Molecular mechanisms of flavonoids and their modulatory effects against breast cancer: A scoping review [version 1; peer review: awaiting peer review]

Background: Breast cancer is the most prevalent malignancy among women. It is a disease whose incidence and mortality rates are on the upsurge globally. Debilitating effects, cost and resistance to available chemotherapeutic interventions render them unideal. Dietary phytochemicals have been shown to have preventive and therapeutic effects. Research continues to affirm the role of flavonoids as potential chemotherapeutic agents in combating the disease. Understanding modulation of key cellular signalling pathways by flavonoids presents promising molecular targets that may be leveraged to develop better chemotherapeutic agents for breast cancer. Methods: To describe the in vitro and in vivo modulatory effects of flavonoids on molecular anti-cancer mechanisms we searched three databases. We included original articles describing modulation of cell signalling processes such as; cell cycle, apoptosis, autophagy,


Introduction
Breast cancer is the most commonly diagnosed disease in females worldwide (Sung et al., 2021). Of all the cancer cases in females, breast cancer leads with incidences and mortality rates of 2,261,419 (24.5%) and 684,996 (15.5%) respectively. According to the global cancer observatory (GLOBOCAN) 2020, these figures are projected to rise (Sung et al., 2021). This, therefore, calls for the urgent unearthing of interventions to help curb the disease.
Breast cancer is categorized into four molecular subtypes namely; Luminal A (estrogen receptor positive (ER+), progesterone receptor positive (PR+) and human epidermal growth factor 2 positive (HER2+)), Luminal B (ER+, PR+ and variable expression of HER2), HER2+ (ER-, PR-and HER2+) and basal like/triple negative (ER-,PR-and HER2-) (Eliyatkin et al., 2015). Treatment regimen of breast cancers is dependent on the type and stage of the disease. It incorporates either; surgery, radiotherapy or chemotherapy (targeted therapy) (Brufsky & Dickler, 2018). Luminal A constitutes 50% of the breast cancer cases, responds favourably to endocrine treatment and has good prognosis. Luminal B constitutes 20%, responds to endocrine treatment (tamoxifene and aromatase inhibitors). However, it has poor prognosis compared to luminal A. HER2+ constitutes 15%, responds to trastuzumab and anthracyclin therapy, and has unfavourable prognosis. The rare TNBC constitutes about 15%, does not show any response to either endocrine or trastuzumab therapy and has the worst prognosis (Eliyatkin et al., 2015). Alarmingly, up to date, no specific treatment has been developed for TNBC (Carels et al., 2016). Grave challenges with regards to disease metastasis, drug resistance and tumor relapse necessitates the search for alternative therapies to aid in combating the disease (Israel et al., 2018).
Breast cancer occurs in a multifaceted fashion. A range of risk factors may contribute to the development and progression of the disease. Among these are; demographics (female age and advanced age), reproductive (early menarche, late age of menopause, pre-term delivery, nulliparity, older age at first full term pregnancy), hormonal (oral contraceptives and post menopausal hormone replacement therapy), hereditary (genetic factors, history of breast cancer in first degree relatives), breast related (benign breast tumors), lifestyle (obesity and overweight, alcohol consumption, smoking, diet) and others (air pollution, higher socioeconomic status and exposure to radiation) (Eliyatkin et al., 2015;Kikuchi et al., 2019).
The mechanisms through which normal cells progress to carcinogenicity as outlined by Hanahan and Winberg in their review of the hallmarks of cancer are bountiful (Hanahan & Weinberg, 2011). Potentially, the hallmark processes present possible target mechanisms that can be leveraged for treatment. Healthy dietary choices such as regular consumption of fruits and cruciferous vegetables which are rich sources of phytochemicals have been reported to protect against various cancers including breast cancer (Vrhovac Madunić et al., 2018).
Phytochemicals are secondary metabolites released by plants in response to environmental cues which may be either biotic or abiotic factors (Ashraf, 2020). Flavonoids are a class of phytochemicals belonging to a group of phenolic compounds. Over 4000 flavonoids have been documented, some of which are known to play a significant role in the prevention and treatment of breast cancer (Liu, 2004). Flavonoids are structurally classified into six sub-types namely; flavanones (hesperetin), flavanols (epichatechin), flavones (apigenin, wogonin, baicalein), flavonols (kaempferol, quercetin), anthocyanidins (cyanidin) and isoflavonoids (genistein) (Abotaleb et al., 2019). They have been reported to modulate signalling mechanisms known to enhance development of breast cancer cells such as; proliferation, cell cycle, anti-apoptosis, angiogenesis, invasion and metastasis (Nkwe et al., 2021).
Although many flavonoids have been widely reported to exert anticancer effects, their mechanisms of action have not been fully elucidated (Kopustinskiene et al., 2020). Therefore, understanding these mechanisms is critical as they can be leveraged as targets for effective prevention and treatment of breast cancer. Secondly, flavonoids may be used to develop novel plant-based chemotherapeutic agents which are believed to harbour less side effects, are less toxic and more effective compared to the conventional regimens (Nkwe et al., 2021). This review therefore, reports a number of flavonoids shown to have chemotherapeutic properties and profiles their mechanism(s) of action in different breast cancer cell lines and animal models.

