Abstract

Graphical Abstract

Open in a separate window

Highlights

• In the rat chemopreventive model, aronia slightly lengthened the latency, decreased tumor volume and improved histopathological characteristics of adenocarcinomas.

• In the syngeneic 4T1 model in mice, aronia significantly decreased tumor volume of treated adenocarcinomas.

• Aronia significantly induced cancer apoptosis, and showed significant antiproliferative, anti-CSC, and antiangiogenic effects in vivo.

• Aronia showed significant beneficial epigenetic effects in vivo in the level of histone chemical modifications, miRNAs expressions, and methylation status of TSG promoters.

• Aronia demonstrated significant and complex anti-cancer properties in the MCF-7 and MDA-MB-231 cell lines and partially improved sensitivity of these cells to epirubicin.

1. Introduction

Breast carcinoma (BC) is currently the tumor disease most frequently diagnosed in women worldwide (1). Acquired resistance or insensitivity of cancer cells to chemotherapeutics represents a severe clinical problem of cytotoxic cancer therapies. One of the possible approaches to reducing the incidence of cancer is chemoprevention. In this context, current research emphasizes the importance of phytochemicals in reducing the risk of developing cancer as part of prevention and tries to increase the effectiveness of chemotherapeutic agents in cancer therapy. Targeting different signaling pathways to improve therapeutic outcomes by increasing the sensitivity of cancer cells and reversing resistance to currently used therapeutic methods represents an essential approach to improving the status of the clinical management of cancer (2). BC is multifactorial and is thus influenced by genetic, environmental, and lifestyle factors, including dietary preferences. Plant-based foods contain complex mixtures of phytochemicals characterized by significant biological activities, including antioxidant, immunomodulatory, anti-inflammatory, and anti-tumor effects, and also significant pro-apoptotic, anti-proliferative, anti-angiogenic, anti-stem-cell activities and epigenetic modulations of the gene expression (3–7). The mixtures of phytochemicals found in plants are potentially helpful in preventing or treating mammary carcinogenesis in humans (8).

Black chokeberry (Aronia melanocarpa L.) is rich in phytochemicals, which contribute to its potent antioxidative properties and various health benefits such as antimutagenic, anticancer, cardioprotective, hepatoprotective, gastroprotective, antidiabetic, anti-inflammatory, antibacterial, antiviral, radioprotective, and immunomodulatory effects (9). A. melanocarpa is a perennial shrub from the Rosaceae family, initially found in the eastern regions of North America. Aronia has also been found in Europe since the beginning of the 20^th century. Aronia fruits are used to prepare juices, syrups, jams, wine, or tea (9). A. melanocarpa is rich in proanthocyanins and anthocyanins. Besides anthocyanins, it contains significant amounts of hydroxycinnamic acids, such as chlorogenic and its isomer neochlorogenic acid, with antitumor activity in vitro (10). In this regard, aronia pomace using pressurized ethanol significantly decreased the viability and proliferation of HCT116 and DLD1 colon cancer cells and showed antioxidant effects (11). A. melanocarpa anthocyanins suppressed cell proliferation and caused cell cycle arrest in colorectal Caco cells. Additional data demonstrated that aronia anthocyanins down-regulated apoptosis via decreasing cytoplasmic β-catenin and the expression of related proteins in the Wnt/β-catenin signaling pathway (12). According to a study conducted by Choi et al. (13), the catechol derived from fermented aronia juice effectively suppressed the growth of BC cells and the formation of mammospheres. The inhibitory effect was observed to be dependent on the dosage of the compound. Furthermore, this catechol compound also reduced the CD44high/CD24low subpopulation, the population of cells expressing ALDH, and the genes nanog, sox2, and oct4 associated with self-renewal. The authors concluded that catechol hinders the formation of BC stem cells by modulating the Stat3/IL-6 signaling pathway. The same authors showed that 3-O-trans-p-coumaroyltormentic acid isolated from A. melanocarpa inhibits breast CSCs via downregulating the c-Myc protein, a CSC survival factor (14). In another study, A. melanocarpa fruit extract showed significant anti-inflammatory effects in LPS‐stimulated RAW 264.7 cells by reducing the expression of the inflammation-related biomarkers TNF-α, IL-6, and NO and the gene expression of iNOS and COX-2 (15). Thani et al. (16) have detailed the increased cytotoxic effects of gemcitabine when combined with polyphenols extracted from A.melanocarpa, as opposed to gemcitabine used alone, in the AsPC-1 pancreatic cancer cell line.

The antitumor properties of A. melanocarpa have not been studied in rodent models of BC. This comprehensive study examined the antitumor effects of orally administered A.melanocarpa in mouse 4T1 syngeneic (therapeutic) and rat chemically-induced (chemopreventive) breast adenocarcinoma models. Based on our extensive previous results with various whole plant substances analyzed in the rodent models, these goals have been formulated (17–24). The study’s subsequent objective was to evaluate the mechanism behind aronia’s anti-tumor properties by utilizing established clinical indicators of apoptosis, proliferation, angiogenesis, inflammation, cancer stem cells, and various epigenetic markers, including histone modifications, expression of oncogenic/tumor-suppressive miRNAs, and the level of methylation in crucial gene promoters linked to carcinogenesis. Furthermore, essential histopathological features of cancer cells, like the high/low-grade carcinoma ratio, mitotic activity index, and tumor necrosis ratio in vivo were also examined. Finally, to validate the mechanisms of the anticancer action of A. melanocarpa found in vivo, markers of proliferation, cell cycle, and apoptosis were realized using two human BC cell lines - hormone-sensitive MCF-7 and metastatic triple-negative MDA-MB-231 cells. Using the same cell lines, we analyzed the sensitization to epirubicin treatment using aronia extract.

