Review

Cytotoxic Effects of Plant Secondary Metabolites and Naturally

Occurring Bioactive Peptides on Breast Cancer Model Systems:
Molecular Mechanisms
Diana Zasheva 1 , Petko Mladenov 2 , Silvina Zapryanova 1 , Zlatina Gospodinova 3 , Mariyana Georgieva 3 ,
Irina Alexandar 4 , Valentin Velinov 3 , Dimitar Djilianov 2 , Daniela Moyankova 2 and
Lyudmila Simova-Stoilova 3, *

1 Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Tsarigradsko Shosse,
73, 1113 Sofia, Bulgaria; zasheva.diana@yahoo.com (D.Z.); silvina_z@abv.bg (S.Z.)
2 Agrobioinstitute, Agricultural Academy, bul. “Dragan Tsankov” 8, 1164 Sofia, Bulgaria;
mladenovpetko@yahoo.com (P.M.); d_djilianov@abi.bg (D.D.); dmoyankova@abi.bg (D.M.)
3 Institute of Plant Physiology and Genetics, Bulgarian Academy of Science, “Acad. Georgi Bonchev” Str.,
Bl. 21, 1113 Sofia, Bulgaria; zlatina.go@abv.bg (Z.G.); stamova@bio21.bas.bg (M.G.);
valentin.velinov82@gmail.com (V.V.)
4 Institute of Molecular Biology “Rumen Tzanev”, Bulgarian Academy of Sciences, “Acad. Georgi Bonchev”
Str., Bl. 21, 1113 Sofia, Bulgaria; ialexandar@yahoo.com
* Correspondence: lpsimova@yahoo.co.uk

Abstract: Breast cancer is the second leading cause of death among women, and the number of
mortal cases in diagnosed patients is constantly increasing. The search for new plant compounds
with antitumor effects is very important because of the side effects of conventional therapy and the
development of drug resistance in cancer cells. The use of plant substances in medicine has been
Citation: Zasheva, D.; Mladenov, P.; well known for centuries, but the exact mechanism of their action is far from being elucidated. The
Zapryanova, S.; Gospodinova, Z.; molecular mechanisms of cytotoxicity exerted by secondary metabolites and bioactive peptides of
Georgieva, M.; Alexandar, I.; Velinov, plant origin on breast cancer cell lines are the subject of this review.
V.; Djilianov, D.; Moyankova, D.;
Simova-Stoilova, L. Cytotoxic Effects Keywords: breast cancer; MCF7; MDA-MB231; cytotoxicity; secondary metabolites; bioactive
of Plant Secondary Metabolites and peptides
Naturally Occurring Bioactive
Peptides on Breast Cancer Model
Systems: Molecular Mechanisms.
Molecules 2024, 29, 5275. https://
1. Introduction
doi.org/10.3390/molecules29225275
The problem of effective cancer therapy is becoming increasingly important. The
Academic Editors: Agata number of patients diagnosed with a tumor and the number of mortal cases among those
Poniewierska-Baran and Maciej
patients have been expanding over the past few years. In 2018 alone, the number of dead
Tarnowski
cancer patients was about 10 million, as reported by the World Health Organization [1].
Received: 30 September 2024 Concerning breast cancer, 1.38 million women were diagnosed in 2012 [2], and by 2020
Revised: 30 October 2024 this number had increased to 2.3 million [3]. Breast cancer is the most common cancer
Accepted: 6 November 2024 among women worldwide by 2021, although about 1% of breast cancer patients are men [4].
Published: 7 November 2024 Breast cancer cases account for 12% of all cancer cases and can be found in teenage girls,
in women of reproductive age, and in menopausal women with preliminarily developed
climax changes [5].
Commonly used methods of breast cancer treatment are surgery, chemotherapy, and
Copyright: © 2024 by the authors.
radiation therapy. Surgical methods are invasive with a long recovery period and usually
Licensee MDPI, Basel, Switzerland.
affect a woman’s quality of life. The surgical method is followed by chemotherapy and
This article is an open access article
radiotherapy or a combination of them. Chemotherapeutic anticancer drugs are usually
distributed under the terms and
conditions of the Creative Commons
synthetic drugs and have cytotoxic effects. They are classified into several groups depend-
Attribution (CC BY) license (https://
ing on the mechanism of action on cells. The following groups are used in anticancer
creativecommons.org/licenses/by/ therapy: microtubule-interacting agents (vincristine, taxol) [6], drugs with a topoisomerase-
4.0/). inhibiting effect (doxorubicin, podophyllotoxin) [7], alkylating agents such as melphalan,

Molecules 2024, 29, 5275. https://doi.org/10.3390/molecules29225275 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 5275 2 of 23

methotrexate, blocking cell metabolism [8], and DNA interacting agents such as doc-
etaxel [9]. The molecular mechanisms of commonly used antitumor drugs are not very
specific; they kill not only cancer cells but their targets could also be normal cells. This fact
explains many side effects of chemotherapeutics. The same is the case with radiotherapy.
These conventional therapeutic methods are associated with loss of normal blood cells, loss
of body mass, and loss of hair; some of them are associated with vomiting and dizziness [2].
Long-term use of drugs in anticancer therapy can lead to the emergence of resistance to
them in cancer cells (multidrug resistance, MDR) [10]. The MDR of cancer cells results from
molecular mechanisms related to the inability of administered drugs to penetrate into the
cells and trigger a mechanism of cancer cell elimination. These facts make drugs ineffective,
and cancer development continues progressively. The probability of the emergence of
multidrug resistance in cancer patients, together with the strong impact of the side effects
of chemotherapy drugs on the treated organism, makes the search for new anticancer
therapeutics so necessary and significant. Possible substances that could be expected to
have little or no side effects are certain substances of plant origin. Plants have been used in
medicine since ancient times. Generally, Chinese traditional medicine is based on them;
Ayurveda in India is based on therapeutic methods using plants and substances of plant
origin. Intensive studies of plant secondary metabolites and bioactive peptides are being
carried out on cancer cell lines, a model system that enables the testing of plant derivatives
on cell lines with characteristics of different types of breast cancer. The mechanisms of their
cytotoxicity and their targets can be established at the cellular and molecular levels. The
subject of this review will be studies of well-characterized substances among secondary
metabolites and polypeptides of plant origin with cytotoxic effects on breast cancer cell
lines with different characteristics and the molecular mechanisms mediating the reduction
in cancer cell number as a result of plant substances application. Breast cancer cell lines are
well-characterized and convenient model systems with the same cell signaling mechanisms
as the primary breast tumors. The possible new search for metabolites with specific targets,
depending on cell-line characteristics and cancer characteristics, will be discussed. The
discovery of plant polypeptides targeting specific proteases with a high impact on cancer
cell proliferation, invasion metastasis formation, and angiogenesis will be elucidated.
Future prospects of searching for new plant metabolites and polypeptides with breast
anticancer effects will be suggested.