Protocol and Registration
The results are reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . Scoping reviews purposes to; establish evidence available, elucidate key ideas, establish how research is done and determine knowledge gaps for a certain topic (Munn et al., 2018).

Eligibility criteria
Articles were included based on the following criteria; 1. The studies primarily focuses on the molecular mechanism of flavonoids in breast cancer 2. The studies to have employed either in vitro and/or in vivo approaches 3. The studies were published from 1 st January 2017 up to 31 st December 2021 4. The studies were in English language 5. The studies' full texts were available Articles were excluded based on the following criteria; 1. The studies were reviews, short communications, editorials 2. The flavonoids were laboratory modified or synthetic 3. The studies were cohort, case-control or in silico 4. The studies were fractions from plant extracts 5. The studies were co-chemotherapies (combined flavonoids with conventional anti-cancers)

Information sources
Articles relevant to the study topic were searched and retrieved electronically from PubMed (https://pubmed.ncbi.nlm. nih.gov/advanced/), Hinari (https://portal.research4life.org/content/ hinari) and Google Scholar (https://scholar.google. com/) using the advanced search builders. The search from the databases was lastly done on 6th January 2022 Search strategy An advanced search in three databases; PubMed, Google Scholar and Hinari were searched to identify peer-reviewed articles on breast cancer, breast cancer therapy using flavonoids and mechanism of action of flavonoids on breast cancer. Specifically, the search queries consisted of relevant medical subject headings (MeSH) and key words relevant to the topic. The search terms included; Breast cancer AND flavonoids OR flavanones OR flavanols OR flavones OR flavonols OR anthocyanidins OR isoflavonoids Selection of sources of evidence Using the search strategy and filters outlined in the search strategy section, the articles obtained were further vetted. Assessment of the resulting articles was done independently by all the reviewers. Disagreements between them were resolved through consensus. First, articles from the initial search were obtained. Duplicate references were removed through manual deduplication. The titles and abstracts of the retrieved articles were screened for relevance to the study topic. Full-text reports were examined for compliance with eligibility criteria.

Data chatting process
Data chatting from the sources of evidence was first assessed independently and then discussed by the team to reach a consensus. The information abstracted was as shown in the table.

Data items
Selection of the review articles based on the molecular mechanism of action as the main outcome domain was guided by the following items;

Synthesis of results
Results from the selected articles were tabulated in the summary of findings (Table 1). The methodologies and molecular mechanisms of action (interventions) were summarized. This enhanced comparison of interventions for the different breast cancer cells.

Search results
Preliminary search done on the three databases yielded 370 articles. Following deduplication, 270 articles were screened through reviewing their titles and abstracts. A total of 175 articles were retrieved for full text review. The other 15 were not retrieved. Full text review against inclusion and exclusion criteria led to elimination of 139 articles. Reasons of exclusion are outlined in the PRISMA flow diagram of the scoping review.

Characteristics of sources of evidence
The characteristics for which data was abstracted from each source of evidence are described in Table 1. They include; article citation, flavonoid name, cell lines and/or animal models, key findings and postulated mechanism of action. Overall, most studies reported induction of apoptosis as the most common molecular mechanism through which different flavonoids exerted their anticancer effects against breast cancer (n=18). Other mechanisms proposed included; inhibition of migration and invasion (n=10), cell cycle arrest at G1 phase (n=4), inhibition of angiogenesis (n=4), inhibition of metastasis (n=4), reduction of cell viability (n=4), reduction of cell proliferation (n=4), cell cycle arrest at G2/M phase (n=3), induction of autophagy (n=2), inhibition of migration and enhancement of motility (n=1), inhibition of intravasation (n=1), inhibition of breast cancer stem cell proliferation (n=1), inhibition of growth (n=1), enhanced pyroptosis (n=1) and reduction of breast cancer stemness (n=1).