2. Materials and methods

The studies received approval from the Ethical Commission of the Jessenius Faculty of Medicine at Comenius University (Protocol No. EK1860/2016) and the State Veterinary and Food Administration of the Slovak Republic (accreditation No. Ro-3239/15-221 and Ro-1640/17-221).

3. Results

3.2. Antioxidant activity of extract obtained from aronia fruit and peel

The methanol and water extracts from aronia dried fruit and peel were tested to evaluate their antioxidant activity in cellular assay on THP-1 macrophages. The results of these extracts were compared with flavonoid quercetin, commonly used as a standard of antioxidant activity. The results are depicted in Figure 1 . The methanol extract from aronia peel showed the most significant activity.

Open in a separate window

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).

3.3. Therapeutic mouse 4T1 model

We observed notable effects of aronia on the reduction of 4T1 tumor volume. The administration of aronia independently led to a 60.5% decrease (P<0.01) and a 51% decrease (P<0.05) in the volume of mouse 4T1 tumors compared to the control group (KONT) ( Figure 2 ). Upon histopathological analysis of the 4T1 tumor model, we observed a significant decrease in the mitotic index activity in tumor cells of the treated groups. The lower dose (ARO 0.3) resulted in a 42.5% decrease (P<0.001), while the higher dose (ARO 3) led to a 53% decrease (P<0.001) compared to the control group (CONT) ( Table 4 ; Figure 2 ). Although the necrosis/whole tumor area ratio evaluation indicated reductions in the treated groups, these changes were not statistically significant due to high standard errors of the means (SEMs) in all experimental groups.

Open in a separate window

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.

Table 4

Histopathological characteristics of 4T1 tumors in Balb/c mice after A. melanocarpa treatment.

Parameter	CONT	ARO 0.3	ARO 3
Necrosis/whole tumor area	7.82 ± 3.84	1.75 ± 0.46	2.04 ± 0.51
Mitotic activity index	20.78 ± 1.25	11.95 ± 0.91***	9.76 ± 0.95***

Open in a separate window

Data are expressed as mean ± SEM. A significant difference, ***P < 0.001 vs. CONT.

3.5. Immunohistochemistry of rat tumors

3.5.1. Markers of apoptosis, proliferation, and angiogenesis

The cytoplasmic expression of caspase-3 was significantly increased by 55.5% (P<0.05) with a higher dose of aronia (ARO 3) compared to the control group (CONT), and by 56.5% compared to the group with a lower dose of aronia (ARO 0.3) (P<0.05). Bax expression was significantly increased by 91.5% (ARO 0.3, P<0.001) with a lower dose of aronia, and by 92.5% (P<0.001) with a higher dose (ARO 3) compared to the control tumor samples. We observed a significant decrease in Bcl-2 expression by 27% with the lower dose of aronia (ARO 0.3, P<0.05), and by 21.5% with the higher dose of aronia (ARO 3, P<0.05) compared to the control group. The expression ratio of Bax/Bcl-2 was significantly increased by 173% (P<0.01) and 142.5% (P<0.001) with aronia, independent of the dose, compared to the control group. Aronia also significantly reduced VEGF expression by 39.5% (P<0.05) and 37% (P<0.01) compared to the control samples. The other evaluated parameters remained unchanged ( Figure 3A ).

Open in a separate window

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.

3.5.2. Markers of cancer stem cells

The assessment of tumor stem cell markers in tumor samples in vivo revealed a significant reduction in CD133 expression by 58.5% in the group receiving a higher dose of aronia (P<0.001) and a less pronounced decrease of 36.5% with a lower aronia dose (P=0.10) compared to the control group. In comparison to the control samples, the reductions in CD44 (by 14%), EpCam (by 6.5%), and ALDH (by 16%) expression after treatment with the higher aronia dose were not statistically significant (P>0.05) ( Figure 3B ).

3.5.3. Markers of histone chemical modification

Figure 3C illustrates the post-translation chemical changes of histones H3 and H4 in rat carcinoma cells after being treated with aronia. The effects of aronia on these histones were dose-dependent. In comparison to the control group, the ARO 3 group showed a significant decrease of 50% in H3K4m3 levels (P<0.001), while the ARO 0.3 group exhibited a decrease of 20% (P=0.10). Both doses of aronia resulted in a reduction of H3K9m3 levels, with the ARO 0.3 group showing a decrease of 37.5% (P<0.05) and the ARO 3 group showing a decrease of 35.5% (P<0.05). Furthermore, the higher dose of aronia led to a mild increase in H4K16ac levels by 19.5% (P=0.21) and H4K20m3 levels by 28% (P=0.16) compared to the control group. However, the lower dose of aronia did not cause any significant changes in H4K16sc and H4K20m3 levels when compared to the control tumor cells in vivo ( Figure 3C ).

The pictures in Figure 4 illustrate the sample expressions of cleaved caspase-3, Bax, Bcl-2, Ki67, VEGFA, VEGFR-2, CD24, CD44, CD133, ALDH1A1, EpCam, H3K4m3, H3K9m3, H4K16ac, and H4K20m3 in rat BC tissue.

Open in a separate window

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.

