Roswell Park Memorial Institute medium RPMI-1640 medium (RPMI-1640), bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Folin-Ciocalteu reagent, propidium iodide (PI), thiobarbituric acid, and RIPA buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and trypsin-EDTA were from Lonza (Milan, Italy). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Tissue culture dishes were purchased from Microtech (Naples, Italy). Annexin V-fluorescein isothiocyanate (V-FITC) Apoptosis Detection kit was purchased from eBioscience (San Diego, CA, USA). Monoclonal antibodies to caspase-9, poly(ADP ribose) polymerase (PARP), Bax, Bcl-2, cyclin D1, p21, p53, β-actin, and polyclonal antibodies to caspases 6 and 7, ERK1/2, and pERK1/2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were obtained from ImmunoReagents, Inc., Raleigh, NC, USA. All buffers and solutions were prepared with ultra-high quality water. All reagents were of the purest commercial grade.

Annurca (Malus pumila Miller cv. Annurca) apple fruits (each weighing approximately 100 g) were collected in Giugliano (Napoli, Italy) in October, when fruits had just been harvested (green peel). Fruits were reddened following the typical treatment for approximately 30 days and then analyzed (15).

APE extraction from Annurca apple was carried out as previously reported by D'Angelo et al (20). Briefly, 40 grams of Annurca apple flesh were homogenized for 5 min by a Tefal Rondo 500 homogenizer using 40 ml of 80% methanol and 20% water plus 0.18 N HCl. After centrifugation (18,000 × g for 25 min), the slurry was dried under vacuum by using the Univapor Concentrator Centrifuge, model Univapo 100 H (Uni Equip). The dried extracts were dissolved in 10 ml of PBS and frozen at −80°C until use.

The total polyphenolic content of apple extract was assessed approximately by using the Folin-Ciocalteu phenol reagent as described by Singleton et al (23). The extracts (100 µl) were mixed with the Folin-Ciocalteu phenol reagent (0.5 ml), deionized water (0.9 ml), and Na₂CO₃ (7.5% w/v, 4 ml). The absorbance at 765 nm was measured 1 h after incubation at room temperature using a Cary ultraviolet-visible spectrophotometer (Varian). Since APE is a mixture of different phenolic compounds, to give an arbitrary APE molar concentration the measurement was compared to a standard curve of prepared catechin solutions and expressed as milligrams of catechin equivalent (EqC) per 100 g of Annurca flesh fresh weight. The chemical characterization was performed in HPLC as reported by D'Angelo et al (24). All experiments were carried out in triplicate. HPLC separation of polyphenols was performed by reversed-phase chromatography on a 5 µm column Kromasil C₁₈ column (150×4.6 mm), using a Beckman Apparatus (Gold-126) equipped with a UV detector fixed at 278 nm. The column was eluted at a flow rate of 1.0 ml/min with 0.2% acetic acid, pH 3.1 (A) and methanol (B) as the mobile phase; the gradient was changed as follows: 95% A/5% B for 1 min, 85% A/15% B in 1 min, 75% A/25% B in 20 min, 0% A/100% B in 15 min, and 95%A/5%B in 3 min. The main o-diphenols were identified on the basis of the retention times of authentic standard references: (+)-catechin, (−)-epicatechin, chlorogenic acid, quercetin, and quercetin glysosides.

Human breast carcinoma cell line (MCF-7) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. The cells were grown in a humidified atmosphere of 95% air/5% CO₂ at 37°C.

MCF-7 cells were seeded in 96-well plates at the density of 3×10³ cells/well in RPMI complete medium. After 24 h incubation, cells were treated with 100, 250, and 500 µM APE EqC for 24 and 48 h. Cell viability was assessed by adding MTT solution in PBS to a final concentration of 0.5 mg/ml. Cells were then incubated at 37°C for 4 h and the MTT-formazan crystals were solubilized in a solution of 4% 1 N isopropanol/hydrochloric acid on a shaking table for 20 min. The absorbance values of the solution in each well were measured at 595 nm using a Bio-Rad iMark microplate reader (Bio-Rad Laboratories, Milan, Italy). Cell viability was expressed as a percentage of absorbance values in treated samples respect to that of control (100%). All MTT experiments were performed in quadruplicate.

