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Current Topics in Medicinal Chemistry, 2015, 15, 000-000 1
1568-0266/15 $58.00+.00 © 2015 Bentham Science Publishers
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer
Višnja Stepanić1,*, Ana Čipak Gasparović1, Koraljka Gall Trošelj1, Dragan Amić2 and
Neven Žarković1
1Ruđer Bošković Institute, Bijenička 54, 1000 Zagreb, Croatia; 2Faculty of Agriculture, The Josip Juraj
Strossmayer University, Kralja Petra Svačića 1d, 31000 Osijek, Croatia
Abstract: Various plant polyphenols have been recognized as redox active molecules. This review dis-
cusses some aspects of polyphenols’ modes of redox action, corresponding structure - activity relation-
ships and their potential to be applied as adjuvan ts to conventional cytostatic drugs. Polyphenols’ anti-
oxidative capacity has been discussed as the basis for targeting oxidative stress and, consequently, for
their chemopreventive and anti-inflammatory activities, which m ay alleviate side-effects on normal cells
arising from oxidative stress caused by cytostatics. Some polyphenols may scavenge various free radicals directly, and
some of them are found to suppress free radical production through inhibiting NADPH oxidases and xanthine oxidase.
Additionally, polyphenols may increase antioxidative defense in normal cells by increasing the activity of NRF2, tran-
scription factor for many protective proteins. To the contrary, the activation of the NRF2-mediated signaling pathways in
cancer cells results in chemoresistance. Luteolin, apig enin and chrysin reduce NRF2 expression and increase the che-
mosensitivity of cancer cells to cytostatic drugs. Their common 5,7-dihydroxy-4H-chromen-4-one moiety, may represent
a starting pharmacophore model for designing novel, non-toxic compounds for overcoming chemoresistance. However,
pro-oxidative activity of some polyphenols (quercetin, EGCG) may also provide a basis for their use as chemotherapeutic
adjuvants since they may enhance cytotoxic effects of cytostatics selectively on cancer cells. However, considerable cau-
tion is needed in applying polyphenols to anticancer therapy, since their effects greatly depend on the applied dose, the
cell type, exposure time and environmental conditions.
Keywords: Adjuvant, anticancer therapy, antioxidative, curcumin, EGCG, flavonoids, NRF2, polyphenols, pro-oxidative.
1. INTRODUCTION
Cancer is the second leading cause of death in the world
[1]. Unfortunately, it is commonly diagnosed in an advanced
stage, despite an improvement in diagnostic procedures. Sig-
nificant improvements in anticancer therapy have been intro-
duced, primarily through designing smart drugs which spe-
cifically target signaling pathways that are centrally involved
in cancer pathogenesis [2,3]. However, conventional, non-
surgical treatment of cancer still relies mostly on the use of
cytostatic drugs and/or radiotherapy. These drugs belong to
several chemical groups: anthracyclines (e.g. doxorubicin,
daunorubicin, epirubicin); platinum-containing complexes
(e.g. cisplatin, carboplatin); alkylating agents (e.g. cyclo-
phosphamide, melphalan); cytotoxic antibiotics (e.g. bleomy-
cin, mitomycin C); podophyllin derivatives (e.g. etoposide);
camptothecins (e.g. topotecan, irinotecan); antimetabolites (5-
fluorouracil); and microtubule targeting mitotic inhibitors
(e.g. vinblastine, vinorelbine, paclitaxel, docetaxel).
For the majority of these chemotherapeutics, the primary
targets are rapidly dividing cancer cells [4]. This targeting
has been primarily achieved through inhibiting the synthesis
of DNA, RNA or proteins. Accordingly, the most significant
therapeutic effects relate to cells that are in S- and mitotic
*Address correspondence to this author at the Ruđer Bošković Institute,
Bijenička 54, 1000 Zagreb, Croatia; Tel: +385 1 457 1248;
Fax: ???????????????; E-mail: stepanic@irb.hr
cell cycle phases. Cytostatic drugs may damage the DNA
molecule either directly, through intercalation, and/or indi-
rectly, through inducing production of reactive oxygen spe-
cies (ROS), resulting in oxidative stress and finally cellular
apoptosis [5]. For example, ROS production and oxidative
DNA adducts were shown to be involved in paclitaxel-
induced apoptosis in hepatoma cells, resulting in G2/M ar-
rest and an increased number of cells with fragmented nuclei
[6]. These changes were joined with altered mitochondrial
membrane permeability and total cellular antioxidant capac-
ity. This last parameter is known to be a critical determinant
of cellular sensitivity to paclitaxel [6].
Anticancer cytostatic drugs are non-selective. They also
target normal, non-cancer cells with high proliferation index,
such as intestinal epithelium, bone marrow and hair follicle
cells, resulting in serious side-effects, like leucopenia and
hair loss [4]. Cytostatic drugs can also be mutagenic and
carcinogenic [4]. Typical examples are alkylating agents,
melphalan and cyclophosphamide. These drugs bind to and
chemically modify the DNA molecule leading to chromoso-
mal abnormality, chromosomal breaks, and an increase in
sister chromatid exchanges [7]. Some cytostatic drugs are
specifically toxic. For example, doxorubicin has cumulative,
dose-dependent cardiotoxicity [8], while platinum deriva-
tives generally induce nephrotoxicity [9].
Thus, discovering highly specific features of cancer cells
to be selectively targeted by novel molecules, represents an
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corresponding author(s)
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2 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić et al.
imperative in the field of cancer treatment. An additional
field of interest is discovering ways of reducing side-effects
of current anticancer therapeutics by combining them with
adjuvants capable to alleviate their toxic effects on normal
cells or/and increase their selectivity against cancer cells.
Recent discoveries point to polyphenols as promising
compounds to be implemented as adjuvants in cancer ther-
apy. Their antioxidative and anti-inflammatory activities
have been well-known [10-12]. Based on these activities, a
combined therapeutic approach, consisting of a polyphenol
and a conventional cytostatic drug, has realistic potential for
alleviating the various side-effects induced by cytostatic
drugs per se. For example, pretreatment with (-)-
epigallocatechin 3-gallate (EGCG) (≤ 50 µM) was found to
significantly attenuate cardiotoxicity mediated by doxorubi-
cin (1 µM or 10 µM) in vitro and in vivo, partially through
attenuating ROS production in cardiomyocytes [8]. Luteolin
significantly reduced pathohistological and biochemical
changes in mouse kidneys induced by cisplatin [9]. Th is
nephroprotective effect was associated with decreased plati-
num accumulation in kidney tissue and suppression of oxida-
tive stress, inflammation and apoptosis.