3.6. miRNA expression in tumor tissue

In order to thoroughly examine the anti-cancer and epigenetic characteristics of A. melanocarpa, we evaluated the expression levels of six clinically validated miRNAs in rat mammary cancer tissue in vivo ( Figure 5 ). A. melanocarpa notably reduced the expression of oncogenic miR155 by 46.5% (P < 0.05) at a lower dosage and by 43% (P < 0.05) at a higher dosage compared to the control group. Higher doses of aronia significantly boosted the expression of tumor-suppressive miR34a by 36.5% (P = 0.05) and slightly increased the expression of tumor-suppressive miR22 by 26.5% compared to untreated tumors. MiR210, known for its dual role in carcinogenesis as both an oncogene and a tumor-suppressor, exhibited a dose-dependent increase in cancer tissue by 77.5% (P < 0.05) and 51.5% (P < 0.05) relative to controls. Following treatment with high doses of aronia, there was an elevation in tumor-suppressive miR145 expression by 76.5% (P < 0.05) compared to the lower aronia dose. Lastly, A. melanocarpa did not induce any changes in the expression of oncogenic miR21 compared to the control group ( Figure 5 ).

Open in a separate window

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.

3.7. Promoter methylation status of tumor suppressor genes in vivo

Following treatment with a higher dose of A. melanocarpa, there was a 30.5% decrease in total methylation of the TIMP3 promoter compared to control samples (P < 0.001). Conversely, aronia significantly increased the methylation status of the PTEN promoter by 155.5% and 148% at both doses, respectively, compared with controls (P < 0.001). Evaluation of three other tumor-suppressor gene promoters revealed no significant changes, although methylation status was reduced by 11% in RASSF1 and ATM genes after higher aronia dosing ( Figure 6 ).

Open in a separate window

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.

3.9. In vitro analyses using MCF-7 and MDA-MB-231 cell lines

3.9.1. Cell viability/metabolic activity testing

An in vitro metabolic test was used to test the potential cytotoxic or growth-inhibitory effect of aronia extract and epirubicin (for combinational treatment) on two different BC cell lines (MCF-7, MDA-MB-231) and two different normal cell line models (MCF-10A, BJ-5ta). All cell lines were tested in the condition of monolayer growth (2D model) or as spheroids (3D model). Based on growth/metabolic activity inhibitory curves analyzed after treatment with ARO and EPI, the IC₅₀ values were calculated, as seen in Table 6 . In both BC cell lines, ARO and EPI showed concentration-dependent inhibition of metabolic activity (data not shown) in both 2D and 3D models. ARO also showed toxicity against normal cell lines, leading to an average selectivity index.

Table 6

Predictive IC₅₀ values of ARO and EPI treatment.

	Predictive IC50 values ± SD (µg/mL or µM)
	MCF-7	MDA-MB-231	MCF-10A	BJ-5ta
ARO 2D	528.0 ± 22.6	482.9 ± 36.2	713.0 ± 44.6	616.7 ± 14.6
ARO 3D	1112.4 ± 4.8	1663.9 ± 115.3	1945.2 ± 130.5	802.4 ± 64.1
Selectivity index MCF-10A (2D/3D)	1.3/1.7	1.5/1.2
Selectivity index BJ-5ta (2D/3D)	1.2/0.7	1.3/0.5
EPI 2D	0.43 ± 0.04	0.07 ± 0.01	0.09 ± 0.01	0.53 ± 0.02
EPI 3D	0.51 ± 0.08	1.34 ± 0.17	1.28 ± 0.21	1.49 ± 0.03

Open in a separate window

For combinational treatment (ARO+EPI), two lower concentrations were prepared from IC₅₀ by half dilutions (IC₂₅ and IC_12.5) and tested for their inhibitory activity. As seen in Table 7 , concentration-dependent inhibition on both BC cell lines is seen as expected.

Table 7

Resazurin metabolic test of several ARO and EPI IC₅₀ dilutions in 2D and 3D models.

2D	MCF-7				MDA-MB-231
	Concentrations
2D	CTRL	IC12.5	IC25	IC50	CTRL	IC12.5	IC25	IC50
ARO	1.00 ± 0.00	0.96 ± 0.02	0.90 ± 0.05*	0.58 ± 0.10***	1.00 ± 0.00	0.72 ± 0.04**	0.62 ± 0.05**	0.50 ± 0.06***
EPI	1.00 ± 0.00	0.94 ± 0.10	0.83 ± 0.05*	0.54 ± 0.06***	1.00 ± 0.00	0.90 ± 0.05*	0.72 ± 0.03**	0.49 ± 0.06***
DMSO a	1.00 ± 0.00	1.07 ± 0.09	0.98 ± 0.07	0.92 ± 0.06	1.00 ± 0.00	1.01 ± 0.06	0.94 ± 0.06	0.91 ± 0.04
3D	CTRL	IC12.5	IC25	IC50	CTRL	IC12.5	IC25	IC50
ARO	1.00 ± 0.00	0.92 ± 0.03	0.72 ± 0.05**	0.61 ± 0.04**	1.00 ± 0.00	0.89 ± 0.03*	0.65 ± 0.06**	0.49 ± 0.05***
EPI	1.00 ± 0.00	0.79 ± 0.05**	0.77 ± 0.03**	0.67 ± 0.03**	1.00 ± 0.00	0.88 ± 0.04*	0.69 ± 0.05**	0.65 ± 0.04**
DMSO a	1.00 ± 0.00	1.01 ± 0.04	1.02 ± 0.04	0.93 ± 0.05	1.00 ± 0.00	1.04 ± 0.02	1.01 ± 0.06	0.91 ± 0.04

Open in a separate window

^aDMSO was used as v/v % equivalent of IC; *p > 0.05, **p > 0.01, *** p > 0.001 vs untreated control. Data are presented as a fold of the control.

