Role of dietary bioactive natural products in estrogen receptor-positive breast cancer

Abstract

Estrogen receptor (ER)-positive breast cancer, including luminal-A and -B, is the most common type of breast cancer. Extended exposure to estrogen is associated with an increased risk of breast cancer. Both ER-dependent and ER-independent mechanisms have been implicated in estrogen-mediated carcinogenesis. The ER-dependent pathway involves cell growth and proliferation triggered by the binding of estrogen to the ER. The ER-independent mechanisms depend on the metabolism of estrogen to generate genotoxic metabolites, free radicals and reactive oxygen species to induce breast cancer. A better understanding of the mechanisms that drive ER-positive breast cancer will help optimize targeted approaches to prevent or treat breast cancer. A growing emphasis is being placed on alternative medicine and dietary approaches toward the prevention and treatment of breast cancer. Many natural products and bioactive compounds found in foods have been shown to inhibit breast carcinogenesis via inhibition of estrogen induced oxidative stress as well as ER signaling. This review summarizes the role of bioactive natural products that are involved in the prevention and treatment of estrogen-related and ER-positive breast cancer.

1. Introduction

Breast cancer is one of the most common cancers diagnosed among women [1]. There are many established risk factors for breast cancer, including age, genetic alterations, family history, mammographic breast density, menstrual and menopausal history, radiation exposure, and life style [2–4]. In particular, the hormones, estrogen and/or progesterone, are known to be capable of increasing breast cancer risk [2, 4]. Both ER-dependent and ER-independent pathways have been proposed for biological responses of estrogen [2, 5]. Estrogen binding to receptors results in their translocation to the nucleus, where they act as transcription factors leading to altered gene expression [6–8]. ER-dependent carcinogenic action of estrogen involves ER-mediated aberrant regulation of estrogen responsive genes that lead to increased cell proliferation and accumulation of DNA damage ultimately causing cell transformation [9]. The ER-independent pathway involves cytochrome P450 (CYP) mediated oxidative metabolisms of estrogen resulting in generation of genotoxic metabolites and ROS [10]. Since a large number of studies have demonstrated an inverse association between consumption of fruits and vegetables and the risk of breast cancer through suppression of ER signaling and reduction of oxidative stress, naturally occurring dietary compounds and phytochemicals have received increasing attention for treatment and prevention of breast cancer [11–15]. This review aims to summarize the potential of bioactive natural products in the prevention and treatment of estrogen-mediated and ER-positive breast cancer.

2. ER-positive breast cancer

ER expression is the main indicator of potential responses to hormonal therapy, and approximately 70–75% of human breast cancer is hormone-dependent and ER and/or progesterone receptor (PR) positive [16–19]. The ER-positive tumors express ER, ER-responsive genes, and other genes that encode characteristic proteins of luminal epithelial cells and, therefore, are termed “luminal group” [16]. Although sub-classification of luminal tumors is still contentious, there seems to be at least two groups of ER-positive breast cancers, commonly defined as luminal A and B tumors depending on the level of expression of the characteristic genes [16, 17]. Patients with luminal A breast cancer (ER/PR-positive and HER-2-negative) have a good prognosis with low histological grade, high survival rates and low recurrence rates [16, 17, 20, 21]. Luminal B tumors are ER-positive and/or PR-positive and HER- positive/negative [20]. At the cellular level, luminal B has low expression of ER and ER-related genes, high expression of proliferation and cell cycle-related genes such as Ki67, cyclin B (CCNB)1 and MYB-like (MYBL) 2 [20, 22]. The histological grade and proliferation index of the luminal B subtype is higher and prognosis is fairly poor than that for luminal A tumors [23]. In addition, patients with luminal B tumors have higher recurrence rates compared to those with luminal A tumors [24].

3. ER-dependent and -independent mechanisms

Estrogens have been implicated in breast carcinogenesis through receptor-dependent or -independent mechanisms [2, 5] (Fig. 1). The ER-dependent mechanism involves the binding of the hormone to nuclear ERs, which then form dimers and bind to estrogen response elements (ERE) in the regulatory regions of estrogen-responsive genes to alter gene expression, referred to as the classical genomic signaling pathway [5, 25, 26]. ER can also regulate cellular functions through nongenomic estrogen signaling [27]. Plasma membrane-associated ER increases the levels of second messengers and activates various tyrosine kinase receptors [28, 29]. The receptor-mediated cell divisions could increase cell proliferation and accumulation of DNA damage with mutational events [30]. In the ER independent pathway, carcinogenic effects of estrogens are believed to occur through the actions of estrogen metabolites [2, 5, 31]. Estrogen metabolism regulated by phase I CYP enzymes mediates the hydroxylation of estradiols and estrones [2, 32]. The formation of catechol estrogens, 2-hydroxyestrone/estradiol (2-OH-E₁/E₂) or 4-hydroxyestrone/estradiol (4-OH-E₁/E₂), are catalyzed by CYP1A1 and CYP1B1, respectively [2, 5]. Further oxidative metabolism of the catechols leads to formation of 2,3- and 3,4-quinone that can cause DNA damage [33]. The 3,4-quinone forms unstable depurination adducts with adenine and guanine, 4-OH-E2/E1-1-N3 adenine and 4-OH-E2/E1-1-N7 guanine, whereas the 2,3-quinone forms relatively stable DNA adducts and depurination occurs uncommonly [9]. These quinones can be reduced to semiquinones by CYP reductase, and this process can establish a redox cycle to produce ROS that can cause oxidative DNA damage [34]. Phase II enzymes, including catechol-O-methyltransferase (COMT), glutathione S-transferase (GST) and quinone reductase (QR), can inactivate estrogen catechols, semiquinones and quinones by preventing formation of adducts and oxidative DNA damage [2, 5].

Figure 1.

Diagrammatic representation of the pathways involved in the effects of estrogen on breast cancer development. The diagram is based on information from references [2, 43–46].

4. Estrogen and breast cancer stem cells

Cancer stem cells, defined by their ability to initiate the formation of tumors, self renew and differentiate, have been suggested to play a role in tumor initiation, maintenance, metastasis and resistance to therapies [35, 36]. Breast cancer stem cells were first identified as CD44⁺/CD24^−/low cell populations with an increased ability to form tumors [37]. Additional stem cell markers, such as high intracellular aldehyde dehydrogenase 1 (ALDH1) and epithelial specific antigen, have also been used in the identification of breast cancer stem cells [38]. Although the importance of estrogen in breast cancer is well-established, the role of estrogen in breast cancer stem cells is not fully understood. Some studies proposed that ER-positive cells are generated from a stem cell compartment that is directly stimulated by hormones, while others suggested that ER-positive cells may stimulate proliferation of a separate stem cell compartment in a paracrine manner [39, 40]. Several studies have reported that estrogen can modulate breast cancer stem cells via multiple pathways (Fig. 1). Major pathways involved in embryonic development, including Wnt/β-catenin, Hedgehog and Notch, have been identified to play pivotal roles in self-renewal behavior of breast cancer stem cells [41, 42]. Estrogen significantly increased the CD44⁺/CD24⁻ stem cell populations in MCF-7 and T47D as well as the size and number of the tumorsphere by targeting ERα36 via the phosphoinositide-3-kinase (PI3K)/Akt/GSK-3β/β-catenin pathway [43]. Fillmore et al. [44] reported that estrogen-induced expansion of cancer stem cells in vitro could lead to a functional increase in breast cancer stem cells and tumorigenic phenotypes in vivo through the FGF/Tbx3 signaling pathway. In addition, estrogen induces the expansion of CD44⁺/CD24^−/low stem-like cells populations and produces larger spheres by up-regulating cancer stem cell-related genes such as ALDH1, glioma-associated oncogene homolog (Gli)-1, Shh, Nanog, SOX2 and Bmi-1 through a noncanonical ligand-independent Shh/Gli signaling pathway [45]. Furthermore, estrogen induces a dramatic increase of OCT4 expression and proliferation of mammospheres as well as the breast cancer stem cell population in MCF-7 mammospheres [46]. These results imply that estrogen can modulate various breast cancer stem cell signaling pathways. The bioactive natural compounds, including curcumin [47–49], sulforaphane [50], soy isoflavone [51], epigallocatechin-3-gallate (EGCG) [52], resveratrol [53], indole-3-carbinol [54], luteolin [55], and 6-shogaol and pterostilbene [56], have the ability to inhibit breast cancer stem cells through regulation of self-renewal pathways such as Notch, Wnt/GSk3β/β-catenin, Hedgehog and PI3K/mTOR pathway. Currently, there are limited data available to conclude whether natural compounds can effectively suppress estrogen-induced breast cancer stem cells. Further studies are needed to identify the mechanisms of estrogen signaling related to breast cancer stem cells and the cancer inhibitory effects of natural compounds.

5. Natural products against ER-positive breast cancer

An increased understanding of the relationship between a healthy diet and lower incidence of a variety of cancers has led many researchers to focus on natural products for the prevention of cancer. A number of different terms are used to describe the many natural products currently being developed for health benefits. These include nutraceutical, functional food, pharmafoods, designer foods, dietary supplements and phytochemicals [57]. The terms functional food and nutraceutical are used interchangeably, and a number of definitions of what is included under these all-encompassing headings have been advanced [58]. Functional foods contain useful ingredients or compounds that have demonstrated physiological benefits; nutraceuticals (coined from “nutrition” and “pharmaceutical”) are naturally derived bioactive ingredients or compounds (usually as a supplement or extract) that provide health benefits [59]. Nutraceuticals and functional foods include many bioactive phytochemicals. Phytochemicals are natural bioactive compounds found in fruits, vegetables, herbal and medicinal plants which act as a defense system against several diseases [60, 61]. Phytochemicals from natural products cover a diverse range of chemical entities such as polyphenols, flavonoids, steroidal saponins, organosulphur compounds and vitamins [62]. Phytochemicals have been shown to exert beneficial effects with good safety profiles on many types of cancers including prostate, colon, breast, ovarian, and liver. Certain bioactive compounds have unique structural similarities to estrogen, and this review, therefore, focuses on phytochemicals that are exclusively involved in prevention and treatment of estrogen-related and ER-positive breast cancer. There are various animal models of ER-positive breast cancer that could be used to test existing and novel chemopreventive agents [63, 64]. The most widely studied are xenograft tumor models of established human breast cancer cell lines transplanted into athymic nude mice [65]. There are three major ER-positive cell lines commonly used, such as MCF-7 [66], T47D [67], and ZR-75-1[68]. In addition, overexpression of genes involved in breast cancer and mutagenesis induced by the mouse mammary tumor virus (MMTV) or polyoma middle-T antigen (PyMT) [69], or treatment with chemical agents, such as 7,12-dimethylbenz(a)anthracene (DMBA) [70], medroxyprogesterone acetate (MPA) [71], or N-methyl-N-nitrosourea (NMU) [72], can induce ER-positive mammary tumors in experimental animals. Furthermore, estrogen-mediated mammary carcinogenesis models are based on long-term subcutaneous administration of estrogen using slow-release pellets or silastic tubes [73]. In Table 1, we review the effects of bioactive natural compounds against ER-positive breast cancer in experimental in vivo models.

