Potential of natural flavonoids to target breast cancer angiogenesis (review)

Abstract

Angiogenesis is the process by which new blood vessels form and is required for tumour growth and metastasis. It helps in supplying oxygen and nutrients to tumour cells and plays a crucial role in the local progression and distant metastasis of, and development of treatment resistance in, breast cancer. Tumour angiogenesis is currently regarded as a critical therapeutic target; however, anti-angiogenic therapy for breast cancer fails to produce satisfactory results, owing to issues such as inconsistent efficacy and significant adverse reactions. As a result, new anti-angiogenic drugs are urgently needed. Flavonoids, a class of natural compounds found in many foods, are inexpensive, widely available, and exhibit a broad range of biological activities, low toxicity, and favourable safety profiles. Several studies find that various flavonoids inhibit angiogenesis in breast cancer, indicating great therapeutic potential. In this review, we summarize the role of angiogenesis in breast cancer and the potential of natural flavonoids as anti-angiogenic agents for breast cancer treatment. We discuss the value and significance of nanotechnology for improving flavonoid absorption and utilization and anti-angiogenic effects, as well as the challenges of using natural flavonoids as drugs.

Abbreviations

C3G

cyanidin-3-glucoside

CAFs

cancer-associated fibroblasts

CAM

chick embryo chorioallantoic membrane

DEL

delphinidin

DMBA

7,12-dimethylbenz(a)anthracene

DOX

doxorubicin

EGCG

epigallocatechin gallate

EMT

epithelial-to-mesenchymal transition

FAK

focal adhesion kinase

GO

graphene oxide

HER2+

HER2-positive

HR+

hormone receptor-positive

ISL

isoliquiritigenin

MEK

mitogen-activated protein kinase kinase

MPA

medroxyprogesterone acetate

mTOR

mammalian target of rapamycin

MVD

microvascular density

OS

overall survival

PLGA

Poly (lactic-co-glycolic acid)

PTK

protein tyrosine kinase

Ptx

paclitaxel

rGO

reduced GO

Src

steroid receptor coactivator

TNBC

triple-negative breast cancer

1 INTRODUCTION

In 2020, breast cancer surpassed lung cancer as the most commonly diagnosed cancer in women globally, constituting 31% of all female cancer cases. It is estimated that there will be an additional 300,000 cases of breast cancer in the United States by the end of 2023 (Siegel et al., 2023; Sung et al., 2021). Breast cancer is classified into three major subtypes based on the expression of oestrogen or progesterone receptors as well as ERBB2 amplification: hormone receptor-positive (HR+), HER2-positive (HER2+) and triple-negative breast cancer (TNBC). The 5-year survival rates for stage I HR+, HER2+ and TNBC are at least 99%, 94% and 85%, respectively. However, for stage IV breast cancer, the median overall survival period for HR+ or HER2+ is approximately 5 years, and the median overall survival period for TNBC is approximately 1 year (Waks & Winer, 2019). Surgery, radiation therapy, endocrine therapy, targeted therapy and chemotherapy are all treatment options for various subtypes and stages of breast cancer (Waks & Winer, 2019). Despite continuous advancements in drug and treatment regimens, primary and acquired resistance in patients with advanced breast cancer receiving multiple lines of therapy results in less effectiveness of anti-tumour drugs and intolerable side effects, ultimately leading to disease progression (Huppert et al., 2023; Waks & Winer, 2019; Zhang, 2021). Through angiogenesis, cancer cells invade the surrounding blood vessels and stroma and enter the circulation via the blood vessels, which is the primary pathway for tumour metastasis to distant organs. Distant metastasis is a leading cause of treatment failure and is associated with a high mortality rate (Chaffer & Weinberg, 2011). Angiogenesis plays a critical role in the development, invasion, and metastasis of breast cancer (Schneider & Miller, 2005). The levels of angiogenic factors, as well as the extent of the vascular network formed, can be used to predict breast cancer survival (Madu et al., 2020).

Anti-angiogenic therapy has been recognized as an important approach in cancer treatment because of the role that angiogenesis plays in cancer initiation and progression. Several cellular and genetic factors, including vascular endothelial growth factor (VEGF) and its receptors, are involved in the regulation of tumour angiogenesis (Banerjee et al., 2007). Bevacizumab is a VEGF-blocking monoclonal antibody that was approved by the Food and Drug Administration (FDA) in 2008 for the treatment of metastatic HER2-negative breast cancer. However, owing to the lack of significant improvement in overall survival (OS) and drug toxicity, the FDA revoked its approval in 2011 (Tanne, 2011). This disappointing outcome is largely due to resistance to antiangiogenic agents and increased drug toxicity (Aalders et al., 2017). This primary or acquired resistance is thought to be associated with a variety of factors. For example, upregulation of non-VEGF angiogenic pathways in breast cancer, heterogeneity of tumour vascular endothelial cells, angiogenesis mimicry, and transdifferentiation of breast cancer stem cells to endothelial cells (Ayoub et al., 2022; Mao et al., 2020). This suggests that anti-angiogenic therapies still face challenges in the treatment of breast cancer. Given the heterogeneity of breast cancer and multiple regulatory pathways involved in angiogenesis, blocking multiple angiogenic signalling pathways at the same time may offer advantages (Aalders et al., 2017; Qin et al., 2019).

Many inexpensive natural products with low toxicity can target cytokines and signalling pathways associated with tumour angiogenesis and have the potential to be developed as anti-angiogenic agents (Li et al., 2021). The anti-angiogenic effect of flavonoids on breast cancer has been widely reported, which has attracted our attention. Flavonoids are a class of natural compounds that are abundant in most human diets; they have a wide range of biological activities, including anti-inflammatory, antioxidant, antiproliferative and oestrogen-modulating properties, and they have shown promise in the treatment of cancer, inflammation, bacterial infections, cardiovascular diseases, and diabetes. Their anti-cancer effects involve a variety of mechanisms and signalling pathways, which has led to an increased interest in flavonoids in recent years (Ahn-Jarvis et al., 2019; Liskova et al., 2020). Most flavonoids have a 15-carbon backbone (C6-C3-C6) consisting of two benzene rings (A and B) and one heterocyclic ring (C). They are divided into several subclasses, including flavanols, flavanones, flavones, anthocyanins, and isoflavones. The ability of flavonoids to downregulate VEGF expression (e.g., baicalin, baicalein and wogonin) is determined by the number of hydroxyl groups, the presence of a C2 = C3 double bond, and structural features with similarity to tyrosine kinase inhibitors (Sun et al., 2022). Natural flavonoids, such as genistein, possess structural advantages that allow them to interact with oestrogen receptors and exert oestrogenic and/or anti-oestrogenic effects. For example, naringenin reduced estradiol binding to oestrogen receptor (ER)α in cancer cells expressing ERα in the presence of 17β-estradiol (Bulzomi et al., 2010). At the same time, these flavonoids can promote as well as inhibit oestrogen synthesis. For example, quercetin inhibits 17β-estradiol biosynthesis by 75% at a high concentration (50 μM), while at low concentrations (1–10 μM), it has a slight promoting effect on 17β-estradiol biosynthesis (Lu et al., 2012). This may be related to the biphasic ability of certain phytoestrogens to regulate angiogenesis, potentially leading to some side effects. Furthermore, nanotechnology and flavonoid-based nanoparticles have shown great potential in treating cancer and improving natural flavonoid bioavailability, among other applications (Kargozar et al., 2020; Khan, Ullah, et al., 2021). In this review, we summarize the mechanisms of breast cancer angiogenesis, the role and mechanisms of action of flavonoids in breast cancer angiogenesis, and the limitations and potential side effects of natural flavonoids. It also explores the use of nanotechnology to improve the bioavailability of natural flavonoids and enhance the anti-tumour and anti-angiogenic ability of natural flavonoids, as well as future challenges.

2 ANGIOGENESIS IN BREAST CANCER (FIGURE 1)

FIGURE 1

In breast cancer, angiogenesis becomes an intricate process. VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; IL, interleukin; TGFβ-1, transforming growth factor β-1; MMPs, matrix metalloproteinases; HIF, hypoxia-inducible factor; CAF, cancer-associated fibroblast; HER2+, HER2-positive.

