Aronia melanocarpa L. fruit peels show anti-cancer effects in preclinical models of breast carcinoma: The perspectives in the chemoprevention and therapy modulation

Abstract

Introduction: Within oncology research, there is a high effort for new approaches to prevent and treat cancer as a life-threatening disease. Specific plant species that adapt to harsh conditions may possess unique properties that may be utilized in the management of cancer.

Hypothesis: Chokeberry fruit is rich in secondary metabolites with anti-cancer activities potentially useful in cancer prevention and treatment.

Aims of the study and methods: Based on mentioned hypothesis, the main goal of our study was to evaluate the antitumor effects of dietary administered Aronia melanocarpa L. fruit peels (in two concentrations of 0.3 and 3% [w/w]) in the therapeutic syngeneic 4T1 mouse adenocarcinoma model, the chemopreventive model of chemically induced mammary carcinogenesis in rats, a cell antioxidant assay, and robust in vitro analyses using MCF-7 and MDA-MB-231 cancer cells.

Results: The dominant metabolites in the A. melanocarpa fruit peel extract tested were phenolic derivatives classified as anthocyanins and procyanidins. In a therapeutic model, aronia significantly reduced the volume of 4T1 tumors at both higher and lower doses. In the same tumors, we noted a significant dose-dependent decrease in the mitotic activity index compared to the control. In the chemopreventive model, the expression of Bax was significantly increased by aronia at both doses. Additionally, aronia decreased Bcl-2 and VEGF levels, increasing the Bax/Bcl-2 ratio compared to the control group. The cytoplasmic expression of caspase-3 was significantly enhanced when aronia was administered at a higher dosage, in contrast to both the control group and the aronia group treated with a lower dosage. Furthermore, the higher dosage of aronia exhibited a significant reduction in the expression of the tumor stem cell marker CD133 compared to the control group. In addition, the examination of aronia`s epigenetic impact on tumor tissue through in vivo analyses revealed significant alterations in histone chemical modifications, specifically H3K4m3 and H3K9m3, miRNAs expression (miR155, miR210, and miR34a) and methylation status of tumor suppressor genes (PTEN and TIMP3). In vitro studies utilizing a methanolic extract of A.melanocarpa demonstrated significant anti-cancer properties in the MCF-7 and MDA-MB-231 cell lines. Various analyses, including Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential, were conducted in this regard. Additionally, the aronia extract enhanced the responsiveness to epirubicin in both cancer cell lines.

Conclusion: This study is the first to analyze the antitumor effect of A. melanocarpa in selected models of experimental breast carcinoma in vivo and in vitro. The utilization of the antitumor effects of aronia in clinical practice is still minimal and requires precise and long-term clinical evaluations. Individualized cancer-type profiling and patient stratification are crucial for effectively implementing plant nutraceuticals within targeted anti-cancer strategies in clinical oncology.

Keywords: Aronia melanocarpa L.; breast carcinoma; epigenetics; in vitro models; mechanism of action; rodent models.

Figure 1

Antioxidant activity of aronia extracts (10 μg/mL) in CAA assay. Quercetin (10 μg/mL) was used as a positive control (PC). All samples were dissolved in DMSO. DMSO alone was used as negative control (NC). A: water extract of fruit, B: methanol extract of fruit, C: water extract from peel; D: methanol extract from peel. The results are expressed as the means ± SE for two independent experiments measured in triplicate (*** Indicates a significant difference in comparison with the vehicle-treated cells (NC) p < 0.001, and **** corresponds to p < 0.0001).

Figure 2

Allograft 4T1 model in mice. (A) The development of the volume of 4T1 mammary adenocarcinomas in mouse allograft model after A. melanocarpa treatment. (B) The mitotic activity index after treatment with A. melanocarpa extract in 4T1 tumors in Balb/c mice. The mitotic figures are highlighted in circles; H&E staining; magnification ×400. CONT—control group, ARO 0.3—a group with aronia administered at a concentration of 3 g/kg in the diet, ARO 3—a group with aronia administered at a concentration of 30 g/kg in the diet. Data are expressed as mean ± SEM. A significant difference, *P < 0.05, **P < 0.01 vs. CONT.