3.9.2. Antiproliferative activity of aronia extract

The cell-permeable fluorescence dye CellTrace™ Yellow was used to analyze the proliferation of cells after ARO treatment. Figure 7 illustrates that treatment with ARO IC₅₀ effectively suppressed the growth of both cell lines at 48 and 72 hours, in contrast to the normal proliferation of DMSO/untreated cells at 72 hours.

Open in a separate window

Figure 7

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

3.9.3. Cell cycle analyses

The effects of ARO IC₅₀ treatment on the cell cycle were examined in cancer cell lines. Both BC cell lines showed G1 cell cycle arrest at 48 h following ARO treatment, with or without continuation up to 72 h ( Figure 8 ). Simultaneously, there was a concomitant decrease in the number of cells in other cell cycle phases and an increase in the apoptotic subpopulation (subG0) at 72 h. SubG0 represented a subpopulation with fragmented DNA.

Open in a separate window

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.

3.9.4. Apoptosis analyses

The externalization of phosphatidyl serine (PS) is an early indicator of cell death, as demonstrated by Annexin V/PI staining, which categorizes the population into four groups: Q4= Live (An-PI-), Q3= Early apoptotic (An+PI-), Q2= Late apoptotic (An+PI+), and Q1= Dying cells (An-PI+). Figure 9 shows that both cell lines ARO showed a gradual increase in the number of early and late apoptotic cells with externalized PS over time. By the 72-hour post-treatment, the early apoptotic cells transitioned slightly towards the late apoptotic phase, contributing to the dying cell population.

Open in a separate window

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).

3.9.5. Mitochondrial potential changes and analyses of caspases activity

The potential pro-apoptotic effects of ARO treatment were examined in MCF-7 and MDA-MB-231 cells, focusing on changes in mitochondrial function and the initiation-execution of apoptotic mechanisms. Figure 10A demonstrates that ARO treatment resulted in the dissipation of mitochondrial membrane potential at all time points tested in both cell lines. Additionally, the caspases activity test revealed that ARO treatment triggered the activation of executioner caspases 3/7, reaching their peak activity at 48 hours in both cell lines ( Figure 10B ). Furthermore, ARO treatment increased the population of cells with damaged membranes, indicating cell death.

Open in a separate window

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.

3.9.6. Western blot analyses of protein expression

The analysis of PARP, p-Rb/Rb, Bax, and Bcl-2 protein expressions through western blot was carried out following ARO IC50 treatment on MCF-7 and MDA-MB-231 cells. Figures 11A, B illustrates that the ARO treatment resulted in the cleavage of PARP due to caspase 3/7 activity. Additionally, a reduction in phosphorylated Rb, a cell cycle regulator in the G1/S transition, was noted after ARO treatment in both cell lines. Total Rb expression was not affected. Furthermore, ARO treatment led to a decrease in anti-apoptotic mitochondrial Bcl-2 protein expression. On the other hand, pro-apoptotic mitochondrial Bax expression was not affected. However, the calculated Bax/Bcl-2 ratio ( Figure 11C ) showed an increase (up to 90% for MCF-7, 110% for MDA-MB-231) in favor of the pro-apoptotic proteins supporting a decreasing survival rate.

Open in a separate window

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.

4. Discussion

Current oncology research has demonstrated the significant anticancer activities of isolated phytochemicals and their mixtures present in whole plant foods (20, 35–38). BC is currently the tumor disease diagnosed most frequently worldwide in women (1). Extensive epidemiological studies show a reduced risk of BC associated with an increased intake of whole plant foods, including fruits, vegetables, and herbs (39–42). In this context, current oncology research emphasizes the importance of phytochemicals in reducing cancer risk through chemoprevention (7).

Phytochemicals, their derivatives or whole plant foods, and medicinal plants also represent promising possibilities for improving the effectiveness of conventional treatment in BC patients or suppressing adverse reactions induced by conventional therapies (43). In addition, the acquired resistance of cancer cells to chemotherapeutics represents a severe clinical problem for cytotoxic antitumor therapies [5]. Targeting the molecular pathways by using phytochemicals to improve therapeutic outcomes by increasing the sensitivity of cancer cells and reversing resistance to currently used therapeutic methods represents an essential preclinical and clinical research approach for improving the management of BC (5). The results of experimental studies have shown a direct connection between the administration of phytochemicals and the suppression or slowing of cancer development. Mechanisms of this effect include modulation of the cellular signaling pathways associated with key events for the initiation, promotion, and progression of BC, including oxidative damage, apoptosis, proliferation, angiogenesis, the formation of distant metastases, or the activity of tumor stem cells (35, 36, 44–46). Animal models of experimental BC represent a logical way of investigating the antitumor effect of phytosubstances in the multistep process of mammary gland carcinogenesis (17–24). Having proposed this idea, we assessed the anticancer potential of A. melanocarpa in therapeutic and preventive rodent BC models and human BC cells in vitro. In our study, the mechanism of action of aronia was analyzed by histopathological, immunohistochemical, and molecular-biological methods, which represent a highly valid approach to evaluating tumorigenesis. The dosing of phytosubstances in this study was optimized according to our previous experience with given animal models. Rodents generally show different pharmacokinetics and pharmacodynamics of many phytosubstances compared to humans.