Of particular interest is polyphenols’ ability to reverse
cancer cell drug resistance, primarily through a significant
decrease of efflux transporters and a consequential increase
in uptake of the anticancer drug. Exposing a cancer cell to a
chemotherapeutic agent represents a stressful event and, in
this context, chemoresistance may be considered as a meas-
urable manifestation of cellular stress defense response. The
first line of cellular defense against drug related stress, is
manifested in an increased activity of such membrane ATP
binding cassette (ABC) transporters as MDR1 (multidrug
resistance protein1, ABCB1, P-gp) and MRP1 (multidrug
resistance-associated protein 1, ABCC1), causing an altera-
tion of drug uptake and/or efflux [13]. For example, the
benefits of combining curcumin with various cytostatics,
primarily through decreased activity of MDR1, have been
shown both in vitro and in vivo and are recently reviewed
[14-16]. Similarly, resveratrol (50 µM) was shown to signifi-
cantly enhance doxorubicin (1 µM) cytotoxicity in MDA-
MB-231 and doxorubicin resistant MCF-7/adr breast cancer
cell lines through a decrease of the mRNA and protein levels
as well as inhibition of ATPase activity of MDR1 efflux
pump [17]. As a result, the presence of resveratrol allows for
increased accumulation of doxorubicin in the treated cancer
cells. These synergistic effects were also shown in a
xenograft mouse model: a daily administration of resveratrol
during four weeks, combined with doxorubicin administra-
tion once p er week, significantly reduced tumor volume and
weight up to 61.5% and 54.7%, respectively, as compared to
the vehicle-treated control group [17]. Another, more pro-
found mechanism of increasing chemosensitivity of cancer
cell by polyphenols, is associated with modulating the activ-
ity of the redox-sensitive transcription factor NRF2 (nuclear
factor E2-related factor 2), which is a master regulator of
various cellular protective genes [18]. The activities of efflux
transporters are frequently induced through activating NRF2
signaling pathways [18]. This relatively novel aspect of
polyphenols’ biological impact is presented in this review as
quite promising, considering their use as chemosensing ad-
juvants to cyto static drugs.
There are several thousand small-molecular-weight natu-
ral polyphenolic compounds [19]. So far, a variety of bio-
logical activities, particularly explored on cancer cell lines in
vitro, have been observed for these plant secondary metabo-
lites [12]. Polyphenols quercetin - the most abundant food
polyphenol, EGCG - the most abundant green tea polyphe-
nol, resveratrol - a polyphenol from the red wine and grapes,
and curcumin - a yellow pigment present in Indian spice
mixture - curry (Fig. 1), have been extensively studied for
their ben eficial health effects as chemopreventive agents
[19]. In this review, we consider them as potential adjuvants
to conventional cytostatic chemotherapeutics from the aspect
of their redox activities. At first, polyphenols’ targets - ROS
and oxidative stress as well as antioxidant defense and their
connections with inflammation and cancer, are described
briefly. Then, the well-known direct and indirect radical
scavenging abilities of polyphenols are summarized. Finally,
recently recognized NRF2 inhibiting potential and the pro-
oxidative effects of polyphenols are discussed as tentative
mechanistic bases for their adjuvant application in anticancer
therapy.
2. ROS AND ANTIOXIDATIVE DEFENSE
Free radicals are molecular species with an unpaired
electron. This characteristic makes them able to readily react
as oxidants with other molecules. Endogenous oxidants are
usually grouped into the two sets: ROS and reactive nitrogen
species (RNS). Common ROS are superoxide radical anion
(O2•-), hydroxyl (•OH), hydroperoxyl radical (•OOH), hydro-
gen peroxide (H2O2), and singlet oxygen (1O2), while RNS
are nitric oxide (•NO) and peroxynitrite (ONOO-).
ROS are produced in both physiological and pathologi-
cal conditions, mostly in the mitochondria, microsomes and
peroxisomes [20]. They are produced either in a controlled
manner (e.g. in thyroid gland, during neutophil’s oxidative
burst or smooth muscle contractions) or in an uncontrolled
manner ("electron leakage" of respiratory chain in mito-
chondria). In the generation of ROS are included various
oxidoreductases such as cytochrome P450 isoenzymes, and
the electron transport chain within the mitochondria mem-
brane mediated by coenzyme Q (ubiquinone), the essential
electron carrier [21]. The superoxide radical anion O2•-, is
generated in the mitochondria by the reduction of molecular
oxygen by NADPH oxidases (NOXs) and xanthine oxidase.
Hydrogen peroxide H2O2, is generated by the dismutation of
O2•- non-enzymatically and enzymatically by superoxide
dismutase (SOD) enzymes, located in the mitochondria and
in th e cytoplasm. It can be also directly produced by a range
of oxidase enzymes [22]. The main deleterious ROS, the
hydroxyl radical •OH, is produced by Fenton and Fenton-
like reactions involving transition metal ions Fe(II) and
Cu(I).
ROS are essential for all processes in a living cell [23].
They take part in signal transduction, regulation of cellular
growth, energy production and biosynthetic reactions. They
are an important part of a cellular defense response which
includes processes of detoxification and destruction of
pathogen microorganisms. In physiological conditions, ROS
also contribute to signals that are very important for cellular
proliferation and apoptosis [23].
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 3
Fig. (1). Some low-molecular-weight polyphenols and the fragments associated with their redox activities. For example, 5,7-dihydroxy-4H-
chromen -4-one moiety is present in compounds that inhibit xanthine oxidase enzymatic activity and down-regulate NRF2 expression.