2. Breast Cancer Types and Signal Pathways Related to the Breast Cancer Development
Depending on cancer origin, the types of breast cancer are ductal (85% of breast
cancer cases; lobular, originating from the lobes (9–14%), and very aggressive atypical
inflammatory forms (1–6% of the cases) [11]. The characteristics of the transformed cells
are also very important. Depending on the expression of cell receptors—estrogen receptor
(ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2),
breast cancer types are divided into four molecular subtypes: basal (triple negative, which
does not express ER, PR, and HER2 (11 % of BC cases with an aggressive phenotype and
lack of response to hormone replacement therapy), luminal A (ER+/PR+/HER2−, 60%
of BC cases), luminal B (ER+/PR+/HER2+, 15% of BC cases), and HER2 overexpressing
(ER-/PR−/HER2+, 14% of BC cases). The cellular transformation of breast cancer is
characterized by changes in signaling pathways as a result of some risk factors such as
genetic and environmental that could be predisposed to it, followed by changes in molecular
mechanisms accompanied by mutations in different groups of genes: tumor-suppressor
genes (p53, NF1, NF2), DNA repair system genes (PTEN, BRCA1 and BRCA2 genes),
oncogenes (RAS, MYC, Bcl2, RAF), and genes involved in cell growth and metabolism [12].
These mutations lead to the acquisition of uncontrollable division of cancer cells, and
invasion of the tissues where they are located and, therefore, can form blood vessels
and metastasize.
The main signaling pathways associated with breast cancer transformation and pro-
gression (Figure 1) depend on the expression of the estrogen receptor and its associated
 PI3K/AKT/mTOR signaling pathway is another one with an impact on brea
development; more PI3K mutations are features of breast cancer predisposition. T
to cellular dedifferentiation of basal and luminal progenitor cells, rendering
Molecules 2024, 29, 5275 multilineage potential [50]. Akt kinase and downstream mTOR hyperactivation 3 of 23
resistance to endocrine therapy [51]. Inhibition of mTOR is a good candidate for
therapy due to the possibility of restoring the antiestrogen sensitivity of cancer
signaling—ER
The knowledge alpha
of (ERα—a membrane
the molecular receptor) andofER
mechanisms beta (ERβ—a
breast cancer nuclear recep-
transformation pr
tor) [13–16]. All of them are linked with the processes of cell development directed by
opportunity to search for new targeted therapeutic drugs that are more effective
changes in cell cycle control and proliferation, cell differentiation and cell death, and cell
invasive.
migration andThemotility
interest
[17].inThe
plant‐derived
pathway associated compounds with anticancer
with cell proliferation is mediated activity
revived in recent years and intense research on the molecular mechanisms
by ERα interaction with cyclin D. Cyclin D is an activator of cyclin-dependent kinases 4 of the
and 6, which mediate cell cycle progression from G1 to S phase
especially targeting cancer signaling pathways, is being performed. Canc[17,18]. This type of cancer
responds to hormone replacement therapy, and estrogen-dependent cell line models such
especially
as MCF7 arebreast
used incancer,
studies toare diseases
search related
for targeted to different
therapeutic blockingchanges
estrogen in the genome,
receptor α
stress,
in high and
levelsaand
change
estrogeninreceptor
immune β infunctions; the search
low levels, whose for molecules
homodimers which
are located in the mod
key elements and
cell cytoplasm, associated with breast
their dimerization cancer
is needed transformation
for activation and nuclear istranslocation.
a challenge in th
They direct the transcription of different genes in the nucleus, or they are cofactors of
related to breast cancer therapy.
various other transcription factors [18–21].

Figure 1. Main signaling pathways associated with breast cancer transformation.

Figure 1. Main signaling pathways associated with breast cancer transformation.
The HER2 receptor signaling pathway characterizes the breast cancer cell overexpress-
3.ing the HER2 receptor (human epidermal growth factor receptor), which is a member of
Plant Secondary Metabolites with Anticancer Effects on Breast Cancer Cell
the endothelial growth factor receptor family of four receptors. They are receptor tyrosine
Groups of Plant
kinases with Secondary
extracellular, Metabolitesand
membrane-binding, and Molecular
intracellular Mechanisms
domains [22–25]. TheMediatin
Cytotoxic
HER2 receptorEffects
is a molecule that forms dimers with other molecules of the HER receptor
families, and the binding of membrane ligands to the dimers enables phosphorylation
of its intracellular domains [26–28]. This activates kinase signaling pathways such as
mitogen-activated protein kinase (MAPK) and phosphatidylinositol 4,5-bisphosphate 3-
kinase (PI3K) associated with breast tumorigenesis through accelerated proliferation and
cell cycle progression [29–32]. High expression of HER2 in breast cancer cells increases the
metastatic potential of breast tumors [30].
The third signaling pathway is the canonical Wnt/β catenin, whose deregulation has
a strong impact on cell transformation, development, and progression of breast cancer. Wnt
proteins are a family of glycosylated secreted proteins with a high impact on embryonal
tissue development and mammary gland development. The interaction of Wnt proteins
with their receptors leads to the maturation of Axin and disheveled proteins to the cell
membrane. The inhibition of glycogen synthase kinase 3β protein is initiated, and its
Molecules 2024, 29, 5275 4 of 23

function as a Wnt protein inhibitor is blocked. The β-catenin ceases to be degraded in

the proteasome and accumulates in the cytoplasm as a result of inhibition of the glycogen
synthase kinase 3β protein, and its translocation into the nucleus is enabled. In the nucleus,
it becomes a cotranslational activator of different oncogenes MYC, CCND1 together with
their translational activator CREB binding protein and T cell factor/lymphoid enhancer
factor transcription factors [33–38]. The Wnt pathway is constitutively activated by an
autocrine mechanism in breast cancer. Mutations in Wnt proteins have not been found in the
genome of breast cancer cells, but the positive regulator of their signaling Dvl is amplified
in 50% of breast cancers, and the Wnt-related inhibitor Frizzled protein 1 is lost in 78% of
malignant cancers diseases of the breast, which correlates with poor prognosis development
of cancer [39,40]. Wnt/β catenin signaling is activated in basal-like breast cancers [41,42],
which is associated with poorer prognosis. The Wnt//β catenin signaling pathway drives
tumor progression in HER2-positive breast cancers, which has been established by in vivo
studies and plays a role in pathways involved in multiple resistance to standard antibreast
cancer drugs [43].
Many other signaling pathways that have a role in normal breast development play an
important role in breast cancer development if dysregulated, such as the cyclin-dependent
kinase pathway that involves cell cycle progression. It is regulated by cyclins, cyclin-
dependent kinases and their inhibitors [44]. Usually, breast cancer development is asso-
ciated with the amplification of cyclin D1 and its high expression, respectively, the high
expression of cyclin E in 60% of breast tumors and a mitogenic effect of cyclin D1 exerted
by an estrogen-dependent mechanism. Decreased expression of the CDKI p27Kip1 has
been established in breast cancers [45].
The Notch signaling pathway consists of five Notch ligands, which are transmem-
brane proteins named Delta-like 1,3,4 and Jagged (Jag) 1,2. Notch signaling is directed by
proteolytic reactions activated by ligand-receptor binding. The result of proteolysis is the
formation of a Notch intracellular domain transcription factor that regulates downstream
target genes. Higher expression of Notch signaling genes leads to poor cancer progno-
sis [46,47]. Breast cancer etiology is linked to sonic hedgehog (SHH) signaling with a role
in mouse glandular development. Disruptions of two of the SHH pathway-related factors,
patched homolog-1 (PTCH-1) or glioma-associated oncogene-2 (GLI-2), are observed in
breast cancer [47,48]. The breast tumor kinase (BTK) signaling pathway is another signaling
pathway with a role in breast cancer. It is a nonreceptor tyrosine kinase overexpressed
in more than 60% of breast cancer cases, and EGFR-regulated signaling is impaired in
these cancers, which significantly increases MAPK activity and thus leads to fast prolifera-
tion and migration of breast cancer cells [49]. The PI3K/AKT/mTOR signaling pathway
is another one with an impact on breast cancer development; more PI3K mutations are
features of breast cancer predisposition. They lead to cellular dedifferentiation of basal
and luminal progenitor cells, rendering them of multilineage potential [50]. Akt kinase
and downstream mTOR hyperactivation leads to resistance to endocrine therapy [51].
Inhibition of mTOR is a good candidate for targeted therapy due to the possibility of
restoring the antiestrogen sensitivity of cancer cells [51]. The knowledge of the molecular
mechanisms of breast cancer transformation provides an opportunity to search for new
targeted therapeutic drugs that are more effective and less invasive. The interest in plant-
derived compounds with anticancer activity has been revived in recent years and intense
research on the molecular mechanisms of their action, especially targeting cancer signaling
pathways, is being performed. Cancers, and especially breast cancer, are diseases related
to different changes in the genome, oxidative stress, and a change in immune functions;
the search for molecules which modulate the key elements associated with breast cancer
transformation is a challenge in the studies related to breast cancer therapy.
Molecules 2024, 29, 5275 5 of 23

3. Plant Secondary Metabolites with Anticancer Effects on Breast Cancer Cell

Lines—Groups of Plant Secondary Metabolites and Molecular Mechanisms Mediating
Their Cytotoxic Effects
Plant secondary metabolites are organic chemical substances that have an essential
role in plant adaptation to changes in environmental factors and for their viability under
stressful conditions. They have a protective role in plants against various stresses—abiotic,
biotic, and oxidative ones. These metabolites are extractable from plants using organic
solvents such as ethanol, methanol, and ethyl acetate [52]. Fractionation of the extracts
on HPLC and LC results in fractions with a mixture of secondary metabolites. These
fractions can be refractionated by a secondary fractionation, and their components can
be characterized by nuclear magnetic resonance (NMR) and mass spectrometric analysis.
These methods enable the use of an integrative multi-omics approach for the prediction
of the potential of a plant metabolite to exert anticancer effects on tumor cells. Plant
secondary metabolites are divided into the following groups: bioflavonoids, unsaturated
fatty acids, tannins, alkaloids, and terpenoids [53]. Components with cytotoxic effects are
usually searched among these groups of plant secondary metabolites, although they are
exceptionally divergent with different structures, depending on the extracted plants and
organs, their areal and living conditions. The anticancer effects of the studied secondary
metabolites could be categorized into the following mechanisms. They usually block the
cell cycle or initiate some of the cell death pathways (necrosis, apoptosis). They can mediate
mechanisms related to the suppression of essential enzymes for cell proliferation or be of a
structure that can block receptors with an essential role in the development, proliferation,
and metastatic potential of cancer.