2.1 Angiogenesis-dependent breast cancer

Accumulating evidence indicates that angiogenesis plays a central role in breast cancer development and metastasis. Angiogenesis occurs before mammary gland hyperplasia transforms into malignant tumours. Studies have found varying degrees of angiogenesis in several precancerous breast cancer lesions that increase with lesion severity. In lesions of florid ductal hyperplasia of the usual type, atypical ductal hyperplasia, and atypical lobular hyperplasia, the average microvascular density (MVD) was higher than that in normal breast tissue [15376259]. MVD is associated with the risk of progression from hyperplasia to breast cancer, and the relative risk of breast cancer increases with the density and concentration of microvessels (Guinebretiere et al., 1994). There is a strong correlation between MVD and OS and relapse-free survival rates in early-stage breast cancer, regardless of the axillary lymph node status (Weidner et al., 1992). A higher vascular count is thought to be linked with a higher rate of axillary lymph node metastasis and mortality (Hansen et al., 2000). Denser blood vessels in breast tumours indicate a higher likelihood of distant metastases. According to Weidner et al., every 10 microvessels in the blood vessel count per 200x field of view increases the risk of distant metastasis by 1.17 times (Weidner et al., 1991). In HER2-positive and HR + breast cancers, high MVD is associated with poor prognosis, and as MVD increases, the time to tumour metastasis decreases (Gojkovic et al., 2011). Furthermore, higher MVD in breast tumours may indicate that bone metastases are more likely to occur, and MVD can predict the likelihood of early bone metastases (Sun et al., 2018). Therefore, targeted angiogenesis is necessary and promising for the treatment of breast cancer.

2.2 Breast cancer subtypes and angiogenesis

Several factors influence breast cancer angiogenesis, including expression of the HER2 oncogene as well as the presence of oestrogen and progesterone receptors. In breast cancer, HER2 overexpression is significantly associated with tumour MVD, and it stimulates the synthesis of hypoxia-inducible factor (HIF)α and the expression of VEGF mRNA via the protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK)/extracellular regulated kinase (ERK) pathways (Alameddine et al., 2013). In human breast cancer cells, HER2 increases VEGF synthesis by activating the mammalian target of rapamycin (mTOR)/p70S6 kinase cap-dependent translation pathway (Banerjee et al., 2007). VEGF expression can be increased by both oestrogen and progesterone (Botelho et al., 2015). Oestrogen can stimulate endothelial cell activity via the ERα pathway, promote angiogenesis, and mobilize bone marrow-derived endothelial progenitor cells, all of which contribute to the neovascularization of breast tumour tissue (Maniyar et al., 2018). Furthermore, oestrogen promotes angiogenesis by inhibiting VEGF receptor (VEGFR)-1 expression (Elkin et al., 2004). HIFα levels can be increased in HER-2-positive and ER-positive breast cancers by upregulating the PI3K/Akt/mTOR signalling pathway, thereby promoting tumour angiogenesis (de Heer et al., 2020).

2.3 VEGF-dominated angiogenesis in breast cancer

VEGF and its receptor family are involved in the main angiogenesis pathway, play a key role in tumour angiogenesis and metastasis development, and are the main factors influencing vascular endothelial cell function (Hoeben et al., 2004). Serum VEGF levels are high in breast cancer, lung cancer, kidney cancer, high-grade gliomas and other malignant tumours (Brogowska et al., 2023). Significant increases in serum VEGF levels in malignancies and breast cancer are usually accompanied by increases in tumour MVD (Thielemann et al., 2008; Tsutsui et al., 2005). Serum VEGF levels are usually higher in metastatic breast cancer than in benign breast disease and localized breast cancer, and often indicate a poor outcome (Adams et al., 2000). As a result, VEGF expression in patients with primary breast cancer can be considered an independent prognostic factor. Higher VEGF levels are associated with lower recurrence-free survival and OS, as well as shorter survival after adjuvant endocrine therapy (Linderholm et al., 2003). The pro-tumour angiogenic capacity of VEGF, which primarily regulates endothelial cell proliferation, migration, adhesion, vascular sprouting, and vascular permeability, is associated with a poor prognosis (Ferrara, 2002). VEGF increases endothelial cell permeability by activating Akt, endothelial nitric oxide synthase (eNOS) and mitogen-activated protein kinase(MAPK)-dependent pathways; VEGF also induces matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA) and other proteins to produce and destroy the basement membrane, thereby allowing vascular endothelial migration and invasion (Lal et al., 2001; Lugano et al., 2020), which in turn prompts the endothelial cells to change their shape and adhere to each other to form a new basement membrane and lumen (Gupta & Qin, 2003). VEGF can also aid neovascular endothelial survival by inducing the anti-apoptotic factors B-cell lymphoma 2 (Bcl-2) and survivin as well as the PI3K-Akt pathway to induce anti-apoptotic signals, and by activating the mitogen-activated protein kinase kinase kinase (RAF)–MEK–ERK pathway to induce endothelial cell growth and maintain the neovascular system vitality (Carmeliet, 2005; Ferrara, 2004). Although VEGF is inextricably linked to tumour angiogenesis, some researchers have found no correlation between VEGF expression and the degree or type of vascularization in breast cancer (Viacava et al., 2004), suggesting that breast cancer angiogenesis is regulated by multiple factors.

Tumour and tumour microenvironment cells, such as cancer-associated fibroblasts (CAFs), macrophages, mast cells and lymphocytes, can be stimulated to release additional angiogenic factors that contribute to tumour angiogenesis, in addition to VEGF (Bouck et al., 1996). For example, CAFs secrete angiogenic factors such as fibroblast growth factors and platelet-derived growth factor (PDGF) to promote endothelial cell proliferation and differentiation and induce breast tumour angiogenesis (Qiao et al., 2016). In breast cancer, inflammatory cells such as mast cells are attracted by stem cell factors secreted by tumour cells producing VEGF, PDGF, basic fibroblast growth factor (bFGF), interleukin-8, transforming growth factor β-1 (TGFβ-1) as well as MMPs and other angiogenic factors that promote tumour vascularization and invasion by promoting extracellular matrix degradation and changing vascular permeability (Ribatti et al., 2016). This suggests that targeting non-VEGF pathways of angiogenesis may be crucial and promising.

2.4 Hypoxic environment in breast cancer promotes angiogenesis

Hypoxia is a characteristic of breast cancer and is one of the most important factors that induces VEGF mRNA expression (Banerjee et al., 2007). Elevated HIF-1α levels are associated with tumour proliferation and metastasis, and are a predictor of poor prognosis for patients with stage T1/T2 breast cancer and positive axillary lymph nodes (Gruber et al., 2004). In an animal model of breast cancer transplanted with MDA/CA-HIF (MDA-MB-231 cells overexpressing constitutively active HIF-1α), Hiraga et al. demonstrated that hypoxia and HIF-1α contribute to the development of osteolytic bone metastases by inhibiting osteoblast differentiation and promoting osteoclastogenesis; bone metastases were significantly increased, and the number of cd31-positive vessels also increased (Hiraga et al., 2007). Furthermore, the abundance of fat around the breast promotes tumour angiogenesis, which aids tumour survival and progression. In hypoxic breast adipose tissue, increased HIF-1α gene expression and defective adipokine secretion create an inflammatory environment for tumour growth and promote angiogenesis in cancerous tissues (Rausch et al., 2017). Progenitor cell populations from white adipose tissue induce breast cancer epithelial-to-mesenchymal transition (EMT) gene expression, promote tumour metastasis, and generate new blood vessels (Orecchioni et al., 2013).

3 ANTI-ANGIOGENIC EFFECTS OF NATURAL FLAVONOIDS ON BREAST CANCER

Natural flavonoids are a class of plant-derived compounds that can be found in a wide variety of fruits, vegetables, herbs, and other plant foods. Flavones, flavanones, flavanols, chalcones, aurones, anthocyanidins, isoflavones and flavonols are the most common flavonoids in the human diet. Natural flavonoids have been shown to exert anti-cancer effects via antioxidant, anti-inflammatory, apoptosis-inducing, cancer stem cell-targeting and anti-angiogenic pathways (Forni et al., 2021; Ververidis et al., 2007). Natural flavonoids exert their inhibitory effects on breast cancer angiogenesis through multiple pathways and targets. For example, natural flavonoids reduce tumour microvascular density by inhibiting signalling pathways such as p38MAPK, PI3K/Akt, focal adhesion kinase (FAK)/signal transducer and activator of transcription (STAT)3, nuclear factor kappa B (NF-κB) and reducing the expression of angiogenic factors such as HIF-α, VEGF, bFGF, and MMPs. Some flavonoids also have the effect of inhibiting tumour growth, reducing tumour invasiveness and enhancing the efficacy of other antitumour drugs. In addition, flavonoids have certain structural advantages to exert anti-tumour angiogenesis effects, especially flavonols and flavones. C2-C3 double bond, C3-OH and C4-oxo group in the C ring, along with the -OH at C5 and C7 in the A ring, the two -OH groups in the A ring, and the additional -OH group at the 4′ position in the B ring, these structural features are critical for the anti-angiogenic activity of flavonoids. Moreover, a double bond between C2 and C3, as well as the position of ring B at C2, along with -OH substituents at position C5 and C7, facilitate the inhibitory effect of flavonoids on tyrosine kinases and the suppression of VEGF expression (Shah et al., 2023; Sun et al., 2022). The following section discusses the anti-angiogenic effects of natural flavonoids on breast cancer (Table 1). Table 2 shows some clinical trials of antiangiogenic drugs in breast cancer.