On the other hand, ROS may cause severe damage in
cells. The most notable cellular targets of ROS are DNA,
proteins and lipids [24]. ROS react with DNA, leading to
single- and double- stranded breaks and oxidation of DNA
nucleobases [25]. As consequence, these changes may lead
to DNA mutations, which may further lead to changes in
cellular control mechanisms and cancer. In membranes, ROS
cause oxidation of lipids, particularly polyunsaturated fatty
acids. Peroxyl radicals, a lipid oxidation intermediates, have
an ability to further propagate lipid oxidation, leading to an
accumulation of reactive aldehyde products, such as
malondialdehyde and 4-hydroxynonenal (4-HNE). Reactive
aldehydes are able to propagate further oxidative stress dam-
age and modify DNA bases leading to mutations [20]. When
discussing proteins as a target of ROS, one should distin-
guish between two types of effects: inactivation and modifi-
cation. ROS can react with proteins resulting in their inacti-
vation and proteasomal degradation [26]. In contrast with
inactivation, modification of thio l (SH) groups of cysteine
(Cys) amino acid residues may take part in redox-signaling
and redox sensing [27]. The SH groups of Cys residues are
the most redox sensitiv e groups in proteins. They are prone
to oxidation by ROS to disulfides that contributes to cross-
linking and the inactivation of various proteins. Only a rela-
tively small subset of Cys amino acid residues of Cys-
proteome are involved in cell signaling [27]. They are acti-
vated through localized oxidant generation (particularly of
H2O2), e.g. at plasma membranes or near mitochondria
[27,28]. These SH groups are often part of the active places
of many enzymes like tyrosine phosphatases, which are key
regulatory components in signal transduction pathways e.g.
the MAPK (mitogen-activated protein kinases) pathway
[29]. However, most Cys residues have redox-sensing roles
and the thiol/disulfide pool status impacts biologic redox
control mechanisms globally regardless of a specific signal-
ing pathway [7].
Maintaining a delicate balance between beneficial and
harmful effects of free radicals, represents a highly evolu-
tionary achievement known as "redox regulation" [20,23]. In
parallel with the evolution of aerobic metabolism, cells have
developed an antioxidant defense system, necessary for at-
tenuating the harmful effects induced by oxygen, including
oxidative stress [30]. The antioxidant defense system is ad-
justed to each cellular compartment and is composed of en-
zymatic and non-enzymatic factors [30,31]. The homeostatic
concentration of ROS is sustained through cascades of en-
zymatic reactions leading to a complete detoxification of the
oxidative substrates. This is the case with the SOD - catalase
cascade, which is well known for detoxifying superoxide
radical anion. SOD enzymes convert O2
•- to H2O2 and cata-
lase further catalyzes the decomposition of H2O2 to H2O and
oxygen, O2. The glutathione peroxidases and the NADPH-
dependent glutathione reductase are important components
of the re-cycling system for glutathione, an important en-
dogenous antioxidant. Glutathione is an abundant cellular
tripeptide (~ 5 mM) with the reducing Cys SH group.
Changes in the r edox state of glutathione also play an impor-
tant role in the regulation of gene transcription. Depletion of
the reduced glutathione sensitizes can cer cells to some che-
motherapeutics by overcoming multidrug resistance (MDR)
[20]. In the process of detoxification and elimination of elec-
trophilic xenobiotic substrates, glutathione-S-transferases
(GST) catalyze their conjugation to reduced glutathione. The
mammalian thioredoxin reductases (TrxR1 and TrxR2) cata-
lyze the NADPH-dependent reduction of the redox active
protein thioredoxin (Trx). Its reduction is important for
maintaining a reduced cellular state and for the proper func-
tioning of other endogenous and exogenous molecules [32].
4 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić et al.
Unlike O2
•-, hydroxyl radicals and the major products of
their biological activity - (lipid) peroxyl radicals, are scav-
enged non-enzymatically, by endogenous and exogenous
antioxidants. Perhaps the best studied endogenous antioxi-
dants, are glutathione and coenzyme Q. The most important
and widespread exogenous antioxidants taken through diet,
are vitamins A and E and their analogs carotenoids and toco-
pherols, respectively, as well as vitamin C and polyphenols.
Their cellular compartmentalization is largely determined by
their physicochemical properties, such as lipophilicity and
charge. For example, vitamin E is lipophilic and is located in
the cellular membrane where it protects lipids from oxidation
[33]. Vitamin C and glutathione are hydrophilic, water solu-
ble molecules. Therefore, they are located mostly in the cy-
tosol. Polyphenols are generally amphiphilic molecules and,
hence, can be localized in both lipophilic and hydrophilic
cellular compartments [12].
Transcription of most of the aforementioned antioxidant
enzymes is directly regulated by NRF2 [31]. This transcrip-
tion factor is also important, indirectly through regulating
metabolic enzymes, for activities of chemical antioxidants.
NRF2 is a transcription factor that binds to antioxidant re-
sponse elements (AREs) and electrophile response elements
(EpREs) of DNA. These DNA sequences are located in the
promoters of many antioxidative, and cytoprotective genes
(phase II response), such as SOD, GST, heme oxygenase-1
(HO-1), NAD(P)H-quinone oxidoreductase (NQO1), UDP-
glucuronosyl transferases (UGT), γ-glutamylcysteine syn-
thetase (γGCS), as well as efflux pumps like ABC transport-
ers MRP2 (ABCC2) and BCRP (breast cancer resistance
protein, ABCG2) [34,35]. Under nonstimulating, resting
conditions, NRF2 is bound to its negative regulator KEAP1
(Kelch-like ECH-associated protein 1). When oxidative
stress or electrophilic stimuli occur, NRF2 translocates into
the nucleus as a consequence of direct oxidation or covalent
modification of SH groups of KEAP1. Nuclear translocation
may also be a consequence of phosphorylation of NRF2 by
redox sensitive kinases such as PKC, PI3K and JNK [35].
NRF2, as well as thioredoxin reductase isoenzymes, are
involved in the antioxidant defense of not only normal cells,
but also of transformed cells [36-39]. These genes are fre-
quently overexpressed in various human cancers. In the con-
text of healthy cell protection, the activity of these enzymes
is beneficial, while in the context of cancer cells, these en-
zymes, along with increased NF-κB (nuclear factor k appa B)
activity, contribute to resistance to chemotherapy [13,18].
There is a rationale for hypothesizing that their selective
inhibition in cancer cells would enhance the effects of oxida-
tive stress and promote apoptotic signaling pathways
[18,39].
3. OXIDATIVE STRESS, INFLAMMATION AND
CANCER
Oxidative stress develops if ROS are overproduced
and/or insufficiently eliminated. An excess of ROS is associ-
ated with the oxidative damage of various macromolecules
and disruption of redox signaling. These effects may induce
either apoptosis or, by contrast, proliferation leading to can-
cer [40]. Persistent oxidative stress is considered to be a
promoter of cancer initiation and development, primarily
through increased cellular proliferation, angiogenesis and
metastasis formation and preventing apoptosis [41,42]. Can-
cer cells are generally characterized by persistent intracellu-
lar ROS production and have higher levels of ROS as com-
pared with normal cells [31,43,44].
The length of exposure time to ROS is an important vari-
able determining their contribution to either carcinogenesis
or cellular apoptosis. For example, the short-term increase of
ROS (24 h) inhibited the growth of breast cancer cells,
MCF-7, in a dose-dependent manner [43]. However, the per-
sistent chronic exposure (three months) to an elevated level
of ROS, increased the growth and tumorigenicity of this cell
line.
