In addition, the use of in vitro digestion and absorption processes in research studies allows the bioavailability of the compounds contained in the product to be taken into account and therefore allows for a more realistic assessment of their combined effect on the body cells.
No comparative results were found in the available literature for the effectiveness of the above-mentioned products before and after digestion on the proliferation and changes in selected cellular parameters responsible for the apoptosis of cancer cells. In addition, no attempt has ever been made to compare the effectiveness of the analyzed beetroot material in relation to human breast cancer cells of the hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) lines.
3.1. Polyphenolic Profile
Polyphenols constitute a structural class of organic chemical compounds possessing at least two hydroxyl groups attached to an aromatic ring. The number and characteristics of these structures determine their physical, chemical, and biological properties. Polyphenols in plants are responsible for color, sensory properties and above all, they exert a chemopreventive effect by transferring protons from hydroxyl groups to reactive oxygen species. Due to their antioxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties, they can prevent chronic non-communicable diseases. Polyphenols can be classified according to their chemical structure and divided into several categories, including phenolic acids, flavonoids, lignans, and stilbenes.
The HPLC analysis performed in the present study enabled detection of phenolic acids and flavonoids in the juice from young shoots of beetroot. Their contents were compared with those obtained in the juice from the root in the full maturity phase. A significantly higher total content of polyphenols was determined in the juice from young shoots compared to the juice from the root of the analyzed cultivar Rywal. In young shoots of beetroot, the major compound was sinapinic acid, from the hydroxycinnamic group; in the root, however, it was catechin, which belongs to the class of flavanols (
Table 1). No anthocyanins were detected in the studied material, due to the lack of the anthocyanin synthetase enzyme involved in the final stage of the pigment formation [
19].
In this study, out of 22 polyphenolic compounds identified in the juice from young shoots of beetroot, hydroxybenzoic acid, hydroxycinnamic acid, flavanols, flavonols, and flavones accounted for 3.73%, 24.67%, 6.27%, 22.36%, and 21.30% of the total polyphenols.
The available literature lacks data on the content of determined polyphenol fractions in young shoots of beetroot. For this reason, the works cited below concern other species of young shoots, including those from the
Chenopodiaceae family, which encompasses
Beta vulgaris. In many plants belonging to the mentioned family, Kyriacou et al. [
20] quantified 28 types of phenolic compounds, such as hydroxycinnamic acids, flavonol glycosides, as well as flavones and flavonol glycosides, representing 7.6%, 24.8%, and 67.6% of total polyphenols.
In studies on young shoots of kohlrabi, pak choi, and coriander, changes in the content of polyphenols were observed, depending on the type of substrate (natural vs. synthetic fiber). Chlorogenic acid and quercetin were found to be the major compounds among the 20 identified phenolics.
Hydroxycinnamic acid and its derivatives, flavonol glycosides and flavone glycosides, accounted for 49.8%, 48.4% and 1.8% of total polyphenols. The results obtained in our study are lower than those cited above.
According to Kyriacou et al. [
21], the content of polyphenols in young shoots and growing conditions is also influenced by species variability.
On the other hand, in beetroot root, out of the 16 identified polyphenolic compounds, hydroxybenzoic acid, hydroxycinnamic acid, flavanols, flavonols, and flavones accounted for 24.83%, 8.48%, 51.31%, 0.48%, and 1.33% of the total content of determined polyphenols.
Using the HPLC technique, Kale et al. [
22] demonstrated the presence of several polyphenolic compounds in the beetroot root, such as gallic acid,
p-coumaric acid, ferulic acid, cinnamic acid, and catechol, but in higher amounts compared to the results presented in this paper.
Values similar to those obtained by Kale et al. [
22] were also presented by Vulić et al. [
23], who, in their experiments, additionally determined flavonoids, such as betagarin, betavulgarine, cochlyophyllin A, and dihydroisoramnetin.
In turn, Jastrebova et al. [
24] determined the following polyphenols in the
Beta vulgaris root: catechin, quercetin,
p-coumaric acid, sinapinic acid, syringic acid, caffeic acid, and chlorogenic acid. The results reported in the cited work were also higher than those determined in the present study. The studies by Amani et al. [
25], analyzing the profile of polyphenols in the beetroot root, showed a higher content of hydroxybenzoic acid, rutin, hesperidin, kaempferol, naringin, apigenin, and isorhamnetin, compared to those presented here. In addition, these researchers detected ellagic acid. Lim [
26] also determined other flavonoid compounds in beetroot: 3,5-dihydroxy-6,7-methylenedioxyflavanone, 5-hydroxy-6,7methylenedioxyflavone, 2,5-dihydroxy-6 and 7-methylenedioxy isoflavone. In turn, Georgiev et al. [
27] showed the presence of 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, ferulic acid, vanillic acid, catechin, and epicatechin in beetroot of the Detroit Dark Red variety as well as rutin, again in amounts higher than those found in the present study.
