Genistein and Cancer: Current Status, Challenges, and Future Directions

Abstract

Primary prevention through lifestyle interventions is a cost-effective alternative for preventing a large burden of chronic and degenerative diseases, including cancer, which is one of the leading causes of morbidity and mortality worldwide. In the past decade, epidemiologic and preclinical evidence suggested that polyphenolic phytochemicals present in many plant foods possess chemopreventive properties against several cancer forms. Thus, there has been increasing interest in the potential cancer chemopreventive agents obtained from natural sources, such as polyphenols, that may represent a new, affordable approach to curb the increasing burden of cancer throughout the world. Several epidemiologic studies showed a relation between a soy-rich diet and cancer prevention, which was attributed to the presence of a phenolic compound, genistein, present in soy-based foods. Genistein acts as a chemotherapeutic agent against different types of cancer, mainly by altering apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis. Targeting caspases, B cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, kinesin-like protein 20A (KIF20A), extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear transcription factor κB (NF-κB), mitogen-activated protein kinase (MAPK), inhibitor of NF-κB (IκB), Wingless and integration 1 β-catenin (Wnt/β-catenin), and phosphoinositide 3 kinase/Akt (PI3K/Akt) signaling pathways may act as the molecular mechanisms of the anticancer, therapeutic effects of genistein. Genistein also shows synergistic behavior with well-known anticancer drugs, such as adriamycin, docetaxel, and tamoxifen, suggesting a potential role in combination therapy. This review critically analyzes the available literature on the therapeutic role of genistein on different types of cancer, focusing on its chemical features, plant food sources, bioavailability, and safety.

Full text

REVIEW
Genistein and Cancer: Current Status, Challenges,
and Future Directions
1,2
Carmela Spagnuolo,
3
Gian Luigi Russo,
3
* Ilkay Erdogan Orhan,
4
Solomon Habtemariam,
5
Maria Daglia,
6
Antoni Sureda,
7
Seyed Fazel Nabavi,
8
Kasi Pandima Devi,
9
Monica Rosa Loizzo,
10
Rosa Tundis,
10
and Seyed Mohammad Nabavi
8
*
3
Institute of Food Sciences, National Research Council, Avellino, Italy;
4
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University,
Ankara, Turkey;
5
Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, United
Kingdom;
6
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, Pavia, Italy;
7
Research
Group on Community Nutrition and Oxidative Stress and CIBERobn (Physiopathology of Obesity and Nutrition), University of Balearic Islands,
Palma de Mallorca, Spain;
8
Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran;
9
Department of
Biotechnology, Alagappa University, Karaikudi, Tamil Nadu, India; and
10
Department of Pharmacy, Health, and Nutritional Sciences, University of
Calabria, Rende, Italy
ABSTRACT
Primary prevention through lifestyle interventions is a cost-effective alternative for preventing a large burden of chronic and degenerative
diseases, including cancer, which is one of the leading causes of morbidity and mortality worldwide. In the past decade, epidemiologic and
preclinical evidence suggested that polyphenolic phytochemicals present in many plant foods possess chemopreventive properties against
several cancer forms. Thus, there has been increasing interest in the potential cancer chemopreventive agents obtained from natural sources,
such as polyphenols, that may represent a new, affordable approach to curb the increasing burden of cancer throughout the world. Several
epidemiologic studies showed a relation between a soy-rich diet and cancer prevention, which was attributed to the presence of a phenolic
compound, genistein, present in soy-based foods. Genistein acts as a chemotherapeutic agent against different types of cancer, mainly by
altering apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis. Targeting caspases, B cell lymphoma 2 (Bcl-2)–associated X protein
(Bax), Bcl-2, kinesin-like protein 20A (KIF20A), extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear transcription factor kB (NF-kB), mitogen-
activated protein kinase (MAPK), inhibitor of NF-kB(IkB), Wingless and integration 1 β-catenin (Wnt/β-catenin), and phosphoinositide 3 kinase/
Akt (PI3K/Akt) signaling pathways may act as the molecular mechanisms of the anticancer, therapeutic effects of genistein. Genistein also
shows synergistic behavior with well-known anticancer drugs, such as adriamycin, docetaxel, and tamoxifen, suggesting a potential role in
combination therapy. This review critically analyzes the available literature on the therapeutic role of genistein on different types of cancer,
focusing on its chemical features, plant food sources, bioavailability, and safety. Adv Nutr 2015;6:408–19.
Keywords: genistein, cancer, source, bioavailability, safety
Introduction
The International Agency for Research on Cancer, which is
part of the WHO, reported that of the estimated 14.1 million
adults worldwide who were diagnosed with cancer in 2012,
8.2 million deaths were recorded (1). Moreover, on the basis
of recent trends in the incidence of major cancers and pro-
jected population growth, >23 million new cancer cases an-
nually are expected by 2030. This means 68% more cases of
cancer than in 2012 (2). The most commonly diagnosed
cancer types worldwide are lung, breast, and colorectal,
whereas those with a higher index of mortality are lung,
liver, and stomach (3).
It is therefore necessary to have new, affordable ap-
proaches to curb the increasing burden of cancer through-
out the world (4). It is widely known that dietary habits have
astrongimpactontheincidenceofchronicdiseasessuchas
cancers (5–8).
High intakes of animal fat, energy, and alcohol increase
the cancer risk (9–12), whereas foods of plant origin exert
their protective effects due to the presence of phytochemi-
cals via different mechanisms of action (i.e., antioxidant ca-
pacity, hormonal activity, stimulation of enzymes, interference
with DNA replication) (13–18). These biological and functional
1
A Sureda was supported by CIBEROBN (CB12/03/30038).