Oxidative stress can develop as a consequence of chronic
exposure to harmful substances or can be induced by strong
inflammatory reactions (e. g. microbial infection, cigarette
and/or alcohol abuse, UV- and ionizing radiation) [41]. In
this context, the border between damaging xenobiotics and
cytostatic drugs is almost invisible. A majority of conven-
tional anticancer drugs boost ROS production, cause cellular
and tissue damage and activate inflammatory response [45].
Many of these drugs have been shown to activate NF-κB, a
key coordinator of innate immunity, inflammation and car-
cinogenesis, in various cancer cell lines and in vivo [45]. An
inflammatory microenvironment represents a particularly
fruitful background for developing the basic hallmarks of
cancer [42]. Inflammatory cells, especially macrophages an d
neutrophils generate an abundance of ROS through oxidative
burst. In this milieu, ROS are produced mainly by NADPH
oxidases [46] and, to a lesser extent, by o ther enzymes im-
plicated in inflammation (e.g. lipoxygenases and cyclooxy-
genases). For all these reasons, chronic inflammation associ-
ated with a persistently high level of ROS makes excellent
ground for cancer development [45,47]. Additionally, ge-
netic events causing neoplasia like oncogene activation, also
generate an inflammatory type of microenvironment, charac-
terized by the presence of ROS, pro-inflammatory cytokines
(TNF-α, IL-1, IL-6, IL-8, etc.), matrix degrading enzymes
and growth factors [42], as well as mitochondrial dysfunc-
tion [41]. Oxidative stress and pathological conditions such
as inflammation and inflammation-associated cancer are
often joined with mitochondrial dysfunction [41,48,49]. Ac-
cumulation of free radicals may induce considerable changes
in mito chondria, in their DNA as well as in membrane per-
meability and potential, changing thus their energy and ROS
production in various ways. "Inflammation - oxidative stress
- cancer" actually makes a self-sustained vicious cycle in
which ROS have the central role. Their increased production
leads to further cellular damage, additional activation of im-
mune responses, stronger inflammatory reaction and even
more ROS production. ROS and products of their oxidative
actions, such as reactive aldehydes, iteratively activate sig-
naling cascades, resulting in the activation of NF-κB which,
in this specific context, activates the transcription of pro-
proliferative genes.
4. ABOUT POLYPHENOLS
Plant polyphenols are well-known antioxidants available
through diets rich in fruits, vegetables and beverages like
green tea and red wine [19]. Following the Latin proverb
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 5
nomen est omen, polyphenols generally posses aromatic ben-
zene ring(s) substituted by hydroxyl (OH) groups. However,
although all natural polyphenols have OH-substituted ben-
zene ring(s), they are structurally d iverse phytochemicals.
They belong to various chemical classes such as small-
molecular-weight phenolic acids, flavonoids, stilbenes, cur-
cuminoids, xanthones, tannins or large up-to-4000 Da lig-
nans [19,50]. In general, the versatile activities observed for
plant polyphenols can be ascribed to often many OH groups
placed at various parts of the conjugated scaffold character-
ized by ex tended π-electron delocalization (Fig. 1). Such OH
groups can take part in free radical scavenging and/or chela-
tion of fr ee metal ions. They make polyphenols potent hy-
drogen bond donors and may be responsible for their binding
affinity for heterogeneous groups of proteins and nucleic
acids. Th e OH groups also considerably affect the physico-
chemical features of polyphenolic molecules. Apart from
phenolic acids, many other polyphenols are also negatively
charged at pH 7.4 due to deprotonation of OH groups [51].
Many in vitro studies have revealed that polyphenols are
pharmacologically versatile compounds, able, at µM concen-
trations, to specifically bind to numerous proteins. This is
illustrated by flavonoids which, in a competitive or non-
competitive manner, with IC50 values reported to be in low
µM range, inhibit the catalytic activities of many kinases,
including those involved in pro-survival or pro-apoptotic
pathways [52]. The reported IC50 values are in the range of
concentrations of polyphenols measured in the plasma [19].
For example, at 2 µM concentration, quercetin was shown to
reduce the catalytic activity of 16 recombinant kinases
(ABL1, Aurora-A, -B, -C, CLK1, FLT3, JAK3, MET,
NEK4, NEK9, PAK3, PIM1, RET, FGF-R2, PDGF-Rα and -
Rß) by more than 80% [53]. The majority of these kinases
are involved in the control of mitosis and mitosis-related
processes. This is a good example, showing that quercetin
even in a low µM dose, influences a number of signal trans-
duction pathways and, consequently, modulates various
processes within the cell.
There are several factors influencing polyphenols' bio-
logical activities. The most important ones are the applied
dose, the cell type, exposure time and environmental condi-
tions. As redox active molecules, polyphenols are prone to
auto-oxidation that may considerably change their structure
[54,55]. This is particularly pronounced under basic condi-
tions. Their structur e may also be significantly inf luenced by
biotransformation, dependent on specific cellular metabolic
machinery. When administrated orally, polyphenols, as
xenobiotics, become extensively metabolized by bacterial
microflora and endogenous metabolic enzymes [56]. In vivo,
the plasma concentrations of parent polyphenol compounds
and their conjugates are, in general, at low µM level [19]. In
the case of consumption of 270 g of fried onions containing
275 µmol of flavonol glucosides, mostly those of quercetin,
the mean human plasma concentration of quercetin-3-O-
glucuronide was 351 nM [19].
The biological effects do not necessarily need to be a
consequence of the action of only the parent polyphenol
aglycon, but can also be asserted by its metabolites [57]. The
metabolic conjugates can even be biotransformed back to the
active aglycon form under specific biological conditions. In
the case of quercetin, one of its main metabo lites in humans -
quercetin-3-O-glucuronide has been found to specifically
accumulate in macrophage-derived foam cells in human
atherosclerotic lesions [58]. Quercetin-3-O-glucuronide ac-
cumulates through the anion binding of its phenolic group(s)
onto the macrophage cell surface proteins. The extracellular
β-glucuronidase secreted from macrophages biotransforms it
back into the quercetin aglycone which then enters macro-
phages and exerts anti-inflammatory and anti-atherosclerotic
effects in the damaged aorta [59]. In addition, quercetin is
transformed to 3'- and 4'-O-methyl quercetin metabolites by
intracellular enzyme catechol-O-methyltransferase (COMT).
