The authors have declared that no competing interests exist.
Conceived and designed the experiments: GFS GB GG. Performed the experiments: MDS MF AB LG. Analyzed the data: GFS MDS. Wrote the paper: GFS MDS GB.
The aim of this study was to evaluate the antiproliferative activity in breast cancer cells and the inhibition of tumorigenesis in pre-neoplastic cells of a new apple cultivar with reddish pulp, called the Pelingo apple.
The antiproliferative activity was evaluated in MCF-7 and MDA-MB-231 human breast cancer cells. The inhibition of tumorigenesis was performed in JB6 promotion-sensitive (P+) cells.
Results showed that Pelingo apple juice is characterized by a very high polyphenol content and strongly inhibited breast cancer cell proliferation. Its antiproliferative activity was found to be higher than the other five apple juices tested. Pelingo juice induced cell accumulation in the G2/M phase of the cell cycle and autophagy through overexpression of p21, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3 beta (LC3B). Remarkably, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells, suppressing colony formation in semi-solid medium and TPA-induced ERK1/2 phosphorylation.
Our data indicate that the Pelingo apple is rich in food components that can markedly inhibit
Several epidemiologic studies suggest that diets rich in fruits and vegetables may reduce the risk or delay the development of chronic diseases such as cancer, cardiovascular disease and diabetes [
Many of these protective effects have been attributed to non-nutrient plant constituents such as carotenoids, phenolic acids and flavonoids [
The anticancer activity of apple constituents has also been documented in rat models. Whole apple extract has been reported to prevent breast cancer in a dose dependent manner [
The antiproliferative properties of apple extracts have been described extensively by
This study focuses on a recently identified apple, named Pelingo apple, characterized by reddish colour and sweet fruity flavour [
Moreover, this apple contains an appreciable amount of polyphenols also in the pulp that showed good antioxidant activity comparable to that of red berries [
This makes the discovery of this new cultivar with potential health benefits quite interesting, and its propagation has been entrusted to a consortium of the breeders.
The purpose of this investigation was to evaluate the antiproliferative activity in both MCF-7 and MDA-MB-231 human breast cancer cell lines and the inhibition of tumorigenesis in JB6 Cl 41-5a promotion-sensitive (JB6 P+) of the Pelingo apple juice. Our results show that Pelingo juice is characterized by a very high polyphenol content, strongly inhibits the proliferation of human breast cancer cells through accumulation in the G2/M phase of the cell cycle, overexpression of p21 and inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity. Moreover, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells.
We have screened six apple cultivars to assess their antiproliferative activity on breast cancer cell lines: the Pelingo apple (
The apples were washed in tap water and dried. The apple core was removed and the remaining parts, cut into small pieces and homogenized using a household juicer (Moulinex JU200045). The solid matrix was then separated by a first centrifugation at 3000 rpm and juice was collected and centrifuged at 10000 rpm for 10 min at 4°C. Finally, the juice supernatant was filtered through a 0.45 μm membrane and stored in aliquots at -20°C.
The total anthocyanin content of the apple extracts was measured using the differential pH method reported by Elisia et al. [
The monomeric anthocyanin pigment concentration in the original sample was calculated as reported by Elisa et al. and Tzulker
MCF-7 and MDA-MB-231 cell lines, diploid human fibroblasts from fetal lung tissue (WI-38), embryo mouse fibroblasts (NIH-3T3) and murine skin epidermal (JB6 P+) cells were purchased from the American Type Cell Culture Collection. All cell lines (with exception of JB6 P+) were cultured in DMEM supplemented with 10% of heat-inactivated FBS, 2 mM glutamine, 0.1 g/L streptomycin, 100 units/ml penicillin and, only for MCF-7, 10 μg/ml insulin. JB6 P+ cells were cultured in EMEM supplemented with 5% of heat-inactivated foetal bovine serum (FBS), 2 mM glutamine, 0.1 g/L streptomycin, 100 units/ml penicillin and 1 mM Na-pyruvate. The cells were grown in a humidified atmosphere at 37°C with 5% CO2. TPA was dissolved in DMSO and used to a final concentration of 10 ng/ml. All cell culture materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).
MCF-7 and MDA-MB-231 cells were plated at a density of 5x104 cells/well in 12-well plate and JB6 P+ cells were plated at a density of 3x105 cells/well in 35 mm dishes, incubated at 37°C with 5% CO2 overnight and treated with increasing concentrations of Pelingo juice (only JB6 P+ were stimulated with 10 ng/ml TPA). After 72 h of treatment cells were washed in PBS and counted using a hemacytometer by Trypan blue exclusion assay or fixed in 4% formaldehyde for 15 min and stained with 0.1% crystal violet for 15 min. Pictures were captured using an optic microscope (20X).