2
Author disclosures: C Spagnuolo, GL Russo, IE Orhan, S Habtemariam, M Daglia, A Sureda,
SF Nabavi, KP Devi, MR Loizzo, R Tundis, and SM Nabavi, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: glrusso@isa.cnr.it (GL Russo),
nabavi208@gmail.com (SM Nabavi).
408 ã2015 American Society for Nutrition. Adv Nutr 2015;6:408–19; doi:10.3945/an.114.008052.
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activities are ascribed both to the micronutrient content of plant
foods, such as vitamins and minerals, as well as plant secondary
metabolites, such as polyphenols, sulfur-containing compounds,
terpenes, and alkaloids (19–25). Among them, the most studied
compounds are polyphenols, which have been investigated for
their potential protective effects on human health over the
past 2 decades (26–29).
Polyphenols are generally subdivided into 2 large groups:
flavonoids and nonflavonoids (30). Isoflavones are flavonoids
in which the B ring is linked to the heterocyclic ring at the C3
instead of the C2 position (31, 32). The most important
isoflavones are genistein and daidzein (Figure 1), followed
by glycitein, formononetin, and biochanin A (Figure 2),
which can occur in foods both in free and esterified forms
(glycosylated, acetyl- and malonyl-glycosylated forms) (33).
The intake of soy products has been attributed to the lower
incidence of breast and prostate cancer in Asian populations,
which is mainly due to the presence of an isoflavone called
genistein. When compared with the other isoflavones, genis-
tein was observed to have structural similarity to 17b-estradiol
and to possess weak estrogenic activity. It competes with 17b-
estradiol for the estrogen receptor (ER),
11
with 4% binding
affinity for ER-aand 87% for ER-b, thereby contributing a
favorable role in the treatment of hormone-related cancers
(34). Moreover, many in vitro and in vivo studies also support
that genistein can be considered a promising chemopreventive
agent for the treatment of different types of cancer. In this
article we review the beneficial role of genistein against differ-
ent types of cancer, which were selected among those that are
more common and with a high mortality rate. We also discuss
the chemistry, plant food sources, bioavailability, and safety of
genistein. Finally, we provide some recommendations that
could be useful in directing future studies on this isoflavone.
Chemistry
Genistein [49,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-
(4-hydroxyphenyl) chromen-4-one] (C
15
H
10
O
5
) belongs
to a multifunctional natural isoflavonoid class of flavonoids
with a 15-carbon skeleton. Similar to other plant constitu-
ents, such as lignans, which possess estrogenic effect, genis-
tein is a typical example of a phytoestrogenic compound. It
was isolated for the first time from Genista tinctoria L. in
1899 and named after the genus of this plant. Since then,
it has been found to occur as the main secondary metabolite
of the Trifolium species and in Glycine max L. (synonym Soja
hispida) (35). As shown in Figure 1, the chemical structure
of genistein is similar to estradiol, leading to its binding abil-
ity to ERs (36–39). It possesses a high solubility in polar sol-
vents including dimethylsulfoxide, acetone, and ethanol,
although its solubility is much lower in water.
Although total organic synthesis of genistein was
achieved in 1928, it has also been obtained by using various
other methods. Chemical synthesis of genistein was per-
formed via cyclization of the corresponding ketones by us-
ing a conventional microwave oven (40). Biotechnological
synthesis of genistein was earlier reported to be achieved
through conversion of (2S)-naringen into genisteinin
NAD(P)H- and oxygen-dependent conditions as well as by
the addition of cytochrome P-450 (CYP) in elicitor-treated
soybean cell suspension cultures (41). Moreover, a metabolic-
engineering approach to genistein synthesis was set up
by using genetically engineered Saccharomyces cerevisiae
yeast cells that contained the isoflavone synthase (IFS)
gene isolated from Glycyrrhyza echinata L. (42). Similarly,
genistein was produced in Nicotiana tabacum L. leaves trans-
formed with IFS, by acting at the phenylpropanoid pathway, al-
though UV-B treatment also enhanced genistein production in
Arabidopsis (43).
On this basis and because of the known beneficial biolog-
ical effects of genistein, chemists have been so far encouraged
to synthesize many derivatives of this compound, with im-
proved pharmacologic profile. For instance, FA-esterified
(44), 6-carboxymethyl (45), nitroxy (46), 7-O-heterocycle
(47), 7-O-b-D-glucoside (48) and 7-O-b-D-glucuronic acid
(49), halogenated (50, 51), deoxybenzoin (50), benzylated
(52), hydroxylated (53), esterified (54), benzosulfonate (55),
dimethylaminomethyl (56), phenoxyalkylcarboxylic acid
(57), glycoconjugate, and alkylbenzylamine (58) derivatives
of genistein have been reported to date. All of these data reveal
that the major structural features of genistein pave the way for
synthesis of new genistein derivatives, which may emerge as
novel types of anticancer, estrogenic, and antiosteoporetic
agents.
Sources
The best known sources of genistein are soy-based foods, such
as soy cheese or soy drinks (i.e., soy milk and soy-based bev-
erages). The content of genistein in mature soybeans has been
shown to range from 5.6 to 276 mg/100 g, and an average
content of 81 mg/100 g is often described for comparative
purposes (59). In addition to genistein, soy foods contain an-
other major isoflavone, daidzein, which differs from genistein
by the lack of the hydroxyl group at position 5 (Figure 1).
