MCF-7 human breast cancer cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA). They were maintained as monolayer cultures in 75-cm² plastic culture flasks in Dulbecco's modified Eagle's medium (DMEM) (Sigma‑Aldrich, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria), penicillin (20 U/ml) and streptomycin (20 μg/ml) (Sigma‑Aldrich) at 37˚C in a humid atmosphere containing 5% CO₂.

The cells were initially cultured for 24 h in DMEM supplemented with 0.5% dextran-charcoal stripped FBS (csFBS) prior to being seeded in 96-multi-well plates in DMEM supplemented with 10% FBS and incubated at 37˚C for 24 h to allow for cell attachment. Melatonin pre-treated cells were incubated for 24 h in DMEM supplemented with 10% FBS containing melatonin (1 nM). Following pre-treatment, both the melatonin-treated and the control cells were seeded into 96-multi-well plates at a density of 3×10³ cells per well, and incubated at 37°C for 24 h to allow for cell attachment. The media were replaced with fresh media with 10% FBS containing docetaxel ranging from 1 μM to 10 pM, plus/minus melatonin (1 nM) and/or the vehicle (ethanol at a final concentration <0.0001%). The cells were cultured for 3 or 6 days and cell proliferation was measured by 3(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, yellow MTT (5 mg/ml in PBS) is reduced to purple formazan in the mitochondria of living cells. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable cells. The formazan crystals can be dissolved by the addition of 4 mM HCl. An increase in cell number is directly related to the increase in absorbance due to the amount of MTT formazan formed (37). After 24 h, the absorbance is read at 570 nm on a microplate reader (Labsystems Multiskan RC 351; Thermo LabSystems Inc. Vienna, VA, USA). MTT solution was obtained from Molecular Probes Inc. (Eugene, OR, USA). Each result represents the median ± standard error of the mean (SEM) of 3 independent experiments and data are presented as a percentage of the untreated control cells.

The cells were initially cultured for 24 h in DMEM supplemented with 0.5% dextran-csFBS prior to being seeded in 96-multi-well plates in DMEM supplemented with 10% FBS and incubated at 37°C for 24 h to allow for cell attachment. The control cells and cells pre-treated with melatonin (1 nM) for 24 h were seeded in 6-well plates at a density of 5×10⁵ cells per well, in DMEM supplemented with 10% FBS and incubated at 37°C for 24 h to allow cell attachment. The media were then replaced with fresh media with 10% FBS containing either docetaxel alone (1 nM) or in combination with melatonin (1 nM) and/or the vehicle (ethanol at a final concentration <0.0001%). After 24 h of incubation, the cells were harvested with trypsin, washed twice with phosphate‑buffered saline (PBS), and fixed in 70% cold ethanol for 30 min. Following the removal of ethanol by centrifugation for 5 min at 300 × g, the cells were stained with a solution containing 50 μg/ml propidium iodide (PI) (Sigma-Aldrich) and incubated in the dark for 30 min at room temperature. In total, 10,000 cells were acquired for each sample on a BD FACSCanto II analyzer (BD Biosciences, San Jose, CA, USA). The cell cycle distribution was determined using a DNA software program (FACSDiva software; BD Biosciences).

The induction of apoptosis was determined using an Annexin V-FITC apoptosis detection kit (Miltenyi Biotec GmbH, Germany), according to the manufacturer's instructions. Briefly, the control cells and cells pre-treated with melatonin (1 nM) were seeded in 6-well plates at a density of 3×10⁵ cells per well in DMEM supplemented with 10% FBS and incubated at 37°C for 24 h to allow for cell attachment. The media were then replaced by fresh media containing docetaxel (1 nM) plus/minus melatonin (1 nM) and/or the vehicle (ethanol at a final concentration <0.0001%). After 24 h of incubation, the cells were harvested, washed twice with PBS, and centrifuged at 300 × g for 10 min; the cells were resuspended in 100 μl binding buffer containing 5 μl of Annexin V-FITC, incubated for 15 min at room temperature, washed twice, and finally resuspended in 500 μl binding buffer containing 5 μl of PI. Cells were immediately analyzed following incubation with the probes using a flow cytometer (BD FACSCanto II analyzer; BD Biosciences). A total of 10,000 events were analyzed using the FL-1 (green; Annexin V-FITC) and FL-3 (red; PI) detectors. Each sample was tested 3 times in independent experiments. Under all conditions tested, the percentages of Annexin-/PI⁻ (alive) and Annexin⁺/PI⁻ cells (early apoptotic) cells were compared.