Summary of evidence
Thirty six studies published between 2017 and 2021 were reviewed. Our findings showed that flavonoids exert anticancer effects through modulation of key cellular signaling mechanisms. The reported mechanisms include; induced apoptosis and pyroptosis, inhibition of angiogenesis, inhibition of invasion and metastasis, cell cyle arrest, reduction of cell viability and proliferation, induction of autophagy, inhibition of inflammation, inhibition of proliferation of breast cancer stem cells, inhibition of growth. Knowledge on the effects of flavonoids on the modulation of cellular signaling mechanisms can be exploited to develop effective chemotherapeutic agents for breast cancer.

Findings
Proposed mechanism(s) 1. (Moradi et al., 2020) Calycopterin was tested in MDA-MB-231, MCF-7, HUVEC (control) cell lines Calycopterin reduced proliferation and cell viability in a dose and time dependent manner. No effects were seen on the HUVEC normal endothelial cells.
It decreased colony formation. However, that of MCF-7 was more evident.

Calycopterin inhibited cell migration
There was increased sub G1 population Calycopterin increased apoptosis -Anti-apoptotic Bcl2 genes were downregulated, pro-apoptotic Bax and caspase-3 genes were augmented in MDA-MB-231 cell lines while caspace-8 was increased in both cell lines It increased the percentage of cells in early and late apoptosis compared to control It increased the percentage population of cells in G1 phase It reduced colony numbers There was a significant decrease in expression of miR21and miR27 genes. However, the expression of miR29 and miR34 genes was enhanced Treatment of mouse fibroblast cells with xanthomicrol resulted to a significant reduction in tumor volume. Lungs and livers of the animal models were normal after 14 days of treatment There was reduced tumor grade compared to control.
Induction of apoptosis and cell cycle arrest: Xanthomicrol induces early and late apoptosis and also causes arrest of the cell cycle at G1 Phase. Cell proliferation is also inhibited in vivo.
Inhibition of angiogenesis and metastasis: Xanthomicrol also enhanced expression of tumor suppressor genes (miR29b and miR34) while repressing expression of tumor promoting genes like miR21, miR27 and miR125b. upregulation of caspase 9 and Bax protein    Cell cycle arrest Cell cycle arrest is a major mechanism through which some potent conventional anticancer drugs exert their cytotoxic action. The cell cycle is a complex molecular process that is closely regulated by several essential molecular compounds such as cyclin-dependent kinases (CDK) and CDK inhibitors such as p21 and p27 (Kikuchi et al., 2019;Lin et al., 2015). Downregulation of some CDK subfamilies and upregulation of p21, p27 and p53 has been associated with cell cycle arrest . Transcription factors such as Forkhead box transcription factor (FOXO3a) are also heavily involved in the modulation of cell cycle progression and apoptosis (Lin et al., 2015;Yuan et al., 2018). Recent research has revealed that upregulation of p21 and p27 causes increased expression of FOXO3a and subsequently, arrest of the MCF-7 cell cycle at G0/G1 phase (Yuan et al., 2018). Further results from studies suggest that FOXO3a is involved in the evolution of breast cancer and could even serve as a prognostic factor (Lin et al., 2015;Yao et al., 2017;Yuan et al., 2018). A few anticancer drugs have been shown to augment the expression of such factors. Cell cycle arrest at G1, G2/M and S can eventually result in apoptosis of the cancerous cells (Lin et al., 2015). Flavonoids such as; xanthomirol, calycopterin, PMF, genistin, quercetin and baicalein were shown to arrest the BC cell cycle at G1 phase (Anaya-Eugenio et al., 2021;Attari et al., 2021;Hwang et al., 2020;Li et al., 2018;Yu et al., 2018). On the contrary, other flavonoids caused arrest of cell cycle at G2/M phase. This observation was evident with sideritoflavone, kaempferol, apigenin (Pham et al., 2021;Sotillo et al., 2021;Zhu & Xue, 2019). These effects were determined using flow cytometry. Sideritoflavone induced DNA double-strand break which consequently led to activation of c-Myc/Max pathway and eventually led to arrest of the cell cycle at G2/M phase (Sotillo et al., 2021). The findings were further evidence of anti-proliferative efficacy of these flavonoids against breast cancer. Findings from the reviewed studies have affirmed that impeding the proliferative activity of cancerous cells ultimately suppresses the tumorigenic and malignant potential of these cells.

