Phytoestrogens as natural prodrugs in cancerprevention: a novel concept
Randolph R. J. Arroo Æ Vasilis Androutsopoulos ÆAsma Patel Æ Somchaiya Surichan Æ Nicola Wilsher ÆGerry A. Potter
Received: 11 October 2007 / Accepted: 28 February 2008 / Published online: 26 March 2008
� Springer Science+Business Media B.V. 2008
Abstract It has been generally accepted that regular
consumption of fresh fruits and vegetables is linked
with a relatively low incidence of cancers (e.g. breast,
cervix, and colon). A number of plant-derived com-
pounds have been identified that are considered to
play a role in cancer prevention. However, at present
there is no satisfactory explanation for the cancer
preventative properties of the above-mentioned com-
pound groups. The current review is an effort to
develop a consistent and unambiguous model that
explains how some plant-derived compounds can
prevent tumour development. The model is based on
recent discoveries in the fields of genomics and drug-
metabolism; notably, the discovery that CYP1 genes
are highly expressed in developing tumour cells but not
in the surrounding tissue, and that a variety of plant-
derived compounds are substrates for the CYP1
enzymes. Our hypothesis is that some dietary com-
pounds act as prodrugs, i.e. compounds with little or no
biological effect as such, but become pharmaceutically
effective when activated. More specifically, we
state that the abovementioned prodrugs are only
activated in CYP1-expressing cells—i.e. cells in the
early stages of tumour development—to be converted
into compounds which inhibit cell growth. Thus, the
prodrugs selectively kill precancerous cells early in
tumour development. The review focuses on the
identification of naturally-occurring prodrugs that are
activated by the tumour-specific CYP1 enzymes and
aims to assess their role in cancer prevention.
Keywords CYP1 � Drug metabolism �Flavones � Natural products � Prevention
Anticancer drug discovery
In the 1950s, the National Cancer Institute (NCI)
started a large-scale empirical anticancer drug screen-
ing program that continued in more or less the same
form until the mid-1970s. In this program, compounds
were tested against a panel of mouse tumour models,
mainly L1210 and P388 leukaemias (Suggit and
Bibby 2005). This screening method led to the
identification of a large number of compound classes
that have formed the basis for most of the anticancer
drugs that are currently in clinical use (Table 1). The
screening program was highly compound-oriented and
resulted in a golden era for phytochemistry, since
natural products formed a major source of lead
compounds. Indeed, a large proportion of the now
conventional anticancer drugs are derived from nat-
ural sources, e.g. Vincristine and Vinblastine from
Catharanthus roseus, Paclitaxel and Docetaxel from
Taxus species, Doxorubicin (Adriamycin) and
R. R. J. Arroo (&) � V. Androutsopoulos �A. Patel � S. Surichan � N. Wilsher � G. A. Potter
Leicester School of Pharmacy, De Montfort University,
The Gateway, Leicester LE1 9BH, UK
e-mail: [email protected]
123
Phytochem Rev (2008) 7:431–443
DOI 10.1007/s11101-008-9093-5
Mitomycin from Streptomycin species, the podophyl-
lotoxin derivatives Etoposide and Teniposide from
Podophyllum species, Camptothecin and its semisyn-
thetic derivative Irinotecan from Camptotheca
acuminata. A recent estimate stated that approximately
60% of drugs in clinical trials for the multiplicity of
cancers are either natural products, compounds derived
from natural products, or drugs containing pharmaco-
phores derived from natural products (Cragg and
Newman 2000).
The use of the mouse tumour models has led to
concerns that the screening may have resulted in
preferential selection of drugs that were only active
against rapidly growing tumours. Indeed, most exist-
ing anticancer agents target DNA replication of
dividing cells, thus inhibiting tumour cell growth
but also normal cell growth. This causes many
undesired side effects such as nausea and vomiting,
alopecia, mucositis, myelosuppression and reproduc-
tive sterility. These side effects of chemotherapy can
have a devastating impact on a patient’s quality of
life. Particularly, the treatment of elderly patients
(older than 70 years) presents characteristics that
make the choice of the correct treatment more
difficult; for this reason, these patients are often
undertreated (Pasetto et al. 2007).