Total RNA was isolated from the MCF-7 cells and purified using the Nucleospin RNA II kit (Macherey-Nagel GmbH & Co,. Düren, Germany) following the manufacturer's instructions. The concentration and purity of the RNA was quantified by measuring the absorbance on a spectrophotometer (Nanodrop 1000; Thermo Fisher Scientific, Wilmington, DE, USA). The absorbance ratio A260 nm/A280 nm was always >1.9. For cDNA synthesis, 0.5 μg of total RNA was used as template using the RT² First Strand kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. First, the genomic DNA was eliminated by incubating the samples 5 min at 42°C. After mixing with the reverse transcription mix, the samples were incubated for exactly 15 min at 42°C in a final volume of 20 μl. The reaction was terminated by incubation at 95°C for 5 min. Subsequently, 91 μl of RNA-free water was added to each reaction and the samples were kept on ice until proceeding with the real-time PCR protocol.

Pathway-focused gene expression profiling was performed using a 96‑well human breast cancer PCR array (RT² Profiler PCR array ‑ PAHS‑131ZA, Human Breast Cancer PCR array, Qiagen, Valencia, CA, USA). In this array, each well contains all the components required and designed to generate single, gene‑specific amplicons, testing the expression of 84 genes related to breast cancer pathways (apoptosis, metabolism, cell cycle and DNA repair), plus 5 housekeeping genes. Each RT² Profiler PCR array plate also includes controls for data normalization, genomic DNA contamination detection, RNA sample quality and general PCR performance.

Briefly, the MCF‑7 cells were seeded in 6‑well plates in DMEM supplemented with 10% FBS and incubated at 37°C for 24 h to allow for cell attachment. The media were then replaced with fresh media with 10% FBS and containing either docetaxel (Sigma-Aldrich) alone (1 μM) or in combination with 1 nM melatonin (Sigma-Aldrich) and/or the vehicle (ethanol at a final concentration <0.0001%). After 6 h of incubation, total RNA was extracted and reverse transcribed as explained in the 'RNA isolation and cDNA synthesis' section. The cDNA template was mixed with the appropriate amount of RT² SYBR-Green qPCR Mastermix (Qiagen GmbH, Hilden, Germany), aliquoted 25 μl to each well of the same plate, and then the real-time PCR cycling program was performed on an MX3005P (Agilent, CA, USA) following the manufacturer's instructions. Amplification was initiated by 1 cycle at 95°C for 10 min and then performed for 40 cycles for quantitative analysis using the following temperature profile: 95°C for 30 sec (denaturation); 60°C for 60 sec (annealing/ extension). Dissociation curves were performed to verify that only a single product was amplified. The Ct data for each gene were analyzed using the Qiagen RT² profiler PCR array data analysis software. Data are represented as the fold-regulation between the experimental groups and the control cells. Fold change values <1 indicate a negative result or downregulation, and the fold -regulation is the negative inverse of the fold change.

The analysis of mRNA gene expression was carried out by RT-qPCR following incubation of the cells for 6 h with docetaxel (1 μM or 1 nM), in the presence or absence of melatonin (1 nM). Total cellular RNA was isolated and reverse transcribed as indicated above and RT-PCR was performed on an Mx3005P QPCR System (Agilent Technologies, Santa Clara, CA, USA) using the same temperature profile. Reactions were run in triplicate and melting curves were performed to verify that only a single product was amplified. Each result represents the median of 3 to 5 independent experiments and data are presented as the fold change between the experimental groups and the control cells. The primers used for amplification (Sigma Genosys Ltd., Cambridge, UK) are listed in Table I.