For this reason, higher doses of the whole plants tested are used in experimental studies to verify the antitumor effects of phytosubstances (17). The content compounds of aronia were analyzed using LC-MS. This analysis confirmed the presence of anthocyanins in aronia. Anthocyanins had been reported previously in aronia (47). They are described as antioxidant and antiradical compounds, and the effects, revealed also in our cellular antioxidant assay, are by previously published studies (9). The content of anthocyanins may also be influenced by the effects observed in our assays on the MCF-7 and MDA-MB-231 cell lines. Anthocyanins are described as compounds interfering with inflammatory pathways (48). They decrease epithelial to mesenchymal transition via Sirt1 promotion and NF-κB inhibition. In addition, they down-regulate the expression of matrix metalloproteinases, which can decrease tumor invasiveness and the potential risk of forming metastasis (49). They interact with estrogenic receptors (50, 51). Furthermore, they possess antiangiogenic effects by effects on VEGF (52). They decrease EGRF/Akt signaling and increase mir-124 expression (52, 53). The effects of different anthocyanins on many other cellular targets were previously summarized by Li et al. (54).

The therapeutic mouse 4T1 model is characterized by high tumorigenicity and invasiveness with the formation of spontaneous metastases from the primary tumor to several distant regions, thus imitating the course of an aggressive form of mammary gland carcinogenesis in humans. In our evaluation of the therapeutic effects of aronia in the syngeneic 4T1 model of mammary carcinogenesis, we specifically focused on tumor volume as a fundamental parameter of mammary carcinogenesis (tumor staging). Additionally, we examined the histopathological assessment of the mitotic index and the proportion of necrosis in the tumor tissue (tumor grading). The results showed that aronia significantly reduced tumor volume in both treated groups compared to the control group. Furthermore, aronia, at both concentrations, demonstrated a dose-dependent reduction in the mitotic activity index compared to the control group. These findings align with the previous research conducted by our working group, where cinnamon (Cinnamomum zeylanicum L.) exhibited similar antitumor effects, including a significant reduction in the volume of 4T1 tumors and a dose-dependent decrease in the mitotic activity index (23). Thyme (Thymus vulgaris L.) significantly reduced the volume of 4T1 tumors in both doses compared to the control group; in addition, thyme significantly reduced the index of mitotic activity and reduced the necrosis/total tumor tissue ratio (22). The current results of our working group have revealed significant beneficial efficacy of Rhus coriaria L (24). and Salvia officinalis L (55). in the same 4T1 model of BC. We can conclude that the current and previous results of our working group confirm the apparent antitumor efficacy of phytosubstances comparable to the therapeutic effects of synthetic drugs evaluated in the same mouse 4T1 model (56–58). Despite the proven antitumor effects of aronia and other plant foods in our laboratory using the 4T1 therapeutic mouse BC model, their potential use in the therapy of human BC requires further intensive investigation at the preclinical and clinical levels. The 4T1 model has several important limitations; for example, t uses only one tumor cell line and has a high degree of uniformity in experimental conditions that cannot be reached in a clinical setting.

The chemopreventive effects of aronia were assessed by examining various parameters of carcinogenesis, such as tumor frequency, incidence, latency, and volume. Additionally, histopathological evaluations of mammary lesions and the ratio of poorly and well-differentiated (high/low grade) carcinomas were conducted. Aronia demonstrated a noticeable, albeit statistically insignificant, decrease in the ratio of poorly and well-differentiated (high/low grade) carcinomas by 34% at the lower dosage and 38.5% at the higher dosage compared to the control group. Furthermore, a slight reduction in tumor volume and an extension of tumor latency were observed following treatment with aronia at the higher dosage compared to the control group. These findings align with our previous robust research using the same chemically induced rat mammary carcinogenesis model. In these studies, chlorella, a blend of dark fruit peels, oregano, cloves, thyme, cinnamon, and sumac, exhibited significant decreases in incidence, frequency, and volume values, as well as an elongation of latency and a reduction in the ratio of poorly and well-differentiated (high/low grade) carcinomas (17, 19–24). Other researchers have also reported significant chemopreventive effects of plant-based foods on the development of mammary carcinogenesis in vivo. Blueberries and blackberries have shown chemopreventive and therapeutic activity in vivo by reducing tumor volume, proliferation, and lengthening latency (25, 26). In another study, rosemary significantly reduced the tumor frequency in DMBA-induced rat mammary carcinogenesis, demonstrating its significant chemopreventive activities in vivo (59). Finally, pomegranate exerted chemopreventive efficacy in carcinogen-induced rat mammary tumorigenesis by proapoptotic and antiproliferative mechanisms of action (60).

Recent research indicates that phytochemicals and their natural combinations play a significant role in regulating the processes involved in cancer development, such as apoptosis, proliferation, angiogenesis, and mechanisms related to cancer stem cells (20). Our study focused on examining the anti-tumor effects of entire plants in a chemopreventive setting by analyzing selected markers of these mechanisms through immunohistochemical and molecular techniques.