Smart anticancer drugs
The main aim of conventional cancer treatment was
systemic, non-specific, high-dose chemotherapy
(Abou-Jawde et al. 2003). The success of these
treatments was outweighed by the systemic toxicity.
However, as more was discovered about the mech-
anisms underlying the initiation and progression of
human cancers, anticancer drug discovery moved
away from the development of classic cytotoxic
agents to the rational design of small molecule
anticancer therapeutics. This development has
prompted a transition from empirical compound-
orientated preclinical screening to target-orientated
drug screening (Suggit and Bibby 2005). The so-
called smart drugs, which have been developed as a
result of the new screening methods, are small
molecules targeted at specific growth signalling
Table 1 Overview of traditional cytotoxic agents, their mode of action and side effects
Class of drug Examples Mode of action Side effects
Alkylating agents Cyclophosphamide
Chorambucil
Melphalan
Mitomycin
Formation of covalent bonds with DNA,
thus impeding DNA replication
Myelosuppression
GI disturbances, hair loss,
Reduced fertility, increased
risk of secondary malignancies
Antimitotic agents Vincristine
Vinblastine
Docetaxel
Paclitaxel
Interference with microtubule assembly
of cells, thereby blocking cell division.
As for alkylating agents;
Neurotoxicity
Antimetabolites Methotrexate
5-Fluoroacil
6-Mercaptopurine
Inhibition of metabolic pathways
involved in DNA synthesis.
As for alkylating agents:
Nephrotoxicity
DNA intercalators Doxorubicin
Mitoxantrone
Prevent DNA replication As for alkylating agents;
Doxorubicin can cause cardiotoxicity
Topoisomerase inhibitors Etoposide
Teniposide
Camptothecin
Irinotecan
Prevent unwinding of DNA strands,
thereby blocking DNA replication.
As for alkylating agents
Heavy metal derivatives Cisplatin
Carboplatin
As metallating agents. As for alkylating agents;
Nephrotoxicity;
Neurotoxicity and potention loss
of hearing
432 Phytochem Rev (2008) 7:431–443
123
pathways. These new drugs are expected to dominate
clinical trials in the years to come either as single-
drug modality or as combination treatment (van der
Poel 2004). The new generation of chemotherapeu-
tics act on specific molecular targets that are
responsible for malignant phenotypes, and thus
provide potentially tumour specific therapy (Table 2).
Most of the compounds are synthetic molecules that
are made to fit particular well-defined targets; very
few of this second generation of anticancer drugs are
based on natural products. Theoretically, the use of
these putatively tumour specific chemotherapeutic
agents, whether alone or in combination, should lead
to an improved clinical response. However, the
expression of the target proteins is not limited to
tumour cells alone; the proteins are over-expressed in
tumour cells, but also expressed at a lower frequency
in normal cells. Thus, problems remained with
unacceptable damage to normal cells and organs, a
narrow therapeutic index, or a relatively poor selec-
tivity for neoplastic cells. A potential strategy to
overcome the limitations of chemotherapeutic agents
is the use of prodrugs, i.e. compounds that need to be
transformed before exhibiting their pharmacological
action (Rooseboom et al. 2004). Prodrugs are often
divided into two groups:
1. prodrugs designed to increase the bioavailability
of antitumour agents, e.g. when the activated
drug has a low chemical stability or is rapidly
broken down in vivo, more stable prodrugs can
be administered.
2. prodrugs designed to locally deliver antitumour
agents (targeting)
CYP1-activated anticancer prodrugs
A recent discovery in the field of oncology was that
the cytochrome P450 enzyme CYP1B1 is expressed
at a high frequency in a wide range of human cancers,
including tumours of the breast, colon, lung, oesoph-
agus, skin, lymph node, brain, and testis (Table 3, 4).
The protein could not be detected in the surrounding
normal tissues (Murray et al. 1997, 2001). Prelimin-
ary data by Stanley et al. (2001a, b) have indicated
that the CYP1B1 protein is also over expressed in
premalignant tissue, which suggests that CYP1B1 has
a role in early tumour development. However, other
authors (Gibson et al. 2003) call for more caution
since they could detect CYP1B1 protein in patient
samples of human colonic tissue specimens, which
were excised C30 cm from the tumour, and did not
show any of the characteristics of premalignant
tissue. A related cytochrome P450, CYP1A1, is
abundant in tumour tissue, but various xenobiotics
(notably aryl hydrocarbons) can induce its expression
in healthy tissue, notably in the epithelial lining of the
lungs (Zhang et al. 2006).