Thiobarbituric acid-reactive species (TBARS) were determined on aliquots (250 µl) of cell culture medium of not treated cells (control) or after 48 h treatment with 100, 250 and 500 µM EqC APE. TBARS were measured as previously described (25). Briefly, samples were incubated with a solution consisting of 0.5 ml of 20% acetic acid, and 0.5 ml of 0.78% aqueous solution of thiobarbituric acid (pH 3.5), heated at 95°C for 45 min, and then centrifuged at 4000 rpm for 5 min. TBARS content was quantified at 532 nm with reference to malondialdehyde (extinction coefficient at 532 nm, 1.56×10⁵ M⁻¹ cm⁻¹). Result is the average of triplicate measurements of each individual experiment performed in duplicate.

MCF-7 cells were cultured in 6-well tissue culture plates (Becton-Dickinson, Franklin Lanes, NJ, USA) at a seeding density of 9.0×10⁴ cells. After overnight attachment, the cells were treated with different concentrations (100, 250, and 500 µM EqC) of APE and the untreated cells were used as control. The cells were cultured for up to 48 h under standard culture conditions and their morphological changes were imaged using a phase-contrast microscope (Axiovert-10 Zeiss microscope).

MFC-7 cells were cultured in 10-cm culture dishes at a seeding density of 5.5×10⁵ cells. After overnight attachment, the cells were treated for 48 h with 100, 250, and 500 µM EqC APE and the untreated cells used as control. After the treatment, the cells were collected by centrifugation, washed twice with ice-cold PBS, and the pellets were lysed using 100 µl of RIPA buffer. After incubation on ice for 30 min, the samples were centrifuged at 18,000 × g in an Eppendorf microcentrifuge for 10 min a 4°C, and the supernatants were quantified for protein content. Aliquots containing approximately 30 µg of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose membranes by Trans blot turbo (Bio-Rad Laboratories). The blots were blocked with 5% non-fat dry milk in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl plus 0.5% Tween-20 (TBS-T). The membranes were subsequently incubated at 4°C overnight in the 5% non-fat dry milk-TBS-T buffer, containing, for each experiment, one of the specific primary antibodies. After three times washing with TBS-T, the blots were incubated for 1 h at room temperature with the HRP-conjugated secondary antibodies. After four times washing, the blots were developed using enhanced chemiluminescence detection reagents ECL (Millipore Corp., Billerica, MA, USA) and exposed to X-ray film. The film was scanned by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell cycle distribution was studied by PI staining and flow cytometry analysis using a FACScalibur (Becton-Dickinson, San Jose, CA, USA) interfaced with a Hewlett-Packard computer (model 310) for data analysis. Briefly, MCF-7 cells were plated in 6-multiwell plates at the density of 9.0×10⁴ cells/well. After 24 h, the medium was changed and APE was added at concentrations of 100, 250, or 500 µM EqC; conversely fresh medium was added to the control well. After 48 h treatment, the cells were recovered by incubation with trypsin-EDTA, washed in PBS, collected by centrifugation, fixed by resuspension in 70% ice-cold methanol/PBS and incubated overnight at 4°C. After fixing, samples were centrifuged at 400 × g for 5 min, washed twice with ice-cold PBS, resuspended in 0.5 ml DNA staining solution (50 µg/ml PI and 25 µg/ml RNaseA in PBS), and incubated at room temperature for 1 h in the dark. Samples were then transferred to 5-ml Falcon tubes and stored until assayed. PI fluorescence was collected as FL2-A (linear scale) by the ModiFIT Cell Cycle Analysis software (Becton-Dickinson). For each sample, at least 20,000 events for each point were analyzed in at least three different experiments giving an SD of <5%.