Programmed cell death is an essential process for the optimal functioning of an organism. Apoptosis can be triggered by either an intrinsic or extrinsic apoptotic pathway. In our research, we examined the impact of aronia on the internal signaling of programmed cell death, specifically through the mitochondria. The activation of caspases plays a pivotal role in apoptosis, with caspase-3 being recognized as an effector caspase involved in the initiation of programmed cell death. Additionally, proteins such as Bax, which promotes apoptosis, and Bcl-2, which inhibits apoptosis, also regulate this process (18). Intracellular responses to various stress signals, such as oxidative stress, DNA damage, and hypoxia, initiate the intrinsic apoptotic pathway. These responses result in the activation of pro-apoptotic proteins and the downregulation of anti-apoptotic proteins. As a result, the permeability of the outer membrane of the mitochondria is disrupted, leading to the release of specific proteins, including cytochrome c, into the cytosol. Cytochrome c, when bound to other proteins, triggers the formation of a complex known as the apoptosome. This complex then activates caspase 9, which subsequently activates caspase 3. Activating caspase 3 cleavages key cell substrates, ultimately resulting in cell death (61). The dysregulation of apoptosis, an intricately orchestrated process, stands as a fundamental mechanism underlying the progression of tumors (35). Phytochemicals play a crucial role in regulating the process of tumor cell apoptosis, augmenting the Bax/Bcl-2 ratio results in heightened caspase-3 activity, ultimately triggering the initiation of cancer cell apoptosis (18). In this study, we evaluated modifications in the expression of three parameters of apoptosis, namely cytoplasmic caspase-3, Bax, and Bcl-2.

At a higher dose, aronia significantly increased the cytoplasmic expression of caspase-3 compared to the control group. Aronia administration at both doses significantly increased Bax expression by over 90% compared to the control group. Additionally, a notable decrease in Bcl-2 expression was observed compared to controls. This substantially increased the Bax/Bcl-2 expression ratio by 173% (ARO 0.3) and 142.5% (ARO 3) versus the control group. Consistent results were also obtained from in vitro experiments. Furthermore, in vitro findings demonstrated a decrease in mitochondrial membrane potential, cell cycle arrest in the G1 phase linked to reduced phospho-Rb checkpoint protein expression, and the initiation of apoptosis following aronia treatment. These effects of aronia may be attributed to its high anthocyanin content, which has previously exhibited pro-apoptotic properties (54).

Previous studies conducted in our laboratory have also demonstrated the pro-apoptotic effect of various whole plants. These studies revealed a significant correlation between an increase in the expression of caspase-3 and an increase in the ratio of Bax/Bcl-2 in BC cells in vivo. This correlation was observed after administering dark fruit peels, oregano, cloves, cinnamon, sumac, and salvia in the NMU-induced chemopreventive model of mammary carcinogenesis in rats (19–21, 23, 24, 55). The findings from our experiments indicate that specific plant foods can potentially induce cancer apoptosis in BC effectively.

Except for apoptosis, cancer cells are characterized by altered signaling, leading to excessive proliferation and the circumvention of signals that in normal cells precisely regulate the cell cycle (62). Naturally occurring phytosubstances can directly regulate the proliferation of estrogen-dependent as well as estrogen-independent mammary tumor cells by influencing essential mechanisms such as the inhibition of COX-2 or the modulation of key signaling pathways (Hedgehog, NF-κB, Nrf2, STAT3, Wnt and others) (63–65). The nuclear protein Ki67 is a good marker of tumor cell proliferation because it is associated with active phases of the cell cycle, whereas it is absent from resting stages (66). Evaluation of tumor cell proliferation in this study showed a dose-independent non-significant reduction of Ki67 expression by 9% and 5% in the aronia groups compared to the control. On the other hand, in our previous chemopreventive studies, we observed a significant reduction of Ki67 caused by a mixture of dark fruit peels, young barley leaves, oregano, cloves, cinnamon, and sumac (18–21, 23, 24). Similar preclinical in vivo data showing the antiproliferative effects of plant foods have also been published by other authors who tested bilberries (67) and Trianthema portulacastrum L (68). A clinical study analyzing the effect of green tea on the expression of Ki67 in BC demonstrated a significant reduction in benign and non-significant decreases in malignant tissues of patients with DCIS or stage I/II BC (69).

Tumor neovascularization is necessary for the progression of cancer disease and the spread of metastases (45). Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR) are important for physiological and pathological angiogenesis processes. Although the VEGF-A binds to both its receptors (VEGFR-1, VEGFR-2), the primary signaling of endothelial cell proliferation and vascular permeability is mediated by the binding to VEGFR-2, and that is why VEGFA/VEGFR-2 represents the most important ligand-receptor complex necessary for tumor angiogenesis (70). In this study, aronia dose-independently decreased the expression of VEGF by 39.5% and 37%, compared to the control group. Similarly, as we described in previous chemopreventive animal studies using dark fruit peels, oregano, cloves, thyme, and cinnamon, we have demonstrated their significant anti-angiogenic potential via the downregulation of VEGF/VEGFR-2 expression (19–23). Natural mixtures of plant secondary metabolites have also shown significant antiangiogenic effects in other experimental BC models (71–73), warranting comprehensive preclinical and clinical analyses on this research topic.

