Resveratrol in breast cancer treatment: from cellular effects to molecular mechanisms of action

Abstract

Breast cancer (BC) is one of the most common cancers in females and is responsible for the highest cancer-related deaths following lung cancer. The complex tumor microenvironment and the aggressive behavior, heterogenous nature, high proliferation rate, and ability to resist treatment are the most well-known features of BC. Accordingly, it is critical to find an effective therapeutic agent to overcome these deleterious features of BC. Resveratrol (RES) is a polyphenol and can be found in common foods, such as pistachios, peanuts, bilberries, blueberries, and grapes. It has been used as a therapeutic agent for various diseases, such as diabetes, cardiovascular diseases, inflammation, and cancer. The anticancer mechanisms of RES in regard to breast cancer include the inhibition of cell proliferation, and reduction of cell viability, invasion, and metastasis. In addition, the synergistic effects of RES in combination with other chemotherapeutic agents, such as docetaxel, paclitaxel, cisplatin, and/or doxorubicin may contribute to enhancing the anticancer properties of RES on BC cells. Although, it demonstrates promising therapeutic features, the low water solubility of RES limits its use, suggesting the use of delivery systems to improve its bioavailability. Several types of nano drug delivery systems have therefore been introduced as good candidates for RES delivery. Due to RES’s promising potential as a chemopreventive and chemotherapeutic agent for BC, this review aims to explore the anticancer mechanisms of RES using the most up to date research and addresses the effects of using nanomaterials as delivery systems to improve the anticancer properties of RES.

Graphical abstract

Introduction

Breast cancer (BC) is the fifth leading cause of death worldwide and in the year of 2020 alone it was responsible for 685,000 deaths globally. In addition, 2.3 million women were diagnosed with breast cancer in the same year [1]. The site of origin for the development of a majority of BC cases are the milk ducts. These cases are categorized as either ductal carcinoma in situ (DCIS) or invasive ductal carcinoma. The mutations occurring in the ductal epithelium can lead to the development of BC. Therefore, understanding the factors that are responsible for BC development can pave the way for the development of novel therapeutics [2–4]. Currently, the most common therapeutic modalities in BC management include chemotherapy, radiotherapy, surgery, and immunotherapy; however, bone marrow suppression and therapy resistance have limited the potential of these therapeutics. These treatment strategies have shown low efficacy in inhibiting triple-negative breast cancer (TNBC) progression, creating a need to discover other therapeutic options or adjuncts [5–7].

Overall, there are two main subtypes of BC, estrogen receptor (ER)-positive and ER-negative BC [8]. ER-positive BC can be further divided into luminal A and luminal B subtypes while ER-negative BC can be divided into the subtypes of human epidermal growth factor receptor-2 (HER2)-enriched and basal-like BC. Interestingly, the metabolism of these cancer types is unique when compared with one another. For instance, lipid metabolism accounts for cancer growth in luminal B subtype, while glycolysis is enhanced in HER2 and basal-like BC [9].

Owing to the advances in therapies and the understanding of the factors responsible for BC progression, the mortality rate has significantly decreased. However, the prognosis of BC patients remains unfavorable and is in desperate need of efforts for improvement [10–12]. The metastasis of BC cells is responsible for the high rate of mortality among BC patients. Metastasis is thought to be the cause of up to 90% of BC deaths with the most common sites of metastasis being bone (30–60% of cases), lung (21–32% of cases), liver (15–32% of cases), and brain (4–10% of cases). The metastatic site seems to be related to BC subtype [13]. Many factors, such as DNA mutations, metabolic changes, and environmental components, contribute to the development and progression of breast cancer. In addition, chemicals contained in pesticides, organic pollutants, beverage containers, and cleaning products may lead to increased susceptibility to cancer development, especially BC [14].

BC is a heterogenous disease and strategies for its treatment depend on the subtype [15–17]. For instance, endocrine therapy is commonly used for the treatment of ER-positive BC [18, 19]. Alternatively, monoclonal antibodies, such as trastuzumab, and tyrosine kinase inhibitors, such as lapatinib, can be utilized for treatment of HER2 BC [20]. Another therapeutic option for BC is the application of genetic tools, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), and the CRISPR/Cas system, for affecting molecular pathways involved in its progression, proliferation, and metastasis [21–23].

In the recent years, attention has been directed towards using plant derived-natural products in the treatment of BC [24–26]. Phytochemicals are affordable and well-tolerated compounds that have minimal side effects, and their multitargeting ability has made them suitable options in cancer suppression [27–29]. Furthermore, phytochemicals can be co-utilized with other chemotherapeutic agents in a synergistic capacity for combination cancer therapy [30–32]. In vitro studies have supported the use of phytochemicals as a potential breast cancer therapeutic agent as evidenced by several cellular mechanisms affecting cell survival. For example, triterpenoids have demonstrated the ability to induce apoptosis in breast cancer cells by preventing the signal transducer and activator of transcription 3 (STAT3) signaling pathway, activating caspases, enhancing the release of cytochrome c, and reducing mitochondrial membrane potential [33]. In addition to STAT3 prevention, phytochemicals prevent cell growth and reduce cell invasion. For example, Parikh et al. [39] found that an oleanane triterpenoid suppressed 4T1 BC cell invasion by inactivating Src and protein kinase B (Akt) and inhibiting c-Myc and Janus-activated kinase-1 (JAK1), resulting in an increased number of cells remaining in the G2-M phase. Sinh et al. [40] demonstrated that tea phytochemicals inhibited phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt-mediated and mammalian target of rapamycin (mTOR) pathways, preventing BC cell proliferation. Other anticancer mechanisms of phytochemicals include modulating the p53 tumor suppressor protein, preventing the aggregation of mutant p53, preventing metastasis and angiogenesis, and improving the sensitivity of cancer cells towards chemotherapeutics via affecting different intracellular pathways [34]. Other research has shown that certain phytochemicals can prevent cell proliferation, induce apoptosis, and increase autophagy in cancer stem cells. These mechanisms of action are supported by the ability to reduce the expression of nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), and matrix metalloproteinase 9 (MMP-9) and by affecting the expression of autophagic factors, such as LC3-II, Atg-7, and Beclin-1 [35]. The main limitation of these natural products is their poor bioavailability that is overcome using nanoparticles for their targeted delivery [36–48].

Among the many phytochemicals, 3,4’,5-trihydroxy-trans-stilbene (resveratrol, RES, Fig. 1) has attracted significant attention in recent years. RES is a nutraceutical agent that was first extracted from the roots of Veratrum album (white hellebore) in 1940 and Polygonum cuspidatum in 1963. It is found in common foods, such as pistachios, peanuts, bilberries, blueberries, and grapes. The biosynthesis of RES is based on the interaction between p-coumaroyl COA and three molecules of malonyl CoA in the presence of the enzyme stilbene synthase. RES exists as two forms, a cis- and a trans-isomer. The cis isoform is unstable and is not commercially available while the trans isoform is more stable but is sensitive to ultraviolet (UV) light and alkaline environments (high pH) that can convert the trans-isomer to the cis-isomer [49].

Fig. 1.

Chemical structure of RES

Owing to its antioxidative, cardioprotective, estrogenic, antiestrogenic, anti-inflammatory, and antitumor properties it has been used against several diseases, including diabetes, neurodegenerative diseases, coronary diseases, pulmonary diseases, arthritis, and cancer. RES may act as a double-edged sword in response to various diseases. For example, while RES promotes cell survival and prevents apoptosis in age-related diseases, it promotes apoptosis and prevents cell proliferation in cancer [50]. Regarding cancer therapeutics, RES acts as an antioxidant, chemotherapeutic, and chemopreventive agent that may affect various cancer types varying from myeloma and lymphoma to skin, lung, ovary, cervix, prostate, liver, pancreas, colon, thyroid, and breast cancer [51]. Anticancer mechanisms of RES include its role in the inhibition of COX-2 and the modification of enzymes involved in tumor prognosis such as ribonuclease, RNA and DNA polymerases, ribonucleotide reductase, and human DNA ligase. RES induces apoptosis by modifying the expression of p53, NF-κB, and Bcl-2 [52]. RES may act as a phytoestrogen agent that has either agonistic or antagonistic effects depending on cancer cell line. By acting as an inhibitor of the enzyme aromatase, RES inhibits the production of estrogen by cancer cells. RES also has the ability to act as a prooxidative agent, antiproliferative agent and as a tumor suppressor agent by affecting the ERα-associated PI3K/Akt pathway and by suppressing telomerase activity [53].