TABLE 1. Natural anti-tumour angiogenesis flavonoids and their chemical structures, experimental models and anti-angiogenic mechanisms and the stages of drug development of these compounds.

Category	Chemical structure	Name	Experimental model(s)	Drug concentration or dose	Anti-angiogenic mechanism(s)	The stages of drug development (cancer-related and last 10 years)	Refs
Flavones		Apigenin	MDA-MB-231 cells	(In vitro) 5, 10, 15 μM	Decreases VEGF and VEGF mRNA	Clinical trials, Phase2, (NCT00609310)	(Jin & Ren, 2007; Mafuvadze et al., 2010, 2011; Seo et al., 2017)
			T-47D and BT-474 cells treated with MPA	(In vitro) 50, 100 μM	Decreases VEGF and VEGF mRNA, reduces progesterone receptor levels
			Progestin-accelerated DMBA-induced mammary tumours in rats	(in vivo) 50 mg·kg^−1	Decreases VEGF and inhibits the expression of VEGFR-2,
			Female nude mice inoculated with BT-474 cells	(in vivo) 50 mg·kg^−1	Reduces VEGF expression and the level of Her2/neu protein
			MCF-7/ADR cells	(In vitro) 80 μM	Inhibits the secretion of STAT3 target genes VEGF and MMP-9
		Baicalin	Female Swiss albino mice transplanted with Ehrlich tumours	(in vivo) 15, 50 mg·kg^−1	Inhibits the activation of the NF-κB pathway and reduces VEGF levels	Preclinical testing	(Shehatta et al., 2022)
		Luteolin	MDA-MB-231 (4175) LM2 cells	(In vitro) 10, 50, 100 μM	Decreases VEGF secretion	Clinical trials, early phase 1 (NCT03288298)	(Cook et al., 2015, 2016, 2017; Sun et al., 2015)
			MDA-MB-231 and MCF-7 cells, HUVECs	(In vitro) 25, 50, 100 μM	Inhibits notch signalling, downregulates MMP-2 and MMP-9
			Sprague–Dawley rat breast cancer model induced by DMBA,	(in vivo) 1, 10, 25 mg·kg^−1	Reduces VEGF levels
			BT-474 and T-47D cells,	(In vitro) 10, 25 μM	Reduces the expression of VEGF and VEGF mRNA
			T-47D xenografted nude mice	(in vivo) 20 mg·kg^−1	Inhibits the expression of VEGF
		Nobiletin	MCF-7 and T-47D cells, MDA-MB-231 cells, HUVECs	(In vitro) 200 μM	Decreases the expression of VEGF and bFGF, inhibits the Src/FAK/STAT3 signalling pathway, downregulates the expression of MMPs	Preclinical testing	(Sp et al., 2017, 2018)
			MCF-7 cells, MDA-MB-231 cells, HUVECs	(In vitro) 200 μM	Inhibits the Cd36/Stat3/NF-κB signalling axis
		Wogonin	MDA-MB-231 and MCF-7 cells, HUVECs, CAM	(In vitro) 10, 20, 40 μM	Reduces HIF-1α expression, decreases VEGF secretion	Preclinical testing	(Song et al., 2013; Zhao et al., 2014)
			Athymic nude mice inoculated with MCF-7 cells	(in vivo) 30, 60 mg·kg^−1	Reduces HIF-1α expression, decreases VEGF secretion
			MCF-7 cells, HUVECs, CAM, Matrigel,	(In vitro) 10, 20, 40 μM, (in vivo) 100 μmol·l^−1	Decreases VEGF secretion, inhibits the PI3K/Akt/NF-κB signalling pathway
		Wogonoside	MCF-7 cells, CAM, HUVECs, MCF-7 xenograft Balb/c nude mouse model	(In vitro) 25, 50, 100 μM (in vivo) 80 mg·kg^−1	Inhibits VEGF expression, inhibits the Wnt/β-catenin signalling pathway	Preclinical testing	(Huang et al., 2016, 2019)
			MDA-MB-468 cells, MDA-MB-231 cells, Balb/c nude mice injected with MDA-MB-231 cells	(In vitro) 25, 50, 100 μM (in vivo) 80 mg·kg^−1	Inhibits VEGF secretion, protein and mRNA expression, inhibits the hedgehog signalling pathway
Flavonols		Jaceidin	Swiss albino mice injected with Ehrlich's ascites carcinoma (EAC)	(in vivo) 50 mg·kg^−1	Decreases VEGF	Preclinical testing	(Elhady et al., 2020)
		Myricetin	MDA-MB-231 and 4T1cells, HUVECs, CAM, female 5-week-old BALB/c nude mice transplanted with 4 T1 cells	(In vitro) 20, 40, 60, 80, 100 μM (in vivo) 25, 50 mg·kg^−1	Inhibits VEGFR2 expression, downregulates VEGFR2 and p38MAPK	Preclinical testing	(Zhou et al., 2019)
		Quercetin	MCF-7 and MDA-MB-231 cells, Balb/c nude mice injected with MCF-7 cells,	(In vitro) 20, 40, 60, 80, 100 μM (in vivo) 50 mg·kg^−1	Inhibits MMP-2, MMP-9, and VEGF expression	Clinical trials, early phase 1 (NCT02989129, NCT05680662), phase 1 (NCT01912820), phase 2 (NCT04733534, NCT03476330, NCT05456022, NCT05724329, NCT03493997), phase 3 (NCT02195232),	(Jia et al., 2018; Oh et al., 2010; Xiao et al., 2011; Zhao et al., 2016)
			MCF-7 xenografted Balb/c nude mice,	(in vivo) 34 mg·kg^−1	Decreases protein and mRNA expression of VEGF and VEGFR2, downregulates the calcineurin/NFA T pathway
			TAMR-MCF-7 cells, CAM, HUVECs	(In vitro) 3, 10, 30 μM	Inhibits VEGF gene transcription and VEGF production,
			MCF-7 and MDA-MB-231 cells	(In vitro) 50, 100, 200 μM	Inhibits COX-2 expression and PGE2 production
		Gold nanoparticle–conjugated quercetin	MCF-7 and MDA-MB-231 cells, HUVECs	(In vitro) 25, 50, 75, 100, 125 μM	Inhibits the EGFR/VEGFR-2 signalling pathway, inhibits MMP-2 and MMP-9 protein levels	Preclinical testing	(Balakrishnan et al., 2016)
		Astragalin	MDA-MB-231 cells, HUVECs, CAM	(In vitro) 1, 10, 20, 50 μM	Targets AKT, ZEB1, VEGF, and MMP-9	Preclinical testing	(Tian et al., 2022)
Flavanones		Naringenin	MDA-MB-231 cells, HUVECs	(In vitro) 10, 30 μM	Downregulates ERRα and inhibits VEGF expression	Preclinical testing	(Li et al., 2016)
		Hesperidin	Mice model with transplanted 4 T1 breast cancer cells	(in vivo) 5, 10, 20, and 40 mg·kg^−1	Reduces the expression of VEGF, MMP-2, MMP-9	Preclinical testing	(Shakiba et al., 2023)
Isoflavones		Genistein	MDA-MB-231 and T-47D cells	(In vitro) 50, 100 μM	Downregulates HIF-1α	Clinical trials, phase 1 (NCT02075112), phase 2 (NCT01985763, NCT02624388, NCT02766478, NCT02499861, NCT01489813, NCT02567799)	(Farina et al., 2006; Mukund et al., 2019; Shao et al., 1998; Valachovicova et al., 2004; Yu et al., 2004, 2012)
			MDA-MB-231 and MCF-7 cells, MDA-MB-231 and MCF-7 xenograft nude mouse model	(In vitro) 20 μg·ml^−1 (in vivo) 0.1, 0.2, 0.5 mg·kg^−1	Inhibits VEGF transactivation, decreases MMP-9 expression and increases TIMP-1 expression, reduces VEGF and TGF-β1 expression,
			MCF-7/HER-2 cells (HER-2/neu high expression)	(In vitro) 1 μM	Reduces VEGF, MMP-9, MMP-2, and uPA expression
			MDA-MB-231 cells	(In vitro) 50, 100 μM	Inhibits uPA secretion, inhibits the NF-κB/AP-1 pathway
			F3II cells, syngeneic mice implanted with F3II cells	(In vitro) 0.1, 10, 20, 50 μM (in vivo) 10 mg·kg^−1	Inhibits uPA secretion
		Formononetin	MDA-MB-231 cells, HUVECs, CAM, Balb/c nude mice injected with MDA-MB-231 cells	(In vitro) 10, 25 μM	Inhibits FGFR2, decreases PI3K, Akt, and STAT3 activities	Preclinical testing	(Wu et al., 2015)
Chalcones		Xanthohumol	MCF-7, male nude mice inoculated with MCF-7 cells	(in vivo) libitum ingestion of 100 μM Xanthohumol solution	Inhibits NF-κB	Clinical trials, phase 1 (NCT02432651)	(Monteiro et al., 2008)
		Isoliquiritigenin	MCF-7 and MDA-MB-231 cells, MDA-MB-231 xenograft nude mouse model	(In vitro) 20, 40, 60, 80, 100 μM (in vivo) 25, 50 mg·kg^−1	Promotes HIF-1α proteasome degradation, reduces VEGF and MMPs levels, inhibits the VEGF/VEGFR-2 signalling pathway,	Preclinical testing	(Wang, Hsia, et al., 2013; Wang, Wang, et al., 2013)
			MCF-7 and MDA-MB-231 cells	(In vitro) 0.1, 0.5, 1, 5, 10 μM	Decreases HIF-1α expression, reduces MMP-2 and MMP-9 levels, inhibits p38 and PI3K-Akt signalling pathway activation
		Cardamonin	MCF-7 and MDA-MB-231 cells, MDA-MB-231 xenograft nude mouse model,	(In vitro) 20, 40 μM (in vivo) 3 mg·kg^−1	Reduces HIF-1α expression	Preclinical testing	(Jin et al., 2019; Shrivastava et al., 2017)
			BT-549 cells	(In vitro) 5, 10, 20 μM	Downregulates VEGF expression
Anthocyanins		Anthocyanins	MCF-7 cells	(In vitro) 400 μg·ml^−1	Inhibits the expression of VEGF, MMPs, inhibits the NF-κB pathway	Clinical trials, early phase 1 (NCT01823991)	(Hui et al., 2010; Paramanantham et al., 2020; Silveira Rabelo et al., 2022)
			Balb/c mouse 4 T1 model,	(in vivo) 150 mg·kg^−1	Inhibits the ERK1/2 and Akt/mTOR signalling pathway
			MDA-MB-453 xenografted Balb/c nude mice	(in vivo) 100 mg·kg^−1	Antagonizes VEGF, and decreases MMP-9, MMP-2, and uPA expression.
		Cyanidin-3-glucoside	MDA-MB-231 and Hs-578 T cells, CAM,	(In vitro) 10, 20 μM	Inhibits the STAT3/VEGF pathway, inhibits VEGF expression	Preclinical testing	(Ma & Ning, 2019)
		Cyanidin-3-O-Sambubioside	MDA-MB-231 cells, CAM, HUVECs,	(In vitro) 1, 3, 10, 30 μg·ml^−1	Downregulates MMP-9	Preclinical testing	(Lee et al., 2013)
		Delphinidin	Wistar-Furth rats injected with MT-450 cells	(in vivo) 1.18 × 10^−5 mol	Inhibits VEGFR-2	Preclinical testing	(Thiele et al., 2013)
Catechins		EGCG	C57BL mice injected with E0771 cells, E0771 cells	(in vivo) 50–100 mg·kg^−1	Decreases VEGF	Clinical trials, early phase 1 (NCT02891538, NCT05680662), phase 1 (NCT04177693, NCT03278925), phase 2 (NCT05758571, NCT02577393, NCT02580279, NCT04553666, NCT06015022, NCT04300855)	(Gu et al., 2008; Gu et al., 2013; Wei et al., 2018)
			Female mice injected with E0771 cells	(in vivo) 50–100 mg/kg	Inhibits VEGF, HIF-1α and NF-κB activation
			Balb/c mice injected with 4 T1 cells	(in vivo) 5, 10, 20 mg·kg^−1	Decreases HIF-1α and GLUT1 expression