The antitumor effect of phytochemicals also includes significant action on the vitality of cancer stem cells (23, 24, 36, 74, 75). Cancer stem cells represent a subpopulation characterized by the possibility of self-renewal and differentiation into different cell types. Cancer stem cells are involved mainly in the multi-step process of carcinogenesis, including tumor initiation, promotion, progression, the formation of metastases, or later resistance to therapy (76). Within the clinical practice and oncology research, several clinically established cancer stem cell markers exist, including CD24, CD44, CD133, ALDH1, and EpCAM (77, 78). In this study, aronia at a higher dose noticeably reduced the expression of CD133 by 58.5% compared to the control group. In our recent experiments using rat mammary carcinomas in vivo, we demonstrated the significant effects of sumac and salvia on CSC markers (24, 55). We found a dose-dependent significant decrease in CD24, ALDH1, and EpCam expression after the sumac treatment. Sage reduced the expressions of ALDH1 and EpCam in rat BC cells. Using the same animal model, we demonstrated a significant effect of oregano, cloves, thyme, and cinnamon on various cancer stem cell parameters (20–23). Preclinical studies pointed to higher antitumor efficacy of whole plants containing mixtures of bioactive phytochemicals (via the modulation of multiple cell signaling pathways and mechanisms, including CSCs) as compared to isolated phytochemicals (36, 37). Data from our laboratory and other authors (79–81) call attention to the need for in-depth research in this area and its consequent translation to clinical oncology.

In recent years, cancer prevention and therapy research has been concentrating on the impact of phytochemicals on specific carcinogenesis processes, particularly the modulation of epigenetic mechanisms. The investigation of abnormal epigenetic mechanisms has garnered significant interest in the BC field due to the reversible nature of epigenetic changes, which occur early in cancer development. Epigenetic mechanisms, such as histone post-translational modifications, specific miRNA expressions, and gene promoter methylation, can influence cellular processes. These mechanisms can affect cell cycle regulators, nuclear receptors, tumor-suppressor genes, transcription factors, and gene products involved in apoptosis or repair mechanisms, ultimately playing a role in cancer development (82–84).

Post-translational modifications of histones and their possible aberrations were an important epigenetic parameter in this experiment with aronia in an animal model of BC. Aronia beneficially decreased H3K4m3 and H3K9m3 levels in cancer cells compared to controls. In addition, apparent but not significant increases in H4K16ac and H4K20m3 were observed in treated groups vs controls. In our recent study, salvia significantly reduced H3K4m3 levels and increased H4K16ac levels in rat BC samples (55). In the same rat BC model, we described positive alterations in post-translation modifications of histone molecules in BC cells in vivo after chemoprevention with clove buds (21), thyme (22), cinnamon (23), and sumac (24). Several researchers have noted comparable epigenetic alterations involving resveratrol, leading to the reactivation of tumor-suppressor genes BRCA1, TP53, and TP21 in MCF-7 BC cells. The hindrance of cancer advancement was linked to a reduction in specific post-translational modifications of histones, such as a decrease in dimethylation or trimethylation of histones H3 and H4 (H4R3m2, H3K27m3), and an elevation in the levels of acetylation of histones H3 (H3K9ac, H3K27ac). These modifications were correlated with the reactivation of the aforementioned tumor-suppressor genes (85). 1. Additionally, the co-administration of sulforaphane and withaferin reduced HDAC enzyme activity, leading to a decrease in acetylation levels. This disruption in histone chemical modifications ultimately caused a decrease in cancer cell viability and triggered programmed cell death in MCF-7 and MDA-MB-231 cells (86). Furthermore, the identical blend of phytochemicals elevated the histone methylation levels, inhibiting the cell cycle in MCF-7 and MDA-MB-231 cells (87).

In general, miRNAs play a crucial role in regulating gene expression and thus can significantly modulate processes of carcinogenesis (88). Specific miRNAs have been introduced as clinical markers in the diagnosis, prognosis, and prediction of therapy for BC. In this regard, we have used in this study well-validated oncogenic miRNAs - miR21, miR155, and miR210 and tumor-suppressive miRNAs - miR22, miR34a, and miR145 as negative/positive regulators of mammary carcinogenesis (89, 90). Some phytochemicals have been documented as efficient tools that affect miRNA expression (91). In this study, aronia showed significant positive regulatory effects using the above-mentioned miRNA markers. Aronia downregulated oncogenic miR155 and upregulated tumor-suppressive miR145 compared to control rat BC samples. An apparent tendency towards the dose-dependent upregulation of miR22 expression was found after treatment with aronia. Oncogenic miR-210 significantly affects cancer by modulating the hypoxia-inducible factor (HIF) signaling (92). On the other hand, miR210 is manifested as a tumor suppressor in normoxic conditions, i.e., during the initiation of cancer (22, 93). In this study, aronia in both doses significantly increased miR210 expression in rat BC specimens compared to controls. We can hypothesize that miR210 behaved as a tumor-suppressor in our aronia study due to the initial stages of the rat BC tumors evaluated (we found only a small average tumor volume < 1 cm³). Significant regulatory effects of the plant nutraceuticals T. vulgaris, C. zeylanicum, and R. coriaria on miRNA expressions were observed in our previous rat chemoprevention studies using the same model (22–24). Anticancer effects of plant-derived molecules on breast carcinogenesis through the modulation of cancer-associated microRNAs have also been observed in other laboratories (94, 95).