The aim of this comprehensive review is to explore the cellular and molecular effects of RES related to its antineoplastic effect in breast cancer. This review introduces the background, metabolism, and uses of RES and addresses its different therapeutic effects on breast cancer. The limitations, such as bioavailability and the effect of nanotechnology on improving its therapeutic effects of RES are discussed.

RES: background, metabolism, and health promoting impacts

RES is a secondary metabolite that is produced in a few plant species, such as mulberries, lingonberries, cranberries, red currants, bilberries, peanuts, pistachios, and grapes [54] and was first isolated from the root of Veratrum grandiflorum in 1940 [55]. RES is categorized as either a polyphenol and/or a stilbene and is partly credible for the “French Paradox” in the 90's, in which the French population had a low prevalence of obesity and cardiovascular disease despite their high fat diet. These beneficial effects may partly be due to a diet that consisted of red wine rich in RES [56]. Finally, the anticancer effect of RES was discovered in 1997 in a mouse model of skin cancer, thereafter its effects against different types of cancer cells was a topic of many research studies [49]. The trans isoform of RES is more abundant than the cis isoform and has potential therapeutic and biological activities. Nevertheless, both isoforms have the capacity to bind to glucose and generate glycosylated forms. Alkaline environments (pH level > 11) or UV irradiation can lead to the transformation of trans-RES to cis-RES [57, 58].

RES is absorbed by enterocytes via passive diffusion or carrier-mediated transport. Following transport, RES undergoes metabolism, leading to the production of either glucuronides or sulfates [59]. Up to 90% of ingested RES then travels to the colon and is fermented and conjugated via methylation, glucuronidation, or sulfation. These metabolites are then used by tissues and cells and the remainder is excreted through the urine [60]. It shows poor bioavailability (less than 12%) after oral administration, which is attributed to its structure, high molecular weight, and metabolism in the liver and intestine, and could restrict its therapeutic application [61].

RES has demonstrated various beneficial health effects, including antitumorigenic, antidiabetic, anti-inflammatory, antioxidant, hepatoprotective, renoprotective, neuroprotective, and cardioprotective activities [62–70]. In a study by Heimesaat et al. [71], RES was found to reduce oxidative stress and immune cell response. These effects were supported by the reduced secretion of nitric oxide by the colon and the improved function of the colonic epithelial barrier. Furthermore, RES displayed its neuroprotective effects via reducing neuronal damage by oxygen/glucose deprivation/reoxygenation (OGD/R) partly through inducing mitochondrial autophagy via the Parkin pathway [72]. Another pathway that is affected by RES is nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, a critical regulator of oxidative stress in cells [73–76]. RES administration resulted in a downregulation of Keap1 expression, therefore, inducing Nrf2 signaling, and leading to a decrease in oxidative damage [77]. RES administration has also been shown to decrease the severity of rheumatoid arthritis by inducing autophagy via p62 downregulation, decreasing the levels of interleukin-1β (IL-1β) and C-reactive protein as well as mitigating angiopoietin-1 and vascular endothelial growth factor (VEGF) pathways [78]. Other therapeutic effects of RES include improved fertility, reduced inflammation in heart failure, decreased hyperoxia-induced brain injury, and DNA damage prevention [79]. One of the most beneficial effects of RES include its anticancer properties which can be attributed to its effects on many different intracellular pathways. For example, RES downregulates the levels of Bcl-2, MMP-2, and MMP-9, and induces the phosphorylation of extracellular-signal-regulated kinase (ERK)/p-38 and FOXO4 [7, 80, 81].

Impacts of RES on BC

The anticancer effects of RES against breast cancer cells were confirmed in several different literature sources [82–84]. It was demonstrated that RES can affect all stages of tumor cells from their initiation to progression via inducing cell cycle arrest and apoptosis. Moreover, RES can affect several intracellular pathways to inhibit cellular proliferation and metastasis and induce tumor suppression [85]. The anticancer effects, different molecular mechanisms of action, and cellular effects of RES will be reviewed in the following sections.

RES effects on cell proliferation and metabolism

Apoptosis and cell cycle modulation

Apoptosis is a physiological mechanism that is responsible for preserving homeostasis, embryonic development, genome integrity, and immune system function [86–89]. Apoptosis occurs through two major pathways, either the intrinsic pathway or the extrinsic pathway. The extrinsic pathway involves death receptors and is activated by extracellular factors. The binding of death ligands to receptors recruits tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) to induce caspase-8 and mediate the extrinsic pathway of apoptosis [90]. On the contrary, the intrinsic pathway involves the mitochondria and is activated by intracellular factors, such as oxidative stress. The intrinsic pathway occurs due to the loss of mitochondrial membrane potential and the release of cytochrome c and other pro-apoptotic factors into the cytosol. Thereafter caspase 9 initiates a caspase cascade, ultimately triggering apoptosis. The anti-apoptotic factor Bcl-2 is downregulated while the pro-apoptotic Bax is upregulated in the intrinsic pathway of apoptosis [91–94].

Investigators have confirmed that RES is a promising agent in impairing the proliferation of MCF-7 breast cancer cells and stimulating apoptosis. Radiation therapy as well as RES can induce apoptosis in breast cancer cells. When combined with radiation therapy, RES enhanced the radiation’s ability to induce apoptosis by downregulating the expression of Bcl-2, and upregulating Bax and caspase-3, leading to apoptosis (Fig. 2) [62]. RES can also regulate the expression of genes affecting apoptosis in TNBC. The administration of RES (3–200 µM) decreased the expression of DNA polymerase delta 1 (POLD1) by binding to its domains (6s1m, 6s1n, 6s1o, 6tny, and 6tnz). POLD1 overexpression abrogated the potential of RES in triggering apoptosis in MDA-MB-231 cells. The POLD1 downregulation by RES results in the upregulation of apoptotic factor caspase-3, as well as a significant decrease in the levels of Bcl-2 and proliferating cell nuclear antigen (PCNA) (Fig. 2). The in vivo experiment involving a xenograft model confirmed the ability of RES to reduce tumor growth via POLD1 downregulation [95].

Fig. 2.

The potential of RES in regulating the proliferation rate of BC cells. Different intracellular factors are affected by RES that led to inhibit the cancer cells proliferation and and initiate/promote cell death via activating apoptosis and autophagy mechanisms. It could induce apoptosis in cancer cells via improving three main mechanisms: increasing the intracellular oxidative stress, enhancing the expression level of p53, and by regulating mechanisms involved in caspase activation. On the other hand, it prevents autophagy via activating the mTOR pathway and preventing compounds related to the activation of autophagosome creation

A comparative study has examined the potential of RES, radiation, piceatannol, and piceid in reducing the progression of MCF-7 BC cells. Among them, a combination of RES and radiation were more efficient in suppressing BC progression compared to piceatannol or piceid. This advantageous effect is attributed to the reduced antioxidant defense system that subsequently promotes reactive oxygen species (ROS) production in the cells. The upregulation of Bax, p53 and caspase-8 by the coadministration of radiation and RES led to apoptosis in MCF-7 cells [96].

The prooxidant activity of RES and its inhibitory impact on BC progression are mediated by regulating gene expression. RES reduces the expression of casein kinase 2 (CK2) and diminishes the viability of MCF-7 cells. Furthermore, RES impairs mitochondrial membrane potential, enhances ROS generation, and induces apoptosis, impairing BC progression (Fig. 2) [97].