TABLE 2. Clinical trials of some anti-angiogenic drugs in breast cancer.

Target	Drug	Mechanism of action	Clinical stage
Growth factors	Bevacizumab	Inhibits VEGF-A	Approval revoked
	Ramucirumab	Blocks VEGFR-2	Discontinued
	Aflibercept	Binds VEGF, and placental growth factor	Discontinued
Tyrosine kinases	Sorafenib	Inhibits VEGFRs, PDGFRs, and Raf	Discontinued
	Sunitinib	Inhibits VEGFRs, PDGFRs, fetal liver tyrosine kinase receptor 3, and c-KIT	Discontinued
	Vandetanib	Inhibits VEGFRs, EGFR, and RET tyrosine kinases	Discontinued
	Axitinib	Inhibits VEGFRs, PDGFRs, and c-KIT	Discontinued
	Pazopanib	Inhibits VEGFRs, PDGFRs, FGFR, and c-KIT	Discontinued
	Cediranib	Inhibits VEGFRs, PDGFRs	Discontinued

3.1 Flavones

3.1.1 Apigenin

Apigenin is a flavone that inhibits aromatase and acts as an ER agonist (Aliyev et al., 2021). Apigenin may inhibit VEGF expression in MDA-MB-231 breast cancer cells by decreasing HIF-α expression (Jin & Ren, 2007). Mafuvadze et al. showed that apigenin blocked progestin-dependent VEGF mRNA and protein expression and reduced progesterone receptor levels in T-47D breast cancer cells, and blocked medroxyprogesterone acetate (MPA)-dependent VEGF secretion in BT-474 cells, resulting in an anti-angiogenic effect (Mafuvadze et al., 2010). Subsequently, Mafuvadze et al. continued their experiments in a rat model of progestin-dependent breast cancer and obtained results similar to the in vitro experiments described above; they found that apigenin blocked the MPA-dependent increase in VEGF and inhibited tumour VEGFR-2 expression, but did not significantly reduce progesterone receptor levels (Mafuvadze et al., 2011). They also discovered that apigenin reduced VEGF expression in breast cancer rat tumours, but not tumour vascular density, and that apigenin treatment was associated with smaller vascular lumens (Mafuvadze et al., 2012). Apigenin overcame drug resistance in adriamycin-resistant breast cancer cells (MCF-7/ADR) by inhibiting STAT3 signalling pathway-induced growth inhibitory activity; it also reduced the expression of the STAT3 target genes VEGF and MMP-9 in MCF-7/ADR (Seo et al., 2017). Because the STAT3 pathway promotes tumour growth and angiogenesis (Chen et al., 2020), the anti-proliferative effect of apigenin, as well as its ability to overcome drug resistance, may be related to decreased tumour angiogenesis.

3.1.2 Baicalin

Baicalin, a natural flavone that inhibits angiogenesis by inhibiting VEGF, bFGF, and other pathways, is found in a wide variety of Scutellaria plants and can be extracted from the Chinese herb Scutellaria baicalensis (Singh et al., 2021). An in vivo study using a mouse model of breast cancer revealed that baicalin, alone or in combination with 5-FU, inhibited NF-κB pathway activation, reduced VEGF and IL-β levels, and inhibited tumour growth and angiogenesis; notably, baicalin enhanced the anti-tumour effect of 5-FU in breast cancer (Shehatta et al., 2022). Another study found that baicalin inhibited tumour growth and lung metastasis in an MDA-MB-231 xenograft model by blocking the p38MAPK signalling pathway, thereby downregulating MMP-2, MMP-9, uPA, and uPA receptor (uPAR) expression (Wang, Zhou, et al., 2013). Taken together, baicalin might have an anti-angiogenic effect on breast cancer cells.