The hypermethylation/hypomethylation of promoter regions of tumor-suppressor genes (TSG) significantly affects processes of carcinogenesis (96). This study analyzed the methylation status of standardized CpG islands of the TSG promoters ATM, PITX2, RASSF1A, PTEN, and TIMP3. These represent clinically well-validated TSG, frequently downregulated in BC patients (97) or in animal models (98) due to promoter hypermethylation. This study revealed that high-dose treatment with aronia significantly reduced the methylation status in the TIMP3 promoter and non-significantly decreased the promoter`s methylation by 11% in both the ATM and RASSF1 genes. However, at both doses, aronia markedly increased the methylation of the PTEN promoter by approx. 150% vs. control samples. Recent data from our laboratory using the same chemoprevention model in vivo documented unambiguous significant hypomethylating effects of natural phytochemical mixtures on the specific TSG promoters´ methylation status after treatment with Syzygium aromaticum L., T. vulgaris, C. zeylanicum, R. coriaria, and S. officinalis (21–24). Demethylation effects of plant nutraceuticals on TSG promoters’ regions have also been described in the cancer models also by other laboratories (99, 100). Despite growing research data, there is still insufficient knowledge of the role of phytochemicals on epigenetic changes in cancer. In addition, most of these data are preclinical and cannot be easily translated into clinical oncology. Future research must resolve several needs in the epigenomics of cancer: (a) more detailed insight into the molecular mechanisms and targets of phytochemicals, (b) a definition of effective (individual) dosing, (c) the evaluation of the combined therapy of plant bioactive agents with conventional chemotherapeutics to target relevant epigenetic mechanisms, (d) innovative methods to improve the bioavailability of phytochemicals in the recipient organisms, and (e) the individual characteristics of the cancer patient (7).

Phytochemicals could also contribute to traditional chemotherapy as cofactors to reduce the dosage of chemotherapy and thus also the unwanted side effects or the sensitizing of chemoresistant cancer cells. The beneficial effects of combination treatment are already widely accepted as part of new strategies in cancer research, as has been reviewed (101, 102). Our preliminary in vitro data showed a beneficial effect of such combinations where IC₂₅ concentrations of aronia and epirubicin showed inhibitory effects similar to or increased as those of a single epirubicin treatment. In addition, several combinations showed synergistic effects. Despite these promising results, further research is needed to optimize the combinational/re-sensitizing therapeutic approach in clinical oncology.

5. Conclusion and future perspectives

Current global trends in BC incidence and mortality require the introduction of more effective approaches to managing this disease. To ensure effective health care for BC patients, modern medicine requires an individual approach and the identification of modern and more effective therapeutic strategies, including increased sensitivity of cancer cells to conventional chemotherapeutics. Plant nutraceuticals are a rich source of bioactive phytochemicals with significant effects on human health, characterized by easy availability and a toxicological profile better than chemotherapeutic drugs. Phytosubstances can demonstrably regulate the multistep process of mammary carcinogenesis via the modulation of numerous cell signalings involved in carcinogenesis. Based on the above discussion, phytochemicals represent potentially effective substances for use in new treatment strategies and the prevention of BC. The incidence of BC can be reduced by timely choice of the right and prevention.

A. melanocarpa exhibited significant antitumor effects in a therapeutic model of 4T1 mouse adenocarcinoma. This was observed through a notable decrease in tumor volume and the mitotic activity index of cancer cells. Additionally, aronia displayed significant antitumor activity in a chemopreventive model of experimental BC in female rats and a model of human BC adenocarcinoma using MCF-7 and MDA-MB-231 cells. This activity was attributed to its proapoptotic, antiangiogenic, antiproliferative, and anti-CSC effects and positive epigenetic changes. Our findings highlight the activation of non-specific signaling pathways in cancer cells, which contribute to the anticancer efficacy of aronia across three distinct BC models. Therefore, in vitro and in vivo experimental BC models are crucial in evaluating novel therapeutic and chemopreventive agents. However, introducing phytochemicals in routine oncological practice requires comprehensive in-depth clinical research, which must precisely define all their effects on the organism, including mechanisms and correct dosage in oncology patients. Using plant nutraceuticals in the clinical management of BC requires comprehensive clinical analyses, which must resolve several key issues within oncology practice:

1. defining pharmacokinetics/dynamics that can determine the effective and safe dosing and modes of administration;.

2. determination of sensitive cancer subtypes respecting the individual characteristics of patients;.

3. it is essential to outline appropriate uses of plant nutraceuticals in conjunction with traditional medications to enhance the re-sensitization of cancer cells, inhibit metastatic growth, and boost the immune system in patients.

The intricate pathways of cellular, subcellular, and molecular mechanisms involved in carcinogenesis are expressed at a comprehensive multi-omics level. As a result, these pathways are regarded as crucial targets for advanced diagnostics and cost-effective management of BC, encompassing therapy and preventive measures (8). 1. The extensively documented anti-cancer properties of phytochemicals and plant nutraceuticals are crucial for enhancing patient outcomes and have the potential to be utilized in primary (preventing cancer development), secondary (preventing possible spread to other parts of the body), and tertiary (preventing additional complications) care within clinical settings (8). In this regard, precise data analyses are crucial to adapting BC management`s algorithms to individual patient profiles. The rapidly growing incidence of BC in the general population and, in particular, the aggressive metastatic BC sub-types found in the young female population prompt clinicians to apply advanced screening methods and targeted individualized and preventive approaches to overall BC management (103). Predictive diagnostics, patient stratification, targeted prevention, and treatments focused on the individualized patient multiomics characteristics represent effective tools for the cost-effective application of plant nutraceuticals to optimize BC management. The primary, secondary, and tertiary approach in BC management within the framework of predictive, preventive, and personalized medicine is considered an advanced strategy to improve both individual outcomes and those of society at large (104–108).

Acknowledgments

Funding Statement

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic under Contract No. VEGA 1/0045/23, 1/0539/21, 1/0498/23, and 1/0145/22.

Abbreviations

References