Both isoflavones may exist in their aglycone or glycoside
forms. The most common glycoside forms of genistein and
daidzein are those of O-b-D-glucoside derivatives at position
7 in both compounds. Because numerous traditional Asian
foods are made from soybeans, the average dietary isoflavone
intake in Asian countries is in the range of 25–50 mg/d,
11
Abbreviations used: ABCG2, ATP-binding cassette subfamily G member 2; AP-1, activator
protein 1; ATO, arsenic trioxide; ATR, ataxia telangiectasia and Rad3-related kinase; Bax,
Bcl-2–associated X protein; Bcl-2, B cell lymphoma 2; BRCA, breast cancer growth
suppressor protein; Cdc2/Cdk1, cell division cycle protein 2 homolog/cyclin-dependent kinase 1;
Cdc25B, cell division cycle 25B; CENPF, centromere protein F; COX-2, cyclooxygenase 2;
CYP, cytochrome P450; dFMGEN, 7-difluoromethyl-5,49-dimethoxygenistein; DKK1,
Dickkopf-related protein 1; EGFR, epidermal growth factor receptor; ER, estrogen receptor;
ERK, extracellular signal-regulated kinase; FoxM1, Forkhead box protein M1; GCSC, gastric
cancer stem cell; Gli1, glioma-associated oncogene family zinc finger 1; GST, glutathione
S-transferase; HCC, hepatocellular carcinoma cell; IFS, isoflavone synthase; INT-1,
integration 1; IkB, inhibitor of NF-kB; KIF, kinesin-like protein; MMP-9, matrix
metalloproteinase 9; PCNA, proliferating cell nuclear antigen; PI3K/Akt, phosphoinositide 3
kinase/Akt; PTEN, phosphatase and tensin homolog; SCLC, small cell lung cancer; THIF,
5,7,39,49tetrahydroxyisoflavone; TNFR-1, tumor necrosis factor receptor 1; TRAIL, tumor
necrosis factor–related apoptosis-inducing ligand; TSA, trichostatin A; UGT,
UDP-glucuronosyltransferase; Wnt/b-catenin, Wingless and integration 1 b-catenin.
Anticancer effects of genistein 409
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whereas in Western countries the estimated intake is as low as
2mg/d(60,61).
Legumes are considered the second most important
source of genistein, at 0.2 to 0.6 mg/100 g, which is present
together with the other related isoflavone, daidzein (62).
The genus Lupinus (commonly known as lupin) represents
a typical example of the legume that is now widely cultivated
for its seeds, which possess a nutritional value similar to soy-
bean. Other important legumes are broad beans and chick
peas, which are known to contain significant amounts of
genistein, although less than soybeans. The content of gen-
istein in fruit, nuts, and vegetables can vary considerably; the
estimated range is from 0.03 to 0.2 mg/100 g (63). However,
in some native cherry cultivars of Hungarian origin, genis-
tein concentrations up to 4.4 mg/100 g have been recorded.
An extended list of foods with their genistein content is
available online in several databases (59).
The biotechnological approach used to maximize the iso-
flavonoid yield by sprouting seeds is the commonest method
used to improve the nutritional and medicinal values of cer-
tain foods. The metabolic processes of seed germination,
which are characterized by degradation of food reserves
and anabolic processes devoted to the developing embryo,
have been shown to enhance nutritional value primarily
by increasing the content of vitamins and plant secondary
metabolites, such as isoflavonoids (59, 64–68). Accordingly,
the increased content of genistein and other isoflavonoid
aglycones has been well documented in germinated soybean
seeds and related products (69). During the process of fer-
mentation of soybean products, the content of genistein
and related aglycones increases (70). Through genetic ma-
nipulation, it is also possible to obtain genistein from non-
legume plant sources, such as rice. Cloning the enzyme IFS
from a genistein-rich soybean cultivar resulted in transgenic
rice lines with 30-fold more genistein content (71). With the
medicinal value of genistein and related isoflavonoids now
well recognized, soy-based meat substitutes, soy milk, soy
cheese, and soy yogurt have recently gained popularity in
Europe and the United States.
Bioavailability
Various experimental models, including in vivo studies, have
shown that genistein from soy extracts, its free form, and its
glycoside genistin are readily bioavailable. For example, in
freely moving unanesthetized rats with a cannula in the por-
tal vein, genistein was readily bioavailable and was detected
in portal vein plasma 15 min after administration with AUC
values (0–24 h) of 54 and 24 mmol $h/L for genistein and
genistin, respectively (72). Several studies, however, indi-
cated that the oral bioavailability of genistin is higher than
that of genistein (73). The limitation of genistein bioavaila-
bility after oral administration is generally due to its poor
water solubility (74). Genistein also has a bitter taste (75),
and formulations to overcome both the limitation of bioa-
vailability and acceptable taste are necessary. Extensive me-
tabolism of genistein in the intestine and postabsorption
has been documented both in humans and experimental an-
imals. Among the several metabolites identified in the
blood and excreta are dihydrogenistein, dihydrodaidzein,
69-hydroxy-O-desmethylangolensin, 4-ethylphenol, glucur-
onoide and sulfate conjugates of genistein and its metabolites,
and 4-hydroxyphenyl-2-propionic aid. The gut microflora is
known to cleave the C-ring of the isoflavonoid skeleton to
give 4-hydroxyphenyl-2-propionic acid and dihydrogenis-
tein (76–78). The metabolism in the gut wall and liver is
also known to yield glucuronide and sulfated products
(79). Generally, the 3 hydroxyl groups (5, 7, and 49)are
available for conjugation, but genistein-7-glucuronide-49-
sulfate and genistein-49,7-diglucuronide byproduct appears
to be the major metabolite in plasma (80). Some reports
also suggest that conjugation plays a role in rapid elimina-
tion by biliary and urinary excretion (81).
