Is There an Answer?

Are Flavonoids Agonists or Antagonists of the NaturalHormone 17b-Estradiol?

Maria Marino and Paola GalluzzoDepartment of Biology, University Roma Tre, Roma, Italy

Is There an Answer? is intended to serve as a forum in

which readers to IUBMB Life may pose questions of the

type that intrigue biochemists but for which there may be

no obvious answer or one may be available but not widely

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Paolo Ascenzi

Flavonoids are part of plant defense mechanisms against

stress of different origins and comprise the most common group

of polyphenolic compounds (Fig. 1). Major dietary sources of

flavonoids include fruits, vegetables, tea, wine, and cereals. Fla-

vonoids consist of two aromatic carbon group rings, namely,

benzopyran (A and C rings) and benzene (B ring), and may be

divided into six subgroups based on the degree of the oxidation

of the C-ring, the hydroxylation pattern of the ring structure,

and the substitution in the 3-position (Fig. 1) (1).

The impact of dietary flavonoids on biologic processes was

first recognized in sheep when an antiestrogenic principle pres-

ent in red clover that caused infertility in sheep in western Aus-

tralia was discovered (2). This adverse effect of flavonoids,

which has been confirmed in laboratory animals, placed these

substances in the class of endocrine-disrupting chemicals (3, 4).

In adult human beings, in contrast to the antiestrogenic effects,

diets rich in flavonoids lead to several estrogen-mimetic effects

such as lowering levels of serum cholesterol, low-density lipo-

proteins, and triglycerides (5), as well as reducing the incidence

of cardiovascular diseases (6) and osteoporosis (7), and improv-

ing cognition and learning (8). Altogether, these data sustain

the dual flavonoid effects in mammalian cell physiology; as

antiestrogen they act as endocrine disruptors, whereas as estro-

gen-mimetic they can maintain the protective effects of the nat-

ural hormones on some degenerative diseases (4, 9). The estro-

gen-mimetic effects of dietary compounds are currently being

explored to prevent the symptoms associated to estrogen defi-

ciency in women during menopause (10, 11).

The molecular basis of flavonoid estrogenicity is particularly

difficult to elucidate, principally because of the 17b-estradiol(E2) mechanism of action which occurs via multiple pathways

upon E2 binding to estrogen receptors a and b (ERa and ERb)(12). ERa and ERb, encoded by two different genes, belong to

the nuclear receptor superfamily (NR3A1 and NR3A2, respec-

tively) of ligand-regulated transcription factors (12). These

receptors mediate different E2-induced effects in live organ-

isms. E2 binding causes ERs to dissociate from heat shock pro-

teins, dimerize, bind to specific DNA sequences [estrogen

response element (ERE)], and stimulate the transcription of

responsive genes (12). Moreover, ERa-regulated gene transcrip-

tion, but not ERb, can occur also through the ERa indirect

interaction with the transcription factors stimulating protein 1

(Sp-1) and activating protein-1 (AP-1) (12). Both in the direct

and indirect action modes, the ligand-activated ERs are not the

transcription controller. In fact, ER needs to interact with core-

gulatory proteins (coactivators or corepressors) which provide a

platform upon which additional proteins are assembled (12).

This ‘‘genomic action’’ of steroid hormones occurs after a time-

lag of about 2 h and explains some of their functions in physio-

logical and pathological situations (12). E2 induces rapid effects

that include the activation of mitogen-activated protein kinase

(MAPK), phosphatidylinositol 3-kinase (PI3K), signal trans-

ducer and activator of transcription, epidermal growth factor

receptor, Src kinase, Shc kinase, protein kinase C, adenylate

cyclase, GTP-binding proteins, and nitric oxide synthase

(12). These rapid effects have been attributed in most cells to

a population of ERs present on the plasma membranes via

Address correspondence to: Maria Marino, Department of Biology,

University Roma Tre, Viale G. Marconi 446, I-00146 Roma, Italy.

Tel.: 139-06-55176345. Fax: 139-06-55176321.

E-mail: m.marino@uniroma3.it

Received 14 December 2007; accepted 20 December 2007

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.34

IUBMB Life, 60(4): 241–244, April 2008

S-palmitoylation which allows ERs anchoring at the plasma

membrane and association to caveolin-1. This accounts for the

ability of E2 to activate different signaling pathways (12, 13).

The action of E2 in living cells is, thus, mediated by various

pathways rather than by a single uniform mechanism. All signal

transduction pathways integrate at different levels and influence

the E2 effects, and these pathways could be modulated by fla-

vonoids. Thus, a reliable evaluation of flavonoid (anti)estroge-

nicity and a correct prediction of their effects on human health

should take into account all E2-induced mechanisms.

Flavonoids bind ERa and ERb, and their affinity is 1,000–

10,000 times lower than that of E2; in addition, flavonoids

show a distinct preference for ERb (4, 14–17).