Assays were carried out in 35 mm dishes. For each dish, the bottom layer consisted of 1 ml of 0.6% agar in DMEM or EMEM (10% FBS) for MDA-MB-231 and JB6 P+ respectively. A total of 5x103 MDA or 5x104 JB6 P+ cells suspended in 1 ml of 0.3% agar in complete medium, were layered on top. Both layers were supplemented with Pelingo juice and, only in JB6 P+, with TPA (10 ng/ml) or vehicle (DMSO 0.01 μl/ml). Weekly, 500 μl of complete medium was added to maintain humidity. Cells were incubated at 37°C for 14 days (MDA- MB-231) and for 21 days (JB6 P+). The colonies after crystal violet staining (0.01%) were counted and photographed with an inverted microscope (5X). Only clusters containing more than 20 cells were counted as colonies.
Cells were plated at a density of 5x103 cells/well in 96-well plate, incubated at 37°C overnight and treated in triplicate with increasing concentrations of apple juice. After 72 h of treatment, cell viability was evaluated using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), based on the ability of viable cells to convert a soluble tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS) to a formazan product, as reported previously [
MCF-7, MDA-MB-231, NIH-3T3 and WI-38 cells were seeded at a density of 1x105 cells in 3 ml of appropriate growth medium in 30 mm dishes for 24, 48, 72 and 96 h. Proliferative indices were evaluated using the formula:
Cell cycles were analyzed by means of the propidium iodide staining procedure previously reported [
Apoptosis, necrosis and autophagy induction was evaluated as previously described [
MCF-7, MDA-MB-231 and JB6 P+ cells were seeded at 5x105 cell in 30 mm diameter dishes, attached overnight and treated with Pelingo juice (only JB6 P+ were stimulated with 10 ng/ml TPA). At indicated times, cells were washed with ice-cold PBS and lysed as reported in [
The results are presented as means ± SEM of at least 3 separate experiments. Data were analyzed using 1- or 2-way ANOVA as appropriate followed by Bonferroni or Dunnet’s
The amounts of polyphenols and anthocyanins measured in apple juice from the six cultivars are reported in
MCF-7 | MDA-MB-231 | Polyphenols (mg/ml) | Anthocyanins (μg/ml) | |
---|---|---|---|---|
Abbondanza white pulp | nd | 5.93 ± 0.21 | 0.294 | 6.70 |
Abbondanza red pulp | 8.15 ± 0.46 | 3.51 ± 0.19 | 0.558 | 186.69 |
Marchigiana pink pulp | nd | 12.24 ± 0.71 | 0.219 | 0.83 |
Annurca | nd | 12.88 ± 0.89 | 0.259 | 0.42 |
Bacci | 12.04 ± 1.24 | 3.24 ± 0.09 | 0.881 | 3.34 |
Pelingo | 3.96 ± 0.23 | 1.81 ± 0.09 | 1.996 | 28.39 |
nd: not detectable
The antiproliferative activity of Pelingo juice was evaluated with anchorage-dependent growth inhibition assay. As shown in
(A) Anchorage-dependent cell growth assay. Cells (5x104) were treated with Pelingo juice for 72 h. Cell growth was evaluated by cell count and crystal violet staining. (B) Cell viability assay. Cells (5x103) were treated for 72 h with six apple juice cultivars: 1, Abbondanza white pulp. 2, Abbondanza red pulp. 3, Marchigiana pink pulp. 4, Annurca. 5, Bacci. 6, Pelingo. Cell viability was evaluated by MTS assay. Data are expressed as means ± SEM of at least three separate experiments. Asterisks indicate significantly differences of Pelingo activity with respect to the same dose of other apples (2-way ANOVA followed by Bonferroni post hoc test. **
The effect of Pelingo juice was then compared with five other apple juice cultivars using the MTS assay. Pelingo juice significantly reduced cell proliferation in a dose dependent manner in MCF-7 and MDA-MB-231 cells (1-way ANOVA followed by Bonferroni
The correlation between the proliferative index and IC50 was also evaluated, using two additional cell lines, WI-38 and NIH-3T3. The results showed that antiproliferative activity was directly proportional to the rate of replication of the cells tested. In fact, the IC50 value was lower (higher activity) in cells with fast growth (MDA-MB-231 and NIH-3T3 cells; proliferative index: 5.45 and 4.23 respectively; IC50: 1.81 and 1.13% v/v respectively), than in cells with slow growth (MCF-7 and WI-38 cells; proliferative index: 0.48 and 0.63 respectively; IC50: 3.96 and 4.34% v/v respectively).
