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Recent Patents on Anti-Cancer Drug Discovery, 2007, 2, 109-117 109
1574-8928/07 $100.00+.00 © 2007 Bentham Science Publishers Ltd.
The Anti-Cancer Charm of Flavonoids: A Cup-of-Tea Will Do!
Amr Amin
1*
and Michael Buratovich
2
1
Biology Department, College of Science, UAE University, UAE,
2
Department of Biochemistry, Spring Arbor University, MI 49283,
USA
Received: October 4, 2006; Accepted: November 23, 2006; Revised: December 4, 2006
Abstract: Hormone-dependent cancers of the breast, prostate and colon have, in the past decade, become the leading causes of morbidity
and mortality. Billions of dollars have been, and still are being spent to study cancers like these, and, in the past three decades, thanks to
work by thousands of dedicated scientists, tremendous advancements in the understanding and treatment of cancer have been made.
Nevertheless, as there is no sure-fire cure for a variety of cancers to date, natural protection against cancer has been receiving a great deal
of attention lately not only from cancer patients but, surprisingly, from physicians as well. Phytoestrogens, plant-derived secondary
metabolites, are diphenolic substances with structural similarity to naturally-occurring human steroid hormones. Phytoestrogens are
normally divided into three main classes: flavonoids, coumestans and lignans. Flavonoids are found in almost all plant families in the
leaves, stems, roots, flowers and seeds of plants and are among the most popular anti-cancer candidates. Flavonoidic derivatives have a
wide range of biological actions such as antibacterial, antiviral, anti-inflammatory, anticancer, and antiallergic activities. Some of these
benefits are explained by the potent antioxidant effects of flavonoids, which include metal chelation and free-radical scavenging
activities. Patent applications regarding flavonoids range from protocols for extraction and purification from natural resources and the
establishment of various biological activities for these extracts to novel methods for the production and isolation of flavonoids with
known biological activities. This review will bring the reader up to date on the current knowledge and research available in the field of
flavonoids and hormone-dependent cancers, and many of the submitted patents that exploit flavonoids.
Keywords: Flavonoids, cancer, estrogen, phytoestrogen, stilbenes, lignans, coumestans, wogonin, isoliquiritigenin.
INTRODUCTION
In their natural environment, plants must have the ability to
cope with an array of stressful conditions. Plant stress factors
include drought, salinity, nutritional deficiency, intense insolation,
adverse climatic conditions, pollutants, pathogens, insects, and
phytophagy. Therefore, the survival of plant species depends on
their genomic plasticity, i.e., the ability of the plant to diversify its
own defense responses against the above mentioned biotic and
abiotic stresses. Additionally, plants are sessile, which makes the
synthesis of phytochemicals one of the major strategies plants use
to counteract unfavorable conditions and adapt to new environ-
ments.
Plant natural products can be roughly classified into three main
classes of compounds, phenylpropanoids, isoprenoids, and alka-
loids, which are widely distributed in plant foods and medicinal
herbs [1-3]. This large array of molecules, derived from plant
secondary metabolism, is of extreme interest to human nutrition and
pharmacology, and the perfumery and cosmetic industries. These
plant-derived products are not pharmaceuticals, but nutraceuticals,
which are dietary components that are able to improve human
fitness [4]. Also, plant-derived foodstuffs and beverages constitute
the so-called functional foods and beverages, which include mainly
fruits, vegetables, herbs, spices, chocolate, tea, beer, and wine.
Hormone-related cancers of the breast, endometrium, ovary,
prostate, testis, thyroid and bone (osteosarcoma) share a unique
mechanism of carcinogenesis. Endogenous and exogenous
hormones drive cell proliferation, and provide the opportunity for
the accumulation of random genetic errors. The emergence of a
malignant phenotype depends on a series of somatic mutations that
occur during cell division, but the specific genes involved in
progression of hormone-related cancers are currently unknown [5].
In this review we discuss the genotoxic potential of phytoestrogens,
which occur in numerous plants and are considered to be potent
candidates in the field of chemoprevention [6-9].
*Address correspondence to this author at the Biology Department, UAE
University, P.O. Box 17551, UAE; Tel: +9713-7134381; Fax: 9713-
7671291; Email: a.amin@uaeu.ac.ae
ESTROGEN RECEPTORS AND SIGNALING
The realization that hormone replacement therapy is not as safe
or effective as previously thought has generated greater interest in
phytoestrogens [10]. Estrogens, steroid hormones produced in the
ovaries and testis, have many biological effects in the body beyond
the reproductive system [8]. The effects of steroid hormones are
normally mediated by specific intracellular receptors in the target
tissue. The first member of what is now known as the steroid and
thyroid hormone receptor superfamily was a protein isolated from
rat uterus that exhibited specificity for 17-estradiol [11]. Although
many other members of this gene family were identified in
subsequent years [12], this protein remained the only estrogen
receptor (ER) known in animal tissues until 1996 when a second
ER subtype was isolated from the rat prostrate and ovary [13]. The
two ERs are products of two independent genes that share an
important degree of homology due to the identity of their common
endogenous ligand [14].