Table 1.

Effects of bioactive natural products against ER-positive breast cancer in vivo

Natural compounds	Experimental model of breast cancer	Effects	Ref
Allyl isothiocyanate	DMBA-treated SD rats	↓ Tumor incidence, tumor volume	[80]
		↓ TBARS, LOOH, CYP450, TC, TG, PL, FFA, LDL, VLDL
		↑ SOD, CAT, GPx, GSH, GST, GR, HDL activities
Apigenin	MPA-accelerated DMBA-induced SD rats	↓ Tumor incidence, VEGF, VEGF receptor-2 expression	[81, 88]
	BT-474 xenograft	↓ Tumor size, ↓ Her2/neu, Ki-67, VEGF, ↑ Apoptotic cells	[87]
Berries	E2 pellet-implanted ACI rats	↓ Tumor incidence, volume, multiplicity	[92]
		↓ CYP1A1, 17βHSD7, COMT expression
	E2 pellet-implanted ACI rats	↓ Tumor volume, tumor multiplicity	[93]
	E2-treated ACI rats	↓ 8-OH-dG, P-1, P-2, PL-1 levels	[94]
	E2 pellet-implanted ACI rats	↓ Tumor volume, tumor multiplicity	[95]
		↓ CYP1A1, ERα, Cyclin D1, PCNA expression
		↓ miR18a, miR20a, miR25, miR34c expression
	E2 pellet-implanted ACI rats	↓ Tumor volume, tumor multiplicity	[96]
		↓ Serum estradiol, prolactin level
		↓ CYP1A1, ERα, PCNA expression
Biochanin A	NMU-treated CD/Crj rats	↓ Tumor incidence, multiplicity, ↓ PCNA labeling index	[102]
	DMBA-treated SD rats	↓ Tumor burden, ↑ Necrosis	[104]
		↑ SOD, CAT, GPx, GST, DTD activities, ↓ MDA activity
	MCF-7 xenograft	↓ Tumor growth, tumor incidence	[103]
Caffeine	DES-treated ACI rats	↓ Total tumor number	[111]
	DMBA-treated SD rats	↓ Tumor incidence	[112, 113]
	PhIP-treated SD rats	↓ Tumor incidence, multiplicity, tumor size	[114]
Coumarin	DMBA-treated SENCAR mice	↓ Cytochrome P450 1A1/1B1 and DNA adduct formation	[119]
		↓ Tumor size, multiplicity and tumor occurrence
	DMBA-treated Wistar rats	↓ Tumor size, multiplicity and tumor occurrence	[120]
		↓ Hepatic drug metabolizing activity
		↑ Serum prolactin level
Curcumin	E2 pellet-implanted ACI rats	↓ Tumor multiplicity, tumor volume	[130]
		↑ Hepatic CYP1A and CYP1B1 level
	DMBA-treated SD rats	↓ Tumor incidence, multiplicity	[127]
		↓ VEGF induction in hyperplastic lesions
	DMBA-treated SENCAR mice	↓ Tumor growth	[128]
		↑ Pro-apoptotic protein, maspin
		↓ Anti-apoptotic protein, survivin
	MCF-7 xenograft	↓ Tumor growth	[129]
		↓ Cyclin D1, cyclin E, cyclin A, CDK2, CDK4, p38
		↑ p21, p27
Diallyl sulfide	DES-treated ACI rats	↓ nDNA adduct formation	[133]
		↑ CYP1A1, CYP1B1, GST, SOD expression	[134]
		↑ p53, Gadd45a, PCNA, DNA polymerase δ expression	[135]
		↓ DES-induced LOOH and ROS production	[136]
	MCF-7 xenograft	↓ Tumor growth	[132]
Diosgenin	NMU-treated SD rats	↑ Antioxidant enzyme activities	[139]
		↓ Peroxidation reaction
	MCF-7 xenograft	↓ Tumor growth	[137]
Ellagic acid	E2-treated ACI rats	↓ Tumor volume, tumor multiplicity	[93]
		↓ Tumor incidence, volume, multiplicity	[92]
		↓ 17β-hydroxysteroid dehydrogenase, COMT expression
		↓ Tumor incidence, burden, multiplicity	[143]
		↓ Plasma prolactin and PCNA levels
		↓ Tumor volume, multiplicity	[96]
		↓ Serum E2 level, CYP1A1 expression
		↓ ERα, Bcl-w, CCND1, CCNG1, RASD	[144]
		↑ FOXO1, FOXO3a
EGCG	E2-treated C57BL/6 mice	↑ Uterine peroxidase activity	[161]
	MCF-7 xenograft	↓ Tumor growth, tumor size	[160]
Ferulic acid	DMBA-treated SD rats	↓ Tumor volume, tumor burden	[165]
		↓ p53, bcl-2 expression and TBARS activity
		↑ GST, GR, DTD, SOD, CAT and GPx activities
Flaxseed	NMU-treated SD rats	↓ Tumor multiplicity	[172]
		↓ Tumor invasiveness, tumor grade
	DMBA-treated SD rats	↓ Tumor incidence, tumor multiplicity	[345]
		↓ Cell proliferation ↑Apoptosis
		↑ BRCA1, p53 expression
		↓ 8-OH-dG levels
	DMBA-treated SD rats	↓ Tumor incidence, size, number, weight	[346]
	MCF-7 xenograft	↓ Tumor growth, tumor size	[168–170]
		↓ Cell proliferation ↑Apoptosis
		↓ ERα, PR, IGF-1 expression
Gallic acid	BT474 xenograft	↓ Tumor volume	[178]
		↓ NFκB p65, pAkt, pPI3K, hypoxia inducible factor 1α, VEGF expression
Genistein	E2-treated SD rats	↓ Bcl-2/Bax ratio	[187]
	DMBA-treated SD rats	↓ Tumor incidence	[185]
		↓ AIB1, erbB2, PCNA expression and PTK activity
		↓ Plasma vascular endothelial growth factor
		↓ Tumor weight, multiplicity, tumor incidence rates	[186]
		↑ Plasma endostatin levels
	MCF-7 xenograft	↓ Tumor weight, volume	[190]
		↓ Smo, Gli1 expression, ALDH staining, Hedgehog-Gli pathway expression
Hesperetin	MCF-7 xenograft	↑ Tumor incidence in long-term genistein exposure	[189]
Indole-3-carbinol	DMBA-treated SD rats	↓ Tumor growth, volume	[205]
	E2-treated SD rats	↑ CYP1A1, 1A2, 3A, 2B activities, ↑ E1 and E2 metabolism	[206]
	MMTV-induced mice	↓ Tumor growth	[188]
	MCF-7 xenograft	↓ Tumor growth	[204]
Luteolin	DMBA-treated Wistar rats	↓ Tumor incidence, volume	[213]
		↑ SOD, CAT, GPx activities
		↓ Lipid peroxidation levels
		↓ Cell numbers	[214]
	T47D xenograft	↓ Tumor volume, tumor incidence rate	[55]
		↓ VEGF expression, blood-vessel density
Lycopene	DMBA-treated SD rats	↓ MDA and NO levels	[220]
		↑ SOD, CAT, GPx activities
	DMBA-treated ACI rats	↓ Tumor incidence	[221]
		↑ SOD, CAT, GPx activities
		↓ Bcl-2 expression and serum MDA, NO, 8-OH-dG levels
	DMBA-treated Wistar rats	↓ Tumor incidence, tumor weight, tumor volume	[222]
		↑ Bax, caspase-3 and caspase-9 expression
Morin	DMBA-treated SD rats	↓ Tumor weight, incidence, size, volume	[228]
		↓ TBARS and hydroperoxides, AFP, CEA, CA-15-3, PCNA
		↑ SOD, CAT, GPx, GST, GR, GSH, G6PD, Vit C, Vit E
Myricetin	DMBA-treated Wistar rats	↓ Plasma TBARS levels, ↑ SOD levels	[233]
Phenylethyl isothiocyanate	DMBA-treated SD rats	↓ Tumor multiplicity, tumor volume	[234]
	NMU-treated SD rats	↓ Tumor incidence, multiplicity	[235]
		↓ Angiogenesis and invasive tumors
	PyMT transgenic mice	↓ Tumor growth	[237]
		↓ ERα, FOXA1 expression
Pomegranate	DMBA-treated SD rats	↓ ERα, ERβ expression, ERα: ERβ ratio	[244]
		↓ β-catenin expression, nuclear translocation
		↓ Cyclin D1 expression
	DMBA-treated SD rats	↓ Tumor incidence, total tumor burden	[347]
		↓ Cell proliferation ↑ Apoptosis
		↑ Bax, Bad, caspase-3, caspase-7, caspase-9, cytochrome C expression
		↓ Bcl-2 expression
	BT474 xenograft	↓ Tumor volume, tumor weight	[249]
		↑ Apoptotic lesions
		↓ Sp1, Sp3, Sp4, VEGF, p65, miR-27a, SHIP-1, pAKT, pPI3K expression
		↑ ZBTB10 expression
Quercetin	DMBA-treated Wistar albino rats	↓ Tumor volume	[257]
		↓ CEA, ACP, cathepsin D levels
	E2-treated ACI rats	No chemopreventive effects	[258]
		↓ COMT activity, tumor latency
	C3(1)SV40Tag mice	↓ Tumor number, volume	[256]
	MCF-7 xenograft	↓ Tumor size	[103, 251]
Resveratrol	DMBA-treated SD rats	↓ Tumor growth ↑ miR-146a expression	[268]
	DMBA-treated SD rats	↓ Tumor incidence, tumor multiplicity	[269]
		↓ NFκB, COX-2, MMP-9 expression
	NMU-treated SD rats	↓ Tumor incidence, tumor multiplicity	[270]
	E2-treated ACI rats	↓ Tumor multiplicity	[271]
		↓ Cell proliferation in mammary terminal ductal structures
		↑ Nrf2-mediated protective pathways
		↓ Nrf2 promoter methylation
Sesamin	DMBA-treated SD rats	↓ Cumulative number of palpable mammary cancer	[275]
		↓ Lipid peroxide, fatty acid, tumor phosphatidylcholine, prostaglandin E2
		↑ PBMC activity
	MCF-7 xenograft	↓ Tumor size, tumor proliferation ↑ Apoptosis	[276]
Sulforaphane	BCSC in NOD/SCID mice	↓ Tumor growth	[50]
	KPL-1 xenograft	↓ Tumor volume, tumor weight, tumor growth	[284]
		↑ Apoptosis
Tangeretin	DMBA-treated SD rats	↓ Tumor growth, total tumor weight, tumor volume	[289]
		↓ NO, LOOH, CEA levels
		↑ SOD, CAT, GPx, GST, GSH, Vit-C, Vit-E levels
	DMBA-treated Wistar rats	↓ Tumor weight	[288, 290]
		↓ Prolactin, estradiol, TSA, LSA, protein carbonyl, NO levels, PCNA, COX-2, Ki-67, cyclin D1, cyclinE, CDK4, CDK2, MMP2, MMP9, VEGF expression	[288]
		↑ Progesterone level and p53, p21, TIMP2 expression
		↓ Tumor weight	[290]
		↓ AST, ALT, ALP, ACP, 5′-ND, GGT, LDH, CytP450, Cytb5, EROD, MROD, PROD, TBARS, CD, AFP, CEA, CA15-3 activities and ER, PR, Her2/neu expression
		↑ GST, QR, SOD, CAT, GPx activities
Thymoquinone	DMBA-treated albino rats	↓ BRCA, BRCA2, Id-1, p53 expression and MDA, LDH, ALP, AST levels	[296]
γ-TmT	NMU-treated SD rats	↓ Tumor burden, multiplicity	[302]
		↓ Bcl-2, XIAP, ERα, PCNA, PKC, p-Akt, Cyclin D1, CDK4, CDK6, Keap1 expression
		↑ Bax, cleaved-caspase 3, cleaved-caspase 9, cleaved-PARP, PPARγ, PTEN, p53, p21, p27, Nrf2, GClm, GSTm1, NQO1, TXN, GPx, HO-1 expression
	E2 pellet-implanted ACI rats	↓ Tumor volume, multiplicity	[301]
		↓ Serum estrogen, 8-OH-dG levels and PCNA expression
		↑ CYP1A1, CYP1B1, Nrf2, NQO1, GClm, HO-1, PPARγ, PTEN and p27 expression
		↓ Serum E2 level, PCNA, ERα, ERβ, COX-2 expression and nitrotyrosine, 8-OH-dG levels	[305]
		↑ Cleaved-caspase3, PPARγ, Nrf2, NQO1, UGT, HO-1, GST, GSTm1, GClm levels
		↓ Nitrotyrosine, 8-OH-dG, 8-isoprostane levels	[306]
		↑ Nrf2, SOD1, GPx, CAT expression
γ-Tocopherol δ-Tocopherol	NMU-treated SD rats	↓ Tumor incidence, multiplicity	[302–304]
		↑ PTEN, p53, p21, p27, NQO1, PPARγ, Nrf2 and phase II detoxifying/antioxidant enzyme expression	[302]
		↓ PCNA, PKC, c-Myc, p-Akt, cyclin D1, ERα expression and nitrotyrosine, 8-OH-dG levels
		↑ p21, p27, PPARγ, cleaved PARP, cleaved caspase-3 expression, ↓ ERα, p-Akt expression	[303]
		↓ PCNA expression	[304]
Xanthohumol	KPL-3C xenograft	↓ Tumor growth, ↓ Ki67, PCNA expression	[307]
		↓ Endogenous BIG3-PHB2 complex
	MCF-7 xenograft	↓ Tumor weight	[315]
		↑ Cell apoptosis in tumor tissues
		↓ Endothelial marker VIII expression and NFκB p65, IκB, IL-1β activities