3.1.3 Luteolin

Luteolin, a flavone, is abundant in commonly consumed foods, including broccoli, onions, carrots, peppers, cabbage and apples (Calabrese et al., 2021). Luteolin inhibits HIF-1α, VEGF, MMP-2 and MMP-9 protein expression in lung cancer and malignant melanoma as well as cell migration and tumour angiogenesis (Li et al., 2019; Pan et al., 2022). In breast cancer experiments, relatively low levels of luteolin significantly inhibit the secretion of VEGF in TNBC cells, and in vivo studies showed that luteolin inhibited the TNBC metastasis to the lungs, which may be because of its ability to inhibit angiogenesis (Cook et al., 2017). Another study discovered that luteolin inhibited TNBC lung metastasis and proposed that this might have occurred by reversing the EMT via β-catenin downregulation (Lin et al., 2017). Luteolin has also been shown to inhibit key Notch signalling components (Notch-1, Hes-1, Hey-1, Hey-2 and Cyclin D) and thus reduce tube formation in human umbilical vein endothelial cells (HUVECs) by decreasing VEGF, MMP-2, and MMP-9 expression in MDA-MB-231 and MCF-7 breast cancer cells (Sun et al., 2015).

Luteolin also has anti-oestrogenic properties. It significantly inhibits oestrogen biosynthesis in human ovarian granulosa KGN cells by inhibiting aromatase (CYP19) (Lu et al., 2012) as well as ER-α protein and mRNA expression in ER-α-positive breast cancer cells by exerting anti-proliferative and migratory effects (Lin et al., 2018). In one study, both low (1 mg·kg⁻¹) and high (25 mg·kg⁻¹) doses of luteolin significantly inhibited progesterone-accelerated DMBA (7,12-dimethylbenz(a)anthracene)-induced breast tumour development and significantly reduced VEGF production and tumour vascular density; however, luteolin had no significant effect on progesterone receptor levels (Cook et al., 2016). Similarly, luteolin has been reported to inhibit progestin-dependent VEGF secretion in breast cancer cells as well as reduce VEGF expression and vascular density in xenograft tumours; it did not block progesterone receptor activation, but inhibited progestin-mediated stem-cell-like properties in breast cancer cells (Cook et al., 2015).

3.1.4 Nobiletin

Nobiletin, a natural flavone, has anti-angiogenic activity; it is found in the skin of numerous citrus fruits in the Rutaceae family (Chen et al., 2022). Nipin et al. demonstrated that nobiletin inhibited VEGF-dependent angiogenesis in ER-positive breast cancer and endothelial cells in vitro, and that nobiletin treatment of MCF-7 and T-47D breast cancer cells resulted in lower VEGF and bFGF expression levels; they also found that nobiletin competitively bound to STAT3-initiating transcriptional sites to inhibit breast cancer tumour angiogenesis by regulating the Src/FAK/STAT3 signalling pathway via paxillin (Sp et al., 2017). Subsequently, Nipin et al. also found that nobiletin could bind CD36, decrease the expression of phosphorylated STAT3 and NF-κB in vitro experiments, and inhibit Cd36-dependent breast tumour angiogenesis through the Cd36/Stat3/NF-κB signalling axis; in addition, they discovered that nobiletin could inhibit STAT3 and NF-κB binding (Sp et al., 2018).

3.1.5 Wogonin and Wogonoside

Wogonin and wogonoside, derived from Scutellaria baicalensis, have a variety of biological activities, including anti-proliferation, apoptosis induction, and anti-angiogenesis activities, with wogonoside being the main in vivo metabolite of wogonin (Ku & Bae, 2014). Wogonin has been found to promote hypoxia-induced HIF-1α protein degradation, inhibit VEGF transcription, reduce VEGF expression, and inhibit cell-induced tube formation of HUVECs in MCF-7 cells in a concentration-dependent manner in vitro; in vivo, wogonin significantly inhibited angiogenesis in MCF-7 xenograft tumours (Song et al., 2013). Wogonin has also been found to inhibit the PI3K/Akt/NF-κB signalling pathway in MCF-7 cells, reduce the secretion, expression and activity of VEGF induced by lipopolysaccharide in tumour cells, and exert a significant anti-angiogenic effect (Zhao et al., 2014).

Wogonoside significantly reduced the vascular density in tumour tissues of xenografted MCF-7 nude mice, with an inhibition rate of 52.3%, and inhibited VEGF secretion, protein expression, mRNA expression, and transcriptional activity in breast cancer cells in vivo, which may be related to wogonoside-induced inhibition of the Wnt/β-catenin signalling pathway (Huang et al., 2016). Huang et al. discovered that wogonoside inhibited MDA-MB-231 and MDA-MB-468 cell-induced angiogenesis in vitro and in vivo by decreasing VEGF expression and inhibiting the Hedgehog signalling pathway, but did not consistently inhibit VEGF secretion from MDA-MB-231 cells (Huang et al., 2019). Furthermore, wogonoside inhibited EMT by downregulating MMP-9 and MMP-2 expression in MDA-MB-231 and MDA-MB-435 cells (Yao et al., 2017). This may be a part of the anti-angiogenic mechanism of wogonosides.

3.2 Flavonols

3.2.1 Jaceidin

Jaceidin is a flavonol extracted from Tanacetum vulgare L. (Kurkina et al., 2011). In vitro experiments showed that jaceidin significantly inhibited the growth of MCF-7 breast cancer cells. Jaceidin significantly inhibited mitosis in mouse Ehrlich tumour cells in vivo. The level of serum VEGF in mice treated with jaceidin was significantly reduced by 61.7% compared to that in the control group, and the angiogenesis-inhibiting activity of jaceidin was confirmed using a molecular docking simulation (Elhady et al., 2020).

3.2.2 Myricetin

Myricetin is a flavonol found in many diets through foods such as Chinese bayberries, blueberries, Camellia sinensis and grape juice (Xing et al., 2021). Zhou et al. discovered that myricetin inhibited angiogenesis by decreasing VEGF/VEGFR2 and p38MAPK expression, that myricetin-treated mice with breast cancer had significantly lower tumour volumes than the controls, and that the MVD of mammary tumours in mice treated with myricetin was significantly reduced. In addition, myricetin significantly decreased VEGF levels in the supernatant of 4T1 cells and the serum of transplanted tumour-bearing mice (Zhou et al., 2019). Furthermore, myricetin has been shown to inhibit the activity of MMP-2 and MMP-9 in MDA-MB-231Br cells (the human breast cancer brain metastasis cell lines), inhibit breast cancer cell migration and invasiveness and suppress breast cancer lung metastasis (Ci et al., 2018).

3.2.3 Quercetin

Quercetin is a natural flavonol found in a variety of foods such as onions, citrus fruits, apples, tomatoes and cherries. In breast cancer cells, it has various pharmacological activities including antioxidant, anti-inflammatory, anti-tumour, anti-viral, anti-proliferative, apoptosis-inducing and anti-angiogenic properties (Ezzati et al., 2020; Wang et al., 2022). Jia et al. discovered that quercetin inhibited MMP-2, MMP-9, and VEGF expression in mice with breast cancer, effectively suppressing the invasive and migratory ability of breast cancer cells and inhibiting breast cancer progression by inducing autophagy to inhibit cell migration and glycolysis via the Akt–mTOR pathway (Jia et al., 2018). Zhao et al. used nude mice transplanted with MCF-7 cells to conduct in vivo experiments and found that quercetin decreased VEGF and VEGFR2 gene expression and protein levels in tumour MVD and tumour tissues, and demonstrated that quercetin regulates the calcineurin/nuclear factor of activated T-cells pathway (Zhao et al., 2016). An in vitro study discovered that quercetin inhibited VEGF gene transcription production in tamoxifen-resistant (TAMR)-MCF-7 cells in a concentration-dependent manner, suggesting that PI3K inhibition is a key anti-angiogenic target of quercetin that reduces Pin expression in TAMR-MCF-7 cells, thereby inhibiting HIF-1α and c-Jun/AP-1 activation and decreasing VEGF secretion (Oh et al., 2010). Quercetin significantly inhibited COX-2 mRNA and protein expression as well as prostaglandin E2 production in breast cancer cells, and significantly inhibited COX-2-mediated angiogenesis in human endothelial cells in a dose-dependent manner, suggesting that COX-2 inhibition in breast cancer cells may be achieved by targeting the p300 signalling pathway (Xiao et al., 2011). By targeting the epidermal growth factor receptor (EGFR)/VEGFR-2 signalling pathway, AuNP-conjugated quercetin was found to inhibit EMT, angiogenesis, and metastasis in breast cancer cells as well as inhibit MMP-2 and MMP-9 protein levels in MCF-7 and MDA-MB-231 cells, thereby affecting EMT and exerting anti-angiogenic effects (Balakrishnan et al., 2016).