The two ERs exhibit different responses to the synthetic anti-
estrogens tamoxifen and raloxifene: Both compounds elicit a partial
agonist activity when interacting with ER, but only antagonism
was observed with ER. Furthermore, the ability of raloxifene to
antagonize estradiol-induced gene expression in an in vitro reporter
system was fifteen times greater with ER compared with ER
[15]. These data strongly suggest that ER has specific roles in the
transduction of hormonal signals that are independent from ER.
The differential tissue distribution patterns of ER and ER
supports this assertion. ER is the predominant ER in the prostate,
lung, bladder, gastrointestinal tract, salivary gland, and developing
pituitary [16-18]. Even in those organs where both estrogen
receptors are present, the identity of the cells expressing each
subtype appears to be different. For example in the ovary, granulosa
cells express mainly ER, whereas ER is restricted to the
surrounding thecal cells [19]. Similarly, the stroma of the prostate
expresses mostly ER and the prostate epithelium contains ER
[13]. Likewise in the uterus, ER is expressed in both the glandular
and luminal epithelia, but ER, is confined to only the glandular
epithelium [19]. Both ER and ER function during normal ovarian
follicular development, and in vascular endothelia, myocardial
cells, smooth muscle, and breast tissue. Also ER is involved in
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110 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 Amin and Buratovich
bone maturation in both males and females, however, only ER
plays a role in bone maintenance in females. ER is more important
in maintaining the concentration of follicle stimulating and
luteinizing hormone in blood, and ER is involved in frontal lobe-
mediated learning and memory [20].
The dominant form of estrogen in the body is 17-estradiol
(Fig. 1A), although any compound that induces estrogen receptor
dimerization and subsequent binding of the receptor to the estrogen
response element (ERE) can be considered an estrogen (Fig. 2).
Antagonistic effects can occur when a compound is able to bind to
the receptor but dimer formation either does not occur or the correct
conformation necessary to bind the ERE and activate transcription
is not induced. Some compounds act as estrogen agonists and
antagonists and are referred to as Selective Estrogen Receptor
Modulators (SERMs). For example, the antiestrogen tamoxifen acts
as an estrogen antagonist in breast tissue but as an agonist in the
uterus, bones and vascular system [21]. These agonist/antagonistic
effects are believed to be responsible for the differential effects of
phytoestrogens as compared to estradiol [6-8,20,22].
FLAVONOIDS
Flavonoids are a large group of phenolic plant constituents. To
date, more than 6000 flavonoids have been identified [9,23],
although a much smaller number is important from a dietary point
of view. The bioactive potential of flavonoids has been long
recognized, but until recently data about their bioavailability,
metabolic fate, and health effects were limited. Interest in these
compounds only emerged in this decade and has grown since then.
Flavonoids are increasingly recognized as possessing a broad
spectrum of biological activities and important therapeutic
applications that include novel features, such as anti-cancer, anti-
tumor, anti-inflammatory, and anti-coagulant activities [24,25].
Flavonoids are typically classified into several groups. Among
them, flavonones occur predominantly in citrus fruits, flavones in
herbs, isoflavonoids in legumes, anthocyanins in fruits and
flavonols in fruits and vegetables. Estimations about the dietary
intake of bioflavonoids vary from 23 to 1000 mg/day [26,27].
Flavonoids are potent antioxidants in vitro, and therefore one of the
main interests in these compounds involves protection against
cardiovascular disease. Antioxidation is, however, only one of the
many mechanisms by which flavonoids can exert their actions. It is
important to keep in mind that the biological and chemical
properties of flavonoids from different subgroups can differ
substantially [28].
CHEMISTRY AND CLASSIFICATION
Flavonoids consist of two benzene rings (A and B) that are
connected by an oxygen-containing pyrene ring (C, See Fig. 1B).
Flavonoids that contain a hydroxyl group in position C-3 of the C
ring are classified as 3-hydroxyflavonoids (flavonols, anthocya-
nidins, leucoanthocyanidins, and catechins), and those lacking it as
3-desoxyflavonoids (flavanones and flavones). Classification within
the two families is based on how additional hydroxyl or methyl
groups are attached to different positions of the molecule.
Isoflavonoids differ from the other groups; the B ring is bound to
C-3 of ring C instead of C-2. Anthocyanidins and catechins, on the
other hand, lack the carbonyl group on C-4 [28,29].