CYP1 enzymes are known to play a role in the
metabolism of estrogens. Notably, CYP1B1 converts
the oestrogen estradiol into 4-hydroxyestradiol. The
Table 2 Overview of targeted cancer treatments, their mode of action and side effects
Class of drug Examples Mode of action Side effects
Hormonal therapies Tamoxifen
Letrozole
Arimidex
Bicalutamide
Prevent biosynthesis of oestrogen,
or block the action of hormones
tumour development.
Endometrial cancer, blood clots, hot
flushes, fatigue, GI disturbances
Signalling inhibitors Imatinib
Gefitinib
Rituximab
Block activation of secondary
messenger molecules; inhibit tyrosine
kinase activity
Fluid retention, skin rashes,
diarrhoea, GI disturbances
Monoclonal antibodies Trastuzumab
Gemtuzumab
Ozogamicin
Antibodies bind to proteins that are
overexpressed in tumours, e.g. CD20 antigen,
or HER2 receptor.
Allergic response
Differentiating agents Tretinoin Induces differentiation and death
of leukaemia cells
Fever, severe respiratory
distress, weight gain
Phytochem Rev (2008) 7:431–443 433
123
latter compound plays a key role in the development
of breast and endometrial tumours. Of particular
interest was the finding that human CYP1B1 activity
is regulated by estradiol via the oestrogen receptor
(For a recent review see: Tsuchiya et al. 2005).
Taken together the findings made CYP1B1 an ideal
molecular target for the development of new anti-
cancer drugs, where the estradiol core could be taken
as a lead structure for drug design. Potter et al.
designed DMU-212 (3,4,5,40-tetramethoxy-stilbene)
which showed promise in in vitro human tumour
assays, and in mouse models (Sale et al. 2004). The
authors noted the close structural similarity of DMU-
212 with resveratrol, a naturally occurring stilbene
found in red wine, which had been shown to have
cancer preventative properties (Jang et al. 1997; Jang
and Pezzuto 1999). Resveratrol is classified as a
phytoestrogen because of its similarity to the endog-
enous oestrogen estradiol. It appeared that resveratrol
is a substrate for the CYP1 family of enzymes
(Fig. 1), and that the enzymes catalyse the conversion
of resveratrol into piceatannol and a second
compound, putatively identified as 3,4,5,40-tetrahydr-
oxystilbene (Potter et al. 2002; Piver et al. 2004).
Both CYP-1 conversion products of resveratrol are
known to inhibit cell proliferation (Ferrigni et al.
1984; Lu et al. 2001).
The role of diet in prevention of cancer
The notion that a diet rich in fresh fruit and
vegetables protects from the risk of most common
epithelial cancers—including those of the digestive
tract, and also several nondigestive neoplasms—is
well-established (e.g. La Vecchia et al. 2001).
When dietary habits of large cohorts were com-
pared and correlated with the occurrence of cancer,
a number of molecules were identified as the most
Table 3 Expression of
CYP1B1 in different types
of tumours and normal
tissue (after Murray et al.
1997)
Tissue Normal (no positive/no tested) Tumour (no positive/no. tested)
Bladder 0/8 8/8
Brain 0/12 11/12
Breast 0/10 12/12
Colon 0/10 11/12
Connective tissue 0/9 8/9
Oesophagus 0/8 8/8
Kidney 0/11 11/11
Liver 0/8 not tested
Lung 0/8 7/8
Lymph node 0/5 9/9
Ovary 0/5 7/7
Skin 0/6 6/6
Small intestine 0/5 not tested
Stomach 0/10 9/10
Testis 0/8 8/8
Uterus 0/7 7/7
Total 0/130 122/127
Table 4 Substitution patterns of selected dietary flavonols
O
O
R1
OH
R2HO
OH
OH
3'
4'
5'
A
B
C
Flavonols R1 R2
Kaempferol H H
Quercetin OH H
Myricetin OH OH
Isorhamnetin OMe H
434 Phytochem Rev (2008) 7:431–443
123
likely chemopreventive agents. At present, there is a
general consensus that plant polyphenols, flavonoids,
catechins, and lignans are the key constituents in
cancer prevention (Kinghorn et al. 2003, 2004). The
latter notion is wide-spread, also in the popular press,
and a number of preparations containing the com-
pounds mentioned above are available commercially
as health-enhancing supplements. Advertisements for
the commercially available preparations often refer to
scientific papers to underpin their health claims.