Safety
There is no clear evidence that the consumption of large
amounts of isoflavones in the diet is harmful in humans, al-
though the multiple and complex effects of these compounds
suggest that the administration of high doses of isoflavones
could induce potentially adverse effects (82). However, mini-
mal clinical toxicity in healthy postmenopausal women was
observed after a single dose that exceeded normal dietary
intakes of purified unconjugated isoflavones (83). The geno-
toxicity of anticancer agents, such as genistein, may be bene-
ficial because they promote cancer cell death by inducing
apoptosis and othercytotoxic processes. However, these agents
FIGURE 2 Chemical structure of glycitein, formononetin, and
biochanin A.
FIGURE 1 Chemical structure of genistein and related
compounds.
410 Spagnuolo et al.
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would also negatively affect normal cells. Genotoxic and po-
tentially adverse effects of genistein (apoptosis, cell growth in-
hibition, topoisomerase inhibition, DNA damage) were
reported in vitro as well as in experimental animals (84–87).
However, genistein concentrations used in these studies were
much higher than the physiologically relevant doses achievable
by dietary or pharmacologic intake of soy foods or supple-
ments. In contrast, in vivo studies generally showed negative
genotoxicity results (88). In a study conducted in postmeno-
pausal women aged 46–68 y, the administration of a purified
unconjugated isoflavone mixture (genistein, daidzein, and
glycitein) showed minimal toxicity at doses as high as 16 mg
genistein/kg body weight (83).
The potential effects of genistein on fertility and fetus
development have been largely investigated. Some studies
showed that therapeutically relevant doses of genistein
have significant negative impacts on ovarian differentiation,
estrous cyclicity, and fertility in the rodent model (89).
However, data from human trials are lacking, and hence
studies on the effect of genistein on the human reproductive
function and/or fetal development need to be considered in
the future. Studies in animal models showed that the expo-
sure to genistein during uterine development caused several
adverse effects (89, 90). The conflicting results are probably
attributable to differences in the timing of exposure, doses,
and experimental endpoints. It is worth noting that human
fetuses can be exposed to isoflavones during the develop-
mental period in the uterus and during infancy via breast
milk (91). Serum genistein concentrations in soy formula–
fed infants are from 10- to >100-fold those of the general
population (92). These concentrations can increase blood
genistein concentrations to values compatible with substan-
tial biological estrogenic effects in children. Further epide-
miologic studies are required to determine the beneficial
(and detrimental) effects of genistein exposure, as well as es-
tablishing its safe therapeutic doses.
Genistein and Cancer
Several epidemiologic studies showed a relation between a
soy-rich diet and cancer prevention. These studies arose
from observations that in Asian countries, such as Japan
and China, where diets are high in soy products, the inci-
dence of breast and prostate cancers is lower than that in
the United States and Europe. In fact, several meta-analyses
suggest that the consumption of soy foods is associated
with a reduction in prostate cancer risk in men (93–95)
and is inversely associated with breast cancer risk among
Asian women. This association was not confirmed in West-
ern women (96–98). Moreover, a recent meta-analysis
found that soy isoflavone intake can lower the risk of breast
cancer in both pre- and postmenopausal women in Asian
countries (99). Furthermore, migration studies showed
an increase in prostate and breast cancer incidence in
Asians after emigration to the United States (100), suggest-
ing that environmental factors and changes in lifestyle, par-
ticularly in dietary practices, affect the etiology of these
types of cancers.
These epidemiologic studies provide the rationale to inves-
tigate at a molecular level how the predominant isoflavone
present in soy (i.e., genistein) is able to prevent cancer with
the use of appropriate cellular and animal models. Because
of its pleiotropic activity, genistein shows promising results
as an anticancer agent in preclinical studies, opening the pos-
sibility to verify its clinical efficacy in clinical trials.
During the biogenesis process, genistein is present essen-
tially in its glycosylated form, mostly with a glucose mole-
cule. Although genistein is ingested as genestein glycoside,
after ingestion a deglycosylation process occurs in the small
intestine and the free genistein aglycone is absorbed by the
body, resulting in different pharmacologic effects including
anticancer effects (101). Apart from genistein, the synthetic
derivatives, such as genistein glycosides, are also reported to
possess anticancer activity when assessed in vitro. The anti-
cancer potency of genistein glycosides varies depending on
the sugar groups attached. For example, the addition of acet-
ylated sugar hydroxyls to genistein resulted in more selectiv-
ity toward tumor cells (102). It is worthwhile to note that the
anticancer potency of genistein and its derivatives differs in
different types of cancer, depending on their selectivity to-
ward the target molecules (Figure 3).
Liver cancer
Epidemiologic data. A recent nested case-control study of a
population-based prospective cohort in Japan, which investi-
gated the preventive role of estrogens in primary liver cancer
development, verified whether isoflavones were associated
with the risk of liver cancer (103). The authors selected pa-
tients with either hepatitis B or C virus infection at baseline
and measured plasma concentrations of isoflavones (genis-
tein, daidzein, glycitein, and equol). The study indicated no
FIGURE 3 Molecular mechanisms mediating the anticancer
effect of genistein: downregulation/suppression, inhibition,
enhancement. AP-1, activator protein 1; Bax, Bcl-2–associated X
protein; Bcl-2, B cell lymphoma 2; EGFR, epidermal growth factor
receptor; IkB, inhibitor of NF-kB; KIF20A, kinesin-like protein 20A;
PI3K/Akt, phosphoinositide 3 kinase/Akt; TRAIL, tumor necrosis
factor–related apoptosis-inducing ligand; TRAIL R, TRAIL death
receptors; Wnt/b-catenin, Wingless and integration 1 b-catenin.