PHYTOESTROGENS
Phytoestrogens are naturally occurring non-steroidal, diphenolic
compounds derived from many of those plants that form part of our
diet. Most phytoestrogens belong to the class of flavonoids that are
structurally characterized by a C
6
C
3
C
6
carbon skeleton (Fig. 1C)
[25]. Phytoestrogens have received increased investigative attention
because of their protective effects against hormone-dependent
cancers (e.g. breast, prostate, colorectal, endometrial and testicular
cancers) [30-43]. The protective effect of phytoestrogens against
cancer may be due to their ability to lower circulating levels of
unconjugated sex hormones. Most of the estrogens that circulate
throughout the bloodstream are inactive, since they are in a
complex with either sex hormone binding globulin (SHBG) or
albumin [44]. Various plant phytoestrogens decrease circulating
estrogen levels through a variety of mechanisms (see below).
In the 1940s it was first realized that some plant-derived
compounds could cause estrogenic effects [45]. For example, sheep
that grazed on pastures rich in red clover (Trifolium pratense)
displayed multiple fertility problems: Immature animals showed
signs of estrus, ewes were unable to get pregnant and those that
were pregnant often miscarried. The clover in these pastures
contained large amounts of the isoflavones, formononetin and
biochanin A [46], which were among the first phytoestrogens
discovered. Currently, four different families of phenolic
compounds produced by plants are considered phytoestrogens: the
isoflavonoids, which are derived principally from soybeans and
include such compounds as genistein, daidzein, and glycitein;
stilbenes; lignans, which are found in flaxseed; and coumestans,
which are derived from sprouting plants like alfalfa. Different
classes of phytoestrogens and the diverse compounds within each
class affect estrogen-mediated responses in different ways [8].
Dietary supplementation with soy isoflavonoids or lignans has
been shown to increase the levels of SHBG [47-50] in
postmenopausal women, which lowers the serum levels of free
estradiol [50-52]. Higher ingestion of isoflavonoids also produces a
significant decrease in the urinary excretion of genotoxic estrogen
metabolites. Three major biochemical pathways that produce
different metabolites can metabolize estradiol and estrone. Two of
these pathways produce 16-hydroxyestrogens and 4-hydroxy-
estrogens, which are known to be genotoxic [53]. 2-Hydroxy-
estrogen metabolites have been proposed to exert a protective effect
against breast cancer, and the ratio of 2-hydroxyestrogens to 16-
hydroxyestrogens is considered to be an important breast cancer
biomarker [54]. The level of serum SHBG negatively correlates
with the urinary excretion of 16 alpha-hydroxyestrone and estriol
[47].
Lower incidences of colorectal cancers (CRC) in Far Eastern
countries may be, in part, attributed to high nutritional intake of soy
and its abundant phytoestrogens [43]. Due to their molecular
similarities to endogenous estrogens, phytoestrogens distinctly
interact with estrogen receptors. Both genomic and nongenomic
mechanisms have been shown to be responsible for the possible
anticarcinogenic properties of phytoestrogens, such as induction of
apoptosis and inhibition of tyrosine kinases and DNA topo-
isomerases. While in vitro and animal studies in general are
somewhat supportive of a protective role of phytoestrogens against
CRC, epidemiological work done to date is presently inconclusive
and is probably confounded by enduring inconsistencies concerning
those concentrations of phytoestrogens most likely to be effective
[55].
The incidence of breast, endometrial and prostate cancer is
lower in Asian populations than in those populations that reside in
Western countries [56-58]. This disparity might be explained by
Asians diets, which tend to be rich in plant protein, particularly soy.
However, genetic factors might also significantly influence the rates
of certain cancers, since ER is expressed in low levels in normal
breast tissue in Japanese women [59]. Conversely, epidemiological
studies have shown that Asians who emigrated to the US also had a
low incidence of breast cancer, whereas the descendants of these
emigrants, who consumed mainly a Western diet, had an increased
risk for breast cancer compared to their parental generation [60-61].
Due to a change in dietary habits and lifestyle in Asian countries,
breast cancer incidence in these countries is presently increasing.
The breast cancer-preventive effects of phytoestrogens are greatest
when isoflavones are consumed in early life [62-64].
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Natural Protection Against Cancer Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 111
ISOFLAVONOIDS
Isoflavones and some of their metabolites are specifically
considered to act as selective estrogen-receptor modulators. They
interact with estrogenic receptors in humans and are capable of
exerting protective effects against hormone-related diseases, even
though several other biological activities and therapeutic uses have
been proposed. Isoflavones are most abundant in soybeans and can
also be found in alfalfa sprouts and legumes. In plants these
phytoestrogens exist primarily as glycosides (isoflavonoides). Many
studies have demonstrated the hormonal activities of isoflavones
[65-66] but studies on their genotoxic activities are still limited.