The enzyme β-glucuronidase is active at an acidic pH. Its
activity has been observed to be promoted by the increased
secretion of lactate in response to the mitochondrial dysfunc-
tion characterized using antimycin-A (a mitochondrial in-
hibitor) and siRNA-knockdown of Atg7 (an essential gene
for autophagy) [60]. Similarly, it may be promoted by War-
burg effect, mitochondrial dysfunction and inflammatory
microenvironment characterizing cancer cells [42].
Polyphenols seem to be very selective in their action, tar-
geting primarily the sites of inflammation and precancerous
or malignant lesions. Such selectivity may be associated with
their accumulation in specific cells, as it has been demon-
strated for quercetin-3-O-glucuronide and EGCG that accu-
mulate in macrophages [59]. Similarly, since in vitro poly-
phenols mostly asserted their activities at µM concentrations,
their localization at specific cellular compartments may also
be of great importance for their activity. For example, in
order to modify the epigenome, polyphenols need to enter
the cell nucleus [12]. Additionally, ROS production and re-
lated altered redox-status may be specifically target by re-
dox-sensitive molecules like polyphenols through mecha-
nisms based on specific flux-based inter- and intra-cellular
transport [27].
When discussing polyphenol supplementation, one must
additionally differentiate between low-dose and high-dose
supplementation (Fig. 2a). Curcumin, quercetin, resveratrol,
piceatannol, EGCG, as well as other natural polyphenols,
show a hormetic dose-response phenomenon, characterized
by U- or J-shaped curves [61]. At low µM concentrations, <
40 µM, their effects are antioxidative and chemopreventive.
They are manifested through modulation of activity and/or
expression level of endogenous antioxidants, sirtuins,
kinases and heat shock proteins, as well as transcription fac-
tors, including redox-sensitive NF-κB and NRF2 [61]. Thus,
they influence the synthesis of molecules which protect cells
and mak e them more resistant to mediators of stress. How-
ever, high doses of polyphenols (usually ≥ 50 µM), show
pro-oxidant properties and are cytotoxic [62].
5. POLYPHENOLS AS SUPRESSORS OF FREE
RADICALS
It has been assumed that an intervention aimed to suppress
the overproduction of ROS and restore normal redox
homeostasis may be of a great benefit in anticancer treatment
[19]. Thus, polyphenols have been intensively investigated for
their antioxidative properties [63]. Their antioxidative and
chemopreventive potentials have primarily been connected
with their radical scavenging activities, direct and indirect.
6 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić et al.
5.1. Direct Radical Scavenging
Direct radical scavenging capacity of polyphenols is re-
lated to the presence of OH groups at specific positions on
the conjugated scaffold, which are able to donate readily a
hydrogen atom to a free radical. This leads to the neutraliza-
tion of free radicals and, consequently, to termination of a
radical chain reaction [64]. Particularly at neutral pH, due to
their low reduction potentials, flavonoids are able to directly
react with different ROS, including O2•-, •OH, peroxyl and
alkoxyl radicals [65]. Polyphenols with catechol (ortho-
dihydroxybenzene) and/or pyrogallol moieties are, generally,
strong in vitro antioxidants (Fig. 1).
A variation of the reaction mechanisms involved in free
radical scavenging by polyphenols has been proposed [66-
68]. These mechanisms can be classified in the two types:
(i) an H-atom abstraction; and (ii) radical adduct formation
(RAF). The H-atom abstraction can occur according to at
least three different mechanisms: single-step hydrogen
atom transfer (HAT), sequential proton loss electron trans-
fer (SPLET) and single electron transfer followed by pro-
ton transfer (SET-PT) [51]. All three mechanisms share the
phenoxyl radical as an intermediate. Its thermodynamic
stability significantly influences the radical scavenging
capacity of a polyphenol. For example, good radical scav-
engers are flavonoids with the conjugated 3-OH group
and/or the catechol group (Fig. 1), since these structural
characteristics increase the stability of the phen oxyl radical
inter med iate. The great radical scavenging potential o f
catechol is attributable to the fact that its semiquinone phe-
noxyl radical formed by donation of one H-atom to free
radical, can be stabilized by an intramolecular hydrogen
bond and the electron-donating properties of the ortho-OH
group. In the case of scavenging small free radicals such as
many of the ROS formed in biological systems, formation
of covalently bound radical adducts (RAF) may also be a
viable mechanism, especially in the case of antioxidants
with extended π-electron conjugated chains like caro-
tenoids and resveratrol [69,70]. Which mechanism will
predominate depends upon many factors, including the free
radical type, solvent type (its polarity and hydrogen bond
accepting capacity) as well as pH and temperature [67]. For
examp le, the SPLET mechanism involving the stable anion
form of a polyphenol is dominant in a polar environment,
while the one-step HAT mechanism of the neutral polyphe-
nol occurs in an unpolar medium [51,71].
Complexes of polyphenols with the transition metal ions
possess even better free radical scavenging activity than
polyphenol aglycons alone [72-75]. It has been shown that
flavonoid-metal ion complexes may also possess anti-
inflammatory activity and cytotoxicity against cancer cells
superior to the parent flavonoid aglycons [73,74]. Such com-
plexes exhibit SOD mimicking activity, with the metal ion
being the main antioxidating center in the following manner
[74,75]:
Me(n+1)+ + O2•- → Men+ + O2
Men+ + O2•- + 2 H+ → Me(n+1)+ + H2O2
where a Me(n+1)+ / Me
n+ pair is often Cu(II)/Cu(I) or
Fe(III)/Fe(II) pair.
5.2. Indirect Radical Scavenging
Polyphenols may also reduce the ROS generation. As ef-
ficient chelators of transition metal ions, polyphenols may
inhibit the ROS generation through Fenton and Fenton-like
reactions [40,74,76]. Polyphenols may also inhibit the key
enzymes - NADPH oxidase isomers and xanthine oxidase.
Various classes of polyphenols have been reported to inhibit
NADPH oxidases [46]. The green tea EGCG and black tea
theaflavin-3,3'-digallate inhibit, in vitro, the ROS formation
through the competitive inhibition of xanthine oxidase, with
low micromolar IC50 values [77]. Planar flavonoids quercetin
and apigenin, and the non-planar protoflavone analog pro-
toapigenone 1'-O-propargyl ether, have been reported as low
µM inhibitors of xanthine oxidase [78]. The common struc-
tural characteristic of these three molecules is the presence of
5,7-dihydroxy-4H-chromen-4-one fragment (Fig. 1).