3.2.4 Astragalin

Astragalin has been isolated from the flowers of Rosa chinensis Jacq. and is the 3-O-glucoside of kaempferol, which has antioxidant, anti-inflammatory, and analgesic effects (Li et al., 2020). According to a molecular docking study, astragalin has high binding activity to VEGF and MMP-9. Subsequently, the researchers found that astragalin treatment diminished the neovascularization capacity and inhibited VEGFR and COX-2 protein expression levels in both HUVECs and chick embryo chorioallantoic membrane (CAM) models. Furthermore, astragalin treatment has been found to significantly reduce MMP-9 activity in MDA-MB-231 cells. Taken together, these results suggest that astragalin has anti-angiogenic activities in breast cancer (Tian et al., 2022).

3.3 Flavanones

3.3.1 Naringenin

Naringenin is a natural flavanone found primarily in citrus fruits such as grapefruit as well as other fruits such as tomatoes and cherries; it has anti-inflammatory, antioxidant, and anti-cancer properties (Motallebi et al., 2022). Li et al. confirmed that naringenin is an Oestrogen-related receptor alpha (ERRα) inverse agonist that inhibits VEGF expression in breast cancer cells by downregulating ERRα to exert an anti-angiogenic effect and reduce pro-angiogenic inflammatory cytokines (IL-6, monocyte chemoattractant protein 1, etc.) (Li et al., 2016).

3.3.2 Hesperidin

Hesperidin is among the most common flavonoids found in citrus fruits, exhibiting various effects including anti-inflammatory, anti-cancer and cardiovascular protective properties. In mice models with transplanted 4T1 breast cancer cells, hesperidin exhibited pronounced capabilities in suppressing tumour proliferation and angiogenesis. Treatment with hesperidin led to a significant reduction in the expression of VEGF, MMP-2 and MMP-9 in the mice, alongside decreased expression of the neovascular marker CD105. And when hesperidin was combined with doxorubicin (DOX) in the treatment of mice with breast tumours, the anti-tumour effect of DOX was significantly enhanced, and it had a stronger ability to inhibit angiogenesis (Shakiba et al., 2023).

3.4 Isoflavones

3.4.1 Genistein

Genistein is a phytoestrogen and isoflavone found in soybeans and soybean products. It can exert oestrogen-like effects through oestrogen receptors because its structure is similar to that of endogenous oestrogen (Peng et al., 2022). Genistein exhibits various biological activities, including antioxidant, anti-inflammatory, and cardioprotective properties (Rasheed et al., 2022). Previous studies have found that the high consumption of soy products is associated with a low incidence of hormone-dependent breast cancer, and that soy flavonoids and genistein contained in soy have anti-angiogenic properties (Varinska et al., 2015). Genistein inhibits angiogenesis by regulating VEGF, HIF-1α, MMPs and uPA levels as well as NF-κB, MAPK and other pathways.

HIF-1α expression levels in MDA-MB-231 and T-47D breast cancer cell lines were significantly reduced after genistein treatment, implying that genistein exerts its anti-angiogenic effects by blocking the transactivation of HIF-1α downstream effectors such as VEGF (Mukund et al., 2019). Genistein treatment reduced tumour vascular density, decreased VEGF and TGF-β1 levels and secretion, downregulated MMP-9, and upregulated tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in a mouse model of MDA-MB-231 xenograft, but had a weaker effect on vascular density in MCF-7 xenograft mouse tumours (Shao et al., 1998). In an in vitro experiment, genistein inhibited HER-2/neu receptor phosphorylation and protein tyrosine kinase (PTK) activity, and downregulated the expression of angiogenesis-related factors (VEGF, MMP-9, and uPA) in breast cancer cells with high HER-2/neu expression (Yu et al., 2004). It has been demonstrated that genistein flavonoids inhibit PTK activity in endothelial cells, VEGF-mediated activation of JNK and p38MAPK, and MMP secretion and activity, thereby interrupting endothelial cell activation (Yu et al., 2012). Genistein also exerts an angiogenic inhibitory effect by inhibiting uPA secretion in MDA-MB-231 breast cancer cells via NF-κB/AP-1 pathway inhibition (Valachovicova et al., 2004). Farina et al. found that genistein inhibits uPA secretion in F3II breast cancer cell monolayers and reduces the number of blood vessels induced in F3II cell tumours implanted in mice (Farina et al., 2006).

3.4.2 Formononetin

Formononetin is a phytoestrogen found in Angelica sinensis, Astragalus membranaceus, and other traditional Chinese medicines and has pharmacological effects such as antioxidant, neuroprotective, anti-tumour and anti-infection properties (Ma & Wang, 2022).

Formononetin has been shown to inhibit FGF2-induced vascular sprouting and proliferation of HUVECs in vitro. In vivo, formononetin significantly reduced downstream PI3K, Akt, and STAT3 activities and decreased MMP-2 and MMP-9 expression by targeting FGFR2, thus inhibiting mouse breast cancer growth and vascular generation. Notably, when combined with sunitinib, a VEGFR2 inhibitor, formononetin significantly enhances the anti-tumour effect of sunitinib (Wu et al., 2015).

3.5 Chalcones

3.5.1 Xanthohumol

Xanthohumol is a prenylated chalcone found naturally in hops (Andrusiak et al., 2021). Monteiro et al. found that xanthohumol-treated mice with breast cancer had reduced tumour MVD and significantly lower endothelial marker VIII levels, suggesting that the anti-angiogenic ability of xanthohumol was associated with NF-κB activity inhibition (Monteiro et al., 2008).

3.5.2 Isoliquiritigenin

Isoliquiritigenin (ISL), a dietary chalcone isolated from liquorice root and many other plants, has a wide range of biological activities, and is a strong antioxidant with anti-inflammatory, anti-tumour and anti-proliferative activities against a variety of cancer cells (Zhao et al., 2019). In vitro, ISL exerts oestrogenic effects on MCF-7 breast cancer cells, with high levels of cytotoxicity and the inhibition of aromatase mRNA expression (Lorusso & Marech, 2013; Maggiolini et al., 2002). Wang et al. demonstrated that ISL significantly inhibited VEGF expression in MCF-7 and MDA-MB-231 breast cancer cell lines by promoting HIF-1α proteasome degradation. Subsequently, Wang et al. showed that ISL inhibited the VEGF/VEGFR-2 signalling pathway, suppressed breast cancer endothelial cell growth, and significantly reduced MVD in mice in vivo. Subsequent molecular docking simulations showed that ISL can stably form hydrogen bonds in the ATP-binding region of VEGFR-2 to interact with aromatics and inhibit VEGF-2 activity (Wang, Wang, et al., 2013).

3.5.3 Cardamonin

Cardamonin, a natural chalcone found in plants of the Zingiberaceae family, including Alpinia katsumadai Hayata, Amomum tsaoko Crevost et Lemarie, and other herbal medicines, has anti-cancer properties such as cell proliferation inhibition and induction of apoptosis (Anqi et al., 2022; Varghese et al., 2020). Cardamonin has been shown to inhibit VEGF-induced angiogenesis in HUVECs in a dose-dependent manner by decreasing ERK and Akt (Jiang et al., 2015). Cardamonin has also been shown to reduce HIF-1α and lactate dehydrogenase A protein levels in vivo in an MDA-MB-231 xenograft model, resulting in a 53.10% reduction in CD31 staining in breast cancer tissues, indicating that cardamonin inhibits tumour angiogenesis (Jin et al., 2019). Cardamonin inhibited catenin protein levels in TNBC mice by downregulating the Wnt/β-catenin signalling cascade, significantly downregulating the expression of its downstream target VEGF and blocking BT-549 cell invasion and migration by reversing EMT (Shrivastava et al., 2017).

3.6 Anthocyanins

Anthocyanins are flavonoids that are abundant in grapes, pomegranates, berries, and other plants, with anti-inflammatory, apoptosis-inducing and anti-proliferative properties. They are also natural pigments.

Anthocyanins extracted from Vitis coignetiae Pulliat fruits inhibited induced NF-κB activation in MCF-7 breast cancer cells and upregulated NF-κB regulatory genes, such as MMP-2, MMP-9, intercellular adhesion molecule (ICAM)1 and VEGF, which are involved in cancer cell proliferation, invasion, adhesion and angiogenesis (Paramanantham et al., 2020). Rabelo et al. found that anthocyanins from dark sweet cherry (Prunus avium) inhibited the abnormal activation of the ERK1/2 and Akt/mTOR signalling pathways in breast cancer, which in turn inhibited cancer cell motility and invasion, with an 85% reduction in the area of angiogenesis in tumour tissue after anthocyanin treatment (Silveira Rabelo et al., 2022). In vivo and in vitro studies revealed that an anthocyanin-rich extract from black rice significantly inhibited tumour growth and angiogenesis by suppressing MMP-9, MMP-2, and uPA expression in MDA-MB-453 breast cancer tumour tissue (Hui et al., 2010). In another in vitro experiment, anthocyanins and proanthocyanidins extracted from dark sweet cherries were found to reduce VEGF protein levels in MDA-MB-453 breast cancer cells (Layosa et al., 2021).