Roughly, two models that may explain the cancer
preventive properties of are cited time and time
again: the antioxidant model and the phytoestrogen
model. However, neither model can fully explain why
certain naturally occurring dietary compounds can
prevent the occurrence of cancer.
Fig. 1 CYP1 catalysed
conversions of estradiol and
resveratrol
Phytochem Rev (2008) 7:431–443 435
123
Antioxidants and cancer prevention
The antioxidant model was proposed in an influential
review by Ames (1983). To date, the paper has been
cited over 2,000 times. In short, reactive oxygen
species, formed as a by-product of oxidative catab-
olism—e.g. singlet oxygen and various radicals—are
possibly involved in DNA damage and tumour
promotion. Antioxidants would prevent the accumu-
lation of reactive oxygen species, and consequently
any cell damage caused by these radicals. Ames lists
vitamin E (tocopherol), b-carotene, and vitamin C
(ascorbic acid) as dietary natural products with
antioxidant activity, but he is reticent about the role
of plant phenols. Nevertheless, plant phenols with
antioxidant activity, e.g. stilbenes, chalcones, and
flavonoids, have been a major focus of attention.
Plant polyphenols have been shown to be highly
effective scavengers of most types of oxidizing
molecules, and the antioxidant properties of these
molecules have often been cited as an explanation for
their cancer preventive properties. Over 50 reviews
have been written on this topic over the past five
years (for a recent example, see Valko et al. 2006).
However, there is a paradox here; supplements of
ascorbate, vitamin E, or b-carotene—known antiox-
idants—do not decrease DNA damage in most studies
(Halliwell 2000, 2007; Halliwell et al. 2005). In
addition, in animal models antimutagens and anti-
carcinogens have been given in unrealistically high
doses, which do not usually reflect the human
exposure situation (Knasmuller et al. 2002).
Intriguingly, Ames’ review also highlights the role
of dietary carcinogens in the onset of cancer. In
particular benzo[a]pyrene, a compound that occurs in
charred meat and in cigarette smoke, has been well-
investigated since. It is now widely accepted that
benzo[a]pyrene is actually a pro-carcinogen which is
activated by the human cytochrome P450 enzyme
CYP1A1. It is the activated compound, benzo[a]pyr-
ene diol epoxide, that causes cell damage (see review
by Alexandrov et al. 2002).
Phytoestrogens and cancer prevention
Oestrogens are hormones responsible for the female
sex characteristics; they are not restricted to females
since small amounts are produced in the male testes.
The principal and most potent example is estradiol.
The aromatic ring (Fig. 2) makes the oestrogen
molecule almost planar and is essential for activity.
Estradiol has been linked to breast cancer etiology,
but the molecular mechanisms underlying the devel-
opment of breast cancer are not completely
understood (Dietel et al. 2005; Russo and Russo
2004).
A variety of plant compounds have been shown to
interact with human oestrogen receptors. Unsurpris-
ingly maybe, the molecules share some structural
Fig. 2 Structural
similarities between the
human hormone estradiol
(1), a steroid, and
phytoestrogens belonging to
the chemical groups of
flavones (2), flavonols (3),
and stilbenes (4). In an
attempt to highlight the
similarities, some structures
are not presented in the
conventional way
436 Phytochem Rev (2008) 7:431–443
123
similarity with estradiol, e.g. the almost planar
configuration and the aromatic rings. Several of these
so-called phytoestrogens have been linked with
cancer prevention, and have been selected as candi-
date drugs in various studies (Usui 2006). Although
to date no clear model exists that explains how
phytoestrogens affect the development of hormone-
dependent tumours, epidemiological data suggest that
the antitumour activity of phytoestrogens has little to
do with their activity as endocrine disruptors. For
example, genistein can stimulate estrogen receptor-
positive (ER+) breast cancer growth and interfere
with the antitumour activity of the endocrine disrup-
tor tamoxifen at low levels. Thus, women who have
ER+ tumours are advised not to increase their
phytoestrogen intake. Several studies suggest an
inhibitory effect on ER- breast cancer cell growth,
and it may be reasonable for women with
ER- tumours to safely consume soy and possibly
other phytoestrogens (Duffy and Cyr 2003).