Inhibition of colony formation
A number of studies assessed the effect of specific flavonoids on colony formation of breast cancer cell lines such as MCF-7, MDA-MB 231, MDA-MB-468. Xanthomicrol, calycopterin and tangeretin exhibited inhibitory effects against colony formation of breast cancer cell lines (Attari et al., 2021;Ko et al., 2020;Moradi et al., 2020). However, sideritoflavone did not affect JIMT1 cell lines colony formation (Sotillo et al., 2021).

Tumour angiogenesis
Angiogenesis is integral to metastasis of breast cancer (De Palma et al., 2017;Ferrara, 2002). Most cancerous cells have the intrinsic potential of producing tumour angiogenic factors that promote the formation of new blood vessels. Tumour cell hypoxia is presumed to prompt the production of chemicals which enhance angiogenesis (Giverso & Ciarletta, 2016;Sp et al., 2017). Several flavonoids were shown to attenuate angiogenesis in breast cancer cell lines. Nobiletin was found to inhibit angiogenesis in ER+ breast cancer cell lines by blocking both the angiogenesis signalling cascade (Src/FAK/ STAT3 pathway) and angiogenic factors such as VEGF and FGF. Further findings from the study revealed that nobiletin inhibited the expression of MMPs that are critical for angiogenesis (Sp et al., 2017). Glabridin was also shown to exert anticancer effects through a similar mechanism (Hsu et al., 2011). Glabridin has also been reported to have antiangiogenic effects on TNBC cell lines. For instance, Mu et al., established that glabridin harboured a significant antiangiogenic effect on MDA-MB-231 cell lines that was mediated through blockade of Wnt/β-catenin signalling pathway (Mu et al., 2017). Previously, Mu et al. (2015) had established glabridin to have antiangiogenic effect on MDA-MB-231 and Hs-578T cells. However, this effect was mediated through inhibition of NF-kB/IL-6/STAT-3 axis which reduced transcription of VEGF (Mu et al., 2015). Xanthomicrol was also found to have appreciable antiangiogenic effect that was exerted through attenuation of angiogenesis promoting factors VEGF and MMP9 as a result of overexpression of miRNA29b. This was modulated through reduced expression of miR27 that further led to inhibition of ZBTB10 and VEGF receptors and enhanced expression of miR29b which inhibits metastasis by modulating angiogenic and metastasis promoting factors (Attari et al., 2021). MEQ inhibited angiogenesis through regulation of MTA2/SerRS/VEGFA axis (Zhang et al., 2020).

Inhibition of migration and invasion (Anti-metastasis)
The ability of tumor cells to migrate and invade normal tissue has profound effects on the capacity of these cells to metastasize and thus affect distant organs which negatively influences the outcome of patients with cancer (Chen et al., 2014). Matrix metalloproteinases (MMPs) play an integral role of breaking down the surrounding extracellular matrix (ECM) that allows the cancer cells to access the circulatory system (Chen & Liu, 2018). Successful metastasis is dependent on the ability of cancer cells to migrate and invade surrounding tissue. This may be achieved through epithelial to mesenchymal phenotypic morphological changes (Xiong et al., 2021). The epithelial-mesenchymal transition (EMT) process can be demonstrated through the expression of biomarkers (E-cadherin, Vimentin, Snail, slug, Twist, FOXC2) and modulation of signalling pathways (NF-κB signaling). Rhoifolin was shown to cause inhibition of ezrin and subsequently decreased interaction between PODXL and ezrin which as a result led to decreased metastatic ability of the breast cancer cells. Moreover, rhoifolin decreased expression of E-Cadherin and also increased expression of vimentin (Xiong et al., 2021). Similarly, liquiritigenin inhibited migration and invasion through a similar mechanism (Liang et al., 2021). Sideritoflavone inhibited JIMT 1 cell line migration but enhanced their motility by causing an increase in p65/NF-κΒ and activation of TGF-β signaling pathway (Sotillo et al., 2021). These findings can be compared to those of Anaya-Eugenio et al who also concluded that PMF inhibited migration of MCF-7 cells through reduced expression of p65/NF-κΒ (Anaya-Eugenio et al., 2021). Yao et al. established that wogonoside exhibited antimetastatic effect through an array of mechanisms which included suppression of EMT in addition to inhibition of invasion and migration of TNF-α induced MDA-MB-231, MDA-MB-435, and BT-474 cells and TNF-α + TGF-β1-induced MCF7 cells (Yao et al., 2017). This was achieved through inhibition of NF-κB signaling. Comparatively, ampelopsin inhibited invasion and migration of MCF-7 and MDA-MB-231 cells through diminished epithelial mesenchymal transition and inhibition of TNF-α/NF-κB signalling pathway (Truong et al., 2021). Wogonin reduced (Lipopolysaccharide) LPS induced metastasis of MDA-MB-231 cells by inhibiting 5-Lipoxygenase/leukotriene B4 Receptor 2 (5-LO-/BLT2) pathway (Go et al., 2018). Baicalein was reported to inhibit morphological changes of MDA-MB-231 cancer cells through interruption of lamellipodia formation (Terabayashi et al., 2018). Further findings by Li et al suggested that calycosin diminished migration and invasive ability of MCF-7 and T47 D cancer cells via down-regulation of FOXP3 and hence down regulation of VEGF and MMP-9 (Li et al., 2017). Consequently, targeting the migration ability of breast cancer cells could provide a critical breakthrough in the development of potent chemotherapeutic drugs. Some studies have further investigated the effects of flavonoids on migration of the cell lines which is an integral characteristic of malignant tumour cells using in vitro tests such as Scrape wound healing assay (Sotillo et al., 2021), AP 48 chamber system to assess vertical migration and Oris™ cell migration assay (Xiong et al., 2021), Gap closure and transwell migration assay (Loung et al., 2019). A similar effect of inhibition of migration of the malignant cell lines was observed in these studies. Pec inhibited migration and invasion of MDA-MB-231, MCF-7 and 4T1 cells in vitro through downregulation of p-stat3, MMP-2 and MMP-9 hence inhibiting stat3 signalling pathway (Li et al., 2019). Corylin reduced migration, invasion and EMT of MCF-7 and MDA-MB-231 cells through miR-34c/LIN00963 target (Liu et al., 2021).