3.1. Flavonoids
Flavonoids belong to the group of substances containing polyphenols, where they
have been described as being about 4000 and they contain 15-carbon derivatives of beta-
gamma-pyrone. They are divided into different classes, such as flavanones, flavans, proan-
thocyanidins [7], coumarins, and coumarin-related compounds [53]. A comparative study
of flavonoids from different subclasses and their effects on breast cancer cell lines estab-
lished the leading role of the 2,3-double bond in the C-ring in cytotoxicity linked with
mitochondrial impairment [54]. Docking analysis of 98 flavonoids with GLUT1 trans-
porter, overexpressed in several carcinomas, has shown that the transporter is a target
for flavonoids. Screening for cytotoxicity on a carcinoma cell line has shown that eight
of them (apigenin, kaempferol, eupatilin, luteolin, hispidulin, isosinensetin, sinensetin,
and nobiletin) reduce cell viability to 50% by inhibition of GLUT1 transporter, the critical
pharmacophores of flavonoids inhibitors being 3′ hydrophobic groups and hydrogen bond
acceptors [55]. The molecular mechanism of glucose metabolism inhibition in tumor cells
by forming the flavonoid–GLUT1 transporter complex is shown in Figure 2. The group of
flavonoids like genistein, which is isolated from soya and soya products, induces cell cycle
arrest in the G2/M phase in the triple-negative MDA-MB231 cell line, and the mechanism
of the arrest is mediated by ERK1/2 kinase pathway activation and downregulation of
Cdk1, cyclin B1, and Cdc25 C [56]. Genistein, in combination with doxorubicin, exhibited a
synergistic effect on MCF-7/Adr drug-resistant cells by an increase in the intracellular accu-
mulation of doxorubicin and inhibition of HER2/neu expression [57]. It was demonstrated
that the bioflavonoid quercetin, which is characterized by low in vivo toxicity, increases the
inhibitory effect of doxorubicin in MCF-7 Adr-resistant breast cancer cells [58]. Combined
again with doxorubicin, quercetin induced rejection of 4T1 breast cancer in mice [59]. The
natural flavonoid naringenin associated with doxorubicin synergistically suppressed the
growth and migration of MCF7 cells [60].
Molecules 2024, 29, 5275 6 of 23
Molecules 2024, 29, 5275 6 of 23

Figure2.2.Molecular
Figure Molecularinteraction
interactionmechanisms
mechanisms ofof suppression
suppression of of breast
breast cancer
cancer survival
survival andand mitigation
mitigation
by natural phenylpropanoid glycosides, flavonoids, terpenoids, and coumarins. Structures of
by natural phenylpropanoid glycosides, flavonoids, terpenoids, and coumarins. Structures of com-
compounds are represented by ball and stick diagrams with their charged groups involved in
pounds are represented by ball and stick diagrams with their charged groups involved in interactions.
interactions. The structure of myconoside was downloaded from Japan Chemical Substance
The structure(Nikkaji);
Dictionary of myconoside was downloaded
the common from Japan Chemical
feature pharmacophores Substance
of flavonoids for Dictionary
interaction(Nikkaji);
with Glut 1
the common feature
represented pharmacophores
with green of flavonoids
and blue spheres for interaction
is according with Glut 1ofrepresented
to [55]; structures with
celastrol and green
conferone
and
wereblue spheres is according
downloaded to [55];Proteins
from PubChem. structuresof of celastrol and
interactions are conferone
represented were
by downloaded from
ribbons downloaded
PubChem. Proteins ofin
from PDB illustrated interactions
the contextare represented
of the processesby andribbons
pathwaysdownloaded from PDB
in breast cancer cells.illustrated
Red dashed
inlines represent
the context of the
the interactions
processes andof pathways
inhibition inof breast
metabolites
cancerwith
cells.target proteins.
Red dashed lines represent the
interactions of inhibition of metabolites with target proteins.
Coumarins
Coumarins
Among flavonoids, coumarins have a very important role in breast cancer research
Among flavonoids, coumarins have a very important role in breast cancer research
on model cell lines. Coumarin is 1,2‐benzopyrone or 2H‐1‐benzopyran‐2‐one. It can be
on model cell lines. Coumarin is 1,2-benzopyrone or 2H-1-benzopyran-2-one. It can be
found in various plant organs (roots, seeds, and leaves). Coumarins are widely available
found in various plant organs (roots, seeds, and leaves). Coumarins are widely avail-
in plants—800 coumarin derivatives are known from about 100 plant families and 600
able in plants—800 coumarin derivatives are known from about 100 plant families and
genera [53,61]. They have a polyphenolic structure and are colorless. The coumarins are
600 genera [53,61]. They have a polyphenolic structure and are colorless. The coumarins
divided
are dividedinto four
into groups—simple
four groups—simple coumarins,
coumarins, composed
composed of of
hydroxylated,
hydroxylated, alkoxilated, and
alkoxilated,
and alkylated coumarin derivatives and their glycosides; pyranocoumarins, which areare
alkylated coumarin derivatives and their glycosides; pyranocoumarins, which
structuresofofsix-furan
structures six‐furanrings
ringsand
andfused
fusedwithwiththethe benzene
benzene ring
ring furanocoumarins,
furanocoumarins, which
which
comprisefurane
comprise furanering
ringand
andcoumarin
coumarinfusionfusion [62];
[62]; and
and pyrone‐substituted
pyrone-substituted coumarins.
coumarins. This
This
group is divided into three subgroups: 4‐hydroxycoumarin, 3‐phenylcoumarin,
group is divided into three subgroups: 4-hydroxycoumarin, 3-phenylcoumarin, and 3,4- and 3,4‐
benzocoumarin. Coumarins have biological effects on various diseases,
benzocoumarin. Coumarins have biological effects on various diseases, including cancer. including cancer.
Themechanisms
The mechanisms of their
of their anticancer
anticancer effectseffects are related
are related to the induction
to the induction of through
of apoptosis apoptosis
through
the the activation
activation of a caspaseofcascade
a caspase cascade
initiated by initiated
caspase 9,by caspase
which 9, whichbyisamediated
is mediated by a
decrease in
decrease in antiapoptotic
antiapoptotic Bcl2 expression Bcl2 expression
levels [63]. Theirlevels [63]. Their
anticancer effectsanticancer
have beeneffects have on
established been
established
the MCF7 breast on cancer
the MCF7 breast
cell model line.cancer cell with
Treatment model line. Treatment
coumarin with coumarin
derivatives increased P21
derivatives increased P21 protein expression and arrested cells in the G0/G1 phase [64],
Molecules 2024, 29, 5275 7 of 23

protein expression and arrested cells in the G0/G1 phase [64], Three synthesized coumarins
derived from triphenylethylene inhibit angiogenesis of breast cancer cell lines and more
precisely, compound TCH-5c changes endothelial cell cytoskeleton organization and migra-
tion of EA.hy926 endothelial cells [64]. Cellular treatment with them affects different cell
signaling and kinase-dependent pathways that direct cancer cells to apoptosis or cell cycle
arrest [63,64]. The coumarin group targets the key cell cycle regulator cdc25 and is a good
option for targeted therapy of breast cancer [65]. Oral uptake of coumarins is effective. They
are absorbed rapidly in the gastrointestinal tract and penetrate cells by passive diffusion
through the lipid membrane. Coumarins are rapidly metabolized in the liver/excreted by
the kidney, and only 2–6% reach the system circulation [66,67]. The toxicological studies of
coumarins are very controversial. Synthetic coumarins have been shown to cause acute
chronic and cancerogenic effects in mice and rats [66]. In contrast, studies on human and
cynomolgus monkey liver fragments or hepatocytes have shown relative resistance to
coumarin toxicity [66]. The use of standard breast anticancer drugs results in a multidrug
resistance phenotype. Some of the plant coumarins suppress Pgp-mediated drug efflux in
the MCF7 cell line, and thus, they are promising for overcoming multidrug resistance [68].
The mechanism of multidrug resistance suppression of conferone is presented in Figure 2.