Annexin V-FITC binds phosphatidylserine residues which are translocated from the inner to the outer leaflet of the plasma membrane during the early stages of apoptosis. A flow-based Annexin V-FITC/PI double staining was used to distinguish apoptotic (Annexin V-FITC-positive, PI-positive) from necrotic (Annexin V-FITC-negative, PI-positive) cells (26). MCF-7 cells were plated in 6-multiwell plates at the density of 9.0×10⁴ cells/well. After 24 h, the medium was changed and APE was added at concentrations of 100, 250, or 500 µM EqC while fresh medium was added to the control well. After 48 h of treatment, the cells were detached by incubation with trypsin-EDTA, washed in PBS, collected by centrifugation, resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂), and then stained with 5 µl of Annexin V-FITC and 10 µl PI (20 µg/ml) for 30 min in the dark, according to the manufacturer's instructions. Analyses were performed with flow cytometry apparatus (Becton-Dickinson). Apoptosis was detected in red and green fluorescence channels with excitation wavelength of 488 nm. For each sample, 2×10⁴ events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

All experiments were performed three times with replicate sample. Data are expressed as mean ± standard deviation (SD). Comparisons between treated samples and control were performed using analysis of variance (ANOVA) plus Bonferroni's t-test. A P-value <0.05 was considered to indicate a statistically significant result.

The total polyphenolic content of the Annurca apple extract measured by Folin-Ciocalteu phenol reagent resulted in 125.2±7.1 mg of catechin per 100 g of sample and is similar to that evaluated in other studies (24,25). The HPLC analysis of APE identified (+)-catechin, (−)-epicathechin, chlorogenic acid, quercetin, and quercetin glycosides as the main Annurca apple o-diphenols (data not shown). This profile confirms the results already described in the literature (16,24).

To evaluate the effect of APE on cell growth, MCF-7 cells were incubated for 24 and 48 h with increasing concentrations of the extract ranging from 100 to 500 µM EqC corresponding to 29–145 µg EqC/ml, and cell proliferation was then assessed by MTT assay. The cells appeared to be quite resistant to APE treatment. The results obtained evidenced that up to 100 µM EqC no appreciable changes in cell viability were observed at any time of incubation. On the other hand, at higher doses, the treatment with APE significantly reduced cell proliferation in a dose- and time-dependent manner (Fig. 1). It has to be noted that after 24 h incubation, 250 µM EqC and 500 µM EqC APE exerted only a poor cell growth inhibitory effect reducing cell proliferation to approximately 98 and 83%, respectively, compared to control cells. In contrast, a prolonged incubation of 48 h resulted in a severe loss of cell viability that decreases to 43.5%, a value near the IC₅₀, in the presence of APE 500 µM EqC. Parallel direct cell counting provided similar results (data not shown). Based on these findings, we selected for further investigations an incubation time of 48 h.

Free radicals are involved in cell death induction in a number of systems and membrane phospholipids represent one of the major target of oxidative stress in cells. It has been shown that lipid hydroperoxides as well as lipid peroxidation initiators, such as ROS, are involved in signaling pathways that control cell proliferation, differentiation, maturation, and apoptosis (1,27). Moreover, it has been proposed that lipid peroxidation may represent a protective mechanism in breast cancer (27).

In order to investigate whether an oxidative stress could be produced under our experimental conditions, we evaluated lipid peroxidation by measuring the extent of lipid degradation products such as, malondyaldehyde and other aldehydes reactive to thiobarbituric acid after 48 h incubation of MCF-7 cells in the presence of increasing amounts of APE. It is interesting to note that, in analogy with the effect exerted on cell viability, APE is able to cause lipid peroxidation only at concentration >100 µM EqC inducing, at 250 µM EqC and 500 µM EqC concentration, respectively, an increase of TBARS cell content of 3- and 6-fold higher than the untreated cells (Fig. 2). These results indicate that, at elevated concentrations, apple polyphenols may act as pro-oxidants and may induce growth inhibition probably enhancing intracellular ROS oxidative stress.