It appears that along with apoptosis induction in BC cells, RES has the capability of triggering cell cycle arrest at S phase and reducing the number of 4T1 BC cells in G0/G1 phase [98]. Furthermore, the ability of RES to stimulate apoptosis can be enhanced via its combination with other types of therapeutic agents. The combination of RES and salinomycin exert a synergistic effect to induce apoptosis of ER-positive BC cells via the suppression of Wnt signaling and the inhibition of epithelial-to-mesenchymal transition (EMT). In the same study, it was also found that the toxicity of RES is concentration dependent, and RES did not exhibit any toxicity in low concentrations (less than 50 µM) [99]. In another study, the combination use of RES (20 µM) and raloxifene (1 µM) promoted the expression levels of Bax, p53, caspase-3, and caspase-8 and triggered apoptosis in BC cells [100]. Furthermore, RES (100 µM) stimulates p53 phosphorylation at serine 20 to sensitize cisplatin-resistant BC cells to apoptosis [101]. The combination of RES and sorafenib, a multikinase inhibitor and a part of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, enhanced ROS generation, cell cycle arrest (cyclin D1 and cyclin B1), and the upregulation of caspase-3, p53, Bax/Bcl-2 expressions, and poly (ADP-ribose) polymerase (PARP) cleavage (Fig. 2) [102]. These studies highlight the fact that RES is a potent regulator of apoptosis and cell cycle arrest in BC cells.

Autophagy regulation

Autophagy and apoptosis are mechanisms to induce programmed cell death (PCD), and the regulation of both processes are important in BC therapy [103–105]. Autophagy can function as a prodeath and a prosurvival mechanism in various cancers, especially BC. The autophagy induction by FAK family-interacting protein of 200 kDa (FIP200) leads to BC progression in a HER2-dependent manner. The inhibition of autophagy paves the way for BC suppression via reducing HER2 expression. This is mediated by HER2 aggregation in endosomes and their release in extracellular vesicles [106]. Furthermore, activation of prosurvival autophagy stimulates brain metastasis of BC cells [107]. The simultaneous inhibition of EMT/hypoxia-inducible factor 1α (HIF-1α) and autophagy mechanisms led to a decrease in BC carcinogenesis [108]. Although these experiments highlighted the tumorigenesis role of autophagy in BC, there are studies that show autophagy is vital for suppressing BC progression. For instance, autophagy flux induction could prevent the proliferation rate and angiogenesis in BC cells [109]. In addition to tumor growth suppression, autophagy induction seems to be vital for augmenting efficacy of chemotherapeutic agents [110]. Based on these studies, autophagy significantly affects growth and progression of BC cells, and its targeting is of importance in cancer therapy [111, 112]. The autophagy-related genes (ATGs), Beclin-1, unc-51 like autophagy activating kinase (ULK1), and AMP-activated protein kinase (AMPK) stimulate autophagy, while mTOR signaling suppresses autophagy (Fig. 2) [113–116].

RES and its combination with other chemotherapeutic agents in BC treatment seem to be a promising tool for inducing autophagy. For example, the combination of RES and doxorubicin was evaluated against BC cells. The results of in vitro studies confirmed the effect of combination therapy on long-term toxicity. Based on the bioinformatics analysis, CCND1, CDH1, ESR1, HSP10AA1, MAPK3, PTPN11, and RPS6KB1 are among the genes affected by RES in augmenting antitumor activity of doxorubicin (DOX) in BC suppression. Notably, inhibiting autophagy, as a prosurvival mechanism, can increase the potential of RES in enhancing cytotoxicity of DOX against BC cells [117]. Beclin-1 is an inducer of autophagy in MCF-7 and MDA-MB-231 cells. RES administration promotes cytotoxicity of DOX against BC cells by downregulating Beclin-1 and subsequently inhibiting autophagy [118].

The activation of prosurvival autophagy prevents apoptosis in BC cells and is in favor of their progression and growth. A combination of RES and rapamycin is involved in mechanistic target of rapamycin complex 1 (mTORC1) inhibition and the suppression of autophagy and Akt signaling. These interactions lead to enhanced apoptosis induction in BC cells. Based on this experiment, autophagy inhibition by RES and rapamycin potentiates apoptosis in BC cells and reduces their viability (Fig. 2) [119]. Another experiment revealed that a combination of RES and salinomycin decreased Beclin-1 expression to inhibit autophagy and impair progression of TNBC cells [120].

RES can induce noncanonical autophagy in a Beclin-1-independent mechanism to stimulate cell death in BC cells and to significantly diminish their progression [121]. Hence, autophagy has both functions in BC and its modulation is of importance for affecting the progression and proliferation rate.

Both apoptosis and autophagy are programmed cell death mechanisms that could regulate different molecular pathways. Increasing evidence has demonstrated a crosstalk between these two mechanisms in tumor cells. The activation of pro-survival autophagy inhibits apoptosis, while pro-death autophagy increases apoptosis in tumor cells, demonstrating their close interaction [122, 123]. Several studies have evaluated the role of RES in affecting both apoptosis and autophagy in breast cancer. An interesting experiment has revealed that RES is advantageous in increasing cytotoxicity of doxorubicin against breast cancer cells. Autophagy induction in breast cancer is a barrier towards doxorubicin-mediated apoptosis in tumor cells. RES administration inhibits inflammation and autophagy in potentiating doxorubicin-mediated apoptosis in breast cancer cells [118]. In respect to pro-survival function of autophagy, a combination of RES and rapamycin suppresses autophagy to potentiate apoptosis in breast tumor cells [119]. However, an experiment has shown that high concentrations of RES induce both apoptosis and autophagy in reducing the viability of breast tumor cells [124].

The overall mechanisms of RES on molecular pathways related to the apoptosis and autophagy of BC cells are presented in Fig. 2. Moreover, Table 1 summarizes the results based on the research studies evaluating the effects of RES on proliferation of different types of BC.

Table 1.

The RES in regulating proliferation rate of BC cells

Cell line	Study design	Signaling network	Effects	References
4T1 cells	50–250 µM	Not available	Increasing cells in S phase Decreasing number of cells in G1/G0 phase Inducing apoptosis via affecting different genes	[98]
MCF-7 cells	50–500 µM (RES) 1–100 µM (curcumin) 1–300 µM (piperine)	Not available	Reducing glyoxalase 1 (GLO1) expression Decreasing the potential of mitochondrial membrane Inducing mitochondrial dysfunction	[125]
MCF-7 cells	100, 200, and 300 µM	miRNA-122-5p	Enhancing the expression of miRNA-122-5p Inducing apoptosis and cell cycle arrest Decreasing the expression levels of Bcl-2, cyclin-dependent kinase (CDK)2, CDK4, and CDK6	[126]
MDA-MB-231, HCC-70, and MCF-7	0.005–5 µM	P53C	Suppressing proliferation and tumorigenesis of cancer cells Inhibiting p53C aggregation	[127]
MCF-7 and MDA-MB-231 cells	10, 25, and 50 µM	SIRT1	Inducing cell cycle arrest Reducing BC cell respiration Triggering inhibition of mitochondrial complex I and ATP synthesis process Inducing mitochondrial alteration Increasing SIRT1 expression Inducing cancer stem cells differentiation	[128]
MDA-MB-231 and MCF-7 cells	10- 40 µM	Not available	Modulating DNA methylation and histone modification via decreasing the activities of DNA methyltransferase (DNMT) and Histone deacetylases (HDAC) Improving the Bax expression and reducing Bcl-2 expression Inhibiting the proliferation of cancer cells and inducing cell apoptosis	[129]
MCF-7 and T47D cells	10–80 µM	ERK1/2/EZH2	Inhibiting cell proliferation and colony formation Enhancer of zeste homolog 2 (EZH2) down-regulating via suppressing ERK1/2 axis	[130]
MDA-MB-231 cells	10–160 µM	Notch1	Decreasing expression levels of Notch1, Dll4, Hes-5, and Jagged1 Reducing the angiogenesis capability of cancer cells Inhibiting cancer cell proliferation	[131]
MDA-MB-231 cells	6.25 and 25 µM	c-Myc/miRNA-17	Suppressing the expression of c-Myc Down-regulating miRNA-17 Promoting the expression level of MHC class I chain related-proteins A/B (MICA/B) Elevating the cytolysis of BC cells by natural killer (NK) Improving the immune response against tumor cells	[132]
MCF-7 and MDA-MB-231 cells	12.5, 25, and 50 µM	HIF-1	Decreasing expression levels of HIF-1 and VEGF at hypoxic conditions Inhibiting the tumor growth Reducing the Ki-67 (an index marker of proliferation) and CD31 (a microvessel density biomarker)	[133]
MCF-10A-Tr	5–100 µM	p21 protein expression	Increasing the expression level of p21 protein Reducing the expression level of antiopototic proteins (like PI3K, AKT, NFκB) Decreasing the expression of cell cycle regulatory proteins and base excision repair proteins Inducing apoptosis	[134]
MCF-7 and MDA-MB-231 cells	10–200 µM	ATP2A3	Decreasing proliferation rate and inducing apoptosis in a concentration-dependent manner Inducing the expression of Sarco/endoplasmic reticulum 3 (SERCA3) Enhancing the expression level of ATP2A3 Decreasing the expression of Bcl-2 and Ki67 genes in MCF-7 cells Increasing the expression of Bim gene in MDA-MB-231 cells	[135]