3.2. Alkaloids in Breast Cancer Therapy

The search for well-characterized and purified anticancer drugs of plant origin has
been very intense in recent years, and some of them are used in therapy, such as the
vinca alkaloids [67]. Their mechanism for arresting the development of cancer cells is
related to the blockage of the microtubule organization of the spindle pole body. They stop
cell division and cancer development by binding it to the tubulin-microtubule-associated
protein structure. A more stable complex between the alkaloid and tubulin-MAP is formed,
and the stability is reflected in the prevention of the formation of the mitotic spindle, which
results in abrogated mitosis and cell proliferation [6,7]. Taxol has the same mechanism
of action. Such effects of taxol have been demonstrated for various cancer cells (breast
adenocarcinoma cells) [6,8], but their application is limited by the multidrug resistance of
cancer cells to taxol. Multidrug resistance to routinely used anticancer drugs is a challenge
for scientists to search for new metabolic plant substances with antitumor potential and
minimal side effects.

3.3. Polyphenols
3.3.1. Curcumin
Curcumin (diferuloylmethane) has a polyphenolic structure and is a yellow powder
derived from a plant extract of Curcuma longa. This substance is the subject of many studies
related to its anticancer effects. Its effects mediate the inhibition of the transcription factor
NFkB in the MCF7 breast cancer cell line [69] after reducing the expression of its target
gene, such as COX2 and cyclin D, leading to apoptosis [6,7]. It can affect cells by binding to
the tubulin-like taxol and thereby block mitosis [69]. It has an effect on normal cells and
is therefore used in combination with other drugs. Paclitaxel and curcumin reduced cell
viability of breast cancer cell lines MCF7 and MDA-MB231 through apoptosis, activation of
caspases 3/7 and protein expression of nuclear NfkB transcription factor [70]. Curcumin
has effects on cell culture models of various origins as an effective inhibitor of tyrosine-
regulated kinase 2, a positive regulator of the 26S proteasome, which disrupts it and leads
to impaired cell proliferation in the triple-negative breast cancer cell line MDA-MB468 [71].
The effect of curcumin on cell viability was favored by binding to jacalin molecules, which
act as a natural ligand of Thomsen–Friedenreich (TF) tumor-associated antigen in this
triple-negative cell line MDA-MB231 [72]. It blocks kinase pathways by NfKb signaling
in combination with taxol in higher doses and can bind to DNA molecules, thus blocking
cell cycle progression; besides, it can block angiogenesis, targeting the VEGF-VEGFR2
signaling pathway [69,70]. Other studies related to the combined treatment of curcumin
with standardly used drugs on breast cancer cell lines show an improvement in the an-
Molecules 2024, 29, 5275 8 of 23

ticancer effects of the chemotherapeutics. Paclitaxel and curcumin combination induced

apoptosis by the NfkB signaling pathway in comparison to single drug treatment [73].
Curcumin reverses doxorubicin multidrug resistance in MCF7 and MDA-MB231 cell lines,
targeting ABCB4 by its ATPase activity [74]. Curcumin inhibits epithelial-mesenchymal
transition induced by doxorubicin by targeting TGFβ and kinase signaling pathways
PI3K/AKT in triple-negative breast cancer cell lines [75]. Studies on MDA-MB-231 cells
reported synergistic combinations of curcumin with different oncotherapeutics, among
which are cisplatin, vinorelbine, and ixabepilone, leading to cell cycle arrest and induction
of apoptosis [76], potentiating the effect of curcumin on the cytotoxicity of paclitaxel against
MDA-MB-435 breast cancer cells, and decreasing the incidence of breast cancer lung metas-
tasis after curcumin and paclitaxel administration in the breast cancer xenograft model
system [77]. In clinical trials with humans and rodents, curcumin was administered orally,
intravenously, transdermally, intraperitoneally, and intratumorally [78]. The combined
treatment of patients with standard therapeutic docetaxel and curcumin shows unchanged
therapeutic outcomes compared to the group of only docetaxel treated patients [79]. Radia-
tion therapy-induced dermatitis was reduced by oral administration of curcumin in clinical
trials on women with noninflammatory breast cancer [80] Curcumin side effects related
to diarrhea, headache, rash, and yellow stool in subjects who have received high doses of
curcumin (500–12,000 mg) [81] and the prolonged use of curcumin (1–4 months) is related
to an increase in serum alkaline phosphatase and lactate dehydrogenase [82]. Curcumin is
usually metabolized in phase I and II biotransformation in the liver (phase I) and in the
intestine and gut microbiota (II), the second phase being more intensive [83].

3.3.2. Saponins
Indicacin is a polyphenol from the group of 3-terpenoid saponins purified from a
methanol extract of Fagonia indika. It has an effect on breast cancer cell lines of different
origins (MDA-MB-468 and MCF7) through PARP cleavage, caspase 3 activation, DNA
fragmentation, and apoptosis activation [84]. Morus alba metabolite lectin has an antiprolif-
erative effect on the MCF7 cell line [85]. Platycodin D from Placticodon grandifloras has a
cytotoxic effect, activates caspases, and induces apoptosis in the MCF7 cell line [86]. The
in vivo activity of platycodin D was established on mice with tumors induced by an injec-
tion of human metastatic breast cancer cells. Oral administration of platycodin D inhibited
cell-induced osteolysis and blocked osteoclast formation and osteoclast-mediated bone
resorption [87]. In combination with docetaxel, platycodin enhanced the antiproliferative
effect in MCF7 and MDA-MB231 cell lines [88]. The side effects of platycodin D, as of
the other saponins, are related to the induction of hemolytic activity [89], which could be
solved by chemical modifications of their structure.

3.3.3. Myconoside
The anticancer effects of the phenyl propanoid glycoside myconoside, identified in the
methanol extract fraction of the resurrection plant Haberlea rhodopensis, were established on
MCF7 and MDA-MB231 breast cancer cell lines. The cytotoxic and antiproliferative effects
of the myconoside-enriched fraction have been demonstrated, and the docking analysis
of myconoside with estrogen receptor, glucose transporter, and MYST acetyltransferase
provides a basis for the explanation of the molecular mechanisms of their anticancer
effects [90] and prospects for the future search of targeted therapy based on myconoside
treatment. The molecular mechanism of the myconoside effect on the MCF7 cell line is
presented in Figure 2. Docking analysis predicts that it blocks estrogen receptors, glucose
transporter GLUT1, and MYST acetyltransferase and, in this way, reduces cell growth and
proliferation. MDA-MB231 triple negative cell line is influenced by linking to the glucose
transporter and MYST acetyltransferase [90].
Molecules 2024, 29, 5275 9 of 23

3.4. Plant Metabolites with Anticancer Effects on Triple Negative Cell Lines
Different groups of plant metabolites have anticancer effects on triple-negative cell
lines that do not express estrogen receptors, progesterone receptors, and the epidermal
growth factor receptor (HER1). These characteristics make them unresponsive to hormone
replacement therapy and with a higher invasive and metastatic potential [91].