Figure 3

Immunohistochemical analyses of rat carcinoma cells in vivo after A.melanocarpa treatment. (A) Immunoexpression of cleaved caspase-3 (cytoplasmic), Bax, Bcl-2, Ki67, VEGFA, VEGFR-2, and MDA in rat tumor samples. (B) Immunoexpression of cancer stem cell markers in rat tumor samples. (C) Immunoexpression of H3K4m3, H3K9m3, H4K16ac, and H4K20m3 markers in rat tumor samples. Data are shown as mean ± SEM. A significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001 vs CONT. The graphs show the protein expression, quantified as the average percentage of antigen-positive area in standard fields (0.5655 mm²) of hotspot areas within the tumor area. At least 60 pictures for each parameter were assessed.

Figure 4

Representative images of the expression of caspase-3, Bax, Bcl-2, Ki67, VEGFA, VEGFR-2, MDA, CD24, CD44, ALDH1A1, EpCam, H3K4m3, H3K9m3, H4K20m3, H4K16ac in rat carcinoma tissue of mammary gland. For detection, polyclonal caspase-3 antibody (Bioss, Woburn, USA), polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA), monoclonal Ki67 antibody (Dako, Glostrup, Denmark), monoclonal VEGFA and VEGFR-2 antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA), polyclonal CD24 antibody (GeneTex, Irvine, CA, USA), polyclonal CD44 antibody (Boster, Pleasanton, CA, USA), polyclonal ALDH1A1 antibody (ThermoFisher, Rockford, IL, USA), polyclonal MDA, EpCAM, H3K4m, H3K9m3, and H4K20m3 antibodies (Abcam, Cambridge, MA, USA), and monoclonal H4K16ac antibody (Abcam, Cambridge, MA, USA) were applied; final magnification: ×400.

Figure 5

Relative miRNA expression of miR21, miR155, miR210, miR22, miR34a, and miR145 in rat mammary carcinoma samples after treatment with A.melanocarpa. MiR-191-5p was selected as the internal control miRNA to normalize the cDNA levels of the samples. Data are expressed as mean ± SEM. Significant difference, * p < 0.05 vs. CONT, + p < 0.05 vs. ARO 0.3.

Figure 6

Promoter methylation status of ATM, PITX2, RASSF1A, PTEN, and TIMP3 tumor-suppressor genes in rat BC specimens after treatment with A.melanocarpa. The methylation level was designated using all evaluated CpG isles of the above-mentioned promoters. The brackets indicate the number of evaluated isles, i.e., ATM comprising six evaluated CpG sites (CpG 1–6), PITX2 six sites (CpG 1–6), RASSF1 three sites (CpG 1–3), PTEN six sites (CpG 1–6), and TIMP3 six sites (CpG 1–6). Twenty rat BC specimens for each experimental group were analyzed. Data are shown as mean ± SEM. A significant difference, *** p < 0.001 vs. CONT group and ⁺⁺ p < 0.01 vs. ARO 0.3 group.

Figure 7

Analysis of MCF-7 and MDA-MB-231 cells proliferation stained with CellTrace™ Yellow after ARO IC₅₀ treatment. Representative histograms.

Figure 8

Cell cycle distribution of MCF-7 (A) and MDA-MB-231 (B) cells after 48 and 72 h of ARO IC₅₀ treatment. Statistical significance: *p > 0.05 vs untreated control. Representative histograms with average data ± SD.

Figure 9

Occurrence of apoptosis in populations of MCF-7 (A) and MDA-MB-231 (B) cells after 48 and 72 h of ARO IC₅₀ treatment. Statistical significance: **p > 0.01, ***p > 0.001 vs untreated control. Representative dot plots with average data ± SD. Legend: Q1 (Death), Q2 (Late apoptotic), Q3 (Early apoptotic), Q4 (Live).

Figure 10

Mitochondrial membrane potential dissipation (A) and caspase 3/7 activity (B) in MCF-7 and MDA-MB-231 cells after 48 and 72 h of ARO IC₅₀ treatment. Statistical significance: *p > 0.05, **p > 0.01, ***p > 0.001 vs untreated control.

Figure 11

Western blot analyses of apoptosis-associated proteins (A) after ARO IC₅₀ treatment of MCF-7 and MDA-MB-231 cells include densitometry band analyses (B) and Bax/Bcl-2 ratio (C). Statistical significance: *p > 0.05, **p > 0.01, ***p > 0.001 vs untreated control.