Abbreviations: AIB1: amplified in breast cancer 1; BCSC: breast cancer stem cells; CA-15-3: cancer antigen 15-3; EROD: ethoxyresorufin O-decarboxylase; GClm: glutamate cysteine ligase modifier subunit; GSTm1: glutathione s-transferase mu 1; G6PD: glucose-6-phosphate dehydrogenase; HDL: high-density lipoprotein; HIF: hypoxia-inducible factor; HO-1: heme oxygenase 1; Id-1: inhibitor of DNA binding 1; IL-1β: interleukin 1β; MROD: methoxyresorufin O-decarboxylase; PAH: polycyclic aromatic hydrocarbons; PKC: protein kinase C; PROD: pentoxyresorufin O-decarboxylase; RASD1: RAS dexamethasone-induced 1; Smo: smoothened; TIMP: tissue inhibitor of metalloproteinase; UGT: UDP-glucuronosyltransferase; XIAP: x-linked inhibitor of apoptosis

5.1. Allyl isothiocyanate

Allyl isothiocyanate, which occurs in many cruciferous vegetables, has been reported to have chemopreventive activities in various cancer models [74]. Bioavailability of allyl isothiocyanate is high and absorbed allyl isothiocyanate is primarily eliminated in the urine in mice and rats [75–77]. Human absorption and disposition of allyl isothiocyanate appear to closely resemble that of animals, as studies showed that about 50% of the dose was recovered in the urine as a metabolite [78, 79]. Allyl isothiocyanate inhibited mammary tumor incidence and tumor growth in DMBA-treated female Sprague Dawley (SD) rats [80]. In this study, allyl isothiocyanate decreased lipid peroxidation-related thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) as well as total cholesterol (TC), triglycerides (TG), glycophospholipid (PL), free fatty acids (FFA), low-density lipoprotein (LDL) and very-low density lipoprotein (VLDL) [80]. Decreased levels of CYP450 and increased activities of phase II and antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), GST and glutathione reductase (GR), have been shown in liver and mammary tissues of allyl isothiocyanate-treated rats [80]. These data suggest that allyl isothiocyanate exerts chemopreventive activity against DMBA-induced mammary carcinogenesis mainly via activation of antioxidant response.

5.2. Apigenin

Apigenin is a natural flavonoid commonly found in fruits and vegetables such as parsley, celery, thyme, and oranges. The chemical structure of apigenin is similar to estrogen, and it may act as a mimic of estrogen. Therefore, extensive studies have shown that apigenin has potent antioxidant and anticancer activities in ER-positive and ER-negative breast cancer [81–83]. Apigenin in its pure form is unstable and insoluble in water or organic solvents. In its natural form, apigenin is present in foods mostly as glycoside conjugates and various acylated derivatives, which are more soluble than the parent compound. Therefore, apigenin bound to β-glycoside provides high bioavailability [84–86]. Several animal studies reported a decrease of estrogen-dependent tumorigenesis with apigenin containing diets. Mafuvadze et al. [87] determined whether apigenin inhibits the growth of aggressive BT-474 xenograft tumors in nude mice exposed to MPA, one of the most common hormone replacement therapies for postmenopausal women. In this study, apigenin inhibited progression and development of BT-474 xenograft tumors by inducing apoptosis, inhibiting cell proliferation, and reducing expression of Her2/neu, Ki-67 and vascular endothelial growth factor (VEGF) [87]. In another study using the DMBA model to examine the chemopreventive effect of dietary apigenin against MPA-accelerated mammary tumors in SD rats, apigenin decreased tumor incidence and tumor multiplicity [81]. Administration of apigenin reduced MPA-dependent tumor incidence and tumor multiplicity as well as MPA-dependent increase in VEGF and VEGF receptor-2 expression [88]. The direct binding of ERα to the ERE in the VEGF promoter region is known to decrease VEGF expression [89], suggesting that apigenin may provide a potent chemopreventive effect via ER-dependent pathway in breast cancer.

5.3. Berries

Dietary berries contain several phytochemicals such as cyanidin, delphinidin, quercetin, kaempferol, ellagic acid, resveratrol and pterostilbene, which have antioxidant properties and help to reduce the risk of breast cancer [90]. Bioavailability of anthocyanin is generally poor compared to other berry flavonoids, usually limited to less than a percent of the amount ingested. Proanthocyanidins also exhibit poor bioavailability. Flavonols in berries show better bioavailability than other flavonoids [91]. Aiyer et al. [92, 93] investigated the effects of dietary berries in estrogen-mediated mammary tumorigenesis. Black raspberry reduced the tumor volume and tumor multiplicity. Blueberry showed a reduction only in tumor volume [93]. The same group evaluated the ability of each of the mixed berries (strawberry, blueberry, blackberry, red raspberry, and black raspberry) and blueberry in reducing oxidative DNA damage in estrogen-treated ACI rats [94]. Their results indicated that 2.5% blueberry diet significantly reduced the estrogen-induced levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG), P-1, P-2 and PL-1 subgroups [94]. Jayabalan et al. [95, 96] tested both preventive and therapeutic activities of diet supplemented with blueberry against estrogen-mediated mammary tumorigenesis. Administration of blueberry delayed the tumor latency and decreased the tumor incidence, tumor volume and tumor multiplicity in both modes. Administration of blueberry diet reduced the expression of CYP1A1, cyclin D1 and proliferating cell nuclear antigen (PCNA) and modulated the levels of miR-18a and miR-34c [95, 96]. These findings indicate that blueberry could modulate major estrogen-associated oxidative stress and proliferative markers, resulting in protective effects against mammary tumorigenesis in animals treated with estrogen.

5.4. Biochanin A

Biochanin A, an isoflavone isolated from red clover, is a natural phytoestrogen [97]. The chemical structure of biochanin A is similar to estrogen, enabling it to bind to ER. The effects of biochanin A include regulation of estrogen receptor, inhibition of cell proliferation, reduction of inducible nitric oxide (NO) synthase, and apoptosis stimulation in breast cancer [98, 99]. Biochanin A has low bioavailability in rats [100, 101]. Treatment with biochanin A decreased the tumor incidence and multiplicity in NMU-induced rat mammary carcinogenesis [102]. One study determined the effect of biochanin A, administrated alone or in combination with the quercetin and epigallocatechin-3-gallate, on the growth of MCF-7 xenograft tumors. Treatment with biochanin A or the mixture of the three compounds resulted in a reduction in tumor incidence and tumor size [103]. Mishra et al. [104] investigated whether biochanin A inhibits mammary tumorigenesis via regulating anti-oxidant and xenobiotic metabolisms in DMBA-treated rats. Treatment with biochanin A suppressed tumor burden by increasing antioxidant enzymes, including SOD, CAT, GPx, GST, DT-diaphorase (DTD), and preventing the peroxidative damage, suggesting that protective effects of biochanin A are mediated by inhibiting oxidative stress and activating antioxidant response.