An alternative explanation for the anticancer
properties of phytoestrogens may be that they prevent
the activation of pro-carcinogens like benzo[a]pyrone
(Tsuji and Walle 2006) since they inhibit the activity
of the enzymes CYP1A1 and CYP1B1. However, at
the same time it has been observed that phytoestro-
gens induce CYP1A1 and CYP1B1 gene expression
(Moon et al. 2006). These two observations seem to
be contradictory.
Dietary prodrugs
In the classical search for anticancer drugs from
botanical sources, plant extracts were fractionated by
various means and the fractions tested for anticancer
properties in a variety of in vitro assays. However,
the compounds that are present in the plant are not
necessarily the compounds that have a pharmacolog-
ical effect in the human body. This has often been
cited as one of the weaknesses of in vitro testing. A
few examples may demonstrate that several plant-
derived compounds act as prodrugs rather than drugs:
1. Contrary to popular belief, the bark of the willow
(Salix alba) does not contain aspirin (acetyl
salicylic acid). Nevertheless, like aspirin, extract
of willow bark has been effectively used to treat
pains and fevers. We now know that the gluco-
side salicin from the bark extract is converted in
the digestive tract and liver (by CYP enzymes) to
form the active ingredient salicylic acid (Fig. 3).
2. Epidemiological investigations showed a corre-
lation between reduced risk of chronic diseases
and increased concentration of the so-called
‘human’ lignans enterodiol and enterolactone
enterolactone in blood plasma (Fig. 4). The
‘human’ lignans play a role in the prevention of
colorectal cancer (Kuijsten et al. 2006). How-
ever, the anticancer mechanism is still unknown.
In fact, the ‘human’ lignans are not formed by
human metabolism at all, but by bacterial
fermentation of plant-derived lignans in the
colon (Rickard et al. 1996).
3. Another example of pharmaceutically active
compounds that arise as the result of fermentation
by gut bacteria are equal and O-demethylango-
lensin (Fig. 5) which are derivatives of the dietary
isoflavones formononetin and daidzein (Atkinson
et al. 2005).
Based on data obtained from literature and observa-
tions in our own lab, we propose that a variety of
naturally occurring compounds (notably phytoestro-
gens belonging to the compound class of the
flavonoids) act as prodrugs with relatively little
biological activity. These compounds are substrates
Fig. 3 Bioconversion of
salicin
Phytochem Rev (2008) 7:431–443 437
123
for enzymes that are highly expressed in tumour cells
(i.e. CYP1A1 and CYP1B1), and the conversion
products inhibit cell growth (i.e. selectively inhibit
growth of tumour cells).
This mechanistic model that may explain the
epidemiological finding that regular consumption of
fruit and vegetables can prevent the development of
tumours. The model also explains why flavonoid-type
antioxidants, but not other antioxidants like ascorbic
acid or tocopherol, seem to play a role in cancer
prevention. In addition, the model explains the
prominence of phytoestrogens in the lists of plant-
derived compounds with possible anticancer
properties.
Flavonoids are substrates for CYP1 enzymes
In an attempt to identify whether or not dietary
flavonoids interact with CYP1 enzymes, the effect of
the flavonoids on CYP1-catalysed 7-ethoxyresorufin
dealkylation (Burke and Mayer 1974) was monitored.
Most of the flavonoids tested inhibited the dealkyla-
tion in a dose dependent manner (Table 5). All the
Fig. 4 Bioactivation of
dietary lignan
secoisolariciresinol
Fig. 5 Bioactivation of
some isoflavones
438 Phytochem Rev (2008) 7:431–443
123
flavonoids tested, except cirsiliol and genkwanin,
inhibited CYP1B1 activity more effectively than
CYP1A1 and CYP1A2. In particular the flavonols
quercetin, myricetin and kaempferol were potent
CYP1B1 inhibitors, whereas they seemed to be less
effective inhibitors for CYP1A1 and CYP1A2.