Glycolysis regulation

There are significant differences in terms of metabolism between noncancerous and cancerous cells. Metabolism is an important process to evaluate in tumor cells due to their ability to rapidly divide, requiring a great amount of energy to carry out this process. The Warburg effect using glycolysis is an alteration in metabolism of BC cells to increase energy production. The high mobility group box protein 1 (HMGB1) upregulation and subsequent activation of fibroblasts stimulate aerobic glycolysis to promote progression of BC cells [136]. The Akt/STAT3 axis induces glycolysis and its upregulation by gremlin-1 leads to an increase in the proliferation rate of BC cells [137]. LINC00346, a long noncoding RNA (lncRNA), and circ-0008039, a circular RNA (circRNA), also affect glycolysis in modulating BC progression [138, 139]. Phytochemicals showed high capacity in suppressing the glycolysis pathway and are therefore promising candidates for BC therapy [140, 141].

To date, only one experiment has evaluated the potential of RES in affecting glycolysis in BC cells, indicating that supplementary research is warranted. Exposure of MCF-7 cells to RES (1–100 µM) significantly decreased their survival rate and reduced their cellular metabolism. The phosphofructokinase (PFK) overexpression seems to be vital for glucose metabolism in BC cells and in enhancing their viability and proliferation. RES’s administration is responsible for decreasing ATP production and glucose metabolism in MCF-7 cells. Furthermore, RES induced the dissociation of PFK from fully active tetramers and reduced its expression. As a result, RES decreased PFK activity, preventing glycolysis and glucose metabolism in BC cells and decreasing cellular growth rate [142]. Further experiments are encouraged to evaluate other molecular pathways that are affected by RES. In addition, the role of RES in glycolysis and its effect on the proliferation of BC cells are also topics that should be further evaluated.

RES effects on modulation of tumor microenvironment

The tumor microenvironment (TME) is the environment of tumor (stem) cells that contains different signaling molecules, extracellular matrix, bone marrow-derived inflammatory cells, immune cells, blood vessels, and fibroblasts. It can directly affect the EMT and improve the invasiveness and metastatic properties of tumor cells [143, 144]. Embryogenesis, tissue formation, wound healing and tissue fibrosis mainly result from a well-known process of EMT [145, 146]. This process, which is reversible, includes the transformation of epithelial cells that have tight junctions and low motility to mesenchymal cells that have an increased ability to migrate [147]. There are three types of EMT, Type-I, Type-II, and Type-III which is responsible for increasing the metastasis of tumors [148]. In addition to morphological alterations, the EMT mechanism shows molecular changes that include the downregulation of E-cadherin, and the upregulation of the mesenchymal markers vimentin, N-cadherin, and desmin. Various factors including Snail, Zinc finger E-box-binding homeobox 1/2 (ZEB1/2) and transforming growth factor-β (TGF-β) can regulate EMT in tumor cells [149]. In addition to promoting the invasion of tumor cells, the EMT mechanism is involved in drug resistance [150]. Therefore, the suppression of EMT may be considered a promising strategy in cancer therapy [151]. This section focuses on the impact of RES on the EMT mechanism in BC cells and related molecular pathways.

As previously mentioned, one of the well-known upstream mediators of EMT is TGF-β. The overexpression of TGF-β stimulates EMT and significantly enhances metastasis and migration of tumor cells [149]. A recent experiment has evaluated the potential of RES in reversing EMT in BC. In this experiment, the administration of RES (12.5–100 µM) inhibited TGF-β signaling and reduced the expression levels of its downstream targets that include Smad2 and Smad3 and as a result impaired the progression of BC cells. Following TGF-β signaling inhibition, a significant decrease in the levels of vimentin, Snail1 and Slug occurred, while E-cadherin levels increased to suppress EMT and metastasis of BC cells. Furthermore, the in vivo experiment demonstrated the potential of RES to inhibit lung metastasis of BC cells [152]. Another study revealed the role of RES in enhancing the sensitivity of BC cells to FL118, a derivative of camptothecin, via its effects on EMT. RES induced apoptosis via caspase-3/7 upregulation and impaired tumor proliferation via inducing G1 arrest. Furthermore, RES administration (0–200 µM) enhanced E-cadherin mRNA and protein levels and decreased the expression of N-cadherin, vimentin, Slug, Snail, Twist1/2, ZEB1/2 and fibronectin, which led to the suppression of EMT and BC metastasis (Fig. 3) [153]. Because of the association between EMT and drug resistance, suppressing EMT can significantly enhance the sensitivity of cancer cells to chemotherapy [154, 155]. A recent experiment revealed that RES can promote the cytotoxicity of cisplatin against BC cells. In this case, RES suppressed various molecular pathways including c-Jun N-terminal kinase (JNK), PI3K/Akt, ERK and NF-κB signaling pathways (Fig. 3). More investigation revealed that by affecting the aforementioned pathways, RES enhanced E-cadherin levels and inhibited TGF-β signaling to suppress EMT and promote CP sensitivity of BC cells [156]. The derivatives of RES have also been able to suppress progression and migration of BC cells. 3,5,4^/-trimethoxystilbene is an analogue of RES and suppresses the PI3K/Akt and Wnt/β-catenin signaling networks to impair metastasis of BC cells [157].

Fig. 3.

Antimetastatic effects of RES against breast cancer cells. RES could exhibit its antimetastatic effects via suppressing both epithelial-to-mesenchymal transition (EMT) and DNA replication through the activation and suppression of different intracellular proteins and pathways. It could suppress molecules and pathways related to improving the EMT and DNA replication from one side and activate the inhibitors of EMT from the other side, that finally leads to the inhibition of cancer cell metastasis and reduce the invasiveness features of the cancer cells

RES effects on metastasis ability of cancer cells

Effects on matrix metalloproteinases

The matrix metalloproteinases (MMPs) play a significant role in both physiological and pathological events. In physiological processes, the most important functions of MMPs include tissue remodeling and degradation of extracellular matrix (ECM). However, MMPs participate in increasing migration and invasion of tumor cells [158–162]. The serum levels of MMP-1 and MMP-3 are significantly enhanced in BC patients [163]. Moreover, the levels of MMP-10 and MMP-3 increase in BC patients and can be considered as diagnostic factors [164]. The downregulation of MMP-1 impairs metastasis and invasion of BC cells [165]. In addition to metastasis, reducing the expression levels of MMPs such as MMP-2 and MMP-9 will induce apoptosis in BC cells [166].