5.5. Caffeine

Caffeine, a natural chemical found in coffee beans, cacao, and tea leaves, has anticancer properties against both ER-dependent and ER-independent breast cancer cells [105, 106]. Several studies demonstrated that caffeine absorption from the gastrointestinal tract is rapid and complete with the bioavailability of 99 to 100% after oral administration [107–110]. Treatment with caffeine decreased the total number of mammary tumors in diethylstilbestrol (DES)-treated ACI rats [111]. Wolfrom et al. [112] assessed the influence of caffeine on the incidence of benign mammary tumors in DMBA-treated SD rats. Caffeine treatment significantly reduced the incidence of benign mammary fibroadenomas as well as the incidence of mammary gland cysts [112]. VanderPloeq et al. [113] explored the effect of chronic caffeine consumption on the initiation and promotion stages of DMBA and NMU-induced mammary tumorigenesis in female SD rats. Caffeine significantly suppressed the initiation stage of DMBA-induced rat mammary tumorigenesis, while there was no inhibitory effect in the promotion stage of NMU-induced mammary tumorigenesis [113]. Another study investigated the chemopreventive effects of caffeine on 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in SD rats. Administration of caffeine reduced tumor incidence and tumor size as well as tumor multiplicity [114]. Taken together, caffeine may reduce breast cancer by modifying metabolism of carcinogens in vivo.

5.6. Coumarin

Coumarin, a naturally occurring fragrant organic compound, is commonly found in many plants such as grass, orchids, citrus fruits and legumes [115]. The pattern of substitutions on the basic chemical structure of coumarin influences both pharmacological and biochemical properties, such as estrogenic activity [116]. However, coumarin has very low bioavailability in humans due to rapid absorption, metabolism, and extensive first-pass hepatic conversion to 7-hydroxycoumarin followed by glucuronidation [117, 118]. Prince et al. [119] investigated the ability of coumarin to block DMBA-induced DNA adduct formation in sensitive to carcinogensis (SENCAR) mouse mammary gland. Coumarin inhibited DMBA-DNA adduct formation through inhibition of CYP450-1A1/1B1 and induction of hepatic GSTs [119], suggesting that coumarin can act via genotoxic pathway. Another study revealed that coumarin suppressed the incidence of adenocarcinomas, tumor growth, tumor size and tumor multiplicity when administered before DMBA. However, there was no effect when coumarin was given after DMBA treatment [120]. These results indicate coumarin as an effective chemopreventive agent against DMBA-induced carcinogenesis by reducing genotoxic pathway.

5.7. Curcumin

Curcumin, a yellow pigment of turmeric, has been reported to be an effective and safe natural compound for the prevention and treatment of breast cancer [121]. Despite much evidence of its efficacy and safety, curcumin has not yet been approved as a therapeutic agent due to its low bioavailability in vivo and in humans [122–124]. The daily dosage of curcumin generally seems safe in the range between 500 and 4000 mg, and no toxicity was observed even up to 8 g per day for 3 months, except for some individuals who had minor gastrointestinal side effects [125, 126]. Carroll et al. [127] revealed that curcumin delayed the first appearance of MPA-accelerated tumors, reduced tumor multiplicity in DMBA-induced and MPA-accelerated tumors, and decreased MPA-induced VEGF induction in hyperplastic lesions. Siddiqui et al. [128] demonstrated that combination of docosahexaenoic acid and curcumin reduced the tumor incidence and tumor growth of DMBA-induced mammary tumorigenesis in SENCAR mice. Zhou et al. [129] also investigated the efficacy of curcumin in combination with mitomycin C in the MCF-7 xenograft model. Curcumin alone and curcumin plus mitomycin C treatment inhibited tumor growth, with increased G1 arrest associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2 and CDK4 via down-regulation of p38 mitogen-activated protein kinase (MAPK) pathway [129]. Bansal et al. [130] tested the potential chemopreventive effect of curcumin against estrogen-mediated mammary tumorigenesis when curcumin was given via diet or implants. Curcumin administered via implants resulted in significant reduction in both tumor multiplicity and tumor volume, while dietary curcumin was ineffective [130]. It may be because dietary curcumin increased hepatic CYP1A and CYP1B1, but not CYP3A4 activities, whereas curcumin given via implants increased CYP1A and CYP3A4, but decreased CYP1B1 activity in estrogen-treated ACI rats, generating non-carcinogenic estrogen metabolites [130]. Overall, these results indicate that curcumin inhibits mammary carcinogenesis by controlling estrogen-metabolism, apoptosis and MAPK pathway.

5.8. Diallyl sulfide

Allyl sulfides, such as diallyl sulfide, diallyl disulfide, and diallyl trisulfide, are major organosulfur compounds derived from garlic. These compounds have been shown to exert potent chemopreventive effects in several types of breast cancer [131, 132]. Oral administration of diallyl sulfide inhibited tumor growth in the MCF-7 xenograft model [132]. Green et al. [133] revealed that diallyl sulfide inhibited the DES-induced mtDNA adducts and caused a decrease in nDNA adduct formation in ACI rats. The same group investigated the effect of diallyl sulfide on the expression of phase I and phase II metabolizing genes involved in estrogen metabolism in the DES-induced ACI rats [134]. Their results indicated that diallyl sulfide treatment increased the expression of CYP1A1 and CYP1B1 as well as GST and SOD [134]. In addition, diallyl sulfide increased the expression of p53, Gadd45a, PCNA and DNA polymerase δ together, which are important in repairing DNA damage thus preventing cancer in DES-treated ACI rats [135]. Gued et al. [136] assessed the effect of diallyl sulfide on DES-induced ROS in ACI rats. Diallyl sulfide attenuated DES-induced LOOH and ROS production in breast and liver tissues. Overall, these data suggest that diallyl sulfide is a modulator of xenobiotic metabolic pathway involved in estrogen metabolism with a potential role in the prevention of breast cancer.

5.9. Diosgenin

Diosgenin, a naturally occurring steroid saponin found in legumes and yams, acts as a potential chemopreventive agent in ER-positive and ER-negative breast cancer [137]. Estrogenic effects of diosgenin have been hypothesized because of its structural similarities to estrogen. However, clinical application of diosgenin is limited due to undesirable pharmaceutical characteristics such as its poor solubility and low bioavailability [138]. An in vivo tumor study indicated that diosgenin significantly inhibited MCF-7 xenograft tumor growth in nude mice [137]. Jaqudeesan et al. [139] revealed that diosgenin has anticarcinogenic activity in NMU-induced mammary carcinogenesis in SD rats by reducing peroxidation reaction marker enzymes and antioxidant enzymes. These data imply that diosgenin exhibits anticarcinogenic activity possibly through enhancing the antioxidant defense system.

5.10. Ellagic acid

Ellagic acid, a dietary flavonoid polyphenol present in berries, grapes, pomegranates and nuts, has anti-cancer effects through modulation of cell proliferation and apoptosis in ER-positive breast cancer cells [140]. Ellagic acid consists of phenolic rings and ortho-dihydroxyl groups, which allow receptor recognition and ER-mediated action [141]. However, the bioavailability of ellagic acid is very low due to poor absorption from the gut, rapid metabolism, and lack of transport to the target organs [142]. Treatment with ellagic acid reduced the tumor volume and multiplicity in estrogen-mediated mammary tumorigenesis in ACI rats, as reported by Aiyer et al [93]. The same group revealed that ellagic acid decreased the tumor incidence, volume and multiplicity as well as CYP1A1 activity in estrogen-induced ACI rats. Ellagic acid decreased 17β-hydroxysteroid dehydrogenase (17βHSD7) and COMT expression at early and intermediate phases of estrogen-induced mammary carcinogenesis [92]. Vadhanam et al. [143] investigated the potent chemopreventive effect of ellagic acid delivered by a subcutaneous implant compared with a dietary route against estrogen-induced mammary tumors. Ellagic acid delayed the first tumor appearance, tumor incidence, tumor burden, and tumor multiplicity by both the implant and dietary routes [143]. In addition, dietary and implant of ellagic acid significantly reduced the plasma prolactin levels and PCNA expression [143]. Ravoori et al. [96] explored the long-term effects of the berries supplemented in diets against estrogen-induced mammary tumorigenesis in ACI rats. Ellagic acid, present in black raspberry, delayed the first tumor appearance as well as reduced the tumor volume and multiplicity [96]. Ellagic acid reduced estrogen-induced serum estrogen and plasma prolactin levels while the expression levels of CYP1A1 and ERα were increased at 3 month and 3-,7- month time points, respectively [96]. Munagala et al. [144] examined the role of ellagic acid on deregulated miRNAs during estrogen-induced mammary tumorigenesis. Administration of ellagic acid down-regulated the expression of ERα, Bcl-w, cyclin D1, cyclin G1 and antiproliferative factor RASD and up-regulated the expression of FOXO1 and FOXO3a. Overall, these data suggest that chemopreventive effects of ellagic acid may be associated with ER-dependent and antioxidant pathways.

5.11. EGCG

EGCG, one of the major polyphenols found in all tea preparations, possesses anti-proliferative, anti-mutagenic, and chemopreventive effects in cell culture and animal models of breast carcinogenesis [145, 146]. EGCG has poor bioavailability when taken orally in animals and humans [147–149]. However, some laboratory studies in mice and dogs have demonstrated the potential hepatotoxicity of acute oral bolus dosing with EGCG or green tea extracts [150, 151]. Although the underlying mechanisms of EGCG-induced hepatotoxicity remain to be investigated, it has been proposed that the acute oral doses of EGCG can result in oxidative stress leading to liver injury [152]. The human case studies also have reported an association between high doses of green tea-containing dietary supplements and hepatotoxicity [153], suggesting that genetic and/or life-style factors may play a role in susceptibility to EGCG-mediated hepatotoxicity. Chronic administration of EGCG or green tea may influence the biotransformation and bioavailability of high doses of EGCG [154, 155]. EGCG can influence ER-dependent expression of estrogen target genes [156], ER-independent signaling pathway [157] as well as cellular signaling pathways [158, 159]. The growth of MCF-7 xenograft tumors was inhibited during the first week of injections of EGCG [160]. Treatment of EGCG increased uterine peroxidase activity compared to estrogen alone in C57BL/6 mice [161], suggesting that the effects of EGCG in estrogen-induced responses in vivo may be due to alterations in the absorption and metabolism of estrogen in target tissues.

5.12. Ferulic acid

Ferulic acid, a polyphenol abundantly present in fruits and vegetables, exerts anti-tumor properties against breast cancer [162]. The bioavailability of ferulic acid has been studied extensively. However, results are quite variable (0.4–98%), in part depending on the food source [163, 164]. Baskaran et al. [165] investigated the chemopreventive effect of ferulic acid on DMBA-induced mammary carcinogenesis in SD rats. Their results indicated that oral administration of ferulic acid prevented the tumor formation and decreased tumor volume. Further, ferulic acid decreased TBARS and enzymatic/non-enzymatic antioxidants, such as SOD, CAT, GPx, reduced glutathione (GSH), vitamin E and vitamin C activities, in plasma and mammary tissues [165]. The treatment with ferulic acid increased the levels of GSH and the activities of phase II detoxification enzymes, such GST, GR and DTD, in the liver, whereas activities of GST, GR and DTD were decreased in the mammary tissues [165]. Oral administration of ferulic acid to DMBA-treated rats significantly down-regulated the expression of p53 and Bcl-2 [165]. Taken together, ferulic acid may alter the apoptosis pathway and detoxification pathway of the electrophilic intermediates that were formed during DMBA-induced carcinogenesis.