3.4.1. Piperine
Piperine from black pepper arrests MDA-MB231 and MDA-MB468 cells by blocking
the activation of the phosphatidyl inositol 3 kinase pathway and triggering the caspase-
dependent mitochondrial apoptosis pathway. Piperine-treated cells showed lower MMP2/9
expression and migration potential [92]. The effects of alkaloids such as piperlongumine
(Piper longum), berberine, a quaternary ammonium alkaloid extracted from Coptis chinensis,
indirubin-3-monoxime found in Indigo naturalis, are associated with the blocking of kinase
signaling pathways and reducing the migratory potential of triple-negative breast cancer
cells [93–95]. Piperine, as other described alkaloid agents in this paragraph, does not have
side effects, and the application of piperine in combination with other conventional drugs
like paclitaxel is very effective. It is absorbed by the intestinal tract and metabolized in the
liver and, kidney, and excreted in bile and urine [96].

3.4.2. Terpenoids
The terpenoid group is another plant metabolite group with established effects on
triple-negative breast cancer cell lines. Tanshinone I and Tanshinone IIA found in the Dan
Shen root of Salvia miltiorrhiza affect the MDA-MB231 cell line by reducing cell growth and
vascular endothelial growth factor (VEGF) expression, thereby reducing the proliferation
level via mTOR/p70S6K/4 E-BP1 signaling pathway [97]. Eupalinolide J (EJ), a sesquiter-
pene lactone found in Eupatorium lindleyanum, astragaloside IV, an active triterpenoid from
Radix astragali found in the roots of Astragalus membranaceus Bunge, Betulinic acid, and Dil-
lenia suffruticosa Martelli root extract KHF16 (24-acetylisodahurinol-3-O-D-xylopyranoside),
a triterpenoid found in the rhizomes of Cimicifuga foetida, the pseudopterosins, a class of
marine diterpene glycosides, extracted from the gorgonian sea whip Antillogorgia elisabethae,
have anticancer effects on triple-negative breast cancer cell lines. The effects of terpenoids
on triple-negative cell lines can be classified based on their molecular mechanisms leading
to cell death. KHF16 (24-acetylisodachurinol-3-O-D-xylopyranoside) triggers cell cycle
arrest and apoptosis in some triple-negative cell lines, promoting G2/M phase cell cycle
arrest and NF-Kb pathway-mediated necrosis [98]. The terpenoids inhibit cell proliferation
by multiple targets. The molecular mechanism of their anticancer activity is related to the
NfkB pathway. Triterpene celastrole inhibits tumor growth of mouse xenographs of the
triple-negative cell lin MDA-MB-435 e to 60% by NfKb inactivation and activates apoptotic
effects induced by tumor necrosis factor α. NfkB inactivation inhibits its DNA binding
capacity (Figure 2). The celastrole molecule suppresses NfkB, targeting its cysteine 179 [99].
The effects of terpenoids on triple-negative cell lines are limited in clinical trial translation
because of their poor absorption and low availability, which implies the need for their
structural modification in synthetic analogs. Their use in combination with standard drugs
like doxorubicin, cis platina, and paclitaxel diminishes multidrug resistance of tumor cells
and increases the effectiveness of chemotherapy [100].
Well-characterized polyphenols with their anticancer mechanisms clarified on cancer
cell lines of different origins are described in Table 1.
Molecules 2024, 29, 5275 10 of 23
Molecules 2024, 29, 5275 10 of 23

Molecules 2024, 29, 5275 10 of 23

Table 1. Plant secondary metabolites with anticancer effects on breast cancer cell lines with established molecular mechanism.
TableTable
1. Plant secondary
1. Plant metabolites
secondary with anticancer
metabolites effectseffects
with anticancer on breast cancercancer
on breast cell lines
cell with
lines established molecular
with established mechanism.
molecular mechanism.
Chemical Structure (PubChem Database‐ Established Established
Chemical Structure (PubChem Database‐ Established Established
Metabolites/ Established
Anticancer Established
Molecular
Metabolites/ Chemical Structure (PubChem Database- Anticancer Molecular References
Metabolites/ https://pubchem.ncbi.nlm.nih.gov/ accessed on 20 September Anticancer Molecular
Plant Origin https://pubchem.ncbi.nlm.nih.gov/ accessed
https://pubchem.ncbi.nlm.nih.gov/ on 20on
Accessed September Activity on Breast Cancer Cell Mechanism of Anticancer References
References
Plant Origin
Plant Origin 2024) 2024) Activity
Activity onon Breast
Breast Cancer Cell
Cancer Mechanism
Mechanism of Anticancer
Anticancer
20 September
2024) Models Activity
Cell Models
Models Activity
Activity
Essential
Essentialchemical
chemical structure
structure
Essential chemical structure

Coumarins‐ Apoptosis activation by

Coumarins‐
Coumarins- Estrogen receptor and Apoptosis activation by
About 800 different substances Estrogen receptor and Apoptosis activation
caspase9 by caspase9
pathway.
About
About 800 800 different
different substances
substances Estrogen receptor and progesterone
progesterone receptor expressing caspase9 pathway. [63,64]
found in various plantsplants progesterone receptor expressing Cell cycle pathway.
arrest in G0/G1 [63,64]
[63,64]
found in various
found in various plants receptor expressing MCF-7 cell line Cell cycle arrest in G0/G1
MCF‐7 cell line Cell cycle arrest in G0/G1 phase
(vegetables, nuts, fruits,
(vegetables, coffee)
nuts, fruits, coffee) MCF‐7 cell line phase
(vegetables, nuts, fruits, coffee) phase

Apoptosis induction,
Vinca alkaloids Group of substances used in Apoptosis induction,
Vinca alkaloids
Vinca alkaloids Group of substances used in Apoptosis binding to DNAbinding
induction, molecules
to DNA
Taxol (Paclitaxel) Groupstandard chemotherapy
of substances that have
used in standard binding to DNA molecules
Taxol (Paclitaxel)
Taxol (Paclitaxel) standard chemotherapy that have and cell
molecules proliferation
and arrest,
cell proliferation arrest, [6–8]
Derived from chemotherapy
effects onthat have effects
different breastoncancer and cell proliferation arrest, [6–8][6–8]
Derived from from
Derived effects on different breast development
cancer of multidrug
development of multidrugresistance
Catharanthus roseusroseus different breast cancer cell lines
cell lines development of multidrug
Catharanthus
Catharanthus roseus cell lines in cells
resistance in cells
resistance in cells
 Molecules 2024, 29, 5275 11 of 23

Table 1. Cont.

Established Established
Molecules 2024, 29, 5275 Chemical Structure (PubChem Database- 11 of 23
Metabolites/
Molecules 2024, 29, 5275 Anticancer Molecular 11 of 23
https://pubchem.ncbi.nlm.nih.gov/ Accessed on References
Plant Origin Activity on Breast Cancer Mechanism of Anticancer
20 September 2024)
Cell Models Activity

Blocking
Blocking activation
activation of
of
Phosphatidyl
Phosphatidyl inositol 33
Blocking activation inositol
of Phosphatidyl
Piperine
Piperine
Piperine MDA‐MB‐231
MDA-MB-231 and and MDA‐MB‐468
MDA-MB-468
MDA‐MB‐231 and MDA‐MB‐468 kinase
inositol pathway
3 kinase
kinase and
pathway
pathway andand [92]
[92] [92]
from Piper Piper longum
fromlongum extract triple-negative cell lines
triple‐negative cell lines triggering the caspase-dependent
triggering the caspase‐
from Piper longum extract
extract triple‐negative cell lines triggering the caspase‐
mitochondrial apoptosis
dependent
dependent mitochondrial
mitochondrial
apoptosis
apoptosis

Blockage
Blockage of
of the
the kinase
kinase signal
signal
Piperlongumine
Piperlongumine Triple‐negative breast cancer
Triple‐negative breast cancer cellcell Blockage pathways,
pathways, signal
of the kinase
Piperlongumine [93]
from Triple-negative breast lines
cancer cell lines pathways, [93] [93]
from Piper
fromlongum
Piper longum extract
Piper longum
extract
extract lines decrease
decrease
decrease
in
in migration
migration
in migration potential
potential
potential