The activity of the Pelingo juice stored at -20°C was stable over a 1-year period (not shown). We also found that the activity of Pelingo juice obtained from peeled apple was not significantly different to that of whole apple (not shown).
Cell cycle analysis was carried out in MCF-7 and MDA-MB-231 cells to evaluate the effect of Pelingo juice on cell cycle progression. Cells were treated with Pelingo juice for 24 and 48 hours at the final concentrations of 2.5 and 5.0% v/v for MCF-7 and 1.5 and 3.0% for MDA-MB-231 and then stained with propidium iodide for flow cytometric analysis. Results showed that Pelingo juice induced a dose-dependent cells accumulation in late S and G2/M phases of the cell cycle in both MCF-7 and MDA-MB-231 cell lines (
Cells were treated for 24 and 48 h with 2.5 and 5% v/v (MCF-7) and 1.5 and 3.0% v/v (MDA-MB-231) of Pelingo juice and stained with propidium iodide. DNA content profiles were analyzed by flow cytometry. Images are representative of one experiment of three replicates. Data are represented as means ± SD.
Moreover, results show that the Pelingo juice did not significantly induces sub-G1 events (
Induction of apoptosis/necrosis/autophagy was simultaneously assessed by colorimetric analyses using Hoechst, propidium iodide and acridine orange dyes. MCF-7 and MDA-MB-231 cells were treated with 2.5 and 5% v/v Pelingo juice for 24, 48 and 72 h, stained as described in the methods section and observed under a fluorescence microscope. Propidium iodide positive nuclei (necrotic cells) show necrosis induction after 24 and 48 h of Pelingo juice treatment in MCF-7 and MDA-MB-231 cells, respectively (
To further investigate the cytostatic activity of Pelingo juice revealed in cytofluorimetric analysis, the level of p21 and activity of ERK1/2 were evaluated. Both proteins are involved in G2 cell cycle arrest and cell proliferation. Results shows that p21 was time-dependent upregulated with about 1.5-fold and 7.0-fold in MCF-7 and MDA-MB-231 respectively after 24 hours of treatment (
ERK1/2 phosphorylation, p21 overexpression and LC3B-II/LC3B-I ratio were analyzed in total cell extracts. (A) Representative images of three experiments giving similar results. (B) Densitometric analysis: p21 and phospho-ERK1/2 are shown as fold changes relative to control; changes in autophagosomes-related protein LC3B are shown as LC3B-II(lipidated)/LC3B-I(non lipidated) ratio. Phospho-ERK was normalized to total ERK; p21, LC3B-II and LC3B-I were normalized to actin. Data are expressed as means ± SEM of three replicates. Asterisks indicate significantly differences respect to control (1-way ANOVA followed by Dunnett's multiple comparison test. *
The effect of Pelingo juice at non-cytotoxic doses (0.125, 0.25, 0.5 and 1% v/v) on MDA-MB-231
(A) Anchorage-dependent cell growth assay. Cells (5x104) were treated for 72 h with 0.125, 0.25, 0.5 and 1% v/v of Pelingo juice. Cell growth was evaluated by cell count and crystal violet staining. (B) Anchorage-independent cell growth assay. Cells (5x103) were growth in soft agar with 0.125, 0.25, 0.5 and 1% v/v of Pelingo juice for 14 days and colonies containing more than 20 cells were counted. Data are expressed as means ± SEM of three separate experiments. Asterisks indicate significantly differences respect to control (1-way ANOVA followed by Dunnett's multiple comparison test. *
The effect of Pelingo juice treatment on the TPA-induced anchorage-dependent and anchorage-independent cell growth was evaluated in JB6 P+ cells (
(A) Anchorage-dependent cell growth assay. Confluence cells (3x105) were stimulated with 10 ng/ml of TPA and treated with 0.125, 0.25 and 0.5% v/v of Pelingo juice for 72 h. Cell growth was evaluated by cell count and crystal violet staining. (B) Anchorage-independent cell growth assay. Cells (5x104) were growth in soft agar with 10 ng/ml of TPA and 0.125, 0.25 and 0.5% v/v of Pelingo juice for 21 days and colonies containing more than 20 cells were counted. Data are expressed as means ± SEM of three separate experiments. Asterisks indicate significantly differences respect to control (1-way ANOVA followed by Dunnett's multiple comparison test. *
The effect on anchorage-independent cell growth was evaluated in soft-agar, monitoring the TPA-induced colony formation of JB6 cells (