Epidemiologic and experimental data suggest that the consumption
of soybean-containing foods may protect against cardiovascular
disease and decrease the risk of developing breast, prostate and
endometrial cancer.
Although concerns have been raised that consumption of
isoflavones may cause potentially adverse effects on the repro-
ductive tract and behavior, a recent study showed that chronic
dietary consumption of a soy-based diet or soy isoflavones has no
adverse effects on the observed reproductive patterns of male
rabbits [67]. The effects of soy protein on benign prostate
hyperplasia and prostate-specific antigen (PSA) have been reported
in some studies [68-73]. Phytoestrogens may prevent prostate
cancer by means of a dose-dependent reduction of serum 17-
estradiol and testosterone levels, as reported in Japanese men [74].
The presence of estrogen receptor in the prostate [75], which
binds the isoflavone genistein with an affinity similar to that of 17
estradiol, suggests that the estrogenic effect of phytoestrogens may
be mediated by interaction with estrogen receptor [76]. Other
suggested mechanisms of action include induction of p21 by a p53-
independent pathway [77-78], and inhibition of epidermal growth
factor autophosphorylation [76]. Various anticancer effects have
been demonstrated including inhibition of tumor formation, growth
factor-stimulated tumor cell growth, and inhibition of proteases,
tyrosine kinases, and angiogenesis. It has also been suggested that
phytoestrogens may reduce cancer risk through their antioxidant
effects [79-81].
Most of the available studies have focused on the isoflavones
genistein and daidzein, but genistein (Fig. 1D) has been studied
more extensively and seems to be a more promising cancer-
protective agent than the closely related compound, daidzein (Fig.
1E). Genistein has been shown to inhibit the growth of cancer cells
through modulation of the genes involved in the homeostatic
control of the cell cycle and apoptosis. Genistein inhibits activation
of nuclear transcription factor, NF-kappa B (NF-B) and the Akt
signaling pathway, both of which are known to maintain balance
between cell survival and programmed cell death (apoptosis) [82-
86]. Genistein can induce responses in cell lines and breast, ovarian,
endometrial, prostate, vascular and bone tissues that are similar to
those induced by estradiol [87-90]. There is a also large body of
epidemiological studies that show that people who consume high
amounts of isoflavonoids in their diets have lower rates of several
cancers including breast, prostate and colon cancer [91]. Food
additives that contain chemical analogues of the flavonoids
genistein, daidzein, formononetin and biochanin A have been
designed to improve the health of patients who suffer from some
cancers, premenstrual syndrome, menopause or hyperchol-
esterolemia [92].
Fig. (1). Chemical structures of A) 17ß-Estradiol; B) Flavane nucleus; C) General structure of phytoestrogens; D) Genistein (isoflavonoid); Daidzein; F)
TRANS-resveratrol (stilbene); G) Matairesinol (lignan); Coumesterol (coumestan), I) 8-Prenyl naringenin, J) Equol, K) Miroestrol; L) Deoxymiroestrol; M)
Wogonin; N) Isoliquiritigenin (ILTG).
O
O
O
OHO
OH O
OH
O
HO
OH
OH
HO
OH
O
O
O
HO
O
HO
O
O OH
OHO
HO
OHO
O
OH
HO
OCH
3
O
O
HO
OH
OH
O
OH
OHO
OH
O
OH
O
OH
OH
H
3
C
H
3
C
H
2
C
O
O
OH
OH
H
3
C
H
3
C
H
2
C
HO
HO
HO
OH
8
5
3
4
6
7
2'
3'
4'
5'
6'
8
2
34
5
4'
5'
2'
3'
6'
6
7
A) B) C) D)
E) F) G) H)
I)
J) K) L)
M)
N)
B
C
A
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112 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 Amin and Buratovich
Isoflavonoids from legumes, which include genistein and
daidzein, exist as glucosides or as aglycones, but the glucoside form
is readily hydrolyzed in the gut to the aglycone form. Aglycones
also are easily transported across intestinal epithelial cells [93].
Glucuronylation of flavonoids might stabilize them and create the
potential for sustained-release forms of flavonoids. The isolation of
the UDP-glucuronyl transferase from Bellis perennis for
commercial preparation of glucuronylated flavonoids potentially
allows for the production of such timed-released forms of
flavonoids [94].