Insulin-like growth factor 1 (IGF-1) is responsible for MMP-2 upregulation and inducing the migration and invasion of BC cells. The activation of PI3K/Akt axis induces IGF-1 expression in promoting the migration of BC cells. RES administration (10 and 20 µM) impaired the migration and invasion of BC cells via inhibiting PI3K/Akt and therefore decreasing IGF-1 expression and preventing the upregulation of MMP-2 (Fig. 3) [167]. In addition to MMP-2, MMP-9 is also involved in increasing the metastasis of BC cells. The activation of heme oxygenase-1 (HO-1) signaling by RES reduced MMP-9 expression and prevented metastasis of BC cells. In a separate study, RES-loaded gold nanoparticles were found to enhance RES’s ability to reduce MMP-9 expression as compared to RES alone (Fig. 3) [168]. In vitro RES decreases MMP-9 expression and metastasis of 4T1 BC cells in a concentration-dependent manner. RES is also capable of impairing BC metastasis in vivo by targeting MMPs. The administration of RES (100 and 200 mg/kg) to BALB/c mice injected with 4T1 cells resulted in a reduced number of lung nodules and expression level of MMP-9 in plasma. These results demonstrate the potential of RES in impairing the migratory ability and therefore the metastasis of BC cells [169].

The heregulin-β-1 (HRG-β1) is another growth factor that can induce metastasis and invasion of BC cells via the upregulation of MMP-9 expression. HRG-β1 triggers ERK1/2 signaling to promote MMP-9 expression. RES administration (2, 5, and 10 µM) downregulated MMP-9 expression by inhibiting HRG-β1 expression to decrease BC migration and invasion (Fig. 3) [170]. NF-κB activation also promotes MMP-9 expression in BC cells [171–174]. RES administration (10–180 µM) inhibited NF-κB signaling and decreased MMP-9 expression to impair metastasis of BC cells [175].

STAT3 upregulation is another mechanism for promoting progression of tumor cells, enhancing their proliferation, preventing apoptosis, and inducing therapy resistance [176–181]. A derivative of RES, known as LYR71, suppresses STAT3 signaling to decrease MMP-9 expression and impair metastasis of BC cells (Fig. 3) [182]. Based on these statements, MMPs are potential downstream targets of RES and its derivatives in BC metastasis suppression [183–185].

Impairment of migration capacity

The previous sections revealed that both EMT and MMPs undergo inhibition by RES to impair metastasis of BC cells. This section focuses on other molecular pathways and mechanisms affected by RES in inhibiting BC migration [186, 187]. The tumor-evoked regulatory B cells (tBregs) are considered a factor involved in BC metastasis and may involve TGF-β signaling. RES administration (20 and 50 µg) decreased STAT3 expression to impair function and generation of tBregs, leading to TGF-β inhibition and the subsequent impairment of BC metastasis [188]. As previously stated, the lungs are one of the sites that is affected by BC metastasis. In a study by Han et al. [189], RES suppressed the migration of TNBC cells to the lung and inhibited metastasis, these effects may be related to the ability of RES to enhance antitumor immunity. RES administration (12.5 and 25 mg/kg) increased the number of CD8⁺ T cells and promoted the levels of IL-2 and IFN-γ. Further investigation revealed that RES inhibited PD-1 expression to promote CD8⁺ T cell activity and enhance Th1 immune responses. Furthermore, RES enhanced M1 polarization of macrophages in promoting antitumor immunity and suppressing lung metastasis of TNBC cells [189]. Another experiment also revealed the role of vaticanol C, as a RES tetramer, in reducing lymph node and lung metastasis of BC cells. However, this experiment has not shown any underlying mechanism responsible for this anti-metastatic activity (Fig. 3, Table 2) [190].

Table 2.

RES and its derivatives in suppressing invasion of BC cells

Cell line	Conc./dose	Signaling network	Effects	References
MDA-MB-231 cells	12.5–100 µM	TGF-β1/EMT	Inhibiting TGF-β/Smad2/3 axis Decreasing the expression level of EMT-related markers and increasing the amounts of E-cadherin Preventing the matrix metalloproteinase (MMP)-2 and MMP-9 secretion Preventing in vivo metastasis to lung	[152]
MDA-MB-436 cells	1–200 µM	Not available	Decreasing the viability of the cells Inhibiting EMT via E-cadherin upregulation and N-cadherin, β-catenin, Snail and Slug down-regulation Increasing the expression level of active Caspase-3/7 Enhancing the apoptosis	[153]
MDA-MB-231 cells	10–250 µM	PI3K/Akt JNK ERK NF-κB	Inhibiting the cell migration induced by TGF-β1 Inhibiting EMT via down-regulating PI3K/Akt, JNK, ERK, and NF-κB pathways Increasing cisplatin sensitivity of tumor cells Reducing body weight loss	[156]
MCF-7 cells	20 µM	Wnt/β-catenin PI3K/Akt	EMT inhibition and down-regulating PI3K/Akt and Wnt to impair progression and metastasis of BC cells Increasing the expression of E-cadherin	[157]
MDA-MB-435 cells	10 and 20 µM	PI3K/Akt/IGF-1/MMP-2	Suppressing PI3K/Akt signaling to down-regulate IGF-1 Reducing MMP-2 expression and impairing BC metastasis	[167]
4T1 cells	10–30 µmol/L	MMP-9	Reducing MMP-9 expression Decreasing the cell adhesion and migration	[169]
MCF-7 cells	2, 5, and 10 µM	HRG-β1/ERK1/2/MMP-9	Inhibiting HRG-beta1/ERK1/2 axis Reducing MMP-9 expression Preventing metastasis of BC cells	[170]
MCF-7 cells	10–150 µM	NF-κB/MMP-9	Down-regulation of MMP-9 via suppressing NF-κB signaling Downregulating Bcl-2 expression	[175]
MDA-MB-231 cells	5–80 µM	STAT3/MMP-9	Suppressing STAT3 signaling and reducing MMP-9 expression Inhibiting tumor migration	[182]
4T1.2 cells	20 and 50 µg	STAT3/tBreg/TGF-β	Suppressing STAT3 signaling Inhibiting tBreg Down-regulating TGF-β for impairing progression of BC cells Preventing cancer metastasis Promoting immunological mechanism	[188]
Murine tumor model	12.5 and 25 mg/kg	PD-1	Inhibiting PD-1 expression Promoting CD8 + T and Th1 cell responses Increasing the amounts of IFN-γ and IL-2 Enhancing antitumor immunity and preventing lung metastasis of BC cells	[189]
MDA-MB-231 and MDA-MB-468 cells	25 µM	RhoA/YAP	Inhibiting Ras homolog family member A/yes-associated protein 1 (RhoA/YAP) Impairing the metastasis of BC cells	[191]
MCF-7 cells	10–50 µM	Rad9	Decreasing the expression level of EMT markers Downregulating Slug Inhibiting cell proliferation Increasing the sensitivity of cancer cells via upregulating Rad9 as tumor-suppressor factor	[192]

Effects of RES on breast cancer stem cells

Cancer stem cells (CSCs) can promote the proliferation of cancer cells, cancer recurrence, and induce resistance to therapy [193–196]. RES has the ability to target CSCs in various tumors. Various events and mechanisms mediated by RES to suppress CSCs and their stemness properties include STAT3 inhibition, trans-differentiation towards endothelial lineage, apoptosis and autophagy induction and reduction of self-renewal capacity [53, 197].

To date, a few studies have evaluated the potential of RES in affecting CSCs in BC treatment. One experiment demonstrated that RES stimulated tumor-suppressor autophagy to diminish the progression of BC cells and prevent stemness by suppressing CSCs. Interestingly, both therapeutic effects were mediated by RES’s effect on Wnt/β-catenin signaling. Restoring β-catenin expression abrogated the potential of RES in suppressing autophagy and impairing CSCs in BC. By upregulating autophagy related 7 (ATG7) and Beclin-1 levels, RES induced autophagy and suppressed BC cell survival. Furthermore, RES inhibited BC stemness via suppressing CSCs. For these anticancer activities, RES inhibits Wnt/β-catenin signaling [198].