The effect of Pelingo juice on TPA-induced tumorigenesis was further evaluated analyzing the activation of ERK1/2, kinase involved in the neoplastic transformation induced by TPA. JB6 P+ cells were pre-treated with Pelingo juice for 24 h and stimulated with 10 ng/ml of TPA for 60 min. The phosphorylation of ERK1/2 was analyzed in total cell lysates, revealing that Pelingo juice partially inhibited the TPA-induced ERK1/2 phosphorylation at 2% v/v (
Cells were pre-treated with 0.5, 1 and 2% v/v of Pelingo juice for 24 h and stimulated with 10 ng/ml of TPA. ERK1/2 phosphorylation was analyzed in total cell extracts. (A) Representative images of three experiments giving similar results. (B) Densitometric analysis of phospho-ERK1/2 respect to control. Phospho-ERK was normalized to total ERK. Data are expressed as means ± SEM of three replicates. Asterisks indicate significantly differences respect to control (1-way ANOVA followed by Dunnett's multiple comparison test. **
During the past decade, breast cancer has become the most common form of cancer among women in Western countries, and the number of cases diagnosed worldwide is increasing [
In this study, have shown that whole juice from a new cultivar of apple with reddish pulp, called the Pelingo apple, is a potent growth inhibitor of human breast cancer cells
Among the isolated polyphenolic compounds, the main flavonoids in apple are quercetin 3-
In agreement with that which was reported by Sun
The cytostatic effect of Pelingo juice it was confirmed by the inhibition of ERK1/2 activity and the upregulation of p21. ERK1 and ERK2 participate in the Ras-Raf-MEK-ERK signal transduction cascade, implicated in the regulation of a large variety of processes including cell adhesion, cell migration, cell cycle progression and proliferation [
Although the induction of apoptosis in cancer cells has been previously shown using total apple extract [
On the contrary, the AVOs’ formation and lipidated LC3B accumulation indicate autophagy induction. This interesting mechanism has recently been proposed as a strategy for cancer prevention because it could be responsible for eliminating damaged proteins, organelles and DNA, all of which can contribute to mutation and initiate transformation in pre-malignant cells [
In this study, we evaluated the effect of Pelingo juice on TPA-induced tumorigenesis of the pre-neoplastic JB6 P+ cells. We found that Pelingo juice is able to block the TPA-induced neoplastic transformation, resulting in a inhibition of colony formation in semi-solid medium. Moreover, since TPA induces neoplastic transformation through activation of the ERKs pathway in various cells [
The approach to developing new chemopreventive agents has changed considerably in recent years and now involves the extensive preclinical mechanistic study of agents, such as inhibition of proliferation, induction of autophagy or apoptosis and differentiation, before clinical trials are initiated [
In conclusion, we have shown that Pelingo apple is characterized by a very high polyphenol content which strongly inhibits the proliferation of breast cancer cells, induces cell accumulation in the G2/M phase of the cell cycle and autophagy, overexpression of p21 and inhibition of ERK1/2 activity. The Pelingo apple also suppress TPA-induced
(TIF)
DNA content profiles of cells exposed for 24 and 48 h to 2.5 and 5.0% v/v, stained with propidium iodide, and analyzed by flow cytometry are shown as ungated cellular events and in a logaritmic scale.
(TIF)
DNA content profiles of cells exposed for 24 and 48 h to 1.5 and 3.0% v/v, stained with propidium iodide, and analyzed by flow cytometry are shown as ungated cellular events and in a logaritmic scale.
(TIF)
Cells were treated with 2.5 and 5.0% v/v of Pelingo juice for 24, 48 and 72 h, and directly stained with Hoechst, propidium iodide and acridine orange. Blue excitation filter was used for acridine orange; the cytoplasm and nucleus fluoresce green, whereas acidic compartments fluoresce orange-red. UV excitation was used for Hoechst and propidium iodide; undamaged cells nuclei fluoresce blue, necrotic cells nuclei fluoresce red.
(TIF)
The authors thank Prof. Timothy Bloom, of the University of Urbino “Carlo Bo”, Italy, for a critical reading of the manuscript.