MCF-7-cells were incubated for 48 h with 100, 250, and 500 µM EqC APE, and the morphology of treated cells was then analyzed with a phase-contrast microscope and compared with the untreated cells (Fig. 3). The results obtained indicated that the cells treated with APE 100 µM EqC (Fig. 3B) maintained the characteristic epithelial morphology and prolific growth as a monolayer, quite similar to untreated MCF-7 cells (Fig. 3A). On the contrary, the cells treated with APE 250 µM EqC (Fig. 3C) and 500 µM EqC (Fig. 3D) displayed morphological alterations such as, shrinkage and cytoplasmic condensation, cell rounding, poor adherence, and cell detachment. Altogether the evidence suggests that APE is able to commit MCF-7 cells to a type of death that mimics apoptosis.

Recent literature data have highlighted the chemopreventive and anticancer properties of polyphenolic compounds and polyphenol-rich nutritional sources. Moreover, several recent investigations have elucidated the mechanisms by which polyphenols are able to modulate some cellular events such as, cell cycle arrest by decreasing cyclin levels, and apoptosis induction (28).

To verify whether APE caused cell cycle perturbation in MCF-7 cells, we evaluated the cell distribution in the cell cycle phases by flow cytometric analysis. In addition, the severe morphological changes observed in APE-treated MCF7-cells under the inverted phase-contrast microscope prompted us to further examine whether the APE-induced cell death was a consequence of activation of apoptosis. Therefore, we also looked at the proportion of cells with hypodiploid DNA content (sub-G1 population), characteristic of cells having undergone DNA fragmentation, a biochemical hallmark of apoptosis.

MCF-7 cells were exposed to increasing concentrations of APE (100, 250, and 500 µM EqC), harvested at 48 h, and examined for DNA content after proper staining with PI. Fig. 4 shows that the treatment of cells with 100 and 250 µM EqC APE does not cause any evident effect on cell proliferation while 500 µM EqC APE induces a cell cycle arrest at G2/M, as evidenced by the significant increase of cell percentage in this phase of the cell cycle (26%) respect to untreated cells (11%). Moreover, a dose-dependent increase of sub-G1 population can be observed that becomes clearly evident at 500 µM EqC APE (34%) suggesting that the cytotoxic activity of APE in MCF7-cells occurs via apoptosis.

To gain further insight into the molecular mechanism of cell cycle arrest induced by APE, we analyzed the expression level of some relevant cell proliferation- and cell cycle-regulators such as, cyclin D1, cyclin-dependent kinase inhibitor 1 (p21) and p53 transcription factor. Thus, MCF-7 cells were exposed to 100, 250, and 500 µM EqC APE for 48 h, after which cell extracts were analyzed by western blotting. As shown in Fig. 5, the treatment for 48 h with APE induced a significant dose-dependent increase in the level of p53 and p21 proteins. It has to be pointed out that the tumor suppressor p53 is a transcription factor that regulates a broad range of processes among which cell cycle arrest, senescence, and apoptosis are the best characterized. In vitro and in vivo studies have shown that polyphenols such as, epigallocatechin, resveratrol, and curcumin as well as nutritional sources of polyphenols are able to upregulate p53 protein in several cancer cell lines (28–32) and that the polyphenol-induced stabilization and expression of p53 is often associated with a G1 or G2/M phase cell cycle arrest (33). It has also been reported that tumor suppressors, like p53 and its analogs, are key molecular targets of polyphenols responsible for their pro-apoptotic effect in human and animal cancer models through the transcriptional regulation of target genes such as p21, Bax, and PUMA (32).

From the western blot analysis reported in Fig. 5 we can observe that concomitantly with the increase of p53 and p21, the level of cyclin D1 was decreased in a dose-dependent manner. In agreement with our results it has been recently reported that epigallocatechin-3-gallate, the major polyphenolic component of green tea, induces apoptosis and cell cycle arrest in colorectal cancer cells by downregulating cyclin D1 and upregulating p21 (34).