The flavonoids which were shown to be potent
inhibitors in this study, might well be potential
substrates for CYP1 enzymes, e.g. resveratrol has
been reported to be a substrate (Potter et al. 2002;
Piver et al. 2004) and an inhibitor (Chun et al. 1999).
The 7-ethoxyresorufin-O-deethylase activity assay
(EROD assay) does not differentiate between the
different types of inhibition, but a molecule which is
a strong competitive inhibitor, would be a likely
substrate, since it will fit in the active site of the
enzymes.
Enzyme assays, where selected flavonoids were
incubated with CYP1 enzymes, confirmed that the
CYP1 enzymes accept a range of flavones and
flavonols as substrates. The reaction products were
analysed by HPLC-DAD, or HPLC-MS. In a number
of cases, the reaction products could be identified by
comparison with authentic reference compounds. We
can confirm that apigenin is hydroxylated to form
luteolin, and that kaempferol is hydroxylated to form
quercetin (Fig. 6, see also Breinholt et al. 2002;
Gradolatto et al. 2004). With other flavonoids, in
most cases reaction products could be observed.
However, due to the absence of authentic references
and the low amounts of product, a conclusive
chemical identification has not been possible. Thus
we can only assign putative structures to these
reaction products. Control incubations, either without
Table 5 Substitution patterns of selected dietary flavones
O
O
R4
R5
R1
R3
B
CA
3'
4'
56
7
R2
Flavones R1 R2 R3 R4 R5
Chrysin OH H OH H H
Baicalein OH OH OH H H
Apigenin OH H OH H OH
Luteolin OH H OH OH OH
Scutellarein OH OH OH H OH
Diosmetin OH H OH OH OMe
Eupatorin OH OMe OMe OH OMe
Cirsiliol OH OMe OMe OH OH
Genkwanin OH H OMe H OH
Fig. 6 CYP1-catalysed
conversion of the flavone
apigenin (R1 = H).
Analogous conversions
have been found for the
flavonol kaempferol
(R1 = OH)
Phytochem Rev (2008) 7:431–443 439
123
CYP1 enzymes or without co-factors, did show
detectable conversion of the flavonoid substrates. In
addition, incubations with a range of other recombi-
nant human cytochromes P450—CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, and
CYP3A5—did not give detectable conversion of the
flavonoids we tested. Only CYP2E1 (Piver et al.
2004) and CYP3A4 (our data) appeared to be minor
catalysts.
Interestingly, the isoflavonoid daidzein, a phyto-
estrogen from soy beans that has long been linked
with prevention cancer, does not inhibit CYP1A1 or
CYP1B1 activity (Chan and Leung 2003), and in our
assays we could not detect any bioconversion prod-
ucts from daidzein.
Phytoestrogens as natural prodrugs in cancer
prevention
Recent developments in smart anticancer drug dis-
covery have led to synthetic agonists of the AhR that
are prodrugs activated by CYP1A1. Phortress (Mac-
Fadyen et al. 2004; Brantley et al. 2006) and
aminoflavone (Kuffel et al. 2002; Pobst and Ames
2006) are both in Phase I clinical trials at the
moment. Flavones have been found to induce
CYP1A1 and CYP1B1 gene expression in animals,
and in in vitro cultivated human cells (Durgo et al.
2007; Hodek et al. 2006), possibly through modula-
tion of the aryl hydrocarbon receptor (AhR) (Pohl
et al. 2006).
Dietary flavonoids naturally occur as glycosides.
During digestion of these conjugated flavonoids, the
sugar moiety is split off allowing absorption of the
aglycone. However, the aglycone is rapidly reconju-
gated and one common characteristic of flavonoids is
that they occur in the bloodstream as glucuronide and
sulfate conjugates (Shimoi et al. 1998; Dupont et al.
2004; Erlund 2004; Chen et al. 2007). The concen-
tration of the conjugates of the more common dietary
flavonoids in the human blood stream is in the
micromolar range, and this is the concentration that
reaches the tumour tissue. Around tumour tissue,
significant extracellular levels of b-glucuronidase can
be found, due to the liberation of this enzyme from
the lysosomes of inflammatory cells (Dodds et al.