Cyanidin-3-glucoside (C3G), a type of anthocyanidin found in many dark fruits, is considered to have anti-inflammatory, antidiabetic, and cardiovascular protective properties (Baster et al., 2020). In vitro experiments showed that C3G attenuated HUVEC angiogenesis induced by MDA-MB-231 and Hs-578 T breast cancer cells in a dose-dependent manner. C3G has also been shown to reduce breast cancer angiogenesis by inhibiting the STAT3/VEGF pathway. Specifically, C3G induces miR-124 to reduce STAT3 expression, thereby inhibiting VEGF expression and secretion (Ma & Ning, 2019).

Cyanidin-3-O-sambubioside, an anthocyanin isolated from the traditional Chinese medicines acanthopanax and hibiscus, has been shown to inhibit angiogenesis in breast cancer cells by inactivating the Akt pathway and inhibiting MMP-9 activity (Lee et al., 2013).

Delphinidin (DEL) is another type of anthocyanin that has anti-inflammatory and antioxidant properties (Zhang et al., 2021). DEL significantly reduced angiogenesis in vivo in the MT-450 syngeneic breast tumour model, and its degradation product, gallic acid, significantly inhibited VEGFR-2; however, in this model, DEL strongly promoted primary tumour growth, resulting in lymph node and lung metastasis, which may have been caused by DEL-induced CD4+ cell reduction (Thiele et al., 2013).

3.7 Catechins

3.7.1 Epigallocatechin gallate

Epigallocatechin gallate (EGCG), which is abundant in green tea, is one of the most studied natural flavonoids, and its numerous biological activities have preventive and therapeutic effects against breast cancer (Romano & Martel, 2021). After EGCG application in mice with breast cancer, tumour MVD was reduced, serum VEGF levels decreased, and tumour progression was inhibited (Gu et al., 2008). In another study, EGCG significantly inhibited VEGF expression by partially suppressing HIF-1α and NF-κB activation and simultaneously reducing the MVD in mouse mammary tumours (Gu et al., 2013). In addition, EGCG regulates the glycolytic process in breast cancer by decreasing HIF-1α and glucose transporter 1 (GLUT1) expression, reducing glucose and lactate levels, and decreasing VEGF expression in breast tumours (Wei et al., 2018). Furthermore, in vitro experiments revealed that EGCG decreased bFGF transcription in HUVECs and MDA-MB-231 cells (Sartippour et al., 2002). Notably, in breast cancer, increased VEGF expression protected against apoptosis after 24 h of EGCG treatment; however, after 72 h, VEGF mRNA levels decreased by 63%, resulting in low cell proliferation levels (Braicu et al., 2013). This may be because of the involvement of other growth factors that induce VEGF signalling.

4 DISADVANTAGES AND SIDE EFFECTS OF NATURAL FLAVONOIDS

Although natural flavonoids have many advantages, we should also be aware of the disadvantages of flavonoid natural products. The majority of natural flavonoids are derived from plant diets and herbal medicines. Orally administered flavonoids have significant absorption barriers, including low solubility, rapid metabolism, poor gastrointestinal tract absorption, low bioavailability and fluctuating pharmacokinetic and pharmacodynamic responses. This is closely related to their composition, subclassification, glycosylation, molecular weight, and esterification, as well as the intestinal environment, microbial actions and other co-actions (Naeem et al., 2022). Nanosystems such as nanoemulsions, nanocapsules, metallic nanoparticles, drug nanocrystals, polymeric nanoparticles, solid lipid nanocarriers and nanoliposomes have the potential to significantly improve flavonoid bioavailability and clinical efficacy (Khan, Ullah, et al., 2021).

While flavonoids generally boast a high safety profile, it is important to also consider potential side effects, such as organ toxicity, genotoxicity, promotion of tumour proliferation, impact on thyroid hormones and sex hormones, and drug interactions. (Figure 2) For example, excessive concentrations of genistein have been found to cause hepatotoxicity in mice, leading to elevated serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate transaminase (AST) (Islam et al., 2021). For instance, quercetin has the potential to exacerbate nephrotoxic effects in pre-damaged kidneys or to facilitate the development of oestrogen-dependent cancers (Andres et al., 2018). While the effect may be mild, it is important to note that certain phytoestrogens can indeed influence thyroid function and the secretion of sex hormones (Rizzo & Baroni, 2018). A mild effect does not necessarily imply its absence. Drug interactions with flavonoids, such as Naringin, quercetin, Genistein, and so on, are also noteworthy, particularly when considering drugs with a narrow therapeutic index. For instance, quercetin increases the bioavailability of drugs like Digoxin and Verapamil, potentially introducing clinical risks (Khan, Deb, et al., 2021).

FIGURE 2

Potential side effects of natural flavonoids.

Natural flavonoids may promote angiogenesis in breast tumours, which requires attention. For example, it has been shown that low doses (0.1 μM) of genistein upregulate VEGF expression in MELN (derived from MCF-7) cells via an ER-dependent mechanism but has no effect on VEGF in TNBC cell lines (Buteau-Lozano et al., 2008). Berndt et al. observed the effects of different concentrations of genistein on angiogenesis regulation using an in vitro three-dimensional HUVEC angiogenesis model; genistein significantly promoted blood vessel formation at low concentrations (0.001–1 μM) and inhibited angiogenesis at high concentrations (25–100 μM) (Berndt et al., 2018). However, in another study, 1-μM genistein had no significant inhibitory effect on VEGFR-2 expression or angiogenesis induced by estradiol and stimulated VEGF secretion (Saarinen et al., 2010). In an in vitro study, low concentrations (0–10 μM) of ISL reduced VEGF, HIF-1α, MMP-9 and MMP-2 expression in MDA-MB-231 breast cancer cells by inhibiting the p38 MAPK, PI3K/Akt and NF-κB signalling pathways, but not in MCF-7 breast cancer cells (Wang, Hsia, et al., 2013). In another study, ISL inhibited VEGF expression in both the MDA-MB-231 and MCF-7 breast cancer cell lines 23861918 (Wang, Wang, et al., 2013). These results might be related to the different ISL concentrations (0–100 μM versus 0–10 μM). Compared to ER-positive breast cancer, TNBC angiogenesis does not appear to be affected by this dose change, and given the affinity of natural compounds such as genistein for ERs, this pro-breast cancer proliferation and angiogenesis effect at low concentrations may be mediated by ERs. The anti-angiogenic effect of high concentrations of phytoestrogens may result from a more potent inhibition of the pathway mechanisms that suppress angiogenesis (Malik et al., 2023).

The biphasic effects of natural flavonoids are highly dependent on compound concentration and may influence the balance between numerous target actions, ultimately leading to either angiogenesis promotion or inhibition. In addition, this biological activity is mediated by multiple pathways that act independently or in combination with each other.

5 NANOTECHNOLOGY FOR FLAVONOID DELIVERY AND ITS EFFECTS ON TUMOUR ANGIOGENESIS

Given the limitations of natural flavonoids, such as insolubility and low bioavailability, the employment of nanotechnology for delivery has emerged as a prominent avenue of investigation. Nanotechnological approaches, including nanoemulsions, nanocapsules, metal nanoparticles, drug nanocrystals, polymer nanoparticles, solid lipid nanoparticles and nanoliposomes, have been extensively studied and hold significant promise. By enhancing the bioavailability of flavonoids, these techniques offer the potential to achieve improved clinical efficacy (Khan, Ullah, et al., 2021). For example, the use of liposomes to deliver hesperidin makes hesperidin stable in plasma for a long time without degradation, has better drug-releasing properties, and has higher antiproliferative activity in breast cancer cells than hesperidin alone (Teng et al., 2023). Nanoparticles loaded with apigenin had higher concentrations of apigenin than free apigenin in both the plasma and liver of mice and had the ability to continuously release the drug with a longer half-life in the plasma and liver, thus inhibiting liver tumour growth more effectively (Bhattacharya et al., 2018). Birinci et al. discovered that quercetin-conjugated titanium dioxide nanoparticles demonstrated greater cell permeability and antioxidant effects in mice compared to regular quercetin (Birinci et al., 2020). There are numerous analogous studies, indicating significant potential within these nanotechnologies for delivering flavonoids, despite the majority remaining at the experimental stage (Peng et al., 2023).

