in Materials and methods. The results showed that the percentage of cells in the G0–G1 phase increased from 56.7 to 77.3% at 24 h after resveratrol treatment, but retained a normal level at 48 h (54.7%) in comparison to the control (ethanol vehicle) in the MDA-MB-231 cells. This change was accompanied by an increment in the proportion of cells in the S phase from 26.4 in the control to 45.3% in the resveratrol-treated cells at 48 h, but not at 24 h (Fig. 3A). However, we did not observed arrest in the G2-M phase in cells treated with resveratrol at 24 and 48 h, in comparison with the control assays using nocodazole a known blocker of G2-M phase transition. In contrast, in the MCF-7 cells the percentage of cells in the G0–G1 phase did not significantly change after 24 h of resveratrol treatment in comparison with the control, whereas at 48 h we observed a slight but significant increase in cells in the G0–G1 phase (Fig. 3B). Taken altogether, these data indicate that resveratrol inhibits cell cycle progression and that its effects are cell line-specific.

Our DNA microarrays and RT-qPCR analysis indicated that the activators of cell cycle AURKA, CCND1 and CCNB1 were significantly repressed by resveratrol at 24 and 48 h, whereas PLK1 was only suppressed at 24 h. Moreover, the cell cycle repressor and AURKA/PLK1 inhibitor, BRCA1, was overexpressed at 24 and 48 h after resveratrol treatment. Thus, in order to confirm whether the alterations in mRNA abundance of these cell cycle genes also occur at the protein level, western blot assays using specific antibodies were performed. Results of the immunoblot analysis indicated that changes in the protein levels of these cell cycle genes were correlated with changes at the mRNA level. Data showed that after 24 h of resveratrol treatment, AURKA levels were markedly decreased (95%) in both the MDA-MB-231 and MCF-7 cells in comparison with the control (Fig. 4A and B). In contrast, no significant changes were detected at 48 h in both cell lines. In addition, PLK1 was slightly but significantly decreased by resveratrol at 24 and 48 h in the MDA-MB-231 cells, but not in the MCF-7 cells (Fig. 4C and D). On the other hand, BRCA1 protein levels increased at 24 h whereas no significant changes were identified after 48 h of resveratrol treatment in the MDA-MB-231 cells (Fig. 4A). In addition, we confirmed the suppression of CCND1 and CCNB1 cyclins, which were previously described as two cell cycle markers modulated by resveratrol in MCF-7 and MDA-MB-231 cells (21).