RES has the ability to prevent self-renewal activity and stemness of stem like-BC cells [199]. Salinomycin is a potent agent for targeting CSCs and its combination with RES can be beneficial in impairing BC progression [200]. Current studies are limited by the lack of findings and published research regarding various signaling networks involved in CSC features in BC and RES’s effect on them. Further experiments should aim to shed more light on this topic and the potential of RES to play a role in these pathways.

RES in reversing breast cancer therapy resistance

Drug resistance is considered a feature in which anticancer drugs have low capacity or are incapable of suppressing tumor progression and therefore lead to chemotherapy failure [201–204]. The process of drug resistance is complicated and various factors, such as tumor microenvironment, cancer cell heterogeneity, CSCs, immune cells, and macrophages participate in its development [205–208]. Other factors including genetic and epigenetic alterations and extrinsic factors including pH, paracrine signaling and hypoxia can result in drug resistance [209, 210]. These factors in addition to EMT, DNA repair, upregulation of tumor-promoting genes, and the enhanced activity of drug efflux transporters contribute to BC resistance to chemotherapy [210, 211]. Therefore, improving prognosis and overall survival of BC patients depends on reversing drug resistance [212]. Notably, tumor cells have developed resistance to radiotherapy. The activation of Nrf2 signaling promotes CSC features in BC via aldehyde dehydrogenase (ALDH) upregulation and mediates radio resistance [213]. The stimulation of STAT3 signaling by heparin-binding growth factor (HDGF) as an upstream mediator, prevents radio sensitivity in BC cells [214]. The epigenetic factors, such as lncRNAs and miRNAs, are also involved in radiotherapy response of BC cells [215]. As therapy resistance commonly occurs in BC, this section focuses on the function of RES in reversing drug resistance and radio resistance.

Resistance to DOX, an anticancer agent, commonly occurs due to EMT induction, apoptosis inhibition, downregulation of tumor-suppressor factors, upregulation of tumor-promoting pathways and the enhanced activity of drug transporters, such as P-glycoprotein (P-gp) [216–218]. It was demonstrated that hypoxia exposed BC cells causes HIF-1α signaling activation, enhancing carbonyl reductase 1 (CBR1) expression and contributing to DOX resistance. RES administration (0–10 µM) reverses DOX resistance in BC via downregulating HIF-1α expression and subsequently decreasing CBR1 expression [219]. The overexpression and increased activity of P-gp can also lead to DOX resistance in tumor cells. RES administration (2–10 µM) inhibits DOX drug resistance by reducing multidrug resistance 1 (MDR1) and P-gp expression to suppress DOX efflux out of BC cells [220]. Another experiment confirmed RES’s ability to increase the influx of DOX into BC cells. RES significantly diminishes MDR1, and multidrug resistance associated protein 1 (MRP1) levels to potentiate DOX’s cytotoxicity via promoting its intracellular accumulation. The combination of RES and DOX inhibits tumor growth by up to 60% in vivo [221]. Interestingly, the enhanced migration and invasion of BC cells resulted from EMT induction, contributing to DOX resistance. RES administration (12.5–200 µmol/L) promotes sensitivity of BC cells to DOX by increasing Sirtuin 1 (SIRT1) expression, and subsequently inhibiting β-catenin signaling and resulting in EMT inhibition [222]. Another important factor involved in DOX resistance is the upregulation of heat shock protein (HSP)-27 in BC cells. RES (250 µM) is capable of reducing HSP-27 expression in BC cells to promote their sensitivity to DOX chemotherapy. Furthermore, RES induces apoptosis via the mitochondrial pathway that consists of the release of cytochrome c into the cytosol [223].

The proliferation of tumor cells largely depends on activation of PI3K/Akt signaling. Phosphatase and tensin homolog (PTEN) is a negative regulator of PI3K/Akt signaling, therefore a PTEN mutation or deletion can result in the upregulation of PI3K/Akt signaling and contribute to tumor progression and therapy resistance [224–226]. RES administration (10–200 mg/L) inhibits PI3K/Akt axis to induce apoptosis and suppress tumor proliferation in vivo, leading to a significant increase in DOX sensitivity of BC cells [227]. In addition to DOX, combination BC therapy of RES and other chemotherapeutic agents has been investigated. Cisplatin resistance is another challenge in cancer chemotherapy [228]. Rad51 upregulation and H2AX downregulation can occur in BC cells to mediate CP resistance. RES administration (50 and 100 µM) reduces Rad51 expression, while promoting the phosphorylation of H2AX at serine 139 to enhance the sensitivity of BC cells to CP and to impair DNA damage repair [229]. Based on these experiments, RES is a potent agent in promoting sensitivity of BC cells to chemotherapy [230–232]. Noteworthy, RES can also be beneficial in improving radiosensitivity of BC cells. Hypoxia is involved in triggering radioresistance in BC cells. An experiment demonstrated that HS-1793, a RES analogue, is able to suppress angiogenesis and prevent hypoxia-mediated BC progression via downregulating HIF-1α expression, an important factor in enhancing radiosensitivity [233]. Other experiments also confirm the potential of RES in providing increased radiosensitivity of cells by exerting antiproliferative activity and triggering oxidative damage (Table 3) [234, 235].

Table 3.

The RES administration promoting therapy sensitivity of BC cells

Cell line	Conc./dose	Signaling network	Effects	Reference
MCF-7 cells	1 and 10 µM	HIF-1α/CBR1	Decreasing HIF-1α level at protein level Reducing hypoxic induction of CBR1 expression Promoting doxorubicin sensitivity	[219]
MCF-7 cells	2–10 µM	MDR-1	Augmenting doxorubicin toxicity against tumor cells Reducing MDR-1 expression Interfering with P-gp activity	[220]
MCF-7 cells	12.5–200 µmol/L	SIRT1/β-catenin	EMT inhibition Providing chemosensitivity Apoptosis induction SIRT1 overexpression and subsequent inhibition of β-catenin Inhibiting cell growth Suppressing cell migration	[222]
MCF-7 cells	250 µM	HSP27	Reducing HSP27 expression and sensitizing cancer cells to DOX Inducting apoptosis via mitochondrial pathway and mediating cytochrome C release Potentiating antitumor activity of doxorubicin	[223]
MCF-7 cells	10–200 mg/L	PI3K/Akt	Suppressing PI3K/Akt axis Preventing doxorubicin resistance Promoting cell apoptosis Inactivating the PI3K/Akt signal pathway Preventing metastasis	[227]
MCF-7 cells	50 and 100 µM	Rad51 H2AX	Preventing DNA damage repair via H2AX phosphorylation Down-regulating Rad51 protein, homologous recombination, and BRCA2 Sensitizing tumor cells to cisplatin chemotherapy	[236]
MCF-7 cells	5–200 µM	TGF-β/Smad/EMT	Inhibiting EMT via suppressing TGF-β and Smad pathway Increasing tamoxifen sensitivity	[230]
MDA-MB-231 cells	2.5–40 µM	MDR1 CYP2C8	Down-regulating MDR1 and CYP2C8 in impairing BC progression Enhancing cell apoptosis and sensitivity toward paclitaxel Preventing cell proliferation and colony formation	[231]
MDA-MB-231 and SK-BR-3 cells	10–25 µM	HER-2/Akt	Inducing apoptosis Promoting docetaxel sensitivity via suppressing HER-2/Akt axis Downregulating docetaxel-induced activation of MAPK and Akt	[232]
Mouse bearing FM3A cells	0.5, 1, and 1.5 mg/kg	HIF-1α	Reducing expression of HIF-1α and vascular endothelial growth factor protein Improving the perfusion and hypoxic status in tumor tissues Preventing angiogenesis Inhibiting the hypoxia-induced cancer stem cell properties Enhancing sensitivity to radiotherapy	[233]
MCF-7 and T47D cells	0.01–1000 ng/ml	Bcl-xl, Bcl-2, and Bax	Reducing the expression level of Bcl-xl and Bcl-2 genes in T47D and MCF-7 cells, respectively Enhancing the expression of Bax in MCF-7 Arresting cells in G2/M phase Improving the cytotoxicity of Herceptin	[237]