For the primers used there were no differences between transcription efficiencies, and the fold change in each sample was calculated using the 2^−∆∆Cq method (38). The fractional cycle at which the amount of amplified target becomes significant (Ct) was automatically calculated by the PCR program. The relative expression of β-actin was used to normalize gene expression.

Statistical analyses were performed using GraphPad Prism software. The data are expressed as the means ± SEM. Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. Results were considered as statistically significant at p<0.05. To confirm whether the effects of docetaxel and melatonin are synergistic as regards gene expression (BAD and BCL-2 expression) and apoptosis, linear regression models were obtained in order to estimate the independent effects of melatonin, docetaxel and their interaction.

Our first goal was to determine whether melatonin potentiates the anti-proliferative effects of docetaxel in MCF-7 cells. As expected, docetaxel inhibited the proliferation of the MCF‑7 cells in a dose‑dependent manner. When the cells were treated with docetaxel at 1 μM, proliferation was decreased by almost 80% after 3 days, indicating that the majority of the cells were dead (Fig. 1A). For this reason, we examined the effect of physiological concentrations of melatonin (1 nM) combined with low concentrations of docetaxel at 1 nM and 0.1 nM.

Consistent with previous findings of our group (1), after 6 days of treatment, melatonin alone induced an inhibitory effect on the proliferation of MCF-7 cells (Fig. 1B). Docetaxel (0.1 nM) also had a slight, but significant inhibitory effect on cell proliferation (8%), whereas docetaxel (1 nM) treatment resulted in a 62% decrease in cell viability. Of note, co-treatment of the cells with docetaxel and a physiological concentration of melatonin (1 nM) significantly potentiated the inhibitory effects on cell proliferation induced at both docetaxel concentrations. When the cancer cells were pre-treated with melatonin for 24 h, a significant further decrease in cell proliferation was obtained. For docetaxel (1 nM), a 77% decrease in cell proliferation was obtained when the cells were pre-treated with melatonin (whereas for non pre-treated cells the decrease in cell proliferation observed was 62%). For docetaxel (0.1 nM), a 27% decrease in cell proliferation was obtained when the cells were pre-treated with 1 nM melatonin, (whereas an 8% decreased was obtained in the in non pre‑treated cells). Moreover, significant differences were observed when melatonin was maintained in the medium following treatment with docetaxel, the effect of the indolamine being a further reduction in cell proliferation.

The effects of melatonin, docetaxel and co-treatment with both molecules on cell cycle phase distribution in the MCF-7 cells are shown in Fig. 2A, and the percentage of cells in the sub G₀-G₁, G₁, S, and G₂-M phases is represented in Fig. 2B. As expected, treatment of the cells with melatonin (1 nM) for 24 h induced an increase in the proportion of cells in the G₁ phase, thus causing a delay in the transition to the S phase. As expected, a higher number of cells in the sub G₀–G₁ phase was evident when the cells were treated with docetaxel (1 nM) for 24 h (27%). When the cells were treated with a combination of melatonin and the taxane, a significant increase in the number of cells in this phase was observed (38%). Of note, treatment with melatonin for 24 h prior to the addition of docetaxel resulted in the highest percentage of cells in the sub G₀–G₁ phase (42%), suggesting DNA fragmentation as a consequence of cell death (Fig. 2B).

We then wished to determine whether the results obtained by flow cytometric analysis correlated with the expression of genes involved in apoptosis. The mRNA expression of the pro-apoptotic genes, BAD and BAX, was stimulated, whereas the mRNA levels of the anti-apoptotic BCL-2 gene were significantly decreased by docetaxel (1 nM). When the cells were treated with both compounds, melatonin further enhanced the effects of the taxane on BAD and BCL-2 expression. However, at this low concentration of docetaxel (together with melatonin), the pineal hormone had no significant effect on BAX expression. Melatonin alone induced a moderate, yet significant downregulation in the levels of BCL-2 (Fig. 2C).