Studies with leaves (chard) of
Beta vulgaris L. var. cicla showed significant amounts of hydroxybenzoic and hydroxycinnamic acid derivatives. Epicatechin, catechin, rutin, vanillic acid,
p-coumaric acid, protocatechuic acid, caffeic acid, syringic acid and proline were determined among the phenolic acids [
28].
The content of polyphenols may vary depending on the variety of beetroot [
29] and on the analyzed site in the root cross-section [
30].
Previous studies have confirmed the antiproliferative effect of polyphenols on T47D, MCF-7, and MDA-MB-231 breast cancer cell lines in a time- and dose-dependent manner [
31]. These compounds affect cellular mechanisms and molecules related to carcinogenesis. Selected polyphenolic compounds have been found capable of suppressing the activity of transcription factors, such as NF-κB and AP-1 [
32]. These protein complexes control many genes that regulate proliferation and apoptosis, and disturbances in their pathways can lead to carcinogenesis. It seems that the inhibitory effect of polyphenols on the aforementioned transcription factors may be due to their antioxidant properties because reactive oxygen species can activate both NF-Κb and AP-1. The anti-proliferative and pro-apoptotic effect of polyphenols is also explained by the inhibition of critical proteins involved in signal transduction pathways from the cell membrane to the cytoplasm and nucleus and in regulation of the cell cycle, as well as in apoptosis through the initiation of caspase-3, activation of p53 protein and of factors affecting cell proliferation and differentiation [
33]. It must be highlighted that polyphenols exert a stronger effect on cancer cells than on normal cells [
34].
3.2. Cell Proliferation
The analysis of the obtained results showed that the juices from the young shoots and root of red beet, both in their native form and subjected to digestion and absorption, inhibited the proliferation of the MCF-7 and MDA-MB -231 breast cancer cell lines (
Table 2) while not affecting the proliferation of MCF-12A normal cell line. Due to the need for effective agents that inhibit the proliferation of cancer cells and at the same time do not show a similar effect on normal cells of the body, these data seem promising.
In addition, it was observed that the juice from young shoots, both in the native form and after digestion and absorption, in most cases reduced the proliferation of cells of both cancer lines to a significantly greater extent than the juice made from root in the full maturity phase.
The difference in the efficacy of the analyzed material on both breast cancer cell lines should also be emphasized; the effect was usually significantly higher in relation to estrogen-dependent cells of the MCF-7 line.
The available literature provides no information on the effect of young shoots of beetroot on the proliferation of cancer cells.
In this study, analyses were carried out using the juice obtained from the native plant material and also subjected to a digestion and absorption process in a model digestive tract in vitro. On the other hand, the papers cited below report on the effects of the direct extract or single isolated components.
Reddy et al. [
35] compared the effects of natural pigments, including betanin, as well as their mutual interactions, on the possibility of inhibiting the growth of cancer cells. In a 48-h experiment, the authors observed growth inhibition of breast cancer cells of the MCF-7 line treated with betanin. Research by Kapadia et al. [
36] showed that beetroot extract strongly inhibited the proliferation of cancer cells, including the breast cells analyzed in this study (line MCF-7). Another study demonstrated a synergistic anti-proliferative effect of doxorubicin and beetroot extract on breast and prostate cancer cell lines [
37]. Nowacki et al. [
38] observed suppressed proliferation of MCF-7 and MD-MB-231 breast cancer cell lines under the influence of a mixture of betanin/isobetanin, with no significant effect on the normal HUV-EC-C cell line. In turn, Das et al. [
39] demonstrated the chemotherapeutic effect of beetroot extract in combination with doxorubicin on the viability of MDA-MB-231 cancer cell line.