Autophagy
Autophagy is a process through which the cell cytoplasmic contents are engulfed in vesicles, bind with lysosomes and then undergo degradation resulting in protein and ATP production (Han et al., 2018;Jain et al., 2013;Karantza & White, 2007). On one hand, autophagy can promote cell survival while on the other, it can contribute to cell death. Therefore, it can either have a positive or negative effect on tumorigenesis (Han et al., 2018;Jain et al., 2013). It is mainly regulated by the mTOR pathway. Han et al reckon that the activated mTOR pathway enhances progression of tumour and as a result has a deleterious effect on the survival of patients with breast cancer. Thus, increased expression of mTOR in breast cancer is a poor prognostic factor (Han et al., 2018). Chen et al concluded that delphinidin augmented protective autophagy through repression of AKT/mTOR/eIF4e/p70s6k signalling cascade and stimulation of the LKB1/AMPK/ ULK1/FOXO3a signalling cascade in HER-2 positive MDA-MB-453 nad BT 474 cells (Chen & Liu, 2018). Quercetin also suppressed glycolysis and cell mobility through Akt-mTOR pathway mediated autophagy (Jia et al., 2018).

Apoptosis
Aberrant control of apoptotic cell death eventually leads to inactivation of apoptosis and has been linked to the pathogenesis of several diseases including cancer (Abotaleb et al., 2019;Kikuchi et al., 2019). Cancer cells usually exhibit resistance to apoptosis through enhanced expression of pro-oncogenes (c-Myc which enhances proliferation and suppresses p53) and anti-apoptotic proteins such as Bcl-2, survivin and livin. In contrast, cancer cells also resist apoptosis by downregulating apoptotic proteins such as caspases, Bad and Bax in addition to enhancing the loss of tumor suppressor function of p53 (Campbell & Tait, 2018). Natural tumor suppressor factors such as p53 gene will exert their action through induction of apoptosis and thus dysregulation of such factors is an important molecular mechanism for the development of malignancies such as breast cancer (Whibley et al., 2009).

Limitations
We acknowledge that we restricted the review to articles only published in English. This might have eliminated some articles with useful information on the scope of the study. Another limitation is the articles reviewed used different methodologies and breast cancer cell lines and/or in vivo models hence making it difficult to compare the findings. The enlisted limitations notwithstanding, we believe that the findings of this study were not significantly affected,

Conclusions
Dietary flavonoids modulate anti-cancer effects through various mechanisms such as; cell cycle arrest, enhanced apoptosis, inhibited proliferation, invasion, angiogenesis and metastasis. Their safety, easy availability and anti-tumour effect may render them as effective alternative strategies for battling breast cancer. We recommend that further research be done to explore the possibility of using flavanoids as lead molecules for anti-cancer drug development.

Data availability
Underlying data All data underlying the results are available as part of the article and no additional source data are required.