5.13. Flaxseed

Dietary flaxseed has numerous biological activities due to its high content of omega-3-fatty acid, α-linolenic acid and lignan, which have shown protective effects against breast cancer [166]. The main lignan in flaxseed is secoisolariciresinol diglucoside, which can modulate estrogen signaling due to its structural similarity to estrogen [167]. Chen et al. [168] determined the long-term effect of flaxseed with or without tamoxifen in ovariectomized athymic mice with MCF-7 xenografts. Flaxseed reduced tumor growth with or without tamoxifen by down-regulating the expression of estrogen-related genes such as cyclin D1, ERα and signal transduction pathways [168]. The same study group revealed that flaxseed inhibited MCF-7 xenografted tumor growth in a dose-dependent manner and enhanced the inhibitory effect of tamoxifen via modulating ER and growth factor signal transduction pathways [169, 170]. In a recent study conducted by Delman et al. [171], treatment with secoisolariciresinol diglucoside normalized several biomarkers in mammary gland tissues that had been altered by DMBA-induced carcinogenesis. Rickard et al. [172] determined the dose-dependent effects of flaxseed on NMU-induced mammary tumorigenesis in SD-rats. Administration of flaxseed decreased the tumor invasiveness and grade, suggesting that flaxseed appeared to delay the progression of NMU-induced mammary tumorigenesis [172]. Overall, these results indicated that flaxseed may be a potential chemopreventive agent against breast cancer via modulation of the ER-mediated signaling pathways.

5.14. Gallic acid

Gallic acid, a natural polyhydroxy phenolic compound, is commonly found in various foods and herbs that are well known as powerful antioxidants [173, 174]. Gallic acid was demonstrated to have anticancer effects by inhibiting cell proliferation and inducing apoptosis against ER-dependent and ER-independent cell lines [174, 175]. A few pharmacokinetic studies show that gallic acid has poor absorption profiles, low bioavailability and rapid elimination in humans and animals [176, 177]. Banerjee et al. [178] determined the molecular mechanisms underlying tumor cytotoxic activities of mango polyphenolics including gallic acid and gallotannins in the BT474 xenograft model. Treatment with mango polyphenolics significantly decreased tumor volume. Moreover, mango polyphenolics decreased the levels of p-PI3K, p-Akt, mTOR, hypoxia inducible factor 1α (HIF-α), VEGF and nuclear factor κB (NFκB) (p65) as well as the expression of cancer-associated miRNAs including miR-126 in the xenograft tumors [178]. Gallic acid has a therapeutic potential against breast cancer, at least in part, mediated through the cancer-associated miRNAs and PI3K/Akt pathway.

5.15. Genistein

Genistein, a soy isoflavone, consists of two aromatic rings linked through a heterocyclic pyran ring. The chemical structure of genistein is similar to that of estrogen [179]. Animal and human pharmacokinetic studies show that genistein has low oral bioavailability [180, 181]. Michael et al. [182] tested the acute and chronic effects of oral genistein administration in rats. In this study, genistein was well tolerated at doses up to 500 mg/kg/day administered for up to 52 weeks [182]. In human studies, the toxicity studies on genistein suggested that it is safe for human consumption, even at the high concentration of 16 mg/kg body weight [183, 184]. Peng et al. [185] investigated the effect of prepubertal exposure to genistein on rat mammary carcinogenesis and the ErbB2/Akt signal pathway in DMBA-treated SD rats. In their study, genistein decreased tumor incidence, and its effect is associated with down-regulation of the expression of erbB2, p-Akt, AIB1 and PCNA as well as with low protein tyrosine-kinase (PTK) activity in the mammary tumors [185]. Kang et al. [186] found that genistein exerted anti-tumor and anti-angiogenic effects, which were evidenced by lower microvascular density, reduced plasma VEGF, and increased plasma endostatin levels in DMBA-treated SD rats. Park et al. [187] examined genistein-induced apoptosis in the presence of estrogen. Bcl-2/Bax ratios were decreased by genistein treatment in both estrogen-sufficient and estrogen-deficient SD rats [187]. Constantinou et al. [188] revealed that treatment with genistein diminished the tumorigenic potential of MCF-7 cells in nude mice. However, Andrade et al. [189] reported that long-term consumption of low doses of genistein (<500 ppm) promotes MCF-7 tumor growth into more aggressive and advanced growth phenotype in the MCF-7 xenograft mouse model. Fan et al. [190] also evaluated the efficacy of genistein suppressing tumor growth in the MCF-7 xenograft model. Genistein reduced tumor development and breast cancer stem-like population through down-regulation of the Hedgehog-Gli1 signaling pathway [190]. Overall data suggest that genistein inhibits tumor growth via multiple mechanisms including Hedgehog-Gli1 stem cell signaling, ErbB2/Akt and ER-dependent pathway.

5.16. Hesperetin

Hesperetin, found in citrus fruits, has potential anticarcinogenic activity through inhibiting cell proliferation, regulating aromatase transcription and inducing apoptosis by triggering accumulation of ROS and inducing cell cycle arrest in breast cancer cells [191–193]. Hesperetin does not bind to the ER, although it belongs to the flavanone, which is able to interact with the ERs [194]. A single hydroxyl group at the 4 position of the B-ring is hypothesized to be the one of the main features required for estrogenicity of a compound [195]. The 4-methoxy group at this very important position in hesperetin is likely to influence its binding to the ER. Although hesperetin is unable to bind to the ERs, it is effective in inhibiting cell proliferation of ER-positive cancer [192]. Ye et al. [196] evaluated the chemopreventive effects of apigenin, naringenin and hesperetin in the MCF-7 xenograft mouse model. Administration of hesperetin significantly decreased tumor volume as well as cell cycle-regulated proteins such as p57^kip2, cyclin A, cyclin E, CDK4 and Bcl-xL, while a null effect was observed in mice treated with apigenin and naringenin. In the same study, hesperetin reduced the serum levels of estrogen and down-regulated estrogen target genes and estrogen metabolism-related genes, such pS2/TFF1 and CYP1A1 [196]. Hesperetin inhibits estrogen-induced breast carcinogenesis via ER-dependent mechanism as well as regulation of oxidative stress.

5.17. Indole-3-carbinol

Indole-3-carbinol, a naturally occurring compound in cruciferous vegetables of the Brassica genus such as broccoli and cabbage, has been indicated as a promising anticancer agent [197–199]. Indole-3-carbinol has poor bioavailability due to its instability in the gastrointestinal tract [200]. The results from short-term and long-term studies indicated that indole-3-carbinol is a relatively non-toxic compound [201, 202]. Indole-3-carbinol decreased the tumor incidence against MMTV-induced mammary tumors [203]. Chang et al. [204] investigated the effect of 3,3′-diindolylmethane (DIM), a major acid-catalyzed product of indole-3-carbinol, on angiogenesis and tumorigenesis in the MCF-7 xenograft mouse model. In this study, DIM decreased the development of estrogen-dependent xenografted tumors in mice. In addition, DIM significantly inhibited mammary tumor growth and tumor volume in DMBA-induced mammary tumors in SD rats [205]. Ritter et al. [206] investigated the effects of indole-3-carbinol on the P450 CYP enzyme activities, P450-dependent metabolism of estradiol (E2) and estrone (E1), and interconversion of E1 and E2 catalyzed by 17β-HSD. In this study, indole-3-carbinol increased activities of P450 enzymes, such as CYP1A1, CYP1A2, CYP3A, and CYP2B, as well as metabolism of E2 and E1 in the liver and mammary gland [206]. Overall, these data suggest that anti-carcinogenic effects of indole-3-carbinol may be associated not only with its alterations in estrogen metabolism, but also with its modulation of P450 enzymes via both ER-dependent and ER-independent pathways.

5.18. Luteolin

Luteolin, a common flavonoid in many types of fruits, vegetables and medicinal herbs, has numerous biological activities [207–209]. The hydroxyl moieties and carbon double bond are important structural features in luteolin that are associated with anti-cancer activity through regulation of estrogen signaling in ER-positive breast cancer cells [210, 211]. Luteolin has low bioavailability due to poor solubility in water [212]. There was no evidence of systemic toxicity in animal models as evidenced by normal food intake and body weight [85]. Samy et al. [213] evaluated the anti-tumor effect of luteolin against DMBA-induced mammary carcinogenesis in Wistar rats. Luteolin inhibited tumor incidence, decreased tumor volume, inhibited lipid peroxide formation, but increased SOD, CAT and GPx activities in the liver, kidney and breast tissues [213]. Another study reported that administration of luteolin led to decrease cells in S phase compared with DES alone in the mammary gland of rats [214]. In addition, luteolin treatment reduced tumor growth through inhibition of MPA-induced VEGF secretion and blood-vessel density in the T47D xenograft model [55]. Because VEGF has been shown to protect cells from undergoing apoptosis [215], loss of VEGF by luteolin may increase tumor cell apoptosis and inhibit angiogenesis. These studies suggest that luteolin exerts chemopreventive and antiangiogenic activity by inducing the antioxidant defense system in DMBA- or DES-induced mammary tumors.

5.19. Lycopene

Lycopene, a natural red colored pigment produced in plants and microorganisms, is potentially effective in the prevention of breast cancer [216]. Lycopene ingested in its natural trans form is poorly absorbed; heat processing induces isomerization of lycopene from all-trans to cis configuration, which increases its bioavailability [217]. Toxicity studies showed that lycopene did not cause any adverse events in animals [218]. In humans, lycopene ranging from 10 to 120 mg was well tolerated with minimal side effects, which were mainly of minimal gastrointestinal toxicity [219]. Several animal studies reported the synergistic effects of lycopene and other natural compounds. Moselhy et al. [220] investigated the anti-tumor potential of lycopene alone or in combination with melatonin in DMBA-induced oxidative stress and mammary carcinogenesis in SD rats. Their results indicated that lycopene alone or combination decreased tumor incidence as well as malondialdehyde (MDA) and NO levels in serum and mammary tissues [220]. The activities of SOD, CAT and GPx in the treatment groups of lycopene alone or combination were found to be significantly higher than the DMBA treatment [220]. Another study conducted by Al-Malki et al. [221] reported that tumor formation and angiogenesis in DMBA-treated rats were ameliorated by lycopene and/or combined treatment with tocopherol. Further, lycopene alone or in combination with tocopherol decreased MDA and NO in serum and mammary tumor tissues but increased SOD, CAT and GPx in mammary tumor tissues [221]. Administration of lycopene and genistein in combination inhibited DMBA-induced mammary tumor development through suppressing oxidative stress markers, including MDA, 8-isoprostane and 8-OH-dG, in DMBA-treated rats [222]. Overall results suggest that supplementation of diet with lycopene or in combination with other bioactive compounds may provide the antioxidant defense system against DMBA-induced mammary carcinogenesis.

