Table 1.
IC50 values (% v/v of fresh juice of all varieties of apple) obtained in MCF-7 and MDA-MB-231 and polyphenol and anthocyanin content of apple juices.
Fig 1.
Effect of apple juice on cell viability in MCF-7 and MDA-MB-231 cell lines.
(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. ** p<0.01; *** p<0.001).
Fig 2.
Cell cycle perturbation in MCF-7 and MDA-MB-231 cells treated with Pelingo juice.
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.
Fig 3.
Western blot analysis of MCF-7 and MDA-MB-231 cells treated for 4, 8 and 24 h with 2.5% v/v of Pelingo juice.
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. * p<0.05; ** p<0.01; *** p<0.001).
Fig 4.
Pelingo juice inhibits in vitro tumorigenesis of MDA-MB-231 cells.
(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. * p<0.05; *** p<0.001).
Fig 5.
Pelingo juice inhibits TPA-induced transformation of 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. * p<0.05; ** p<0.01).
Fig 6.
Western blot analysis of JB6 P+ cells treated with Pelingo juice and TPA.
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. ** p<0.01).