STILBENES
Some of the stilbene-related heterocyclic compounds that have
been analyzed display anti-HIV activity through interference with
NF-B and Tat function [95]. Stilbenes, like other flavonoids, are
produced through the phenylpropanoid-acetate pathway. The main
dietary source of phytoestrogenic stilbenes is resveratrol from
grapes and peanuts (Fig. 1F). Although there are two isomers of
resveratrol, cis and trans, only the trans form has been reported to
show estrogenic activity [96]. The type of grapes examined and the
post-harvest processing they undergo has a large effect on the
resveratrol content in the final product. This is evidenced by the
fact that purple grape juice contains more resveratrol than white
grape juice. White grape juice is made by cold pressing the grapes,
while a hot extraction method is used for purple grape juice [97].
This is also true for peanuts, since boiled peanuts contain more
resveratrol than peanut butter or roasted peanuts [98]. As peanuts
mature, their resveratrol 4 content declines, with smaller peanuts
having higher levels of resveratrol 4 than larger ones [98]. In
peanuts, resveratrol 4 is found throughout the nut, but the seed-coat
has the highest levels by weight [99]. Dried roots of Polygonum
cuspidatum (Japanese Knotweed or Mexican Bamboo), which are
used in traditional Chinese medicine for a variety of therapeutic
purposes, contain resveratrol levels as high as 377 mg /100 g dry
Fig. (2). Estrogen signaling mechanism. The estrogen receptor (ER) is a cytoplasmic monomer that is bound to Hsp90 orthologs and other proteins when not
bound to its ligand. However when bound by ligand, the estrogen receptor sheds the associated proteins and dimerizes. Phosphorylation by Protein kinase A
facilitates dimerization. Dimerization is followed by translocation to the nucleus where it binds estrogen response elements (ERE) in the upstream regulatory
regions of estrogen responsive genes (consensus sequence: 5´-GGTCAnnnTGACC-3´ where n is any nucleotide). Once bound, transcriptional co-activators
bind and activate transcription of the gene, which generates mature mRNAs. These mature mRNAs are transported to the cytoplasm where they are translated
by ribosomes into protein. In some cases, the estrogen receptor acts as a transcriptional repressor instead of a transcriptional activator. Various flavonoids and
phytoestrogens either antagonize estrogen synthesis, mimic estrogen activity by binding to the estrogen receptor and activating it or by binding to the ER and
preventing ER activation. Genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-phenylnaringenin, coumestrol and resveratrol do not necessarily inhibit
nuclear transport of dimerized estrogen receptors, but they do prevent the activation of estrogen-dependent transcription.
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Natural Protection Against Cancer Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 113
root [100]. In addition to these food products, resveratrol 4 has been
isolated from several grass species [101], pine bark [102], ivy and
lilies [97].
Resveratrol 4 has a greater capacity to activate the ERß receptor
than the ER receptor [103]. When the genes that encode the
human ERß and ER proteins are transfected into MCF-7 cells or
the hamster ovarian cell line, CHO-K1, treatment of these cells with
resveratrol 4 elicits both agonistic and antagonistic estrogenic
activities [8, 104-106]. Similarly, resveratrol has some therapeutic
effects that are due to its antioxidant potential. It apparently inhibits
the oxidation of human LDL and reduces the propensity of human
plasma and LDL to undergo lipid peroxidation [107,108]. In
addition to its antioxidant potential, it has also been reported to
have a variety of anti-inflammatory, anti-platelet, and anti-carcino-
genic effects [109,110]. The beneficial effects of red wine
consumption, which have been purported to reduce the risk of
cardiovascular disease, have been attributed to the multiple effects
of resveratrol and its high concentration in grape skins [111].
Recently, resveratrol was shown to inhibit cellular events
associated with tumor initiation, promotion, and progression [112].
Furthermore, it has been reported that resveratrol has the potential
to inhibit DNA polymerase and cyclooxygenase [109] and also has
a direct antiproliferative effect on human breast epithelial cells
[113]. Based on its antimutagenic activity [114,115], it has been
suggested that resveratrol might hold potential as an effective
cancer chemopreventive agent in humans [116].
LIGNANS
Lignans are natural plant compounds with estrogenic properties
that are widely distributed in human daily diets, and predominantly
occur in whole grain products (Fig. 1G). High amounts of
phytolignans are found in flaxseed, whole grain breads, vegetables,
and tea. Fruits have low levels of these lignans with the exception
of strawberries and cranberries. After ingestion dietary lignans are
converted by gut microflora to the enterolignans enterodiol (End)
and enterolactone (Enl). Because of their resemblance to
endogenous 17-enterolactone, these compounds have also been
described as phytoestrogens, and anticarcinogenic potentials have
been suggested for them as well [117]. A high plasma concentration
of the mammalian lignan, Enl is correlated with a reduced risk of
breast cancer [118,119], and women in the highest quintile for
circulating enterolactone concentrations have half the risk of
developing breast cancer than those who occupy the lowest quintile
[118]. Similar correlations have been found between dietary intakes
of isoflavonoids and lignans and thyroid [120], ovarian [121], and
breast [122,123] cancers in pre- and post-menopausal women. In
contrast a large study that examined dietary factors and incidence of
breast cancer, which followed a cohort of 111,526 women in
California, found no association between phytoestrogens and breast
cancer rates [124]. A nested case control study in Finland found no
correlation between serum Enl concentrations and the risk of
developing breast cancer in pre- or post-menopausal women [125].