2002). Thus, tumour cells are likely to be met by
flavonoid aglycones at micromolar concentrations.
In order to screen for the presence of CYP-1
activated prodrugs, a test panel of human breast cell
cultures had been designed (Potter and Butler 2004).
This panel was used to test our hypothesis that
phytoestrogens are selectively activated in CYP-1
expressing tumour cells. The panel consists of the
human breast adenocarcinoma cell line MCF-7—
which normally does not express CYP-1 enzymes but
can be induced with the dioxin TCDD, the human
breast adenocarcinoma cell line MDA-MB-468—
which constitutively expresses CYP1s-, and the non-
tumorigenic epithelialbreast cell line MCF-10A—
which was used a model for normal breast cells. The
efficacy of a pro-drug can be expressed by determin-
ing its IC50 value (concentration needed to inhibit cell
proliferation by 50%) for the different cell lines, and
then calculating its activation factor or its tumour
selectivity factor. When the activation factor is
greater than one, cells expressing CYP1 enzymes
are inhibited more by the test substances than cells
not expressing CYP1 enzymes. When the tumour
selectivity factor is greater than one, tumour cells are
more inhibited than non-tumour cells (Tables 6, 7)
shows that some common dietary flavonoids have
activation factors and tumour selectivity factors
ranging from 2 to 10. The activation and tumour
Table 6 Inhibition of recombinant human CYP1 enzymes by
dietary flavonoids. CYP1 (CYP1A1, CYP1A2, CYP1B1)
microsomes (5 pmol/ml) were incubated with various con-
centrations of flavonoids
Compound CYP1B1 CYP1A1 CYP1A2
Flavonols
Quercetin 0.7 ± 0.3 7.0 ± 0.0 12 ± 3.5
Myricetin 0.9 ± 0.2 4.0 ± 0.0 13 ± 0.7
Kaempferol 1.3 ± 0.2 8.0 ± 1.4 9.0 ± 2.1
Flavones
Apigenin 2.0 ± 0.0 4.2 ± 0.0 6.0 ± 0.0
Luteolin 1.8 ± 0.4 10 ± 0.7 15 ± 1.4
Diosmetin 0.5 ± 0.1 1.2 ± 0.3 18 ± 4.0
Baicalein 7.0 ± 1.1 20 ± 0.0 20 ± 0.0
Chrysin 0.5 ± 0.1 0.5 ± 0.1 2.0 ± 0.0
Eupatorin 1.1 ± 0.1 2.3 ± 0.1 25
Genkwanin [25 [25 [25
Cirsiliol [25 [25 [25
Results are calculated from percentage of inhibition of
7-ethoxyresorufin-O-deethylase activity compared to control,
and expressed as IC50 (lM). Each value corresponds to
mean ± standard deviation for n = 4 determinations
440 Phytochem Rev (2008) 7:431–443
123
selectivity factors presented in Table 7 indicate that
the dietary flavonoids tested cannot be used in cancer
therapy; the dose that stops tumour growth is too
close to the dose that stops normal cell development.
However, micromolar concentrations of these dietary
compounds in the blood stream are sufficient to
selectively inhibit the proliferation of tumorigenic
cells expressing CYP1-enzymes, and thus prevent
development of tumours at the very earliest stages.
Thus, our hypothesis of phytoestrogens as natural
prodrugs explains how micromolar amounts of die-
tary flavones may prevent the onset of a wide range
of cancers. More research is needed to identify
dietary prodrugs and characterise their activated
products, and to establish the exact mechanisms by
which these compounds selectively inhibit the pro-
liferation of tumour cells.
Conventional screening methods for anticancer
compounds have led to a number of clinically used
drugs for cancer therapy. Although, new compounds
will still be discovered using conventional screening
methods, it now seems to be past its peak. Most of the
smart drugs, and probably none of the pro-drugs,
would have been picked up by conventional screen-
ing. Many drug companies have abandoned major
conventional screening programmes in favour of
novel methods for smart drug development using
tools that have become available the post-genomic
era, i.e. specific in vitro enzyme assays or even
in silico data mining.
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