5.20. Morin

Morin is a flavonoid from Moraceae that exhibits anti-proliferative and antitumor effects in ER-positive, ER-negative and PR-negative human breast cancer cell lines [223, 224]. Morin, a phytoestrogen, has the 7- and 4- positions occupied by hydroxy groups; these two positions correspond to the 3- and 4- positions of DES [225]. The bioavailability of morin after a single oral dose is very low due to low aqueous solubility and low intestinal permeability [226, 227]. Nanhakumar et al. [228] evaluated the anti-carcinogenic effect of morin against DMBA-induced mammary carcinogenesis. Administration of morin showed a decrease in PCNA expression and the number of AgNOR/nuclei [228]. Morin improved the enzymic and nonenzymic antioxidant activities, including SOD, CAT, GPx, GST, GR, GSH, G6PD, vitamin C, and vitamin E, and considerably decreased the lipid peroxidation and tumor markers, such as CA15-3, α-feto protein (AFP) and carcinoembryonic antigen (CEA), in serum as compared to DMBA-treated rats [228]. These data suggest that morin inhibits mammary carcinogenesis possibly through the antioxidant pathway.

5.21. Myricetin

Myricetin, a member of the flavonoids, is commonly derived from many fruits and vegetables that are known as powerful antioxidants [229]. Myricetin has low oral bioavailability in rats and in humans due to poor aqueous solubility [230, 231]. The structure of myricetin is similar to estrogen, and it acts as an agonist by activating the agonist-dependent activation of ERα and ERβ, inducing the ERα nuclear translocation and regulating the expression of estrogen target genes in ER-dependent breast cancer cells [232]. Jayakumar et al. [233] assessed the protective effect of myricetin against oxidative damage in DMBA-treated Wistar rats. Myricetin decreased TBARS levels in plasma and mammary tissues and increased SOD levels in erythrocyte lysates and mammary tissues [233], indicating that myricetin was effective in preventing oxidative damage induced by the carcinogen DMBA.

5.22. Phenethyl isothiocyanate

Phenethyl isothiocyanate, a naturally occurring isothiocyanate found in some cruciferous vegetables, has cancer prevention effects in breast cancer [234, 235]. Phenethyl isothiocyanate has high bioavailability, and it is likely that phenethyl isothiocyanate undergoes modest first-pass metabolism as metabolites of isothiocyanates can be generated by intestinal as well as hepatic enzymes [236]. Futakuchi et al. [234] revealed that phenethyl isothiocyanate exerts chemopreventive effect on the promotion and/or progression stage of DMBA-induced rat mammary carcinogenesis. In addition, phenethyl isothiocyanate reduced the tumor incidence and tumor burden in the NMU-induced breast cancer animal model [235]. McCune et al. [237] investigated the chemopreventive activity of phenethyl isothiocyanate in the PyMT transgenic model. The PyMT transgenic mouse model serves as an excellent model for studying progression of luminal subtype tumors [238]. Phenethyl isothiocyanate inhibited tumor initiation and delayed progression by regulating ERα, forkhead box protein A1 (FOXA1) and GATA binding protein 3 (GATA-3) transcriptional factors [237]. As these factors constitute a functional transcription factor network that determines estrogen dependence and response to anti-estrogen treatment [239], phenethyl isothiocyanate exerts chemopreventive effects possibly via the ER-dependent pathway.

5.23. Pomegranate

Pomegranate (Punica granatum L.) contains various bioactive phytochemicals that exert anti-proliferative [240], anti-angiogenic [241], anti-aromatase [242], proapoptotic [243] and estrogenic activities in breast cancer cell lines and animal models [244, 245]. Despite many biological properties, punicalagin and ellagic acid, the major polyphenols in pomegranate, have poor absorption, low bioavailability and short retention time [246, 247]. In addition, pomegranate seeds are known to contain the estrogenic compounds, estrone and estradiol, that are chemically identical to those biosynthesized in human body [248]. Treatment with pomegranate polyphenolics significantly decreased tumor volume and weight by decreasing Sp1, Sp3 and Sp4 as well as miR-27a in the BT474 xenograft model [249]. Recently, Bishayee et al. [250] reported that pomegranate emulsion reduced the tumor incidence, total burden and tumor weight in DMBA-treated rats with a concurrent inhibition of cell proliferation, induction of apoptosis, up-regulation of proapoptotic protein Bax, and down-regulation of antiapoptotic protein Bcl-2 in mammary tumors. The same group investigated the effects of pomegranate emulsion on ER and Wnt/β-catenin signaling as well as expression of cyclin D1, a downstream target for both ER and Wnt signaling, in DMBA-treated mammary tumorigenesis [244]. Pomegranate emulsion downregulated the expression of ERα, ERβ, β-catenin and cyclin D1 via ER and Wnt/β-catenin signaling pathways [244]. These studies suggest that pomegranate exerts chemopreventive activity by inducing apoptotic and estrogen-dependent pathways.

5.24. Quercetin

Quercetin, a major representative of the flavonol subclass of flavonoids, is commonly found in fruits and vegetables including onions and apples [251]. The structure of quercetin is similar to estrogen, including two hydroxyl groups and a phenol ring [252]. Therefore, quercetin acts as a phytoestrogen that binds to the ER and participates in the regulation of cell growth and gene transcription through estrogen dependent pathway [253, 254] as well as via ER-independent pathway [157]. Quercetin has low bioavailability in humans [255]. In addition, the bioavailability of quercetin differs among food sources, depending on the type of glycosides they contain [255]. Tao et al. [251] evaluated the anti-tumor effects of quercetin in the MCF-7 xenograft model. Tumor volume was decreased by quercetin compared with control mice, and expression of miR-146a was up-regulated after treatment with quercetin [251]. Moon et al. [103] determined the effect of quercetin in combination with biochanin A and EGCG in the MCF-7 xenograft model. Mixture of these three compounds reduced the tumor incidence, whereas quercetin and EGCG administration did not affect tumor size [103]. In in vivo study by Steiner et al. [256], quercetin reduced tumor number and tumor volume in the C3(1)/SV40Tag transgenic mouse model of breast cancer. Quercetin suppressed tumor growth, decreased the serum levels of CEA, a potent marker for tumor growth and invasion, and reduced activities of acid phosphatase (ACP) and cathepsin D in DMBA-treated Wistar-albino rats [257]. However, Singh et al. [258] reported that quercetin does not confer protection against estrogen-mediated mammary carcinogenesis, does not inhibit estrogen-induced oxidative stress and may exacerbate breast carcinogenesis in estrogen-treated ACI rats, possibly due to the inhibition of COMT activity. Overall effects of phytoestrogens, such as quercetin, on breast cancer remain unclear. Depending on the doses, quercetin may differently affect antioxidant pathway in breast cancer.

5.25. Resveratrol

Resveratrol, a polyphenol naturally occurring in grapes and red wine, is structurally similar to the DES [259]. Resveratrol suppresses the proliferation of breast cancer cells, induces apoptosis, inhibits the breast cancer stem cells and induces autophagy via suppressing the Wnt/β-catenin signaling pathway [53, 260, 261]. Resveratrol has low water solubility, leading to low bioavailability. However, resveratrol absorption can vary depending on food ingested [262]. Crowell et al. [263] reported that adverse effects occurred with higher doses and consisted mainly of nephrotoxicity in rats. In a recent study conducted by Williams et al. [264], low and high doses of resveratrol are well tolerated and non-toxic in rats. In addition, consumption of high-dose resveratrol did not cause serious adverse effects in humans [265, 266]. Resveratrol is well absorbed and rapidly metabolized into sulfo- and glucuronide conjugates which are eliminated in urine [267]. Therefore, resveratrol seems to be well tolerated without any marked toxicity. Administration of resveratrol significantly reduced the tumor number and increased the time to onset of DMBA-induced mammary tumors [268]. Treatment with resveratrol reduced cell proliferation and induced apoptotic index for epithelial cells in mammary terminal ductal structures [268]. Banerjee et al. [269] investigated the chemopreventive potential of resveratrol against DMBA-induced mammary carcinogenesis in SD rats. Treatment with resveratrol suppressed the DMBA-induced tumor incidence and tumor multiplicity, which correlates with down-regulation of NFκB, cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 expression [269]. Treatment with resveratrol at high concentration delayed tumorigenesis and decreased the tumor multiplicity in NMU-treated SD rats [270]. Histomorphology of mammary tumors showed that resveratrol increased alveolar and adipocyte differentiation of mammary tumor cells [270]. Singh et al. [271] reported that resveratrol inhibited estrogen-induced breast carcinogenesis via regulation of nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant genes, NAD(P)H dehydrogenase quinone 1 (NQO1), SOD3, oxidative DNA damage repair gene, 8-oxoguanine DNA glycosylase 1 (OGG1), and detoxification genes, aldehyde oxidase 1 (AOX1) and flavin monooxygenase 1 (FMO1). In this study, treatment with resveratrol also induced apoptosis and inhibited estrogen-mediated increase in DNA damage in estrogen-treated mammary tissues [271]. These results suggest that resveratrol inhibits estrogen-induced breast carcinogenesis via induction of Nrf2-mediated protective pathways as well as ER-dependent mechanisms.

5.26. Sesamin

Sesamin, a lignan isolated from sesame seeds, possesses chemopreventive effects against breast cancer and other chronic diseases through ER-dependent pathway and NFκB pathways [272–274]. Treatment with sesamin significantly reduced the cumulative number of palpable mammary tumors in DMBA-treated SD rats [275]. The levels of lipid peroxides, fatty acid compositions, tumor phosphatidylcholine and prostaglandin E2 were decreased while peripheral blood mononuclear cells (PBMC) were increased by sesamin [275]. Truan et al. [276] revealed that sesamin reduced MCF-7 xenograft tumor growth at high levels of circulating estrogen in athymic mice. In this study, sesamin increased apoptosis via regulating human epidermal growth factor receptor 2 (HER2) and endothelial growth factor receptor expression and MAPK pathway [276]. The inhibitory effect of sesamin against mammary carcinogenesis may be due to increased antioxidant pathway and several cellular defense pathways in addition to ER-dependent mechanism.