Most recently, the effects of the mammalian lignans END, ENL,
and a particular soy isoflavone, GEN, both alone and in combi-
nation, on bone and uterus health were examined in a postmeno-
pausal breast cancer mouse model. GEN acts estrogenically on the
uterus to increase its weight, and on bone to increase femoral bone
mineral density and yield load in comparison to negative controls.
Lignans, however, did not act estrogenically on either bone or
uterus. Mixed phytoestrogens induced no effects on bone when
compared to negative controls but increased the weight of the
uterus beyond that induced by GEN alone. This study has clinical
implications since many breast cancer patients use alternative
therapies to supplement breast cancer treatments and to reduce
menopausal symptoms [126, 127]. Although most studies have
been based on the possible estrogenic action of lignans, it now
seems clear that non-estrogenic mechanisms should be also
investigated to explain the reported relationship between the
consumption of lignan-rich foods and the lowered risk of such
chronic diseases as cardiovascular disease [128,129] and breast
cancer [130].
COUMESTANS
Although there are many coumestans, only a few show a potent
estrogenic activity, and of those that show such activity, coumestrol
3 and 4 methoxycoumestrol are the most noteworthy. The main
dietary source of coumestrol, is legumes, but low levels have been
reported in brussel sprouts and spinach [131-133]. Clover and
soybean sprouts are reported to have the highest coumestrol
concentrations (28 and 7 mg/100 g dry wt., respectively); even
higher than mature soybeans (0.12 mg/100 g dry wt) [8].
Epidemiologic data suggest a relationship between dietary intake of
phytochemicals and a lower incidence of some cancers. Modulation
of steroid hormone metabolism has been proposed as the basis for
this effect, since the steroid biosynthetic enzymes aromatase, 3-
hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydro-
genase (17-HSD) are inhibited by the isoflavones, genistein and
daidzein, and coumestrol [134]. In a recent study, the estrogenic
activities of the following eight phytoestrogens were assessed in a
variety of MCF7 cell system-based assays: genistein (Fig. 1D),
daidzein (Fig. 1E
), coumestrol (Fig. 1F), resveratrol (Fig. 1H), 8-
prenylnaringenin (Fig. 1I), equol (Fig. 1J), miroestrol (Fig. 1K) and
deoxymiroestrol (Fig. 1L). The abilities of these phytochemicals to
antagonize the action of estradiol in both reporter gene expression
and cell growth assays were documented. The rank order of potency
of these phytoestrogens was shown to be very similar for all cell-
based assays. However, the rank order of potency based on cell-
based assays did not match the rank order determined by estrogen
receptor binding assays, even though all binding assays used the
same receptor. Poor correlation between the relative binding
affinity of the ligand to its receptor and the cellular response
elicited by the chemical has also been observed for polychlorinated
biphenyls in MCF7 cells and several xenoestrogens in rat
adenocarcinoma cells. Explanation of such discrepancies may lie
either in the relative ability of the ligand–receptor complex to
transactivate gene expression or in the fate of the ligand within the
cell, which could be influenced by cellular uptake and/or other
metabolic processes [135].
Extracts of black bean (Phaseolus vulgaris) contain a mixture
of coumarins, tannins, flavonoids and polyphenols and such
compositions show some efficacy in the treatment of hormone-
dependent and –independent cancers. These same extracts, which
include raw extracts, HPLC refined extracts and fractionated
extracts also lowered serum cholesterol levels by inhibiting
cholesterol synthesis [136,137].