Anticancer effects of genistein 411
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association between isoflavones and the occurrence of pri-
mary liver cancer risk in middle-aged Japanese women and
men with hepatitis virus infection.
In vitro studies. In vitro studies support the efficacy of gen-
istein as a chemopreventive and/or chemotherapeutic agent
against liver cancer. It induces apoptosis in the following
hepatocellular carcinoma cells (HCCs): Bel 7402 (104),
HuH-7 (105), Hep3B (106), and HepG2 (107). Genistein
may affect HCC progression as a result of its activity on apo-
ptosis and cell cycle regulation (104, 108), acting as a promising
inhibitor of the metastatic process in HCCs. In fact, genistein
has been shown to inhibit the migration of 3 cell lines
(HepG2, SMMC-7721, and Bel-7402 cells) (109). Moreover,
it promotes anti-invasive and antimetastatic effects against
12-O-tetradecanoylphorbol-13-acetate-mediated metastasis
via downregulation of matrix metalloproteinase 9 (MMP-9)
and epidermal growth factor receptor (EGFR) and subsequent
suppression of NF-kB and activator protein 1 (AP-1) transcrip-
tion factors through inhibition of MAPK, inhibitor of NF-kB
(IkB), and phosphoinositide 3 kinase/Akt (PI3K/Akt) signaling
pathways (110).
Several studies also reported the synergistic effect of genis-
tein when administered together with other anticancer drugs.
For example, TNF-related apoptosis-inducing ligand
(TRAIL) is a member of the TNF superfamily, and it has
been shown that many human cancer cell lines are refractory
to TRAIL-induced cell death. The treatment with nontoxic
concentrations of genistein, sufficient to inhibit MAPK acti-
vation, sensitizes human hepatocellular carcinoma Hep3B
cells to TRAIL-mediated apoptosis (111, 112). Genistein
also potentiates the cytotoxic effect of arsenic trioxide
(ATO) against human hepatocellular carcinoma. ATO pos-
sesses limited therapeutic benefit in the treatment of solid tu-
mors; genistein, by inhibiting Akt and NF-kB, potentiates the
proliferation-inhibiting and apoptosis-inducing effect of ATO
on human HCC cell lines in vitro (15–20 mMgenistein)and
dramatically increases its suppressive effect on both tumor
growth and angiogenesis in nude mice (50 mg genistein/kg
body weight) (113).
In vivo studies. Tumor growth in male BALB/C nu/nu mice
injected with Bel 7402 cells was significantly retarded when
treated with 50 mg genistein/kg body weight in comparison
with control mice; genistein also significantly inhibited the
invasion of Bel 7402 cells into the renal parenchyma of nude
mice with a xenograft transplant by altering cell cycle, apo-
ptosis, and angiogenesis (104). In a different animal model
of liver cancer, HCC-bearing rats (male Wistar rats induced
with N-nitrosoiethylmine by single intraperitoneal injec-
tion and promoted with phenobarbital), it was reported
that genistein efficiently inhibited cell proliferation and in-
duced apoptosis. In fact, the administration of a 15-mg gen-
istein/kg body weight suspension in olive oil stimulate d
caspase-3 activity and remarkably decreased proliferat-
ing cell nuclear antigen (PCNA) in these HCC-bearing rats
(114).
Gastric cancer
Epidemiologic data. The beneficial role of soybean pro-
ducts against gastric cancer remains debatable from an inter-
ventional point of view. A nested case-control study within
the Korean Multicenter Cancer Cohort suggested that high
serum concentrations of isoflavones were associated with a
decreased risk of gastric cancer (115); on the contrary, a par-
allel nested case-control study within the Japan Public
Health Center–Based Prospective Study indicated a null as-
sociation between isoflavone intake and gastric cancer risk
among Japanese men and women (116).
In vitro studies. In preclinical models, genistein was able to
induce apoptosis in primary gastric cancer cells (20 mMfor
24–72 h) by downregulating the expression of the antiapop-
totic protein B cell lymphoma 2 (Bcl-2) and upregulating
the expression of proapoptotic Bcl-2–associated X protein
(Bax) (117). A similar modification of the Bcl-2:Bax ratio
was considered responsible for the ability of genistein (0.5,
1, and 1.5 mg/kg) to induce apoptosis in SG7901 cells trans-
planted into subcutaneous tissue of nude mice (118). In the
human gastric cancer cell line BGC-823, genistein treatment
inhibited cell proliferation and induced apoptosis in a dose-
and time-dependent manner. In this model, the molecule
exerted a significant inhibitory effect on activation of the
transcription factor NF-kB, causing a reduction in cycloox-
ygenase 2 (COX-2) protein concentrations (119).
The ability of genistein to induce G2/M cell cycle arrest
was tested in SGC-7901 and BGC-823 cells. Here, genistein
(20–80 mM) inhibited Akt activation by upregulation of
phosphatase and tensin homolog (PTEN). This event re-
sulted in the decreased phosphorylation of Wee1 on Ser642
and increased phospho-activation of cell division cycle protein
2 homolog/cyclin-dependent kinase 1 (Cdc2/Cdk1) on Thr15,
leading to G2/M arrest (120).