Apoptosis
Apoptosis activation
activation by by
inhibition
inhibition of
of NfkB
NfkB
transcription
transcription factor
factor followed
followed
by
by reduction
reduction of
of its
its target
target
Curcumin
Curcumin Effects
Effects on
on MCF‐7
MCF‐7 cell
cell line
line and
and genes
genes expression
expression
isolated
isolated from
from extract
extract of
of triple‐negative
triple‐negative cell
cell lines
lines like
like (COX2,
(COX2, cyclin
cyclin D),
D), [6,7,68–72]
[6,7,68–72]
Curcuma
Curcuma longa
longa MDA‐MB‐231, MDA‐MB‐468
MDA‐MB‐231, MDA‐MB‐468 Inhibition of tyrosine‐
Inhibition of tyrosine‐
regulated
regulated kinase
kinase 2, 2,
perturbation of
perturbation of 26S 26S
proteasome
proteasome andand cell
cell cycle
cycle
arrest
arrest
 Molecules 2024, 29, 5275 12 of 23
Blockage of the kinase signal
Piperlongumine Triple‐negative breast cancer cell pathways,
Table 1. Cont.
[93]
from Piper longum extract lines decrease in migration
potential
Established Established
Chemical Structure (PubChem Database-
Metabolites/ Anticancer Molecular
https://pubchem.ncbi.nlm.nih.gov/ Accessed on References
Plant Origin Activity on Breast Cancer Mechanism of Anticancer
20 September 2024)
Cell Models Activity

Apoptosis activation by
inhibition of NfkB
transcription
Apoptosis factor
activation by followed
inhibition of
NfkBby reduction factor
transcription of its followed
target by
Curcumin Effects on MCF‐7 cell line and reduction
genes expressiongenes
of its target
Curcumin Effects on MCF-7 cell line and
expression
isolatedisolated
from extract of of
from extract triple‐negative
triple-negative celllike
cell lines lines like (COX2, cyclin D), [6,7,68–72]
[6,7,68–72]
(COX2, cyclin D),
Curcuma
Curcuma longalonga MDA-MB-231,
MDA‐MB‐231,MDA-MB-468
MDA‐MB‐468Inhibition Inhibition of tyrosine‐
of tyrosine-regulated kinase
regulated
2, perturbation kinase
of 26S 2,
proteasome and
cell cycle arrest
perturbation of 26S
Molecules 2024, 29, 5275 12 of 23
proteasome and cell cycle
arrest

Myconoside *
Myconoside * MCF‐7
MCF-7 Cytotoxic and
Cytotoxic and antiproliferative [90] [90]
from Haberlea rhodopensis
from Haberlea rhodopensis MDA‐MB‐231
MDA-MB-231 antiproliferative effectseffects

Cytotoxic
Platycodin D ** effect
MCF‐7 [86]
from Placticodon grandifloras activation of caspases and
apoptosis

Reduction of cell growth

Tanshinone I Tanshinone IIA and VEGF expression,
Molecules 2024, 29, 5275 12 of 23
Molecules 2024, 29, 5275 12 of 23
Molecules 2024, 29, 5275 12 of 23

Molecules 2024, 29, 5275 13 of 23

Myconoside **
Myconoside MCF‐7 Cytotoxic and
and
MCF‐7 Cytotoxic [90]
from Haberlea rhodopensis
Myconoside * Table 1. Cont. MDA‐MB‐231
MCF‐7 antiproliferative
Cytotoxic effects
and [90]
from Haberlea rhodopensis MDA‐MB‐231 antiproliferative effects [90]
from Haberlea rhodopensis MDA‐MB‐231 antiproliferative effects
Established Established
Chemical Structure (PubChem Database-
Metabolites/ Anticancer Molecular
https://pubchem.ncbi.nlm.nih.gov/ Accessed on References
Plant Origin Activity on Breast Cancer Mechanism of Anticancer
20 September 2024)
Cell Models Activity

Cytotoxic
Cytotoxic
Platycodin D
Platycodin D **
** effect
Cytotoxic
Platycodin MCF‐7 effect
Cytotoxic [86]
Platycodin
from Placticodon ** D **
Placticodon grandifloras
grandifloras
D MCF‐7
MCF-7 activation of caspases and
effect
effect
activation of caspases and [86] [86]
from from Placticodon grandifloras MCF‐7 [86]
from Placticodon grandifloras activation of apoptosis
activationcaspases and apoptosis
of caspases and
apoptosis
apoptosis

Reduction of
Reduction of cell
cell growth
growth
Tanshinone Reduction of cellof
growth and VEGF
II Tanshinone
Tanshinone
Tanshinone
Tanshinone IIA IIA
I Tanshinone
IIA and VEGF
and VEGF
Reduction
expression,
expression,
cell growth
expression,
decrease in proliferation
Tanshinone
the Dan
the Danthe
Shen
Shen
I Tanshinone
Danroot
root Salvia
Shenofroot ofIIA
of Salvia Salvia MDA‐MB‐231
MDA-MB-231
MDA‐MB‐231 decrease in proliferation
and VEGF proliferation
expression, via [97] [97]
via decrease in
mTOR/p70S6K/4 via
E-BP1 signaling [97]
miltiorrhiza
Shen root of Salvia
the Danmiltiorrhiza MDA‐MB‐231 mTOR/p70S6K/4
decrease in E‐BP1
proliferation via [97]
miltiorrhiza pathway E‐BP1
mTOR/p70S6K/4
miltiorrhiza signaling pathway
signaling pathway
mTOR/p70S6K/4 E‐BP1
signaling pathway

Blockage of
of the kinase
kinase signal
signal
Berberine
Berberine Triple‐negative breast
Triple‐negative breast cancer cell Blockage
cancer cell Blockage of the
the kinase signal
Berberine
Triple-negative breast lines
cancer cellcancer
pathways,
Blockage
lines cell pathways, of
pathways, thedecrease
kinase
decrease
decrease
in
signal
in
in migration
[94,95]
[94,95]
[94,95]
from Coptis
Berberine
from Coptisfrom chinensis
Coptis chinensis
chinensis Triple‐negative breast
lines
Molecules 2024, 29, 5275 migration
pathways, potential
decrease
potentialin
potential
migration [94,95]
13 of 23
from Coptis chinensis lines
migration potential

Eupalinolide
Eupalinolide Triple‐negative breast cancer cell Triggering
Triggering
of cellofcycle
cell arrest
cycle and
Triple-negative breast cancer cell lines [98] [98]
from Eupatorium
from Eupatorium lindleyanum
lindleyanum lines apoptosis
arrest and apoptosis

Triple‐negative breast cancer cell

Cell cycle arrest in G2/M
KHF16 *** lines MDA‐MB‐231, MDA‐MB‐
phase, NF‐Kb pathway‐ [98]
From Cimicifuga foetida 468, estrogen receptor‐expressing
mediated necrosis
lines MCF‐7 and T‐47D
Molecules
Molecules 2024,
2024, 29,
29, 5275
5275 13
13 of
of 23
23
Molecules 2024, 29, 5275 14 of 23

Table 1. Cont.
Eupalinolide
Eupalinolide Triple‐negative
Triple‐negative breast cancer cell Triggering of
of cell
cell cycle
Establishedbreast cancer cell Triggering
Established cycle [98]
[98]
from Eupatorium
from lindleyanum Chemical Structure (PubChem Database- lines
lines arrest and
and apoptosis
arrestMolecular
apoptosis
Metabolites/ Anticancer
https://pubchem.ncbi.nlm.nih.gov/ Accessed on References
Plant Origin Activity on Breast Cancer Mechanism of Anticancer
20 September 2024)
Cell Models Activity

Triple‐negative breast cancer cell

Triple‐negative
Triple-negative breast
breast cancer cancer
cell lines cell Cell
KHF16 *** lines MDA‐MB‐231, MDA‐MB‐ Cell cycle
cycle arrest
arrest in
in G2/M
G2/M
KHF16KHF16
*** *** lines MDA‐MB‐231,
MDA-MB-231, MDA-MB-468,MDA‐MB‐ Cell cycle arrest in G2/M phase,
phase,
phase, NF‐Kb
NF‐Kb pathway‐
pathway‐ [98]
[98] [98]
From Cimicifuga
From foetida foetida
From Cimicifuga 468,
468, estrogen
estrogen estrogen receptor‐expressing
receptor-expressing lines
receptor‐expressing NF-Kb pathway-mediated necrosis
mediated
MCF-7 and T-47D
lines MCF‐7 and T‐47D mediated necrosis
necrosis
lines MCF‐7 and T‐47D