5.3. Comparison with Other Free Radical Suppressing
Adjuvants
While combining polyphenolic compounds and cytostatic
drugs may sound reasonable, more research and evidence-
based data is needed [5]. This is especially important in light
of studies showing that the concomitant use of antioxidants
and chemotherapeutic agents may have not only favourable
outcomes, but also harmful effects [79]. Antioxidants, as
adjuvants, may alter the efficacy of cancer chemotherapy
either antagonistically or agonistically [5].
Prior to recognizing polyphenols as potential additives in
combined anticancer therapy, large clinical studies were per-
formed based on various combination of cytostatic drugs and
high doses of various natural antioxidants (glutathione, mela-
tonin, N-acetylcysteine and vitamins A (β-carotenoids), E
and C). Their primary focus was on alleviating the toxic ef-
fects of anticancer drugs, commonly induced by exaggerated
oxidative stress [5,79]. The reports were contradictory. Some
results indicated that these pharmacological combinations
resulted in either increased survival times, increased tumor
responses to therapy, or both, as well as in reduced tox-
icities.. Other studies recorded significant reduction of the
anticancer activity of chemotherapeutic agents, known in-
ducers of oxidative stress [5,80]. However, such a dimin-
ished anticancer activity may not be a consequence of the
radical scavenging activities of antioxidants. The following
example with vitamin C is particularly illustrative [80]. Vi-
tamin C, in a dose-dependent manner, attenuated the cyto-
toxic activity of diverse classes of chemotherapeutic com-
pounds such as doxorubicin, cisplatin, vincristine,
methotrexate, and imatinib in leukemia (K562) and lym-
phoma (RL) cell lines. However, this effect was not medi-
ated by ROS. Vitamin C (~ 10 mM, i.e. ~ 2 mg/L) given
before these cytostatics, which are all mitochondrial mem-
brane depolarization inducers, antagonized their therapeutic
efficacy in a model of human hematopoietic cancers, by pre-
serving mitochondrial membrane potential.
The polyphenol resveratrol strongly diminished the cyto-
toxic effect of paclitaxel in several breast cancer cell lines
(MDA-MB-435s, MDA-MB-231 and SKBR-3), but not in
the cell line MCF-7 [81]. Resveratrol strongly suppressed
paclitaxel-induced death of MDA-MB-435s cancer cells
grown in athymic nude mice as xenografts. In cultured
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 7
MDA-MB-435s cells, resveratrol suppressed ROS formation
induced by paclitaxel, a well-known effect that significantly
contributes to the therapeutic efficacy of this cytostatic drug
[82]. However, part of the opposing effect of resveratrol on
paclitaxel was a consequence of resveratrol’s inhibition of
paclitaxel-induced G2/M cell cycle arrest. The application of
resveratrol alone, resulted in an accumulation of cells in the
S-phase of cell cycle. These changes subsequently led to a
resveratrol-related strong inhibition of paclitaxel-induced
Bcl-2 and Bcl-xL inactivation and, finally, to avoidance of
apoptotic cell death [81].
The final antioxidant effect on anticancer therapy de-
pends on many factors. For example, during 16 years, 1993-
2009, very meaningful results were obtained through 11
large clinical/epidemiological studies. Some of them were
very interesting in the context of cancer prevention and
treatment. The Linxian trial (China; 29584 subjects; dura-
tion: 1986-1991) was conducted in the Linxian region,
known for an extremely high incidence of gastric and eso-
phageal cancers. The subjects were receiving various combi-
nations of antioxidants (#1+#2, #1+#3, #1+#4, #2+#3,
#2+#4, #3+#4, #1+#2+#3+#4; #1: retinol palmitate and zinc;
#2: riboflavin and niacin; #3: vitamin C and molybdenum;
#4: β-carotene, selenium and α-tocopherol), during more
than five years (63 months) [83,84]. While there was no sta-
tistically significant results that would indicate the preven-
tive potential of these compounds, the lethality of cancer was
significantly decreased in a group that was receiving combi-
nation #4. However, this study did not address the possibility
of continuous exposure to a harmful agent. Accordingly, it
seems to be hard to observe a beneficial effect of antioxi-
dants without the prior eradication of an agent that may be
included in cancer occurrence. Indeed, almost ten years after
these studies were published, a very high incidence of H.
pilory infection in this population was shown [85]. If the
bacterium, a well-known promoter of NF-κB associated sig-
naling pathways, was eradicated before antioxidants applica-
tion, the question is whether these results would be the same
[86]. Recently published paper showed an increased synthe-
sis of nitric oxide (consequential of a positive effect of NF-
κB on nitric oxide synthase) in H. pilory infected tissue,
leading to an increased activity of DNA methyltransferase
(DNMT) and hypermethylation of E-cadherin promoter with
sequential silencing of this tumor suppressor gene [87].
6. POLYPHENOLS AS ENHANCERS AND INHIBI-
TORS OF ANTIOXIDATIVE DEFENSE
Apart from the radical scavenging activities, various
other types of redox bioactivity have been revealed for plant
polyphenols [12]. In subtoxic, low µM doses, polyphenols
represent mild cellular stressors, able to activate stress resis-
tance programs characterized by the up-regulation of anti-
oxidant systems, increased resistance to oxidative stress and
suppression of excessive inflammation [61]. Resveratrol,
curcumin, flavonoids, and green tea catechins have been
reported to induce such adaptive responses in normal cells
and this represents one of the basic mechanisms of their can-
cer chemopreventive activity [88]. These compounds may
also minimize the damaging effects of free radicals through
participation in the regeneration of antioxidants (e.g. α-
tocopherol).
Many polyphenols stimulate defensive cellular mecha-
nisms through activating NRF2 (Fig. 2b) [40]. The final ef-
fect on NRF2 can be positive or negative, depending in large
part on the polyphenol type and concentration as well as the
cell type. For example, in human hepatocellular carcinoma
cells HepG2, quercetin (≤ 40 µM) enhanced NRF2/ARE
activity through up-regulation of NRF2 at both the mRNA
and protein levels [89]. At the same time, quercetin repressed
protein level of KEAP1. These effects were both found to be
essential for the transcription activation of the NQO1 gen e
which codes for a quinone reductase [89]. This antioxidative
enzyme contributes to maintaining ubiquinone and α-
tocopherol quinone in their reduced forms and prevents oxi-
dative DNA damage by ROS [32]. Another example shows
that treatment of HepG2 cells with sub-toxic doses of quer-
cetin (10 and 25 µM), significantly increased the nuclear
translocation of NRF2 and the nuclear content of phosphory-
lated NRF2 [90]. However, the treatment with 50 µM quer-
cetin, decreased the level of phosphorylated NRF2 and the
nuclear/cytosolic NRF2 ratio. In HepG2 cells, quercetin was
found to modulate NRF2 activity through targeting the p38-
MAPK pathway [90].