It was shown that betanin, isolated from red beets, depending on the dose, inhibited the proliferation of the lung cancer cells NCI-H460, the CNS (central nervous system) line, as well as gastric AGS and large intestine HCT-116 lines (gastrointestinal tract) [
40]. Another study showed an antiproliferative effect of beetroot extract against breast cancer cells (MCF-7) and additionally a beneficial effect on lung (A-549), kidney (Caki-2), prostate (PC-3), pancreas (Panc-1), and chronic myelogenous leukemia (K-562) cancer cell lines [
41]. In addition, betanin and betaine, extracted from beetroot, exhibited antiproliferative activity against liver cancer cells of the HepG2 line [
42]. Inhibition of proliferation under the influence of betalains extracted from beetroot root was also observed in studies on bladder cancer cells of the T24 line [
43]. In the latest 48-h experiment, Saber et al. [
44] showed the inhibitory effect of betanin and beetroot extract on HT-29 colorectal cancer cells and Caco-2, as measured by proliferative capacity.
As described above, several in vitro studies have confirmed the effectiveness of an extract or individual phytonutrients most commonly isolated from beetroot root in reducing the proliferation of various types of cancer cells.
In addition, the literature provides the results of research on the antiproliferative effects of young barley [
45] or young shoots of cruciferous vegetables [
46]. Interesting results were obtained by Drozdowska et al. [
47], who assessed the effect of the juice from young shoots of white head cabbage (
Brassica oleracea var.
capitata f. alba) on the proliferation of the MCF-7 breast cancer cell line. This study showed a decrease in cell viability depending on the analyzed juice’s concentration and duration of action. In subsequent research, Drozdowska et al. [
48] compared the effectiveness of young shoots of red head cabbage (
Brassica oleracea var.
capitata f. rubra) with the effect of the plant in full maturity. The authors demonstrated a tendency for the inhibition of the proliferation of DU-145 prostate cancer cell line, both under the influence of juice from young shoots of red head cabbage and from the mature form of this vegetable.
The results obtained in this study concerning the impact of beetroot juice in the full maturity phase, undigested and digested, are generally consistent with the results reported by other authors quoted above who, as already emphasized in this paper, investigated the effectiveness of direct extracts or single components isolated from the red beet.
3.5. Expression of Selected Genes and Proteins
In the present study, the most beneficial effect was demonstrated for the juice from young shoots of beetroot, subjected to digestion and absorption in a gastrointestinal tract model in vitro, against breast cancer cells of the MCF-7 and MDA-MB-231 lines, in comparison with undigested and digested root juice.
Digested root juice was also effective but to a significantly lesser extent. Therefore, to explain the mechanism responsible for reducing proliferation, the level of expression of both genes and proteins related to cell apoptosis under the influence of the mentioned most effective factor, as well as digested root juice, was discussed and confirmed in the previously described analyses using flow cytometry.
The available literature lacks information on the profile of changes in the expression of the genes and proteins discussed below in breast cancer cells of both analyzed lines under the influence of the juice, not only from young shoots but also from vegetables in the full maturity phase. On the other hand, the quoted literature items refer to changes in mRNA and/or protein levels under the influence of beetroot root extract, a single isolated compound, or a mixture thereof, often also against other cancer cell lines.
One of the main pro-apoptotic factors is the
TP53 gene, the product of which is the so-called guardian of the genome. As a transcription factor, the p53 protein is involved in the regulation of many cellular processes, in particular in the activation of DNA repair mechanisms, and in their absence, in the introduction of the cell into the path of apoptosis [
53].
The present study results show a significantly higher expression of the
TP53 gene, both under the influence of the digested juice from young shoots as well as from the root, in MCF-7 (
Figure S7) and MDA-MB-231 (
Figure S8) cell lines compared to control cells. The gene expression results were correlated with the protein expression results determined by Western blot (
Figure 3 and
Figure 4). In addition, the study demonstrated a tendency (
Figure S7) for the enhanced expression of the
BBC3 gene, also known as
Puma, in the MCF-7 cell line and significantly higher expression in the MDA-MB-231 cell line under the influence of digested young shoot juice (
Figure S8). In turn, digested root juice did not affect the expression of the above-mentioned gene in any of the analyzed cell models (
Table 7).
Nowacki et al. [
38] also showed that betanin, isolated from beetroot, induced apoptosis in MCF-7 breast cancer cell line by activating the p53 protein.
The p53 protein plays a key role in the induction of apoptosis, including by regulating the Bcl-2 family proteins when DNA repair is impossible [
40]. The Bcl-2 family includes pro-apoptotic proteins (BID, BAX, BAD, BIM, PUMA) and anti-apoptotic proteins (BCL-2 and BCL-XL). Their effect on apoptosis induction depends on the activity and ratio of pro- and anti-apoptotic proteins [
54].