Cell
Cell cycle
cycle arrest
arrest inin G2/M
G2/M
phase
phase mediated
mediated by
by ERK1/2
ERK1/2
Genistein
Genistein
Genistein
Cell cycle arrest in G2/M phase
kinase
kinase
mediated bypathway
pathway activation
activation
ERK1/2 kinase pathway [56]
isolated from
from soya
isolatedisolated fromand
soya soyasoya
and soya
and soya MDA‐MB‐231
MDA‐MB‐231
MDA-MB-231 [56] [56]
and
activationdownregulation
and downregulation
and downregulation of
of of
products
products
products Cdk1, cyclin
Cdk1,
Cdk1, cyclinB1
cyclin B1and
B1 and
andCdc25
Cdc25
Cdc25C
C
C

Note: The
The chemical
Note: Note: chemical structures
structures of plant metabolites
metabolitesareare from PubChem: https://pubchem.ncbi.nlm.nih.gov/ accessed onon 2020 September 2024.
2024. ** The myconoside
The chemical structuresofofplant
plant metabolites are from
from PubChem:
PubChem: https://pubchem.ncbi.nlm.nih.gov/
https://pubchem.ncbi.nlm.nih.gov/ accessedaccessed
on 20 September September
2024. * The myconosideThe myconoside
structure from Haberlea
structure
structure from
from Haberlea
rhodopensis Haberlea
as rhodopensis
rhodopensis
determined by MS/MS as determined
determinedinby
as identification by MS/MS
MS/MS
reference identification
identification
[90]. in
in reference
** The Platycodin D structure[90].
reference [90]. **
** The Platycodin
Thefollowing
is described Platycodin D
D structure
structure
the link: is
is described
described following
following the
the link:
link:
https://www.selleckchem.com/products/platycodin-
https://www.selleckchem.com/products/platycodin‐d.html
d.html accessed on 20 September 2024. *** KHF 16 structure accessed
https://www.selleckchem.com/products/platycodin‐d.html is described
accessed on 20
20 September
onfollowing the [98].2024.
September 2024. *** KHF 16
*** KHF 16 structure
structure is
is described
described following
following thethe [98].
[98].
Molecules 2024, 29, 5275 15 of 23

4. Naturally Occurring Plant Bioactive Peptides and Mechanisms of Their Cytotoxic

Effects on Breast Cancer Cell Lines
Other compounds of plant origin with a potential in cancer therapy are plant bioactive
peptides, which are also a subject of intense research and, due to the different chemical
nature compared to secondary metabolites, could have different targets and may present
synergism if combined with conventional drugs or phytochemicals. Recently an increasing
interest has manifested in the application of bioactive peptides in cancer therapy as an
alternative or supplement to conventional drugs, especially in diminishing the severe side
effects and reversing multidrug resistance [101]. Anticancer peptides are usually composed
of 10–100 amino acids linked by peptide bonds in a linear or cyclic manner [102,103]. They
present better biocompatibility and biodegradability compared to conventional drugs, as
well as structural variability and high affinity binding, which makes them versatile tools
for selective tumor targeting; however, some immunogenicity or in vivo degradability by
proteases cannot be excluded [101]. Bioactive peptides could be obtained from virtually
all living organisms, including more than 3000 plant species; plant families of Fabaceae,
Asteraceae, Solanaceae, Cucurbitaceae, and Brassicaceae are particularly rich in anticancer
peptides [101,102]. Based on their source, bioactive peptides can be natural (well-defined
molecules or protein hydrolysates), artificially modified (for example, to resist proteolysis
or to carry some cargo molecules), or in vitro synthesized. Lately, artificial intelligence has
been actively used for screening possible adverse effects, immunogenicity, or peptidase
degradability [102]. In the application of bioactive peptides, an advantage is taken of the
different properties of cancer cells compared to normal ones, such as higher membrane
fluidity with increased cell surface area and higher net negative charge and certain overex-
pressed receptors on tumor cell surface [104]. According to the mode of anticancer action,
peptides could disrupt cell membrane integrity by forming pores, could bind to receptors
and activate downstream signaling, and could penetrate the cell and exert their cytotoxic
effects inside [101–104]. There are several reviews summarizing the current research on
anticancer peptides, including protein hydrolysates [101,102,104,105]. It is essential to
know the exact mechanism of anticancer action and to relate structure to function, which is
possible only for peptides with well-known amino acid sequences. In the present review,
the attention is focused on two structurally well-characterized kinds of peptides—soybean
lunasin and Bowman–Birk type legume protease inhibitors, whose cytotoxic effects on
breast cancer model cell lines have been thoroughly studied. Moreover, it has been estab-
lished that a co-occurring protease inhibitor protects lunasin from proteolytic degradation,
thus increasing its bioavailability [106].

4.1. Lunasin
Lunasin is a soybean-derived bioactive peptide of 5.5 kDA (43 amino acids, SKWQHQQ
DSCRKQLQGVNLTPC-EKHIMEKIQG-RGD-DDDDDDDD, four structural fragments).
It was discovered in 1987, and its antimitotic and cell death effects were established
12 years later [107]. The RGD motif (Arg-Gly-Asp) competes with integrins, suppressing
the integrin-mediated signaling pathway [108]. This motif mediates the binding of the
peptide to receptors and its internalization into tumor cells; besides, binding through
the RGD motif could directly activate caspase-3 and promote apoptosis. The C-terminal
aspartic acid residues can interact with chromatin, and the second fragment of nine amino
acids is responsible for binding to the histone core [107]. Lunasin structure presents highly
disordered and flexible features typical for intrinsically disordered proteins, which partic-
ipate in transcription and translation regulation, DNA condensation, cell cycle, mitosis,
and apoptosis [107]. Lunasin has no inhibiting effects on the normal breast cancer cell
line MCF-10A, whereas 50% inhibition of proliferation in the triple-negative MDA-MB-231
breast cancer cell line is achieved at a twice lower concentration than that of the hormone-
responsive MCF-7 cell line. In both cancer cell lines, lunasin treatment decreased aromatase
gene expression and activity, vascular endothelial growth factor (VEGF) secretion and
cell vitality, and induced cell apoptosis [107]. Estrogen receptor (ER)α gene expression
Molecules 2024, 29, 5275 16 of 23

was decreased by lunasin treatment, and ERβ gene levels were significantly increased in
MDA-MB-231 cells [109]. Transcriptomic and proteomic analysis of breast cancer cell line
MDA-MB-231 treated with synthetic lunasin and lunasin from overexpressing transgenic
maize demonstrated apoptosis activation by significant upregulation of cysteinyl aspartate
specific proteinase (CASP) 3, CASP 7, and CASP 14, almost 10-fold increase in Bax/Bcl-2 ra-
tio, and down-regulation of DNA replication genes [110,111]. In the MCF7 cell line, lunasin
up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome
ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced
nuclear PTEN localization, and the induced apoptosis was p53-independent [112]. In
both MCF7 and MDA-MB-231 cell lines, 10–20 µM lunasin inhibited the expression of
matrix metalloproteinase (MMP)-2/-9, the phosphorylation of focal adhesion kinase (FAK),
Src, Akt, ERK and nucleus translocation of NF-κB, thus demonstrating the possibility of
metastasis suppression in breast cancer cells through integrin-mediated FAK/Akt/ERK
and NF-κB signaling pathways and downregulation of MMP-2/-9 [108]. The anticancer
action of lunasin is preserved by a protease inhibitor Bowman–Birk type [106].