WOGONIN
Wogonin (Fig 1M) is a flavone that has a C-8 methoxy group
(5,7-dihydroxy-8-methoxyflavone), and is found in Scutellaria
baicalensis [138]
. This compound was previously shown to possess
interesting biological activities, in that it potently down-regulates
cyclooxygenase isoform-2 (COX-2) and intracellular nitric oxide
synthetase (iNOS) in activated macrophages [139-141]. In addition,
when topically applied, wogonin reduces prostaglandin E
2
(PGE
2
)
concentrations in vivo and down-regulates COX-2 induction in an
animal model of skin inflammation provoked by multiple 12-O-
tetradecanoylphorbol 13-acetate (TPA) treatments (sub-chronic skin
inflammation) [142]. Further work has shown that the same
compound potently inhibits COX-2 and TNF- induction, but it
shows a less pronounced effect on intercellular adhesion molecule-
1 (ICAM-1) and interleukin-1 (IL-1) induction in this same
animal model [143]. This suggests that some differential
suppressive activities depend on the genes and animal models
studied. These results also indicated that wogonin is a potential
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114 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 Amin and Buratovich
anti-inflammatory agent when applied to skin, since topical
application of wogonin suppresses proinflammatory enzyme
expression of cyclooxygenase-2 (COX-2). This seems to be the
main anti-inflammatory activity of wogonin. However, the detailed
effect on each skin cell type is not understood. It was recently found
that wogonin (10–100 DM) clearly down-regulated COX-2
expression from NIH/3T3 cells treated with 12-O-tetradecanoyl-
phorbol 13-acetate, interleukin-1 or tumor necrosis factor-, but
the expression levels of COX-1, interleukin-1 and fibronectin were
not significantly affected. These data correlate well with the
significant reductions in prostaglandin E
2
(PGE
2
) production caused
by wogonin. As a comparison, NS-398 (a selective cyclooxygenase
2 inhibitor) did not suppress COX-2 expression or the expression of
other genes, while PGE
2
production was potently reduced by
wogonin concentrations of 0.1–10 DM. All these results suggest that
COX-2 down-regulation of skin fibroblasts may be, at least in part,
one of the anti-inflammatory mechanisms of wogonin [144].
Most recently a cancer-specific apoptosis-inducing activity of
wogonin was reported. In this study, a methanol extract of cultured
Scutellaria baicalensis cells inhibited the proliferation of the human
monocytic leukemia cell line THP-1 and the human osteogenic
sarcoma cell line HOS. It should be noted that wogonin did not
show an inhibitory effect on the normal diploid human fetal lung
cell line TIG-1, in comparison to the inhibition observed in cancer
cells. Physiological analyses in THP-1 cells showed that wogonin
induced cell cycle arrest at the G
2
/M checkpoint and, subsequently,
apoptosis [145].
ISOLIQUIRITIGENIN
Isoliquiritigenin (ILTG, Fig 1N), is a simple chalcone-type
flavonoid, found in licorice (legume) and shallot (liliaceae). It is a
potent antioxidant with anti-inflammatory, anti-platelet aggregation
and cancer-preventing properties [146-150]. It exhibits an
inhibitory effect on carcinogenesis in skin and colon, and
suppresses the proliferation of pulmonary, prostate, breast, gastric
and melanoma cancer cells [151-155]. ILTG suppresses the level of
COX-2 protein in the murine macrophage cell line RAW264.7, and
cell proliferation via induction of apoptosis in colon cancer cells
[156]. Cellular damage induced by chronic inflammation is a well
known cause of colon carcinogenesis. Cyclooxygenase-2 (COX-2),
the enzyme that converts arachidonic acid to prostanoids, is known
to play an important role in inflammation. ILTG has previously
been reported to be a strong suppressor of the COX-2 pathway as
well as an inducer of apoptosis. It has recently been reported that
ILTG-mediated apoptosis in mouse colon adenocarcinoma Colon
26 cells is COX-2-dependent. This dependency is enhanced by the
blockage of the lipoxigenase-mediated metabolic pathway and
attenuated by the addition of several prostaglandins and
thromboxanes. Taken together, these findings indicate that ILTG-
induced apoptosis is negatively regulated by the level of COX-2
expression. Although a strict correlation between the down-
regulation of COX-2 and growth suppression has not yet been
demonstrated, ILTG is currently accepted as a potential chemo-
therapeutic agent for several diseases whose induction requires the
COX-2 pathway, including colon cancer [157].
A recent study has
shown that ILTG-induced apoptosis is negatively regulated by the
expression level of COX-2 [157]. In addition, ILTG has recently
been shown to be a potential chemopreventive agent against liver
cancer [158].
CURRENT & FUTURE DEVELOPMENTS
The medicinal uses of herbs rich in phytoestrogens have been
widely recognized in folk medicine for some time even though the
active components of these medicinal plants were unknown until
relatively recently [159,160]. Since then, a large body of
epidemiological data and observations from in vitro and in vivo
systems has corroborated the potential medicinal uses of plant
phytoestrogens. Despite the vast amount of information about the
biological efficacy of these compounds, much work remains and
still significant questions linger.
It is still unclear how the data generated by in vitro systems
might apply to whole organisms. Since flavonoids like genistein
and daidzein have been primarily studied by means of in vitro
systems, the ability of these compounds to elicit favorable respon-
ses without causing deleterious side effects remains uncertain. Also,
it is equally unclear how to successfully use flavonoids and other
phytoestrogens as part of a treatment regime. Needless to say, these
pending questions have not prevented enterprising researchers from
designing dietary supplements and pharmaceutical agents that
include flavonoids. Furthermore patent applications have been
submitted for several compositions that purportedly treat or help
prevent a wide variety of medically significant conditions.