5.27. Sulforaphane

Sulforaphane, an isothiocyanate naturally rich in cruciferous vegetables such as broccoli, broccoli sprouts, cabbage and kale, has been shown to reduce the risk of developing many common cancers [277]. Sulforaphane has anti-cancer activity against various breast cancer cell lines via regulating cytochrome P450 involved in estrogen metabolism [278], Nrf2-related antioxidant pathway [279], Akt/mTOR-S6K1 pro-survival pathway, ERK1/2-IKKα and NAK-IKKβ [280], ErbB2/ER-PI3K/Akt-mTOR-S6K1 signaling [281] and cancer stem cell pathway [50, 280]. Sulforaphane has shown high bioavailability, as determined by significant intracellular and plasma concentrations in rats and humans [282, 283]. Kanematsu et al. [284] revealed that sulforaphane inhibited cell growth, induced apoptosis and reduced primary tumor growth, tumor volume and tumor weight in the KPL-1 breast cancer xenograft model. Furthermore, sulforaphane decreased breast cancer stem cells in vivo, thereby abrogating tumor growth after the reimplantation of primary tumor cells into the secondary mice via Wnt/β-catenin self-renewal pathway [50]. These results indicate that sulforaphane inhibits the mammary tumor growth through the antioxidant pathway as well as Wnt/β-catenin self-renewal pathway.

5.28. Tangeretin

Tangeretin, a flavonoid found in the peel of citrus fruits, induces the apoptosis of ER-dependent cells mainly through caspase-dependent or -independent pathways [285]. Despite its widely documented bioactivities, tangeretin has relatively low bioavailability due to its low water solubility, which is a major factor limiting its application as a nutraceutical [286]. A safety assessment indicated that oral administration of tangeretin did not show any adverse effects [287]. Arivazhagan et al. [288] investigated the therapeutic effect of tangeretin against DMBA-induced rat mammary carcinogenesis. Treatment with tangeretin decreased tumor weight and reduced serum levels of estradiol, progesterone, prolactin, lipid bound sialic acid (LSA), total sialic acid (TSA), NO and protein carbonyls. Oral administration of tangeretin also effectively reduced the tumor cell proliferation markers such as PCNA, COX-2, Ki-67 and inhibited metastasis by suppressing MMP-2, MMP-9 and VEGF [288]. Periyasamy et al. [289] also investigated the chemopreventive and therapeutic effect of tangeretin on DMBA-induced oxidative stress in SD rats. The levels of oxidative stress markers, such as NO and LOOH, and breast cancer marker CEA were decreased, whereas the antioxidant enzymes such as SOD, CAT, GPx, GST, GSH, vitamin C and vitamin E were increased in tangeretin pre-treated and post-treated animals [289]. Tangeretin significantly suppressed DMBA-induced mammary tumors in rats as reported by Lakshmi et al [290]. In this study, tangeretin decreased the activities of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP), 5′-nucleotidase (5′-ND), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) and levels of tumor markers in serum as well as TBARS and conjugated dienes (CD) in plasma and mammary tissues [290]. The levels of enzymatic/non-enzymatic antioxidant and phase II detoxification enzymes were increased significantly by the administration of tangeretin [290]. DMBA-treated rats showed higher expression of ER, PR and Her2/neu, whereas tangeretin treatment to tumor bearing animals significantly decreased the expression of the hormones and their receptors [290]. These results suggest that the chemopreventive effects of tangeretin in vivo could be mediated by ER-dependent pathway as well as ER-independent mechanism.

5.29. Thymoquinone

Thymoquinone, a phytochemical compound found in plant Nigella sativa, has been shown anti-cancer effects via regulation of apoptosis, estrogen metabolism, MAPK and Akt signaling pathways in breast cancer [291–294]. Although thymoquinone has these biological effects, its administration remains problematic partly due to its poor water solubility, leading to low absorptivity and bioavailability [295]. Thymoquinone regulates the expression of genes involved in estrogen metabolism including CYP1A1 [293]. Linjawi et al. [296] reported that thymoquinone reduced tumor markers, the levels of MDA and lactate dehydrogenase as well as alkaline phosphatase (ALP) and AST activities. In addition, treatment with thymoquinone decreased the expression of breast cancer (BRCA)1, BRCA2, Id-1 and p53 mutations in mammary tissues of DMBA-treated rats [296]. These data suggest that thymoquinone exerts a protective effect against mammary carcinogenesis by regulating the antioxidant pathway.

5.30. Tocopherols

Vitamin E consists of eight different forms which include four tocopherols (α-, β-, γ-, and δ-forms) and four tocotrienols (α-, β-, γ-, and δ-forms) [297]. The major dietary sources of tocopherols are vegetable oils, such as corn, soybean, sesame and canola oils [297]. α-Tocopherol has a higher bioavailability than other forms of vitamin E. Because the acetate form is less susceptible to oxidative destruction, α-tocopherol acetate is widely used as source of vitamin E [298]. In addition, the bioavailability of natural-source vitamin E is approximately twice that of synthetic form of vitamin E [299]. Iuliano et al. [300] studied the bioavailability of tocopherols in relation to food intake. Consumption of tocopherols after food intake increases its bioavailability. γ-Tocopherol-rich mixture of tocopherols (γ-TmT), a byproduct of refined vegetable oil, is a naturally occurring tocopherol mixture rich in γ-tocopherol [301]. γ-TmT, containing 57% γ-tocopherol, 24% δ-tocopherol and 13% α-tocopherol, inhibited tumorigenesis in NMU-treated animals through antioxidant dependent pathway [302]. Dietary administration of 0.1%, 0.3%, or 0.5% γ-TmT suppressed mammary tumor growth and tumor multiplicity through the regulation of ER, peroxisome proliferator-activated receptor γ (PPARγ) and Akt signaling [303, 304]. Smolarek et al. [305] reported that treatment with dietary γ-TmT inhibited cell proliferation, down-regulated the expression of ERα, ERβ, PCNA, COX-2 and level of serum estrogen, while γ-TmT up-regulated the levels of cleaved-caspase 3, PPARγ, Nrf2 and Phase II detoxifying enzymes in hyperplastic mammary tissues of ACI rats with sustained release estrogen pellets. In addition, Das Gupta et al. [306] demonstrated that dietary administration of 0.3% γ-TmT reduced estrogen-induced oxidative/nitrosative stress markers including nitrotyrosine, 8-OH-dG and 8-isoprostane and up-regulated Nrf2 as well as Nrf2-dependent oxidative response genes such as SOD1, GPx and CAT in early mammary hyperplasia. A recent study determined the efficacy of dietary γ-TmT on estrogen-induced mammary tumors in two different in vivo models [301]. In the first study, γ-TmT up-regulated the estrogen-metabolizing enzyme, CYP1A1, Nrf2-dependent antioxidant enzymes as well as PPARγ and its downstream genes while down-regulated oxidative stress and cell proliferation markers at early, intermediate and late stages of mammary tumorigenesis in ACI rats [301]. In a second study, 0.05% to 0.5% dietary doses of γ-TmT inhibited the mammary tumor growth in MCF-7 xenografted mice [301]. Smolarek et al. [302] reported that γ- and δ-tocopherol, but not α-tocopherol, reduced levels of nitrotyrosine and 8-OH-dG stress markers while increased levels of phosphatase and tensin homologue (PTEN), p53 pathway, PPARγ, Nrf2/phase II detoxifying and antioxidant enzymes in NMU-treated SD rats. Taken together, γ-TmT as well as γ- and δ-tocopherol work through antioxidant and/or ER-dependent mechanisms, leading to the inhibition of mammary tumorigenesis.

5.31. Xanthohumol

Xanthohumol is a type of flavonoid isolated from the hops and beer [307]. Several studies demonstrated that xanthohumol is able to modulate estrogen synthesis and estrogen metabolism in breast cancer [308–310]. Xanthohumol has poor absorption, low systemic bioavailability and short retention time [311, 312]. Toxicological studies in animals revealed that xanthohumol did not affect the major organ functions, sex hormones or cause blood clotting [287, 313, 314]. Monteiro et al. [315] investigated the effect of oral administration of xanthohumol in the MCF-7 xenograft model. Xanthohumol reduced the inflammatory cell number and focal proliferation areas by increasing percentage of apoptotic cells and decreasing microvessel density [315]. Decreased immunostaining for NFκB, phosphorylated-inhibitor of κB and interleukin-1β was also observed [315]. In addition, Yoshimaru et al. [307] determined the antitumor activity of xanthohumol in KPL-3C orthotopic breast cancer xenografted mice. Their results indicated that xanthohumol inhibited estrogen-induced tumor growth by inducing G1-arrest via targeting the guanine nucleotide-exchange protein 3 (BIG3)-tumor suppressor prohibition 2 (PHB2) interaction [307]. BIG3-PHB2 complex play a pivotal role in estrogen signaling modulation in ERα-positive breast cancer [316], suggesting that xanthohumol may be a promising natural compound to regulate estrogen receptor signaling and NFκB signaling pathway.

6. Bioactive natural products targeting estrogen-mediated mechanisms

Based on bioactive natural products reviewed in experimental in vivo models of ER-positive breast cancer, we summarized bioactive natural products targeting estrogen-mediated mechanisms in ER-positive breast cancer cells in Table 2. The estrogen-responsive breast cancer cell lines, such as MCF-7, T47D, BT474 and ZR-75-1, have been used to test the estrogenicity as well as anti-estrogenic activities of various bioactive natural compounds. Among many bioactive natural compounds, genistein and resveratrol have been the most widely studied, and reports showed that both compounds inhibited estrogen-induced DNA synthesis and proliferation of ER-positive breast cancer cells [317–326]. However, genistein and resveratrol could differently modify ERE-dependent transcription and expression of ERE-mediated genes, such as ER, PR, and pS2/Trefoil factor 1(TFF1) in the presence and absence of estrogen [97, 270, 319, 324–327]. These effects of genistein and resveratrol could be mediated through both ER-dependent and ER-independent pathways. Shao et al. [328] reported that curcumin inhibits the expression of ER downstream genes including pS1 and transforming growth factor (TGF)-β and ERE activity induced by estrogen in MCF-7 cells, suggesting that curcumin inhibits MCF-7 cell proliferation via an ER-dependent pathway. Anthocyanidins, delphinidin and pelargonidin in berries bind to ERs and significantly reduce estrogen-induced ERE-luciferase activity, similar to the action of antiestrogens [329]. Several studies clearly demonstrated that indole-3-carbinol exerted anti-estrogenic effect by intervention of estrogen and/or ER signal transduction pathways, by alterations in estrogen metabolism, and by regulation of the estrogen-responsive genes such as pS2, cathepsin D via an ER-dependent pathway [330–334]. Sesamin also significantly decreased the estrogen-induced ERE activation and pS2/TFF1 gene expression [274]. Pomegranate extract inhibited the binding of estradiol to ER and suppressed the estrogen-induced proliferation as well as estrogen-responsive receptor gene expression via an ER-dependent pathway in MCF-7 cells [335]. The dietary carotenoids, lycopene and β-carotene inhibited estrogen signaling pathways by decreasing estrogen-induced transactivation of ERE and regulating both ERα and ERβ [336]. γ-Tocopherol, δ-tocopherol and γ-TmT suppressed the estrogen-induced cell proliferation and ERα expression and increased the transactivation of PPARγ in both MCF-7 and T47D cells [303]. Since ERα binds to the PPAR response element and represses transactivation of PPARγ [337], it is likely that activation of PPARγ may mediate the anti-estrogenic action of γ- and δ-tocopherol. Xanthohumol treatment prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα [316]. This event led to the suppression of the estrogen signaling pathways by inhibiting estrogen-induced ERE activation and pS2/TFF1 and CCND1 expression [316]. Several compounds, including apigenin, biochanin A, EGCG, ferulic acid, quercetin, and α-linolenic acid in flaxseed were distinct, in that their antiestrogenic activity did not appear to correlate with binding to ER [317, 338] and, therefore, their suppression of estrogen-induced cell proliferation may occur independent of direct antagonism of the receptor. Taken together, cancer preventive activities of bioactive natural compounds may occur through ER-dependent and ER-independent mechanisms.