Cyclin D1 is a key factor regulating cell proliferation, this cyclin links the extracellular signals to cell cycle advancement (35). Cyclin D1 levels must be elevated during G1 phase to start DNA synthesis, but then they must be suppressed during S phase to allow DNA synthesis. If the cell has to continue to proliferate, cyclin D1 levels must be induced once again during G2 phase. This induction depends upon the activity of proliferative signaling molecules, and ensures that the extracellular environment continues to be conducive for growth (36). For its prominence, cyclin D1 is an intriguing target in anticancer agent development. Many anticancer drugs induce cyclin D1 degradation in several tumor cell lines. Moreover, increased cyclin D1 degradation in cancer cells, is achieved also by various natural compounds such as polyphenols (37). Altogether our results indicate that cyclin D1 degradation and p53 and p21 overexpression contribute to APE-induced growth inhibition in MCF-7 cells.

MAPK cascade is a major cell signaling pathway triggered by ROS, leading to activation of numerous transcription factors which control DNA repair, cell cycle, apoptosis, or the immune system, among other processes. Major MAPKs can be grouped into four subfamilies: extracellular signal-regulated kinases (ERKs), c-Jun amino terminal kinase (JNK), p38 MAPK, and big-1 kinases (BMAPK-1). The whole pathway is regulated by various extracellular signals and, in this manner, it regulates distinct and even opposing cellular phenomena, such as proliferation, differentiation, survival and apoptosis (38). Moreover, in human hepatocellular carcinoma cell lines, multiple anticancer effects such as inhibition of cellular proliferation as well as induction of cell cycle arrest and apoptosis have been achieved by blocking ERK signaling (39).

In order to investigate whether the antiproliferative effect induced by APE in MCF-7 cells was related to ERK, we analyzed the expression and activation (phosphorylation) of ERK by western blotting. As reported in Fig. 5, APE caused a strong dose-dependent inhibition of ERK phosphorylation and a decrease of pERK/ERK ratio while no variations in the total amount of ERK1/2 protein levels occur at any of the concentrations tested. The obtained results suggest that ERK inactivation may contribute to the G2/M cell cycle arrest and to the antiproliferative activity of APE even if the detailed molecular mechanisms underlying the APE-induced ERK signaling modulation needs to be further investigated. In agreement with our data it has been reported that red wine polyphenols inhibit the proliferation of colon carcinoma cells by modulating MAPK intracellular signal transduction pathways (40) and that quercetin may induce apoptosis in HepG2 cells by direct activation of caspase cascade and by inhibiting survival signaling (41).

The occurrence of apoptosis in MCF-7 cells upon APE treatment was assessed by FACS analysis after double labeling of cells with Annexin V and PI. Representative pictures are shown in Fig. 6 together with the percentage of apoptotic cells. The exposure of cells for 48 h to Annurca apple polyphenols caused a dose-dependent increase of apoptotic cells respect to the control. A low percentage of early apoptotic cells (4.9%) is already evident after exposure to 100 µM EqC APE. This value significantly increases in the cells treated with APE 250 µM EqC (19%) and 500 µM EqC (46.2%).

The APE-induced lipid peroxidation and the highly increased expression levels of p53 after cell incubation with APE led us to hypothesize that the apoptotic process in MCF-7 cells occurs through a ROS-dependent mitochondrial pathway.

The balance of the expression of anti- and pro-apoptotic members of the Bcl-2 gene family is one of the major mechanisms that regulate this type of apoptotic process in mammalian cells (42). Therefore, to determine whether APE-induced apoptosis in MCF-7 cells was associated with the modulation of members of this protein family, we examined the expression of Bcl-2 and Bax proteins. The results obtained, reported in Fig. 7, show that APE treatment resulted in the increased expression of pro-apoptotic Bax while the level of the anti-apoptotic Bcl-2 was decreased. We then tested the effect of APE on the cascade of caspases that are crucial initiators or effectors in this cell death pathway. Western blot analysis shows that the increased Bax/Bcl-2 ratio coincides with the activation of the initiator caspase-9 and of the executioner caspase-6 and -7 and with the increased PARP cleavage (Fig. 7). Altogether these results indicate that the activation of the mitochondrial pathway is involved in the apoptosis of MCF-7 cells induced by APE.