Nanotechnological approaches to improve bioavailability and therapeutic efficacy

Owing to the low bioavailability and hydrophobic nature of RES, the use of a delivery carrier could improve its activity. Among the most attractive methods used for this purpose is the application of nanotechnology [236, 239]. Accordingly, different types of nanoparticles have been introduced for the delivery of RES in BC treatment [240–245]. A recent experiment prepared polymeric micelles for RES delivery. The micelles were prepared using an emulsion method with Pluronic F127 block copolymer and D-α-tocopheryl polyethylene glycol succinate (or vitamin E-TPGS). The resulting nanocarriers had a particle size of 179 nm and were spherical in shape. Their encapsulation efficiency (EE) was 73% with drug loading of 6.2%. These nanoparticles had high biocompatibility and exerted no toxic impact on normal cells. The nanoparticles increased cellular uptake and significantly decreased the viability of BC cells [246]. The in vivo experiment also exhibited the use of micelles as a potential delivery system for RES to decrease cancer growth and volume [247]. Interestingly, polymeric micelles have been utilized for co-delivery of RES and docetaxel in BC chemotherapy. The application of polymeric micelles significantly decreased the IC₅₀ value of drugs (23 µg/ml and 10.4 µg/ml for RES and docetaxel, respectively). Due to their prolonged release, these two antitumor compounds exerted a synergistic effect on MCF-7 cells to decrease tumor cell viability [248]. Overall, lipid nanostructures appear as promising nanoscale delivery systems for RES [249]. The interesting features of lipid nanoparticles include their low particle size and high EE. A recent experiment prepared targeted lipid nanoparticles with a particle size of 88.3 nm and EE of 88% for the treatment of BC. The folate receptors, upregulated on the surface of BC cells, were utilized for selective targeting to these malignant cells. The modification of lipid nanocarriers with folic acid selectively targets BC cells overexpressing folate receptors. Increased cellular uptake of RES enhanced its cytotoxic effect on BC cells. The in vivo experiment revealed the ability of lipid nanoparticles to increase the bioavailability of RES from 6.37 µg/ml to 57.92 µg/ml, a ninefold increase [250].

A liposome, a vesicle that contains a phospholipid bilayer, can also be used as a delivery vehicle for RES. In one study, liposomes with antibodies targeted to HER2-positive BC cells were formed and contained RES and curcumin within them. The results of this study not only showed that the nanoliposome acted as a carrier for the delivery of the chemotherapeutic drug, but also as a therapeutic agent itself. The antiproliferative activity of the drug-loaded immunoliposome was dramatically higher when compared to the normal liposomes and free drugs [251]. Liposomes were also utilized for the co-delivery of RES and p53 gene. Positively charged nanoliposomes were synthesized using peptide-cationic lipid (CDO14). The CDO14 liposomes increased apoptotic activity of BC cells, confirming their potential as an anticancer agent [252]. Targeted liposomes containing black phosphorus (BP), catalase (CAT), and RES were applied for the combination of photothermal, photodynamic, and chemotherapy. In this study, utilizing folic acid enhanced the cellular internalization of loaded liposomes. Following cellular uptake, when the cells are exposed to near infrared light, the BP photothermal property led to the release of both drugs, RES and CAT with involvement in chemotherapy and photodynamic therapy, respectively. In this study, both targeting agent and photothermal effects led to an enhancement in the intracellular amounts of RES and improved its therapeutic effects [253].

Solid self-nanoemulsifying drug delivery system is another type of carrier that could be used for the combination delivery of RES with other therapeutic agents. The combination of RES and tamoxifen were delivered by this carrier into the breast cancer cells. This carrier could provide oral delivery of drugs to breast cancer cells via improving different features of drugs, including their bioavailability and anticancer and anti-inflammatory effects as well as providing P-gp inhibition and better cellular internalization [254].

The layer-by-layer (LBL) nanoparticles are another kind of delivery system used for RES in BC treatment. A lipid-based nanoparticle modified with different layers of chitosan (positive charge) and hyaluronic acid (negative charge) showed high biocompatibility and enhanced selectivity towards tumor cells [255, 256]. These effects can partially be attributed to the use of hyaluronic acid, which provides specificity by targeting the CD44 receptor, a receptor found on the surface of cancer cells [216]. As a result, the proliferation rate of MCF-7 tumor cells decreased significantly, while no adverse impact was observed on noncancerous healthy cells. The use of this delivery system resulted in an increase in cellular uptake and therefore an increase in the apoptosis of breast cancer cells [257].

Polymeric nanoparticles, which can be formed using either natural or synthetic polymers, are another type of carriers that can be used for RES’s delivery. For instance, albumin nanoparticles were used for co-delivery of paclitaxel (PTX) and RES to overcome drug resistance of tumors. This nanocarrier with a size of about 150 nm provided synergistic anticancer effects against multidrug resistance (MDR) cancer cells [258]. Green synthesized core–shell nanoparticles of acrysol oil coated with poly(lactic-co-glycolic acid) (PLGA) polymeric shell were also used for delivering RES and for improving RES’s anticancer activity. The fabricated nanoparticles with a size of about 375 nm showed high biocompatibility, controlled drug release capability, and improved cytotoxicity against MCF-7 breast cancer cells [259].

Oxidized mesoporous carbon nanoparticles (oMCNs) with a size of less than 200 nm were used for the encapsulation and delivery of RES. oMCNs displayed high loading capacity, excellent biocompatibility, and high percentages of cellular uptake. This nanoformulation enhanced the cytotoxic and pro-apoptotic effect of RES with the cleavage of PARP and caspase-3 [260].

Another interesting type of nanoparticle are nanoparticles that are stimuli-responsive which act in a specific location, for example, at the tumor site by releasing the anticancer drug based on the pH level, redox status, enzyme activities, etc. [261–263]. One of the features specific to tumor microenvironments is the increased level of glutathione (GSH) (0.5–10 mM) as compared to noncancerous cells (2–20 µM) [264]. Using this feature, a carrier can be designed that is responsive to GSH levels to target cancer cells [265]. The cyclodextrin nanosponges (NSs) possess disulfide bonds that provide them with the ability to respond to GSH levels [266]. The cyclodextrin NSs were used for delivery of RES with an EE of 80.64% and a loading efficiency of 16.12%. The RES-loaded NSs showed cellular uptake by BC cells instead of noncancerous cells. These nanoparticles were GSH-responsive and released RES at the tumor site to promote its cytotoxicity against BC cells [267]. However, there is no research regarding pH- or enzyme-sensitive nanoparticles for RES delivery in BC suppression and should be a focus in future research.

The composite formulation of nanomaterials can also be applied for the delivery of RES, alone or in combination with other therapeutic agents/methods. For instance, in situ hydrogels containing RES and dopamine-reduced graphene oxide (photothermal nanoagent) were used for the chemo-photothermal treatment of MCF-7 cancer cells. The results of this study confirmed the synergistic effect of combination therapy via enhancing the toxicity of the composite more than 2-folds (from 25% to about 70%) [268].

The high biocompatibility and increased bioavailability of RES using nanostructures as delivery carriers enhance RES’s anticancer effects and are benefits of using this delivery system (Fig. 4, Table 4) [269–271].

Fig. 4.

RES loaded nanomaterials for BC treatment. RES could be encapsulated in or loaded on the surface of different types of nanomaterials. This could improve the therapeutic effects of RES via improving its bioavailability, delivering it to its targeted site, and releasing it in a controlled manner

Table 4.