NRF2 is overexpressed in many cancer types where it
acts as a promoter of tumor growth [91]. This is even more
important in light of a discovery showing that some onco-
genes (K-Ras, B-Raf) induce NRF2 transcription, promote
ROS elimination and enhan ce tumorigenesis [92]. The acti-
vation of the NRF2-mediated signaling pathway has also
been associated with developing chemoresistance in many
different cancer models and the inhibition of NRF2 is re-
garded as a novel therapeutic approach to target for over-
coming resistance to chemotherapy [18].
Recently, flavonoids have been reported to downregulate
the NRF2 signaling pathway in cancer cells. This discovery
may be promising for their use as chemosensitizers, to pre-
vent or reverse resistance to cytostatic drugs. Luteolin (Fig.
1) has been found to inhibit, in a redox-independent way,
NRF2/ARE-driven gene expression in human lung adeno-
carcinoma cells A549 [93]. In these cells, which possess
constitutively active NRF2, luteolin elicited a dramatic re-
duction in NRF2 at both mRNA and protein levels, leading
to decreased NRF2 binding to AREs, down-regulation of
ARE-driven genes, and depletion of reduced glutathione.
Consequentially, luteolin significantly sensitized A549 cells
to various anticancer drugs including oxaliplatin, bleomycin,
and doxorubicin. Luteolin (40 mg/kg) has also been found to
enhance the anticancer effect of cisplatin in vivo [91]. In
these experiments, non-small-cell lung cancer (NSCLC)
cells, originating from cell line A549, were subcutaneously
inoculated into nude mice and cisplatin was administrated
intraperitoneally. Inhibition of the tumor growth and de-
creased cellular proliferation, joined with reduced expres-
sions of NRF2 and antioxidant enzymes, were observed in
the tumor xenograft tissues.
Luteolin (1, 5 and 10 µM) has also been reported to re-
store sensitivity to oxaliplatin, cisplatin and doxorubicin in
oxaliplatin - resistant colorectal cell lines (HCT116-OX and
SW620-OX) [94]. The effect was achieved through a dose-
dependent inhibition of NRF2, which was adaptively acti-
vated in resistant cell lines as compared with parental ones.
8 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić et al.
Fig. (2). (a) Multiple anti- and pro-oxidative activities of polyphenols have been demonstrated so far. (b) Modulation of NRF2 signaling is
one of the major actions associated with polyphenols and should be explored in the future.
Finally, luteolin (40 mg/kg) was shown to inhibit protein
expression of NRF2 target genes (NQO1, HO-1 and GST
α1/2) and decrease level of the reduced form of glutathione
in vivo, in wild type mouse small intestinal cells.
Similarly to luteolin, apigenin (Fig. 1) was shown to re-
store sensitivity to doxorubicin in doxorubicin-resistant he-
patocellular carcinoma BEL-7402 cells, BEL-7402/ADM
[95]. This phenomenon was associated with decreasing
NRF2 at both mRNA and protein levels through down-
regulation of the PI3K/Akt pathway upon treatment with
apigenin (10 µM). In BEL-7402 xenografts, apigenin and
doxorubicin acted synergistically, as shown through the
stronger inhibition of tumor growth, decreased rate of cellu-
lar proliferation and an increased rate of apop tosis.
Chrysin (Fig. 1) was also shown to enhance the che-
mosensitivity of BEL-7402/ADM cells to doxorubicin [96].
Mechanistically, in BEL-7402/ADM cells pretreated with
chrysin (10 - 20 µM) for 24 h, both mRNA and protein levels
of NRF2 were found to be significantly decreased through
the down-regulation of PI3K-Akt and ERK pathways. Con-
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 9
sequently, there was reduced expression of NRF2-
downstream genes HO-1, AKR1B10 (aldo-keto reductase
family 1 member B10) and MRP5.
Luteolin (3',4',5,7-tetrahydroxyflavone), apigenin (4',5,7-
trihydroxyflavone) and chrysin (5,7-dihydroxyflavone) share
a 5,7-dihydroxy-4H-chromen-4-one scaffold (Fig. 2b),
which may correspond to a pharmacophore important for
their common mode of action - down-regulation of NRF2.
All three polyphenols are only moderately strong to poor
free radical scavengers [50].
The effects of polyphenols on NRF2-regulated cellular
defense, including responsiveness to chemotherapy, may
depend on the basal cellular level of NRF2 [97]. Chemopre-
ventive agents of various chemical classes, including the
polyphenol EGCG, increased NRF2 and detoxification en-
zymes level in breast cancer cell lin e MCF-7, a cell line with
a very low basal NRF2 level. This increase was shown to be
inversely correlated with a chemosensitiv ity to cytotoxic
drugs. These effects were less prominent in breast cancer cell
line MDA-231 with intermediate amount of NRF2. In lung
cancer cells A549 with high NRF2 basal level, neither of
chemoprev entive compounds studied, had effect on NRF2,
but did enhance chemoresistance.
The antioxidant enzymes thioredoxin reductases are also
very often upregulated in cancer cells [39]. Curcumin (Fig.
1) has been found to irreversibly, and in NADPH-dependent
manner, inhibit the antioxidant activity of both cytosolic and
mitochondrial variants of thioredoxin reductase by the cova-
lent modification of the active site amino acid residues
Cys496 and selenocysteine Sec497 [39]. The irreversibly
modified enzyme had an increased NADPH oxidase activity,
and due to related increased ROS production, cancer cells
may become more sensitive to anticancer agents.