A stable isotope labeling by/with amino acids in cell cul-
ture quantitative proteomics approach was used to identify
the genistein-regulated factors and to investigate the anti-
cancer mechanisms of the molecule. In SGC-7901 cells
treated with 40 mM genistein for 48 h, the expression of 86
proteins involved in the regulation of G2/M transition, cel-
lular growth, and proliferation resulted modulated by genis-
tein, with 49 being upregulated and 37 being downregulated.
In particular, 4 kinesins [kinesin-like protein (KIF) 11,
KIF20A, KIF22, and KIF23] and a KIF, centromere protein
F (CENPF), were found to be significantly downregulated
by genistein, with KIF20A playing an important role in
genistein-induced mitotic arrest (121).
Increasing evidence suggests that gastric cancer stem cells
(GCSCs), a subpopulation of tumor cells capable of self-re-
newal and resistant to chemotherapeutic drugs, are responsi-
ble for the relapse of the disease. Gastric cancer cells treated
with a low dose of genistein (15 mM) inhibited the GCSC-
like properties such as self-renewal ability, drug resistance,
and tumorigenicity, which were associated with the inhibition
of ATP-binding cassette subfamily G member 2 (ABCG2) ex-
pression and extracellular signal-regulated kinase (ERK) 1/2
412 Spagnuolo et al.
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activity (122). In GCSCs, genistein can also inhibit glioma-
associated oncogene family zinc finger 1 (Gli1), an activator of
Hedgehog signaling, involved not only in oncogenesis but
also in cancer stemness and overexpression of CD44, a typ-
ical GCSC surface marker. In more detail, it was shown that
the levels of Gli1 and CD44 expression are downregulated
by genistein in GCSCs sorted from MKN45, a human gas-
tric cancer cell line, according to CD44 expression. In
addition, the high cell migration capacity of CD44
+
cells
was blocked by genistein, suggesting that it can be an effec-
tive agent for gastric cancer therapy by targeting cancer
stem cell-like features (123).
In vivo studies. Tatsuta et al. (124) used, as an in vivo model
of gastric cancer, Wistar rats induced with N-methyl-N’-ni-
tro-N-nitroso guanidine and treated with sodium chloride
to enhance induction of gastric carcinogenesis. They showed
that, after 25 wk of the carcinogen treatment, daily injections
of genistein (30 mg/kg body weight) decreased the labeling
index and vessel counts of the antral mucosa and signifi-
cantly reduced the incidence of gastric cancers, inducing in-
creased apoptosis and decreased angiogenesis of antral
mucosa and gastric cancers.
Moreover, to investigate the development of cancer ca-
chexia and malignant progression of human stomach cancer,
MKN45cl85 and highly metastatic 85As2mLuc (2 cachexia-
inducing sublines) cells were isolated from the human
stomach cancer cell line MKN-45. These 2 cell lines
induce cachexia at high frequency in mice. It has been
shown that a long duration of treatment with isoflavones in-
duced tumor cytostasis, attenuated cachexia, and prolonged sur-
vival in rats (the antitumor effect was graded as AglyMax >
daidzein > genistein) (125).
Lung cancer
Epidemiologic data. Estrogens have been shown to have
mitogenic effects in lung cells and interact with growth factor
pathways in tumorigenesis; epidemiologic studies have pro-
duced conflicting results regarding the association between
lung cancer risk and isoflavone intake (126–128). However,
prospective studies carried out in Asia indicated an inverse as-
sociation in never smokers (129). A nested case-control study
within a large-scale, population-based prospective study in Jap-
anese women with different isoflavone intakes and a high prev-
alence of never smokers revealed an inverse association between
plasma isoflavone concentration and lung cancer risk (130).
In vitro studies. Several in vitro and in vivo studies showed
a protective effect of genistein on lung carcinogenesis when
this compound was either used alone or in association with
other compounds (131–134). Genistein showed anticancer
effects on the small cell lung cancer (SCLC) cell line H446;
the molecule induced cell cycle arrest and apoptosis, dereg-
ulating Forkhead box protein M1 (FoxM1) and its target
genes [e.g., cell division cycle 25B (Cdc25B), cyclin B1,
and survivin] (135). Several articles have also shown a syn-
ergistic effect; for example, in A549 lung cancer cells
genistein (5–10 mM) enhanced apoptosis induced by tri-
chostatin A (TSA) and increased the expression of the death
receptor TNF receptor 1 (TNFR-1), which mediates extrin-
sic apoptosis pathways (134, 136). Patients with non-SCLC
treated with tyrosine kinase inhibitors developed an ac-
quired resistance to this therapy. In a non-SCLC cell line
carrying the T790M mutation in EGFR, genistein associated
with gefitinib, an EGFR tyrosine kinase inhibitor, showed a
synergistic anticancer effect due to apoptosis induction and
inhibition of the key regulators of growth signaling path-
ways, such as Akt (131). The synergistic effect was also con-
firmed in in vivo experiments.
In vivo studies. Gu et al. (104, 108) investigated, in vitro and
in vivo, the inhibitory effects of genistein on the invasive po-
tential of HCCs (Bel 7402 and MHCC97-H). The authors
first proved the ability of genistein (10 mg/mL) to significantly
inhibit the growth of HCCs in vitro; subsequently, Bel 7402
or MHCC97-H cells were injected in BALB/C nu/nu mice
before the administration of 50 mg genistein/kg body weight.