In the present research, a significantly increased expression of
BAD gene was observed in both breast cancer cell lines (
Table 7 and
Figure S7) and a significantly increased expression of
BID gene in the MDA-MB-231 cell line (
Table 7 and
Figure S8) under the influence of digested juice from young beetroot shoots.
In turn, a tendency for enhanced expression of
BAD in the MCF-7 cell line (
Figure S7) and significantly higher expression of this gene in the MDA-MB-231 cell line (
Figure S8) was demonstrated as a result of the action of digested root juice. In addition, this juice did not affect the changes in
BID expression in either of the analyzed research models (
Figures S7 and S8). The protein products of these genes are usually located in the cytoplasm, but during the induction of apoptosis, they are activated and transferred to the outer mitochondrial membrane.
Nowacki et al. [
38] also showed that betanin, isolated from beetroot root, increased the level of BAD protein expression in MCF-7 cell line. No studies have been found in the available literature regarding the impact of products from beetroot on the expression of the
BID gene and protein.
In this study, we observed a tendency for downregulation of the expression of the
BCL-2 gene, encoding an anti-apoptotic protein, under the influence of digested juice of young shoots and a significant decrease in the level of this gene as a result of the action of digested root juice (
Table 7 and
Figure S7) in the MCF-7 cell line. The RT-qPCR conducted in the present study partially confirmed earlier observations made using a flow cytometer.
Opposite results were published by Das et al. [
39], who reported a decrease in the expression of the anti-apoptotic protein BCL-2 under the influence of the combined action of beetroot extract and doxorubicin, which in turn increased the BAX:BCL-2 in the MDA-MB-231 cell line, making the cells more sensitive to this mixture. In studies on colorectal cancer cells, Saber et al. [
44] showed that both red beetroot extract and betanin induced apoptosis pathways (internal and external) by reducing the expression of the anti-apoptotic gene
BCL-2 and increasing the expression of the pro-apoptotic gene
BAD.
As a result of apoptosis, cytochrome c is released, which is generally located on the inner side of the mitochondrial membrane and acts as an electron transmitter; Smack/DIABLO and HtrA2/Omi proteins are also transferred from the mitochondria to the cytoplasm. An increase in the expression of the proteins mentioned above, confirmed in our own research using the Western blot technique, was observed in the MCF-7 (
Figure 3) and partly in the MDA-MB-231 cell lines (in the case of HtrA2/Omi, there was a decrease) (
Figure 4).
Release of cytochrome c, lowering the potential of the mitochondrial membrane along with the activation of the intrinsic apoptotic pathway. after exposure to betanin, was demonstrated by Sreekanth et al. [
41], but in studies with human cells (chronic myelogenous leukemia cells of the K562 lineage).
In the cytoplasm, cytochrome c interacts with the Apaf1 protein and procaspase-9, forming the so-called apoptosome [
53] that activates procaspase-9 [
55], followed by effector caspases, including caspase-3 and -7 [
56].
The present study showed a significant increase in the expression of the
APAF1 gene in MCF-7 (
Figure S7) and MDA-MB-231 (
Figure S8) cell lines under the influence of digested juice from young shoots, and a tendency for increased or a significant increase in this gene expression in MCF-7 (
Figure S7) and MDA-MB-231 (
Figure S7) cell lines, treated with digested root juice. Although the results presented in the paper clearly indicate the induction of apoptotic events as a result of the action of digested juice from young beetroot shoots, the expression of the
CASP3 gene was not detected in the MCF-7 line, which may be explained by the presence of a functional deletion [
51]. In addition, some studies have indicated that apoptosis can proceed without the activation of caspase-3 but with caspase-7 [
52]. The presented results confirm this finding because after treating the cells with the analyzed material (both digested juice of young shoots and the root), a tendency was shown for the enhanced expression
CASP7 gene in the MCF-7 cell line (
Figure S7).
The gene expression results correlated with increased protein expression in the cells of the estrogen-dependent line (
Figure 3). The second analyzed cell line showed a significantly increased expression of
CASP3 and partially caspase-7 (no significant changes in the case of digested root juice) at the level of active protein (
Figure 4).