We note that the use of nanotechnology can not only improve the bioavailability of natural flavonoids, but also enhance the anti-angiogenesis, inhibition of tumour growth and anti-invasion and metastasis effects of flavonoids. For example, compared with free quercetin, gold nanoparticle-conjugated quercetin can better reduce the expression of VEGFR-2 protein in HUVECs, inhibit the expression of breast cancer MMPs, and more effectively inhibit angiogenesis in vivo and in vitro. In in vitro experiments utilizing the MCF-7 cell line and the CAM model, the nanoparticle quercetin showed better anticancer and anti-angiogenic activity than bulk quercetin, with the former having twice the anti-angiogenic score of the latter (Bansode et al., 2019). EGCG has low bioavailability. In one study, a combination of paclitaxel (Ptx) and EGCG encapsulated within a targeted core/shell PLGA-casein nanoparticle released the drug sequentially in a controlled manner and effectively sensitized Ptx-resistant breast cancer cells to Ptx by inhibiting NF-κB activation to downregulate key genes associated with angiogenesis, tumour metastasis, and survival (Narayanan et al., 2015). CD11, MMP-9 and TGF-β1 expression in the tumour tissue of breast cancer mice was significantly reduced after treatment with sophorolipid-coated silibinin and curcumin co-loaded nanoparticles, leading to anti-angiogenesis and reduced EMT effects. This nanoparticle improves drug stability in the intestinal environment and rapidly penetrates the mucosal layer in a bioresponsive manner to improve absorption. In terms of pharmacokinetics, it increases the maximum concentration (C_max) and area under the curve of silibinin by 32.31-fold and 11.48-fold, respectively (Liu et al., 2022). Shukla RP et al. developed a spermine (SPM) tethered lipid-polymeric hybrid nanoconstruct targeting tumour-specific heparan sulfate proteoglycan (HSPG). This facilitates the synergistic release of DOX and genistein, exhibits stronger anti-angiogenic effects in HUVECs and MDA-MB-231 cells in vitro and Balb/c mouse models in vivo, and inhibits the expression of VEGF pathway and MMPs (Shukla et al., 2020). However, it is important to note the bimodal effect of many nanostructures, which can stimulate angiogenesis at low doses or concentrations, and also inhibit angiogenesis at high doses or concentrations (Kargozar et al., 2020). For example, the nanomaterials graphene oxide (GO) and reduced graphene oxide (rGO) promote angiogenesis at low doses (<100 ng·ml⁻¹) and inhibit angiogenesis at high doses (>100 ng·ml⁻¹). This was attributed to the intracellular production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the activation of phospho-eNOS and phospho-Akt at low doses of GO and rGO, which promoted the proliferation of vascular endothelial cells. In contrast, at high doses GO and rGO produced excess ROS generating cytotoxicity and thus inhibited angiogenesis (Mukherjee & Patra, 2016). The dual role of ROS in angiogenesis and cytotoxicity, depending on the concentration, has been widely recognized and established (Nethi et al., 2019). Ideally, this could enable selective modulation of angiogenesis to treat different diseases with different doses of the same preparation, such as angiogenesis-promoting treatment of revascularization in stroke-affected areas, and anti-angiogenic effects against tumour growth. However, this task is exceedingly challenging as it involves the precise delivery of ROS to specific body areas and the intricate control of ROS levels. Under normal conditions, cellular antioxidant systems work to mitigate disturbances caused by ROS. However, if ROS concentrations exceed a certain threshold, they may compromise the effectiveness of cellular antioxidants and trigger oxidative stress. Moreover, elevated ROS levels pose an increased risk of carcinogenicity, cytotoxicity, and mutagenicity (Augustine et al., 2019).

6 SUMMARY AND FUTURE PERSPECTIVES

The potential of natural flavonoids for the treatment of breast cancer angiogenesis has been explored in this review. In cancer treatment, angiogenesis inhibition is a key strategy. Natural flavonoids have shown anti-angiogenic effects in breast cancer via various mechanisms (Figure 3), demonstrating the anti-angiogenic potential of flavonoid agents. Their low toxicity is advantageous for patients who are suffering from severe side effects of chemotherapy or targeted therapy. One of the most important anti-angiogenic mechanisms of natural flavonoids is VEGF and HIF-1α inhibition. These compounds reduce angiogenesis by inhibiting signalling pathways such as MAPK, NF-κB and PI3K/Akt. To address breast cancer heterogeneity, researchers must simultaneously develop drugs and identify biomarkers that can predict clinical outcomes for a more precise and individualized treatment.

FIGURE 3

The antiangiogenic mechanism of natural flavonoids in breast cancer. VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; PGR, progesterone receptor; VEGFR, VEGF receptor; FGFR, fibroblast growth factor receptor; HIF, hypoxia-inducible factor; COX, cyclooxygenase; JNK, c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; STAT, signal transducer and activator of transcription; Src, steroid receptor coactivator; FAK, focal adhesion kinase; AP-1, activator protein 1; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; u-PA, urokinase-type plasminogen activator; uPAR, uPA receptor; EGCG, epigallocatechin gallate.

We are also concerned about the limitations and potential side effects of natural flavonoids. The development of nanocarriers and other technologies has improved the bioavailability and efficacy of flavonoids, which is conducive to solving these limitations. It is noteworthy that nanotechnology enhances the antitumor and anti-angiogenic abilities of flavonoids. Furthermore, certain nanoflavonoids have demonstrated the potential to reverse treatment resistance in anticancer therapies and mitigate chemotherapy-related toxicity. For example, nanoparticles using a combination of EGCG and DOX can protect cardiomyocytes and reduce cardiotoxicity caused by DOX, potentially reversing multidrug resistance (Aiello et al., 2021). Tian et al. used nanotechnology to combine the flavonoid quercetin with chemotherapy drugs to achieve combined chemotherapy/angiostatic therapy and multidrug resistance reversal targeting breast cancer (Tian et al., 2018).

Although flavonoid anti-angiogenic agents have shown promise in animal studies, and nano-preparations have many advantages, particularly in improving drug aqueous solubility, there are still many challenges to overcome before they can become clinically useful. These challenges include, among other factors, the large gap between animal models and humans, the inability of animal trial data to accurately predict clinical efficacy, tumour heterogeneity, and individual differences (Park, 2017). All of these variables can have an impact on the efficacy of flavonoid anti-angiogenic agents in clinical trials. With the development of gene-array technology, metabolomics and proteomics, the use of these technologies to study a large number of samples and prospectively conduct relevant biomarker studies is conducive to the selection and development of natural products for tumour heterogeneity (Baracos, 2018). Promisingly, the use of systems biology and machine learning to establish computer system models, humanizing the data obtained from animal experiments, will provide strong support for clinical trials (Brubaker & Lauffenburger, 2020). Notably, some flavonoids have both anti-angiogenic and pro-angiogenic properties. This phenomenon may be related to the experimental method, drug concentration, or other factors. Therefore, a conclusion based solely on the findings of a study using cultured cells may not reflect the true effect in vivo. Further animal testing and a deeper understanding of the underlying mechanisms are necessary to establish a robust foundation for subsequent clinical trials.

In conclusion, natural flavonoids have the potential to target breast cancer angiogenesis, especially in combination with nanotechnology. Unfortunately, there do not seem to be any relevant clinical trials at this time, and we would like to see more research.

6.1 Nomenclature of Targets and Ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander, Cidlowski et al., 2021; Alexander, Fabbro et al., 2021a,b; Alexander, Kelly et al., 2021a,b).

AUTHOR CONTRIBUTIONS

Yuetong Wang: Conceptualization (lead); data curation (lead); investigation (lead); writing—original draft (lead). Mengge Huang: Data curation (equal); investigation (equal); writing—original draft (supporting). Xintong Zhou: Investigation (equal). Huayao Li: Writing—review and editing (equal). Xiaoran Ma: Writing—review and editing (equal). Changgang Sun: Conceptualization (lead); data curation (lead); supervision (lead); validation (lead); writing—original draft (supporting); writing—review and editing (supporting).

ACKNOWLEDGEMENTS

Thanks to all authors for their hard work on this study.

CONFLICT OF INTERESTS STATEMENT

The authors have declared that there is no conflict of interest.

Open Research

DATA AVAILABILITY STATEMENT

Data availability is not applicable to this article as no new data were created or analysed in this study.