The tumor growth in genistein-treated nude mice was signif-
icantly lower than that in control mice. The molecule signif-
icantly inhibited the invasion of Bel 7402 cells into the renal
parenchyma of nude mice with xenograft transplant. More-
over, in the in situ xenograft transplantation of MHCC97-H
cells, the number of pulmonary micrometastatic foci after
genistein treatment were significantly lower than in the con-
trol group.
Because of the low bioavailability of genistein in vivo, there
is a growing interest in its derivative, 7-difluoromethyl-5,49-
dimethoxygenistein (dFMGEN), which possesses a better in
vivo bioavailability. An in vitro study showed the efficacy of
dFMGEN in reducing the viability of lung carcinoma A549
cells through induction of G1 phase arrest (137). Moreover,
dFMGEN suppressed tumor growth in vivo and was well
tolerated, confirming its therapeutic potential in the treat-
ment of human lung cancer (137).
Colorectal cancer
Epidemiologic data. The consumption of soy has been
found to reduce colon cancer risk in human and animal
studies (138, 139). Epidemiologic evidence indicates that
phytoestrogens may protect against the development of co-
lorectal cancer (140, 141).
For example, a case-control study evaluated the associa-
tion between dietary phytoestrogen intake (isoflavones, li-
gnans, and total phytoestrogens) and colorectal cancer risk
among healthy subjects and those belonging to the popula-
tion-based Ontario Familial Colorectal Cancer Registry. It
was reported that dietary lignin and isoflavone intake was
associated with a significant reduction in colorectal cancer
risk; moreover, it was observed that, with respect to phytoes-
trogen intake, polymorphic genes encoding enzymes in-
volved in the metabolism of phytoestrogens [CYP, catechol
O-methyl transferase, glutathione S-transferases (GSTs),
and UDP-glucuronosyltransferase (UGT)] were not subject
to modifications (142).
Anticancer effects of genistein 413
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In vitro studies. Numerous in vitro studies have shown an-
ticancer properties of genistein against colorectal cancer, and
the mechanisms whereby it exerts anticancer effects have been
widely investigated. Genistein efficiently suppresses colon
cancer cell growth by attenuating the activity of the PI3K/
Akt pathway (143, 144), which has a critical role in the regu-
lation of colon cancer progression. In colon cancer cells, gen-
istein also affects the expression of ERs and tumor suppressor
genes (145, 146). In addition, it can block uncontrolled cell
growth in a DLD-1 cell line by inhibiting the Wingless and in-
tegration 1 (Wnt) signaling pathway (147). In particular, gen-
istein enhanced gene expression of the Wnt signaling pathway
antagonist, Dickkopf-related protein 1 (DKK1), through the
induction of histone acetylation at the promoter region in
an SW480 human colon cancer cell line (148).
In vivo studies. An in vivo study that used azoxymetha ne as
a chemical inducer of colon cancer in male Sprague-
Dawley rats showed that rats fed 140 mg genistein/kg body
weight from gestation to 13 wk of age showed a downregu-
lation of Wingless and integration 1 b-catenin (Wnt/b-cat-
enin) signaling and a reduction in total aberrant crypts,
confirming the role of this isoflavone in preventing the de-
velopment of early colon neoplasia (149).
Clinical trials. A phase I/II pilot study of genistein use in the
treatment of metastatic colorectal cancer is currently recruit-
ing participants; because of the promising results of the in
vitro and in vivo studies it is expected to have interesting
findings.
Breast cancer
Epidemiologic data. Several case-control studies (150, 151)
showed an inverse relation between soy intake and breast
cancer risk; and a prospective cohort study (152) found
that frequent miso soup and isoflavone consumption was as-
sociated with a reduced risk of breast cancer in Japan.
Clearly, the chemopreventive effects of soybeans and soy-
containing foods are related to their isoflavone content.
In vitro studies. Genistein induced apoptosis in several
breast cancer cell lines and promoted synergistic inhibitory
effects when combined with anticancer drugs. For example,
genistein was shown to induce apoptosis in the low-invasive
(ER-positive) MCF-7 and in the high-invasive (ER-negative)
MDA-MB-231 breast cancer cell lines (10–100 mM) (153,
154). Synergistic proapoptotic effects were also described
when genistein was used in combination with adriamycin
and docetaxel in MDA-MB-231 cells (155) and with tamox-
ifen on BT-474 breast cancer cells (156). The main molecu-
lar targets of the molecule in breast cancer cells appear to be
NF-kB (157) and Akt pathways (158). Moreover, genistein
induces in breast and prostate cancer cells the expression
of breast cancer growth suppressor protein (BRCA) 1 and
BRCA2 tumor suppressor genes and the overexpression of
many genes involved in the BRCA1 and BRCA2 pathways
(159). However, it is important to underline the paradoxical
effect of genistein, which stimulates proliferation and
estrogen-sensitive gene expression of the ER-positive breast
cancer cell lines at concentrations of 1–10 mM (160). At
these low concentrations, genistein abrogates tamoxifen-
associated mammary tumor prevention, but its effect is null
on ER-negative and tamoxifen-resistant breast cancer cells
(161).
In vivo studies. The in vitro observations have been con-
firmed in in vivo studies, suggesting that genistein exposure
early in life may reduce the risk of breast cancer (162). On
the contrary, in a preclinical mouse model that resulted in
17b-estradiol blood concentrations similar to those found
in postmenopausal women, dietary genistein in the presence
of low concentrations of circulating E2 acted in an additive
manner to stimulate estrogen-dependent tumor growth
in vivo (163). Results from this study suggest that the con-
sumption of products containing genistein may not be safe
for postmenopausal women with estrogen-dependent breast
cancer.