In the study with the human lung cancer A549 cell line, Zhang et al. [
57] showed that betanin induced apoptosis by activating caspases-3, -7, and -9. In the research with colorectal cancer cells of the HT-29 and Caco-2 lines, Saber et al. [
44] found that the combined action of beetroot extract and betanin resulted in the induction of apoptosis pathways, among others, by increasing
CASP-3 and
CASP-9 gene expression.
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme essential for maintaining genome integrity. During apoptosis, it is one of the first proteins to be degraded by the action of caspase-3 and -7 in order to prevent cell survival. Cleavage of the PARP protein and the occurrence of its fragments (the so-called cut form) of low molecular weight is a characteristic marker of the apoptosis process [
58].
In the present study, a significantly increased activity of the cleaved form of this protein was demonstrated in the MCF-7 cancer cell line under the influence of both types of juice (
Figure 3). In turn, the MDA-MB-231 cell line showed a significant increase in PARP cut as a result of the action of only digested juice from young shoots (
Figure 4).
Zhang et al. [
57] showed that betanin induced apoptosis by increasing the expression of the PARP protein in the lung cancer A549 cell line. Das et al. [
39] also showed that a mixture of beetroot root extract and doxorubicin significantly increased the expression of the cleaved form of the PARP protein.
Another pro-apoptotic protein released from mitochondria during apoptosis is AIF, which moves to the nucleus and causes DNA fragmentation and chromatin condensation [
59]. The results obtained in this study showed a significant increase in mRNA expression of the gene encoding the AIFM1 protein in cancer cells of both lines as a result of the action of digested juice from young shoots (
Figures S7 and S8). In turn, a significant increase in the expression of this gene under the influence of digested root juice was shown only in the estrogen-independent cells (
Figure S8).
Two different pathways leading to the process of programmed death, that is, intrinsic via mitochondrial proteins and extrinsic via death receptors, are often characterized by common elements such as activation of effector caspases and release of the Smac/DIABLO group of proteins from the mitochondrion during an apoptotic signal derived from the receptor [
60].
The p53 protein, activated after cell DNA damage, also affects the mitochondrion, releasing the BAX protein, and affects the transcription of the Fas ligand gene, stimulating its production [
61].
During the binding of the ligand to the membrane death receptor, caspase-8 may also be activated and, as a result, trigger the external apoptosis pathway. The initiating caspase-8 is responsible for forming the DISC complex and activating procaspase-3, -6 and -7. Active caspase-8 cleaves the BID protein, creating the tBID protein, which, by increasing the permeability of the mitochondrial membrane, releases cytochrome c and, consequently, links receptor-based and mitochondrial apoptosis.
In our own research, a significantly higher expression of the
CASP8 gene was demonstrated in the MCF-7 cell line under the influence of digested juice from young shoots and under the influence of both types of juices in the MDA-MB-231 cell line (
Table 7 and
Figures S7 and S8). The increase in
CASP8 gene expression correlated with the increase in the level of caspase-8, assessed by Western blot (
Figure 3 and
Figure 4).
Scarpa et al. [
43] also showed an increased expression of caspase-8 under the influence of betalains, except in bladder cancer cells of the T24 line. On the other hand, in studies with colon cancer cells of the HT-29 and Caco2 lines, Saber et al. [
44] showed that a mixture of beetroot extract with betanin enhanced the expression of the
CASP8 gene.
In the MDA-MB-231 cell line, a significantly reduced expression of the
FADD gene was demonstrated due to the action of both types of digested juice analyzed (
Table 7 and
Figure S8). In addition, the MCF-7 cell line also showed reduced expression of the
Fas gene when exposed to the digested juice from young shoots, while no changes were observed in the cells treated with digested root juice (
Table 7 and
Figure S7).
Opposite results were obtained by Nowacki et al. [
38], who observed the increased expression of the
Fas protein under the influence of beetroot extract enriched in betanin in the MCF-7 cell line. In turn, in studies with colon cancer cells of the CaCo-2 line, Saber et al. [
44] showed that red beetroot extract, in combination with betanin, increased the expression of the
Fas gene, inducing the extrinsic apoptosis pathway.
In the present study, a significant reduction in the expression of RIP protein was additionally demonstrated based on Western blot analysis in both analyzed research models (
Figure 3 and
Figure 4) under the influence of both types of digested juice. RIP is essential for TNF-α-induced activation of NF-κB. This study showed a significant reduction in
NF-κB gene expression in the MCF-7 cell line under the influence of digested juice from young shoots of beetroot and no effect of digested root juice (
Table 7 and
Figure S7). Interestingly, in the second analyzed line, a significant increase in the expression of this gene was demonstrated under the influence of both discussed factors (
Table 7 and
Figure S8).