The isoflavonoids daidzein and genistein, the favanone narin-

genin, and the flavonol quercetin increase the activity of ERE-

luciferase reporter gene construct in cells expressing ERa or

ERb (18–21). Whereas, flavonoids impair the interaction of ERs

with other trancription factors (e.g., Sp-1 and AP-1) (20, 22,

23). The engineering of DNA microarrays has enabled investi-

gators to examine the effects of a putative estrogen and not just

the effects on their favorite gene or protein. Cluster analysis

performed in a mammary gland cancer cell line (MCF-7) indi-

cated that genistein induces gene expression profiles very simi-

lar to that of E2 (24–26). On the other hand, 227 genes of the

uterus of immature rats were affected by genistein, the majority

of which were not estrogenic (27). Such discrepancies are par-

tially due to the analysis of gene array data that suffer from

having many factors and few replicates, and this leads to poor

statistical power and mostly observed changes may be false

(28). Moreover, gene expression levels are not indicative of

Figure 1. Subdivision of bioactive compounds from plants present in foods (top panel). The white, grey, and black boxes are repre-

sentative of phytochemical families, flavonoid classes, and demonstrative compounds, respectively. General structure and number-

ing pattern for common food flavonoids (bottom panel). For details see text.

242 MARINO AND GALLUZO

cellular effects. Gene arrays and other broad approaches in pro-

teomics and metabolomics are now increasingly used to address

the question of what an estrogen does and what a flavonoid

does.

The ability of flavonoids to evoke the membrane starting

activation of specific rapid phosphorylation cascades is largely

unknown. Quercetin and naringenin impair ERa-mediated

rapid activation of signaling kinases (i.e., ERK/MAPK and

PI3K/AKT) and cyclin D1 transcription which are important

factors for the progression of cell proliferation (20). This

effect is observed only when carcinoma uterine cells (HeLa),

devoid of any ER isoforms, are transiently transfected with the

human ERa expression vector demonstrating the ER-dependent

mechanism. In the same cell system, naringenin activates the

rapid phosphorylation of p38/MAPK and, in turn, the induc-

tion of a proapoptotic cascade (i.e., caspase-3 activation and

PARP cleavage). Thus, naringenin decouples the ERa action

mechanisms, preventing the activation ERK/MAPK and PI3K/

AKT signal transduction pathways and drives cells to apopto-

sis (19). Flavonoids could induce different conformational

changes of ER, also precluding the activation of rapid signal-

ing cascades. Indeed, naringenin reduces ERa localization at

the plasma membrane and the receptor association to caveolin-

1, impairing the activation of rapid signals (Marino, unpub-

lished results). On the other hand, naringenin does not impair

the ERa-mediated transcriptional activity of an ERE-contain-

ing promoter (19, 20). Thus, naringenin modulates specific

ERa mechanisms and can be considered as ‘‘mechanism-spe-

cific ligand of ER’’ (29); it acts as an ERa antagonist of cer-

tain pathways in all organs. In addition, some flavonoids (e.g.,

quercetin, naringenin, and daidzein) act as E2-mimetic in the

presence of ERb and rapidly activate p38/MAPK and the apo-

ptotic cascade (19, 21). Thus, nutritional compounds could act

as antagonists of ERa-evoked rapid responses and agonists of

ERb-dependent proapoptotic signaling (4). Remarkably, the in-

terference with the ERa-associated PI3K pathway has recently

been proposed as a mechanism underlying the antiestrogenic

inhibition of survival and proliferation by 8-prenyl-naringenin

in MCF-7 cells (30).

In conclusion, flavonoids, like other polyphenols, induce

various effects in E2-target cells: antiestrogenic actions, E2-

mimetic actions, E2-mediated target gene expression, and E2-

dependent target kinase modulation (4, 31). However, dissimilar

results regarding the E2-like potency of the flavonoids have

been reported. This may be due to the variety of cell system

employed, the different techniques, and the different cellular

context. Therefore, care should be taken when defining the

estrogenic/antiestrogenic effects of a compound. The intrinsic

estrogenic status and the dose should be considered, especially

in the context of using a compound to prevent symptoms asso-

ciated with estrogen deficiency during menopause or to prevent

hyperestrogenic effects in E2-related cancer. Therefore, it is

essential to assess the flavonoids effects at multiple levels,

in vitro and in vivo, to obtain a full picture which appears to be

relevant in different physiological or pathological conditions

(e.g., menopause, premenopause, and cancer).

The challenges in the near future are to continue identifying

the discrete actions of flavonoids and their metabolites on each

intracellular pool of ERs and to define the classification of the

flavonoid-dependent gene expression. Moreover, considering the

great variety of dietary flavonoids, it appears extremely unlikely

that any one substance is responsible for all of the associations

seen between plant foods and human health protection. The spe-

cific mechanisms of most flavonoids and isoflavonoids appear

to be varied, complementary, and/or overlapping. Further inves-

tigations into the potential role of flavonoids and isoflavonoids

in cancer prevention and/or therapy are warranted.

ACKNOWLEDGEMENTS

The authors thank the past and present members of their labora-

tory teams, who have contributed with data and discussion to

the ideas presented here. This work has been made possible by

grants from the Ministry of University and Research of Italy

(PRIN-COFIN 2006 to M.M.).

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New Questions

1. Is the susceptibility to phytochemicals gender-dependent?

2. Are phytochemicals safe for human health?

244 MARINO AND GALLUZO