4.2. Protease Inhibitors

Proteases have a crucial role in maintaining proteostasis in normal cells and the extra-
cellular matrix [113,114]. Key proteases in this respect are the ubiquitin-proteasome system
in the cytoplasm, the human caseinolytic protease p (hClpP) in mitochondria, which is a
serine-type protease, the cysteine protease cathepsin B of the autophagy lysosomal system,
and matrix metalloprotease family which participates in extracellular matrix remodel-
ing [113]. The 26S proteasome coordinates the regulation and degradation of redundant,
unwanted, and misfolded proteins, as well as short living regulatory proteins critical for
cell growth, cell cycle progress, signaling pathways, and pro-apoptotic and antiapoptotic
proteins. Tumor growth, invasion and metastasis are associated with upregulation of the
main proteolytic activities, which were revealed as promising drug targets. Numerous
small-molecule protease inhibitors have been developed, some of them being in clinical
practice or the preclinical stage; however, the therapeutic effect has been not as strong as
expected based on in vitro results on cell cultures [93]. One possible reason could be the
complex role of certain proteases, which could be dual—pro- and anti-autofagy [115], as
well as the complex interplay between various proteases, including both up- and downreg-
ulation [114].
Other sources of therapeutic agents targeting proteolysis in tumor cells are the protease
inhibitors of plant origin, such as Kunitz trypsin inhibitors (18–24 kDa), Bowman–Birk
inhibitors (6–9 kDa, 60–80 amino acids), phytocystatins (10–23 kDa) recently reviewed
by [116]. Leguminosae and Gramineae seeds are particularly rich in protease inhibitors [117].
Of special interest is the two-headed trypsin-chymotrypsin inhibitor from soybean, origi-
nally described by Bowman in 1946 and characterized by Birk in 1960. This inhibitor has a
characteristic tightly packed molecular structure with seven disulfide bonds, providing
exceptional thermo- and pH-stability, and two opposite active centers, enabling indepen-
dent inhibition of two target serine proteases [116,117]. A serine-type protease activity has
been identified in the cytosol of cancer cells which was inhibited by soybean Bowman–Birk
protease inhibitor (BBPI) [118]. The mechanisms of the BBPI antitumor effect have been
studied in detail in breast cancer cell lines. In MCF7, it was established that Bowman–Birk
inhibitor specifically inhibits the proteasomal chymotrypsin-like activity and suppresses
cell proliferation through the accumulation of MAP kinase phosphatase-1, accompanied by
accumulation of ubiquitinated proteins and the proteasome substrates, p21Cip1/WAF1 and
p27Kip1, downregulation of cyclin D1 and cyclin E and cell cycle arrest at G1/S phase, and
decrease of phosphorylated extracellular signal-related kinases (ERK1/2) [119]. BBI had no
inhibitory effects on EGF-stimulated activation of ERK1/2 and Akt [119]. A similar anticar-
cinogenic effect has been established for BBPI from Vigna unguiculata seeds on MCF7 cell
line—cell cycle arrest in S and G2/M phase, proteasome inhibition, apoptosis and lysosome
membrane permeabilization; the cytostatic effects were accompanied by alteration in nu-
Molecules 2024, 29, 5275 17 of 23

clear morphology, plasma membrane fragmentation, cytoplasm disorganization, presence

of double-membrane vesicles, mitochondrial swelling, and an increase in the size of lyso-
somes, DNA fragmentation, mitochondrial membrane potential reduction, and cytoplasm
acidification [120]. In another study on the effects of purified BBPI from Vigna unguiculata
seeds on cell lines MCF7, MDA MB 231, and MCF10A, a cytostatic effect at the G2/M
phase of the cell cycle was found along with apoptosis induction in a caspase-dependent
manner through mitochondrial impairment and oxidative damage, following proteasome
20S inhibition, with no cytotoxic effect on normal mammary epithelial cells [121]. The
Bowman–Birk inhibitor affected NF-kB target gene expression in both MCF7 and MDA MB
231 breast cancer lines [121]. Soybean BBI inhibited the cell growth of the MDA-MB-231
cell line in a dose-dependent manner, with an IC50 of 200 µg/mL, altered the expression of
autophagy-related genes Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased
the Bax/Bcl2 ratio. Thus, Bowman–Birk protease inhibitor-induced apoptosis by changing
the Bax/Bcl2 expression ratio [122]. A limited number of studies have reported enhanced
cytotoxicity of the combined treatment with cisplatin and BBI on MCF7 cells without signifi-
cant adverse effects on normal cells [123,124]. It is interesting to note that the Bowman–Birk
protease inhibitor has been reported to possess radioprotective activity, which is related to
the chymotrypsin-inhibitory region of its molecule [125]. The radioprotective effect of BBPI
was established for normal fibroblasts but not for the transformed fibroblasts [126].

5. Conclusions and Future Directions

The search for convenient breast cancer treatment has become increasingly important
due to the development of multidrug resistance and the severe side effects of conventional
chemotherapy [127]. In this respect, plant natural resources in terms of a variety of sec-
ondary metabolites and bioactive peptides could be of great value. Plant secondary metabo-
lites have a wide distribution, and some of them have been used in traditional medicine for
centuries. They present a broad spectrum of health promoting activities—antioxidant, an-
timicrobial, antidiabetic, antihypertensive, neurostabilising, etc. This fact makes them good
additives to the standard anticancer treatment for the improvement of the general condition
and quality of life of the patients. On the other side, they show different anticancer effects
targeting various molecular receptors and, in this way, modulating signal pathways in can-
cer cells towards cell death. Some of these substances are lipid soluble; they pass through
the membranes and target key molecules, increasing apoptosis rate or abrogating kinase
signaling pathways, which are essential in breast cancer transformation. Accumulated
experimental evidence supports that some plant-derived substances have the capacity to
overcome multidrug resistance and in combination with standard anticancer drugs, could
increase treatment effectiveness. They interfere with the complex signaling pathways in
tumor development and exert cytotoxic effects by various mechanisms [128]. An added
value is that these plant-derived substances differentiate nonmalignant and malignant cells,
and in the majority of cases, they do not exert cytostatic/cytotoxic effects on the normal
cell lines [95]. Another advantage is the possibility of inhibiting the highly aggressive
triple-negative breast cancer forms, which cannot be influenced by hormonal therapy.
Disadvantages in applying plant secondary metabolites in combined cancer treatment
are linked to their low bioavailability, difficulties in accumulation of therapeutic doses of
some of them because of their insolubility in water, not well-known pharmacokinetic and
pharmacodynamics properties, and possible toxicity at higher doses. Low bioavailability
could be solved by the creation of synthetic homologs with the right modifications of their
active groups or the use of different carriers targeting them to breast cancer by recognizing
markers of breast cancer cells. Some plant metabolites could have side effects but data
are controversial and depend on the experimental animals used. The disadvantages of
applying bioactive peptides are linked to their susceptibility to protease digestion, some
immunogenicity, and undesired reactivity.
Despite disadvantages, plant anticancer compounds are a valuable additional resource
in cancer treatment, and ¼ to ½ of the patients diagnosed with cancer complement treatment
Molecules 2024, 29, 5275 18 of 23

with conventional drugs with herbs [129]. In this respect, it is important to know the
molecular mechanisms of action in the active constituents and to predict the interactions
with a chemotherapeutic, which could be of three kinds—synergism, an additive effect,
or antagonism (if two compounds bind to the same target). Knowledge of the molecular
mechanism of cytotoxic action, target sites, doses, and pharmacokinetics will be at hand to
go beyond the trial-and-error approach and predict possible interactions. In this respect,
the use of model cell lines with defined molecular characteristics close to those of in vivo
tumor cells is of enormous importance to check the effectiveness of certain combinations;
however, the experiments on cell lines cannot be extrapolated directly to cancer situation
in vivo [129].
Knowledge of the exact molecular mechanisms of the observed cytotoxic effects
permits combined treatments with substances targeting different pathways of cytotoxic
action. Despite recent progress, a largely unexplored field is the use of drugs, which target
the activated proteolytic machinery in tumor cells [113] and the combined treatment with
natural substances and/or drugs with different molecular targets, which could additionally
destabilize malignant cells and could be a way to reverse the development of multidrug
resistance [101,114].

Author Contributions: Conceptualization and writing—original draft preparation, D.Z., P.M., Z.G.
and L.S.-S.; writing—review and editing, S.Z., M.G., I.A., V.V., D.D. and D.M.; visualization, D.Z.,
P.M. and M.G. All authors have read and agreed to the published version of the manuscript.
Funding: The research was funded by NSF of Bulgaria, Grant number KΠ-06-H41/6.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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