Clinical trials. These controversial results have been con-
firmed in human clinical studies, in which, in some cases, pu-
rified genistein did not exert any adverse estrogenic effects on
breast tissue when consumed at a dose of 54 mg/d (164, 165),
whereas others found proestrogenic effects of dietary soy sup-
plementation on breast tissue (166–168). Thus, considering
the agonist activity of genistein against ER-a,itsusein
women with established ER-positive breast cancers must be
carefully considered. In this regard, 2 clinical trials based on
the use of genistein in breast cancer, a phase II study entitled
“Gemcitabine Hydrochloride and Genistein in Treating
Women with Stage IV Breast Cancer,”and a phase I study en-
titled “Genistein in Preventing Breast or Endometrial Cancer
in Healthy Postmenopausal Women,”have been completed,
although the results are not yet published (169).
Genistein Metabolites and Cancer
Although genistein is reported to be metabolized mainly
through oxidation, sulfation, glucuronidation, hydroxyla-
tion, or methylation (170), the influence of genistein metab-
olites on its anticancer property is not understood clearly.
Metabolites such as 5,7,39,49-tetrahydroxyisoflavone
(THIF) and 2 glutathinyl conjugates of THIF were identified
in T47D tumorigenic breast epithelial cells that were treated
with genistein. Because THIF has been shown to inhibit
angiogenesis and endothelial cell proliferation (171), it is
worthwhile to note that the formation of THIF during gen-
istein treatment may play a major role in cell cycle arrest, in-
hibition of cellular proliferation, and activation of signaling
pathways such as p38 MAPK, which was observed in T47D
cells. Furthermore, oxidation of THIF to o-quinone along
with formation of hydrogen peroxides and reactive oxygen
species induces DNA strand breakage. This leads to the ac-
tivation of the ataxia telangiectasia and Rad3-related kinase
(ATR) signaling pathway, which activates the kinases in-
volved in DNA damage check-point control (172).
414 Spagnuolo et al.
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Conclusions and Recommendations
We reviewed the available evidence on the promising role of
genistein against cancer. Several experimental and clinical in-
vestigations suggest a therapeutic role of genistein on different
types of cancer. The emergence of negative phenomena in can-
cer treatment is well known, such as drug resistance, high risk
of relapse, and the unavailability or poor outcome of therapies,
such as surgery, chemotherapy, phototherapy, and radiother-
apy. Therefore, attention has been paid in recent years to nat-
ural remedies possessing the capacity to improve the efficacy of
chemotherapeutic treatment and to lower adverse effects. Gen-
istein can be included among these compounds because the
molecule shows synergistic behavior when associated with
well-known anticancer drugs, such as adriamycin, docetaxel,
and tamoxifen, suggesting a potential role in combination ther-
apy. However, genistein, as well as other bioactive phytochem-
icals, benefits and, at the same time, suffers from 2 apparently
opposite features: high pleiotropy and low bioavailability. The
former refers to the ability of a given compound to act at sev-
eral levels in the cells, triggering at the same time several bio-
chemical pathways involved in the occurrence and
development of cancer (i.e., cell cycle arrest, apoptosis, cell
death). The net result is a synergistic effect that may enhance
the efficacy of a specific drug, even if present in the cells at rel-
atively low concentrations. In this regard, in the previous par-
agraphs, we reviewed several molecular targets of genistein,
such as ER, tyrosine kinases, and pro- and antiapoptotic fac-
tors. Bioavailability was also discussed above, which brings
us to the concept that “what we adsorb”from food is even
more important than what we eat: the plasma concentration
of genistein present in the diet (similar to other bioactive com-
pounds) is significantly lower than the concentrations needed
in experimental models (cell lines and animal studies) to trig-
ger an anticancer response. Therefore, it is reasonable to pre-
dict a significant clinical outcome of genistein when applied
at pharmacologic doses (hundreds of micromolars) and weak
or null effects when the same molecule is administered at che-
mopreventive doses (<1 mM). The reality is probably more
complex than we can expect. In fact, when genistein is ad-
sorbed at low concentrations together with other bioactive
compounds present in the diet, we can postulate pleiotropic
anticancer effects that result from synergistic mechanisms at-
tributable to the plethora of individual compounds (or their
metabolites) deriving from the diet. Alternatively, we can also
hypothesize that genistein possesses, at low doses, effects differ-
ent than those measured at high doses, depending on the cel-
lular background and the molecular target investigated. In this
respect, the ability of genistein to inhibit cell growth in both
hormone-dependent and -independent cancer cells is dose de-
pendent (173). In fact, when genistein concentration increases
from a low nanomolar concentration to hundreds of nanomo-
lars, preferential activation of ER-bis lost and genistein acti-
vates both ERs (aand b); therefore, at least in different
breast cancers, the ratios of the ER subtypes and the concentra-
tions of genistein strongly influence the final effect on hor-
mone-regulated gene expression and cell fate (174).
Future studies are necessary to clarify the potential ther-
apeutic and chemopreventive use of genistein. In particular,
it will be important to investigate the following:
·The pharmacodynamics and pharmacokinetics of genistein
and related compounds in experimental and clinical studies
·Possible strategies to increase the bioavailability of genistein
·The ideal therapeutic dose for treatment of specific types of cancer
·Other molecular mechanisms explaining the anticancer effects
of genistein (e.g., microRNAs)
·Possible interactions between genistein and well-known anti
cancer drugs, by both experimental and clinical studies
Despite the promising results reported in literature, there is
still a long way to go.
Acknowledgments
All authors read and approved the final manuscript.
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