NFκB enters the cell nucleus and initiates anti-apoptotic gene transcription that promotes survival. This is consistent with the results of our own work described above. In most cases, lower effectiveness of reducing the proliferation of MDA-MB-231 breast cancer cell line was demonstrated compared with the MCF-7 line, under the influence of the tested material.
In this work, the impact of the analyzed material on the expression of selected proteins was observed in several cellular processes, regulating proliferation, differentiation and the cell cycle. One of the most important pathways of signaling in the cell, involved in the process of oncogenesis, is the AKT kinase pathway. Although activation of AKT kinase alone is not believed to be sufficient for the onset of cancer, it plays an essential role in its progression by preventing apoptosis, promoting changes in the metabolism of the diseased cell, and regulating processes related to cell proliferation and migration.
The AKT1 gene encodes a protein with serine/threonine kinase activity and is involved in signaling pathways related to survival and proliferation.
In turn, the PHLPP1 gene encodes a phosphatase involved in the dephosphorylation of the AKT1 kinase.
In this work, it has been shown that the expression of the
AKT1 gene, responsible for proliferation enhancement due to the activation of the PI3K/Akt pathway, was significantly reduced in the cells of both analyzed lines under the influence of digested juices from both young shoots and the root (
Table 7,
Figures S7 and S8).
Another kinase evaluated in this study was p38 mitogen-activated kinase. The study showed that the juice from young shoots and beetroot root, subjected to digestion and absorption, significantly increased the expression of p38 MAPK in the MDA-MB-231 cell line (
Figure 4). On the other hand, a significant increase in the expression of the discussed protein in the MCF-7 estrogen-dependent breast cancer cell line was demonstrated as a result of the action of digested root juice, while no significant changes were observed as a result of cell treatment with digested juice from young shoots (
Figure 3).
According to Swat et al. [
62], mitogen-activated kinase p38 can inhibit cell proliferation by antagonizing the JNK/c-Jun pathway, involved in the regulation of proliferation and apoptosis. One of the most important functions of the p38 protein is the promotion of cell differentiation. Research also shows that the activation of the p38 kinase pathway in different types of tumors may be downregulated. Genetic modifications of the proteins involved in this pathway showed that p38 may act as a tumor suppressor. Research shows that p38 can also inhibit cell proliferation by modulating the expression of epithelial growth factor receptors (EGFR) [
63]. Overexpression or mutation of EGFR receptors in cancer cells may lead to dysregulation of the mechanism of signaling through these receptors, resulting in excessive proliferation, increased angiogenesis, and, consequently, migration of cancer cells to the surrounding tissues. This information is crucial in the light of the research results presented in this paper, according to which the analyzed juice modulated the expression level of p38 kinase in the MDA-MB-231 cell line, characterized by overexpression of the EGFR receptor.
The above-described effect of the juice from young shoots of beetroot, subjected to digestion and absorption, is also important in activation of the p53 transcription factor by the p38 mitogen-activated kinase, and consequently inhibition of cell proliferation.
Another gene analyzed in this work was
MYC, encoding a protein C-MYC, which is also involved in regulating proliferation and apoptosis. Our study results showed that digested juice from both young shoots and beetroot root significantly increased the
MYC gene expression in the cells of both analyzed lines (
Table 7,
Figures S7 and S8).
The described mechanism of action of digested juice from young shoots in the MCF-7 breast cancer cell line indicates the involvement of the internal apoptosis pathway with the participation of executive caspases and the p53 transcription factor. The second signaling pathway whose activity changed under the influence of the analyzed juice was the pathway in which AKT proteins are involved. Reducing the activity of these proteins resulted in the inhibition of cell proliferation and survival. In turn, in the MDA-MB-231 cell line, MAP kinases and the NF-κB factor were additionally activated. The increase observed in the expression of caspase-8 in the cells of both research models, with the simultaneous lack of an increase in the expression level of the analyzed death receptors, requires further research.
In turn, by analyzing the results of own research on the impact of digested beet root juice, it can be concluded that similar mechanisms were activated in MCF-7 and MDA-MB-231 cell lines, resulting in the release of cytochrome c and activation of the Smak/DIABLO protein (only in the MCF-7 line), caspases-7, -8 and PARP, but to a lesser extent compared to the effect of digested juice from young shoots.