The role of nanoparticles for targeted delivery of RES in BC treatment

Nanovehicle	Particle size (nm) Zeta potential (mV) and encapsulation efficiency (%)	Study type	Cell line/animal model	Effects	Reference
Polymeric micelles	179 nm 73%	In vitro	MDA-MB-231 and MCF-7 cells	Spherical shape and good particle size High encapsulation efficiency High cellular uptake and biocompatibility Reducing survival of cancer cells	[246]
Polymeric micelles	Not available	In vitro In vivo	MCF-7 cells Rat	Co-delivery of RES and docetaxel in combination cancer therapy IC50 value of 23 µg/ml for RES and 10.4 µg/ml for docetaxel Sustained drug delivery Synergistic impact between RES and docetaxel	[248]
Polymeric micelles	Not available	In vitro In vivo	MCF-7 cells Rat	Co-delivery of RES and docetaxel in combination cancer therapy IC50 value of 23 µg/ml for RES and 10.4 µg/ml for docetaxel Sustained drug delivery Synergistic impact between RES and docetaxel	[248]
Layer-by-layer nanoparticles	Not available	In vitro In vivo	MCF-7 cells Mice	Increasing cellular uptake of RES and tamoxifen Apoptosis induction High biocompatibility and lack of toxicity of major organs, such as heart, lung, liver, spleen, and kidney	[257]
Cyclodextrin nanosponge	Less than 200 nm	In vitro	MDA-MB-231 cells	Selective toxicity towards tumor cells Release of drug at tumor site in a GSH-dependent manner	[267]
Lipid nanoparticles	88.3 nm − 42.15 mV 88%	In vitro In vivo	MCF-7 cells Rat	Surface modification of nanoparticles with folic acid promotes their selectivity towards tumor cells High cytotoxicity Increasing bioavailability of RES in vivo	[250]
Mesoporous carbon nanoparticles	Less than 200 nm 24.8%	In vitro	MDA-MB-231 cells	High biocompatibility and cellular uptake Increasing solubility of RES Apoptosis induction via upregulating PARP and caspase-3	[261]
Microneedle-assisted RES loaded lipid nanoparticles	200 nm Up to -12 mV Less than 70%	In vitro In vivo	MDA-MB-231 cells Rats	High localization in breast tissue Suppressing migration and invasion of breast tumor cells	[250]
Gold nanoparticles	170 nm Up to 80%	In vitro	MCF-7 cells	The gold nanoparticles have no capacity in inducing cell death in tumor cells, but their conjugation with RES shows anti-tumor activity against BC cells High biocompatibility	[85]
Graphene oxide nanoparticles	221.7 nm − 20.3 mV 86.9%	In vitro In vivo	MCF-7 cells Xenograft	High specificity and encapsulation efficiency High stability Providing both chemotherapy and photothermal therapy Decreasing cancer cell viability	[271]

Clinical trials

As previously discussed, RES is metabolized into glucuronide and sulfate conjugates in the liver and intestine. These conjugates are then utilized by tissues and cells with the remainder excreted via the urine. In addition, RES exists in a complex form with plasma proteins (ex. albumin) and low-density lipoproteins. While a small amount can be found in a free form in systemic circulation [85].

Despite several experimental and preclinical research performed on the application of RES for breast cancer treatment, the number of clinical studies is limited in this context. It has been revealed that the toxicity of RES depends on its dosage and duration of treatment. Utilizing doses more than 0.5 g/day could lead to symptoms, such as vomiting, diarrhea, flatulence, nausea, headache, rash, and/or abdominal cramps. However, this quantity varies amongst different resources, and some others mention doses lower than 1 g/day could be tolerable [272]. The dose-dependent effect of RES was evaluated in a clinical study in which two different doses of trans-RES (5 and 50 mg) were administered twice a day for 12 weeks to 39 different female volunteers with an increased breast cancer risk. Results of this study showed that increasing the amount of RES led to a decrease in the methylation of RASSF-1α, a tumor suppressor gene. A decrease in RASSF-1a methylation was directly associated with the downregulation of the expression of prostaglandin E2 (as a type of tumor promotional gene) [273].

The other main challenge that restricts the clinical application of RES is its low water solubility. To overcome this issue, a study evaluated the effect of soluble galenic form trans-RES on 15 volunteers. They divided volunteers into two groups, one group received a powder form of RES, while the other group received a soluble form. Their results showed significantly higher amounts of the soluble form of RES in the blood plasma in comparison to the powder form, confirming the advantage in bioavailability and tolerability of the soluble formulation of RES [274].

Conclusion, challenges, and future perspectives

Among the various kinds of cancers in women, lung cancer and BC are the most common and malignant. This review article focused on BC treatment by RES by evaluating several in vitro and in vivo studies. RES is capable of impairing the proliferation and viability of BC cells via apoptosis induction (mainly through the intrinsic pathway), autophagy regulation (both induction and inhibition), glycolysis inhibition, CSCs suppression via affecting different molecular pathways, such as Wnt/β-catenin, Akt and mTOR suppression. Besides, it has the capability of diminishing the metastasis and invasion of BC cells through the regulation of MMPs, EMT, and related molecular pathways, such as TGF-β, STAT3, and NF-κB. Owing to RES’s potential in inhibiting growth and invasion of BC cells, this plant derived-natural compound can be beneficial in increasing therapy sensitivity (chemotherapy and radiotherapy) of BC cells. However, the hydrophobicity of this compound, along with its nonselectivity, photolytic and chemical instability, and low permeability through the stratum corneum restrict the wide application of this therapeutic compound [82, 275]. These limitations could be addressed by examining the use of nanodelivery agents that could increase the bioavailability of RES, improve its stability, enhance its accumulation in its targeted site, and ultimately enhance its therapeutic impact on BC cells. Polymeric micelles, LBL nanoparticles, cyclodextrin conjugates, mesoporous silica nanoparticles, lipid, and gold nanoparticles as well as graphene oxide nanocomposites have been utilized in RES delivery and augment its suppressive activity against BC cells via promoting its accumulation at the tumor site.

One of the other main limitations of RES is the limited number of in vivo and human trial studies performed using this phytochemical and its different nanoformulated forms that restrict our knowledge about their distribution, metabolism, and probable side effects. Indeed, the nontargeted distribution could not only affect other organs, but also limits the therapeutic performance of RES [276]. Renal toxicity is another concern of RES, reiterating the importance of using targeted delivery formulations to prevent renal accumulation of RES [49]. Accordingly, it is critical to use targeted nanoparticles to deliver the drug directly to the cancerous tissue, which improves the efficacy of treatment, reduces the dosage of drug, and prevents the deleterious effects on other tissues. Based on this, different generation of nanomaterials are introduced, among the newest of them are smart targeted nanomaterials, which release their therapeutic cargo in response to a specific intrinsic or extrinsic factor. However, there are few clinical trials regarding the application of nanomaterials. This may be due to the novelty of this field of science and the lack of knowledge about the probable effects of these materials on cell organelles and genome, as well as its nonacceptance by the public. Indeed, despite several efforts and research performed in this context, there is a need of further preclinical and clinical research to explore the biocompatibility of these different nanoformulations and the effectiveness of their therapeutic compound (RES) against breast cancer cells. The research surrounding RES and its many effects on cellular response is in its early years, and further studies are further stressed to completely evaluate its potential role in other intracellular pathways.

Author contributions

MB: conceptualization, investigation, writing—original draft; MD: methodology, investigation, writing—original draft; AZ: conceptualization, investigation, writing—original draft; DK: methodology, investigation; SJ: investigation, writing—original draft; FHS: methodology, writing—original draft; MH: conceptualization, writing—original draft; TT: investigation, writing—original draft; MR: investigation, writing—original draft; SM: methodology, writing—original draft; AZ: investigation, writing—review and editing; AZ: writing—review and editing, supervision; DDG: writing—review and editing; AB: writing—review and editing, supervision, project administration.

Funding

None.

Availability of data and material

Not applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest.

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