Table 2.

Bioactive natural products targeting estrogen-mediated mechanisms

Natural compounds	Dose of estrogen used	Effects	Ref
Apigenin	100 pM	↓ Cell proliferation	[348]
Biochanin A	100 pM	↓ Cell proliferation	[349]
Curcumin	1 nM	↓ Cell proliferation	[328]
		↓ pS2/TFF1, ER, TGF-β expression
		↓ ERE activities
EGCG	10 nM	↓ Cell proliferation	[350]
Ferulic acid	10 pM	↓ Cell proliferation	[351]
Flaxseed	1 nM	↓ Cell proliferation	[338]
		↓ ABCG2, GATA3, KRT8, MAPK1, SNAl2, SRC, BRCA1, FOXA1 expression
Genistein	100 pM - 50 nM	↓ Cell proliferation, DNA synthesis	[317–322]
		↑ Apoptosis	[97, 187, 319, 320, 326, 352, 353]
		↓ TERT, Myc, ras, Bcl-2, human complement 3 expression	[97]
		↓ FADD, cytochrome C, truncated Bid, caspase-9, caspase-3 expression	[319]
		↑ CCNB1, p21, p53 expression	[187]
		↓ ERE-dependent transcriptional activity	[326]
		↓ ERα expression	[97]
Indole-3-carbinol	1 nM	↓ Cell proliferation	[331]
		↓ ER and E2 metabolites	[331–333]
		↓ pS2/TFF1, cathepsin-D expression	[332]
Lycopene	100 pM	↓ ERE transactivation	[336]
Pomegranate	10 nM	↓ Cell proliferation	[335]
		↓ ERE-dependent transcriptional activity
		↓ ERα, pS2/TFF1, PR expression
Quercetin	10 nM	↑ PCNA, PI3K, p-Akt, ERα expression	[319]
		↓ FADD, cytochrome C, truncated Bid, caspase-9, caspase-3 and ERβ expression
Resveratrol	1nM, 10 nM	↓ Cell proliferation	[323–326]
		↑ Apoptosis	[319, 323, 326]
		↓ p53, p21 expression	[323]
		↓ FADD, cytochrome C, truncated Bid, caspase-9, caspase-3 expression	[326]
		↓ NFκB activation
		↓ PR, pS2/TFF1 expression	[270, 324, 325]
		↓ ERβ expression	[319]
		↓ ERE-dependent transcriptional activity	[324, 326]
Sesamin	1 nM	↓ ERE activity	[274]
γ-Tocopherol	10 pM	↓ Cell proliferation, ERα expression	[303]
δ-Tocopherol		↑ PPARγ transcription activity
Xanthohumol	10 nM	↓ BIG3-PHB2 complex	[307]
		↓ ERα, ERE transcriptional activity
		↓ Akt, p42/44 MAPK, ERα, pS2/TFF1, CCND1 expression

Abbreviations: ABCG: ATP-binding cassette, subfamily G member; FADD: FAS-associated death domain; KRT 8: keratin 8; SNAI2: snail homolog 2; SRC: v-src sarcoma viral oncogene homolog; TERT: telomerase reverse transcriptase

7. Clinical trials of bioactive natural products in breast cancer

According to the in vivo and in vitro experimental studies, numerous natural products have been reported to have anticancer activities against ER-positive breast cancer. The current information about completed or ongoing clinical trials of bioactive natural products on breast cancer is summarized in Table 3. Bayet-Robert et al. [339] investigated the tolerability of the combination of curcumin and docetaxel in phase I clinical trial. Curcumin was given orally for seven consecutive days, and docetaxel (100 mg/m²) was administered as intravenous infusion every 3 week for six cycles. Based on this study, the recommended dose of curcumin is 6,000 mg/day for seven consecutive days every 3 week in combination with a standard dose of docetaxel [339]. Thompson et al. [166] conducted a randomized, placebo-controlled, double-blinded, prospective study. Patients with breast cancer in the treatment group (n = 19) ate one muffin daily containing 25 g of flaxseed included in their usual diet. The results of this study showed that daily intake of 25 g flaxseed has the potential to reduce tumor growth by reducing cell proliferation and increasing apoptosis in patients with breast cancer [166]. A clinical trial was conducted to evaluate the effects of high oral dose of soy isoflavone administered daily for 84 days to healthy postmenopausal women. Genistein appears to be safe and well tolerated in healthy postmenopausal women at doses of 900 mg per day [340]. Recently, Shike et al. [341] investigated the effect of soy supplementation on modulating gene expression in one hundred-forty patients with early-stage breast cancer. Treatment with soy protein supplementation upregulated the expression of cell cycle transcripts, including those that promote cell proliferation, such as fibroblast growth factor receptor 2, E2 factor 5, BUB1, CCNB2, MYBL2, CDK1, and CDC20 [341]. Gregory et al. [342] conducted the phase I clinical trial of indole-3-carbinol in seventeen women from a high-risk breast cancer cohort. Patients received 400 mg and 800 mg of indole-3-carbinol per day for four weeks. Daily administration of indole-3-carbinol at doses of 400 and 800 mg were tolerated well by all subjects [342]. In addition, a randomized, placebo-controlled, double-blind, crossover trial was conducted to evaluate whether tomato-derived lycopene supplementation decreases serum levels of total IGF-I in premenopausal women with a history of breast cancer and a high familial breast cancer risk [343]. They received 30 mg of lycopene daily for 2 months. In this trial, lycopene effects were discordant between the two study populations showing beneficial effects in women with a family history of breast cancer but not in breast cancer survivors [343]. Nesaretnam et al. [344] conducted a double-blinded, placebo-controlled pilot trial study to examine the effect of adjuvant tocotrienol therapy in combination with tamoxifen for five years in women with early breast cancer. Two hundred-forty women with either tumor node metastasis Stage I or II breast cancer with ER-positive tumors were assigned to two groups and then administrated tocotrienol-rich fraction plus tamoxifen [344]. The results indicated no association between adjuvant tocotrienol therapy and breast cancer specific survival in women with early breast cancer [344]. Currently, limited intervention studies are on-going or completed to determine the beneficial effects of natural products in patients with breast cancer. With a better understanding of the mechanisms of action of bioactive natural products in estrogen-mediated breast cancer, future intervention studies with bioactive natural products could be designed to evaluate their roles in the treatment and prevention of breast cancer in humans.

Table 3.

Clinical trials of bioactive natural products in breast cancer

Compounds	Subject number	Intervention	Trial phase	Current status	Numbers of clinical trial/Ref
Curcumin	30	2 g of curcumin three times per day orally for 4–7 weeks	Phase 2	Completed	NCT01042938
	695	2g of curcumin three times daily orally throughout course of radiation treatment plus one week	Phase 2/3	Completed	NCT01246973
	14	500 mg of curcumin orally daily for seven consecutive day	Phase 1	Completed	[339]
EGCG	40	Green tea extract daily for 6 months in the absence of disease progression or unacceptable toxicity	Phase 1	Completed	NCT00516243
	1084	800 mg of Epigallocatechin-3 gallate two times per day for 12 months	Phase 2	Completed	NCT00917735
	20	3 tea capsules daily orally for 3 weeks	Phase 1	Ongoing	NCT00949923
Flaxseed	32	25 g of flaxseed-containing muffin daily for 39 days	Unknown	Completed	[166]
Genistein	30	Genistein two times per day for 84 days	Phase 1	Completed	NCT00099008
	19	100 mg of genistein (Novasoy) two times per day for 7 and 21 days	Phase 2	Completed	[340] NCT00244933
Indole-3-carbinol	18	Indole-3-carbinol two times per day for 12 and 16 weeks	Phase 1	Completed	[341] NCT00033345
	17	400 mg and 800 mg of indole-3-carbinol daily orally for 4 weeks	Phase 1	Completed	[342]
Lycopene	60	30 mg of lycopene daily for 2 months	Unknown	Completed	[343]
Sulforaphane	54	Sulforaphane supplement three times per week for 2–8 weeks	Phase 2	Completed	NCT00843167
	34	Broccoli sprout extract daily for 14 days	Phase 2	Completed	NCT00982319
	32	Sulforaphane topical application three times a week for 5 weeks	Phase 2	Ongoing	NCT00894712
Tocotrienol	240	200 mg tocotrienol Rich Fraction daily for five years	Unknown	Completed	NCT01157026

8. Conclusion and future directions

Current chemopreventive strategies for ER-positive breast cancer have targeted the ER, estrogen, or estrogen metabolites. It is well-established that many bioactive natural products have beneficial effects on estrogen driven breast cancer. These natural products have shown promise in preventive and therapeutic settings and point toward tumor initiation and progression as points of intervention. However, use of natural products and derivatives has many hurdles as chemopreventive agents because of low bioavailability, poor absorption and lack of specific cellular targets. Due to the role of breast cancer stem cells in tumor initiation, maintenance, and progression, stem cells have become a new area of interest in the study of estrogen-mediated breast carcinogenesis. Recent studies are identifying possible estrogen-mediated mechanisms of stem cell expansion. However, the currently available reports provide only limited information whether natural compounds may inhibit estrogen-induced cancer stem cells. As cancer stem cell signaling pathways become more evident, it will be important to identify inhibitors of estrogen-mediated signaling to effectively target the breast cancer stem cell subpopulation. The bioactive natural products identified in this review serve as good starting points for further investigation since their effects on estrogen signaling in breast cancer have already been established. In order to effectively prevent or treat estrogen-driven breast cancers, it is important to consider not only targeting classical estrogen signaling but also identifying natural compounds effective in inhibiting breast cancer stem cells.