To examine whether the anti-proliferative effects and the changes in the cell cycle phase distribution were related to the induction of apoptosis, we treated the MCF-7 cells with docetaxel (1 nM), alone or in combination with 1 nM melatonin for 24 h and used an Annexin V-FITC apoptosis detection kit, which enables the differentiation of viable cells from apoptotic and necrotic cells. The effects of melatonin pre-treatment for 24 h were also examined. The results of Annexin V-FITC assay are shown in Fig. 3A and the quantification of the results obtained are shown in Fig. 3B. Following treatment with melatonin alone, we observed a slight decrease in the percentage of live cells compared to the control cells. Similarly, treatment with docetaxel (1 nM) resulted in a decrease in the number of viable cells parallel to an increase in the percentage of cells undergoing early apoptosis. Of note, when the cells were simultaneously treated with docetaxel and melatonin, the number of viable cells was further decreased and the number of cells undergoing early apoptosis was augmented, suggesting that melatonin enhanced the apoptotic effects of docetaxel. Melatonin pre-treatment seemed to sensitize the MCF-7 cells to this chemotherapeutic agent, significantly further increasing the number of cells undergoing early apoptosis.

The changes in gene expression induced by chemotherapeutic agents employed at clinical doses remain largely unknown. In this study, we employed the human breast cancer RT² Profiler PCR Array, to determine the changes induced by docetaxel (1 μM) either alone or in combination with melatonin (1 nM) on the gene expression profiles in MCF‑7 cells. The RT² Profiler PCR Array allows the simultaneous analysis of 84 genes relevant to a specific pathway or disease state and includes genes involved in one or more processeses, such as angiogenesis, adhesion, proteolysis, cell cycle progression and apoptosis. Melatonin alone significantly enhanced the expression of some genes, such as TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A), cadherin 13 (CDH13) or PGR, whereas it diminished the expression of other genes, such as c-MYC, interleukin (IL-6) or BCL-2 (data not shown). Establishing as criteria a change of at least 1.5-fold either with docetaxel alone or in combination with melatonin (in comparison with expression of the untreated cells), we found that treatment with docetaxel upregulated the expression of 8 genes and downregulated the expression of 36 genes. When docetaxel was used in combination with melatonin, we observed the upregulation of 15 genes and the downregulation of 29 genes (Table II). TP53, BAD and CDKN1A were upregulated >3-fold and mucin 1 (MUC1), BCL-2 and c-MYC were downregulated >3-fold (Fig. 4A). The following genes were selected for further analysis by specific RT‑qPCR: TP53 and CDKN1A as cell cycle regulators, and BAD and BCL-2 as genes involved in apoptosis. Docetaxel (1 μM) significantly inhibited the expression of TP53 and CDKN1A (Fig. 4B), and treatment with melatonin (1 nM) counteracted this inhibitory effect. The expression of the pro-apoptotic gene BAD was stimulated by docetaxel and co-treatment with melatonin further increased its expression, whereas the expression of the anti-apoptotic BCL-2 gene was significantly decreased. Treatment with melatonin enhanced this inhibitory effect (Fig. 4C). We analyzed the expression of BAX, another pro-apoptotic member of the family; similar to BAD, BAX expression was stimulated by docetaxel and melatonin enhanced this stimulatory effect (Fig. 4C).

Among the genes classified as transcription factors or involved in angiogenesis and/or cell adhesion, MUC1, GATA binding protein 3 (GATA3) and c-MYC were downregulated (>3-fold), and TP53 and CDH13 were upregulated (>4-fold) when the cells were treated simultaneously with melatonin and docetaxel in comparison with the cells treated only with the taxane (Fig. 5A). Specific RT‑PCR analyses confirmed that docetaxel (1 μM) stimulated the expression of the transcription factors, MUC1, GATA3 and c-MYC, whereas a physiological concentration of melatonin counteracted this stimulatory effect of the chemotherapeutic agent (Fig. 5B). The expression of CDH13 (involved in angiogenesis) was inhibited by docetaxel and melatonin reversed this effect (Fig. 5C).