Molecular mechanisms of flavonoids and their modulatory effects against breast cancer: A scoping review

Protocol and Registration

The results are reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Page et al., 2021). 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;

Articles were excluded based on the following criteria;

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;

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.

Synthesis of results

Thirty-six studies evaluating the modulatory effects of flavonoids on different breast cancer cell types were reviewed. The majority of them used in vitro assays (n=35). Most studies utilized MDA-MB-231 cell lines (n=24) followed by MCF-7 cell lines (n=20). Other cell lines used included; BT 474 (n=3), 4T1 (n=3), MDA-MB-468 (n=2), BT 549 (n=2), T47D (n=2), JIMT 1 (n=2) MDA-MB-435 (n=1), MDA-MB-231/IR (n=1), MCF-10A (n=1), SKBR3 (n=1) and MDA-MB-453 (n=1). A few of the studies used in vivo methods such as nude mouse model (n=2), xenograft mouse model (n=2), sub-cutaneous homotransplant mouse model (n=1) and mice bearing MCF-7 or SKBR3 xenografts (n=1). 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.

Cell viability

Cell viability using the MTT assay seemed to form a baseline against which a metabolite qualifies for further investigation. The studied flavonoids mainly displayed a dose and time-dependent effect on the variant breast cancer cell lines tested (Anaya-Eugenio et al., 2021; Attari et al., 2021; Go et al., 2018; Hallman et al., 2017; Hart et al., 2018; Hwang et al., 2020; Jia et al., 2018; Karimi et al., 2017; Li et al., 2017, 2018; Liang et al., 2021; Moradi et al., 2020; Qiu et al., 2019; Wang et al., 2021; Yu et al., 2018). The sensitivity of the flavonoids differed based on flavonoid type, concentration and cell line used. For instance, although both MCF-7 and MDA-MB-231 were sensitive to apigenin in a dose-dependent manner, MCF-7 cells were slightly more sensitive (Vrhovac Madunić et al., 2018).

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 (Fan et al., 2014). 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).

In most of the papers reviewed, induction of apoptosis was showed to be a major mechanism by which breast cancer progression was attenuated by flavonoids. Flavonoids including calycopterin, apigenin, KARP, tangeretin, xanthomycrol, PMF, kaempferol, aviculin, genistin, PEC, hyperoside, nobiletin, quercetin, liquiritigenin, isoquercitrin, ampelopsin, MEQ induced apoptosis in different BC cell lines. The mechanisms involved included reduction in expression of antiapoptotic genes such as Bcl2, XIAP and increased expression of Bax, PARP 1, caspases 3, 7, 8, 9. Other mechanisms included increased mitochondrial oxidative stress, downregulation of mitochondrial membrane potential, inhibition of NF-κB, modulation of miR-200b/JAZF1 pathway and activation of PAX8-AS1-N/miR-17-5p/PTEN, CDKNIA,ZBTB4 regulation axis (Abbaszadeh et al., 2018; Anaya-Eugenio et al., 2021; Attari et al., 2021; Harrath et al., 2021; Hwang et al., 2020; Ko et al., 2020; Lee et al., 2020; Liang et al., 2021; Moradi et al., 2020; Pham et al., 2021; Qiu et al., 2019; Truong et al., 2021; Vrhovac Madunić et al., 2018; Wang et al., 2021; Xu et al., 2019b; Yu et al., 2018; Zhang et al., 2020; Zhu & Xue, 2019).

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,

Underlying data

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

Reporting guidelines

Harvard Dataverse. Murithi, Mary, 2022, “Replication Data for Molecular mechanisms of flavonoids and their modulatory effects against breast cancer: A scoping review”, DOI: https://doi.org/10.7910/DVN/V5YGQP, Harvard Dataverse, DRAFT VERSION.