Several patent applications describe combinations of flavonoids
with tocotrienols (members of the vitamin E family) to comprise a
variety of pharmaceutically useful concoctions. Combinations of
polymethoxylated flavonoids like hesperetin, nobiletin, tangeretin
and naringenin with tocotrienols have been generated for treating
patients at risk for cardiovascular disease [161]. Similar ingredients
have also been proposed to aid in the prevention of neoplasias and
inflammation [162], and might also serve to lower serum choles-
terol levels [163]. Other mixtures of tocotrienols with limocitrin
derivatives, which naturally occurs in the peel of lemons, and the
flavonoid quercetin may also lower total serum cholesterol [164].
Several patent applications have described the extraction of
flavonoid-rich extracts from particular plants. For example, purified
aqueous reflux extracts of honeysuckle (Ionicera japonica) stems
contain flavonoids, tannins, saponins and a powerful anti-inflam-
matory and analgesic compound called sweroside. These extracts
are purported to be quite safe and effective anti-inflammatory
agents [165]. Extracts of Ginkgo biloba are reported to increase the
activity of tyrosine hydroxylase, which boost dopamine levels in
the brain and ameliorate the symptoms of Parkinson’s disease
[166]. Also cocoa extracts rich in polyphenols and procyanidins
have been described that reportedly can treat atherosclerosis [167].
The exploitation of the medicinal properties of flavonoids
underscores the efficacy of these compounds and their potential to
join the large cadre of pharmaceutical and nutritional agents already
available to fight human disease and raise the quality of human
health worldwide. Unfortunately, how well these compounds might
work in the inhibition of cancer or lowering serum cholesterol
levels has yet to be rigorously tested in Phase IV tests. Likewise
there is little known about the interactions of flavonoids with other
drugs presently prescribed by physicians and used by patients for
the treatment of cancer or other ailments. Thus our present
knowledge has many gaps in it that will require filling before such
compounds can receive approval by regulatory agencies, broad
acceptance by the medical community and join other pharma-
ceuticals on drugstore shelves.
Nevertheless, the potential to fill these gaps is presently
increasing. Important advances in optical biosensor instrumentation
now allow drug screens based on whole-cell sensing in both high-
throughput and high-content fashions [168]. Additionally, drug
screens that utilize non-optical detection systems circumvent the
limitations of optical detection systems. Several start-up and
established biotechnical companies have developed non-optical
detection technologies that utilize impedance-assay systems for
cell-based screening, acoustic systems, calorimetric systems and
microelectromechanical sensors. Impedance-assay systems either
measure electrical impedance changes across microelectronic cell
sensor arrays directly integrated into the bottom of a microtitre
plate or slide to detect changes in cells or use cellular dielectric
spectroscopy to measure cell-surface receptor responses in live
cells. Acoustic systems like piezoelectric quartz crystal technolo-
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Natural Protection Against Cancer Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 2 115
gies that measure the build-up of drug molecules on an oscillating
surface and dissipative quartz crystal microbalance that examine
protein or polymer thin-film formation and the attachment of cells
to surfaces provide real-time binding information on molecular
interactions between drugs and their targets. Calorimetric systems
consist of nanocalorimetric platforms that measure the enthalpic
changes that ensue when receptors are bound by ligands. Micro-
electromechanical sensors are submicron-sized mechanical devices
that are built into semiconductor chips. These include things like
microcantilevers which are tiny sensors that deform in response to
changes in binding or surface stresses during molecular recognition
events or nanowire sensors that can be used to grow individually
addressable drug-screening arrays. These drug-screening techno-
logies are commercially available and provide innovative ways to
perform post-high-throughput screening hit confirmation and mode-
of-action studies [169]. The application of such methodologies to
flavonoid research would greatly accelerate our knowledge of the
clinical usefulness of these molecules.
In the meantime, flavonoids are available in many different
foods and supplements and are well worth ingesting on a regular
basis. Recombinant bacteria have been made that possess the
biosynthetic genes for flavonoid synthesis. Thus pure, comercially
available flavonoids will probably be available for consumption in
the near future [170,171]. Until that time, curl up and have a cup of
tea - it will do you good.
ACKNOWLEDGEMENTS
This review is dedicated to the memory of Dr. Hazem Kataya, a
dear colleague and a respected scientist, who passed away in June
2006. Authors also thank Ms. Huda Ateek and Ms. Kami Moyer for
their valuable assistance preparing this manuscript.
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