7. POLYPHENOLS AS PRO-OXIDANTS
Some polyphenols have an ambivalent redox character,
acting as antioxidants and pro-oxidants (Fig. 2a). Which
function will be active depends on many factors. Polyphe-
nols are prone to act as pro-oxidants when applied at high
concentrations and in the presence of an excessive availabil-
ity of transition metal ions [98,99]. In the latter case, poly-
phenols can induce a pro-oxidative effect by electron dona-
tion to Fe(III) or Cu(II), thus supporting the generation of
H2O2 and free radicals that have the potential for inducing
DNA damage [98,100-102]. Since in vivo polyphenols target
cancer cells selectively, their pro-oxidant action may thus
enhance the cytotoxic effects of some anticancer drugs and,
accordingly, permit reducing the administered dose of a cy-
tostatic [31]. Pro-apoptotic features of some polyphenols
against cancer cells may also be ascribed to the formation of
the pro-oxidant quinone forms and their quinone methide
isomers (Fig. 3). The polyphenolic quinones and quinone
methides (Fig. 3) are reactive, electrophilic (bio)chemical
agents. They may induce severe cellular damage through
creating adducts with various nucleophilic biomolecules
(glutathione, DNA and proteins) and also through ROS pro-
duction in the process of their redox cycling [103-105]. It is
also possible that a polyphenol, as shown for quercetin, de-
creases the intracellular production of ROS and, at the same
time, produces peroxides in the cell culture medium, promot-
ing ROS influx from the medium and, thus, disturbing intra-
cellular redox homeostasis and signaling [105].
The antitumor activity of many chemotherapeutic drugs
is actually mediated by reactive quinones. For example, oxi-
dation to a reactive quinone is the mechanistic basis of action
of etoposide, a phenolic anticancer drug [106]. The carcino-
genic propensity of (xeno)estrogens has also been associated
with the formation of electrophilic and redox active quinones
[107]. On the other hand, mitomycin C is already a quinone
and it is reductively activated to semiquinone free radical,
which is further biotransformed into DNA alkylating me-
tabolites with strong cytotoxic potential [108]. Bioactivation
of mitomycin C is also joined with production of ROS, in-
cluding the •OH radical.
Polyphenols that act as pro-oxidants through the forma-
tion of (semi)quinones, are primarily those with catechol or
pyrogalloyl rings, such as quercetin and EGCG, respectively
(Fig. 1) [109, 110]. By donating an H-atom, such a polyphe-
nol is transformed to a semiquinone form which can further
donate a second H-atom and convert to quinone (Fig. 3) [70].
Such molecules are generally very efficient radical scaven-
gers, able to reduce two and/or more free radicals [111].
Their quinone forms can also readily resume their parent
reducing aglycon forms by redox-cycling, thereby producing
ROS like O2
•- and •OH. Additionally, polyphenols can be
metabolised to quinone forms by oxidative enzymes such as
cytochrome P450 and peroxidases [112]. In some types of
cells, the pro-oxidant effect of polyphenols may be very pro-
nounced due to the presence of enzymes which may
biotransform phenols to catechols, such as tyrosinase in
melanoma cells [112].
Both the reactivity and mechanism of biological action, in-
cluding cytotoxicity, of the ortho-quinones derived from the
catechol fragment, depend largely upon the rest of the molecu-
lar scaffold. If there is a possibility for extended π-electron
conjugation, ortho-quinones (3,5-cyclohexadien-1,2-dione)
may isomerize to quinone methides (4-methylene-2,5-
cyclohexadien-1-one) (Fig. 3) which may have different mode
of action, acting primarily through alkylation. For example,
human melanoma cells UIC-SO-MEL-2, which have high
levels of tyrosinase, were treated by two sub-groups of
catechols [112]. The lifetime of ortho-quinones which did not
isomerize to quinone methides, significantly linearly corre-
lated with cytotoxicity. The catechols which formed long-
lived ortho-quinones, were the most cytotoxic. In difference,
catechols with ortho-quinones which readily isomerize to
para-quinone methides, were equally cytotoxic, but no corre-
lation between the lifetimes of these ortho-quinones and the
cytotoxic potency was observed. Similarly, by studying the set
of five flavonoids: eriodictyol, luteolin, 4′-hydroxyflavanone,
3′,4′-dihydroxyflavone and genistein, it was suggested that the
quinone forms of the flavonoids with the double C2=C3 bond
in C-ring (luteolin, 3′,4′-dihydroxyflavone and genistein) (Fig.
1), can be rapidly hydrated to the unreactive species and,
hence, are unlikely to damage important biological macro-
molecules [113]. However, the quinone forms of the flavon-
oids with the single C2-C3 bond and without extended π-
electron conjugation, were observed to have relatively higher
aqueous stability and longer half-lives (> 1 s), and thus are
able to modify biological macromolecules.
10 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić e t al.
Fig. (3). Quercetin can be transformed into several quinone forms of different concentrations and reactivity. Luteolin can form only ortho-
quinone.
Quercetin, a flavonoid with the catechol moiety conju-
gated with double C2=C3 bond in C-ring and also 3-OH
group (Fig. 1), can act as a cytotoxic pro-oxidant after its
metabolic activation to semiquinone and various quinone
products (Fig. 3). These metabolites can undergo redox-
cycling associated with free radical productions [109]. At the
cellular level, quercetin’s activity has been shown to depend
on the presence of H2O2 [114]. Quercetin induces the forma-
tion of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of
oxidative DNA damage, in HL-60 cells. In their H2O2-
resistant clone, HP-100 cells, this effect does not occur.
Luteolin does not produce such damaging effects. It, as
compared with quercetin, misses the 3-OH group and can be
transferred only into the ortho-quinone form (Fig. 3) which
is readily inactivated by hydration [113].
At low concentrations (10-25 µM), quercetin exerted pro-
tective effects against H2O2-induced cytotoxicity in rat hepa-
toma cells H4IIE [115]. When present at high concentrations
(> 50 µM), it showed a cytotoxic effect, characterized by
DNA strand break and fragmentation, as well as caspase
activation. Similarly, when applied to hepatocellular carci-
noma cell lines HA22T/VGH and HepG2 at a concentration
of 80 µM, quercetin induced the production of O2
•- and, to a
lesser extent, H2O2 [103]. At the same time, it increased the
levels of malondialdehyde (that is induced membrane lipid
peroxidation) during the apoptotic process. Pretreatment of
HA22T/VGH cells with quercetin (> 40 µM) 24 h before the
addition of microtubule-targeting paclitaxel (0.1 µM), sig-
nificantly enhanced the cytotoxic effects [103]. Since ROS
production was already shown to be involved in paclitaxel-
induced apoptosis in hepato ma cells [6], these observations
may be ascribed to the synergetic pro-oxidant-ROS generat-
ing activity of both substances.
Similarly, in A549 cells, quercetin has a protective role at
concentrations below 10 µM [105]. It increases the propor-
tion of living cells, reduces the rate of apoptotic and necrotic
fractions and increases total antioxidant capacity. However,
at concentrations ≥ 50 µM, quercetin produces concentra-
tion-dependent cytotoxic effects, manifested through in-
creased amounts of apoptotic and necrotic cells.
The redox effect of quercetin on the cancer cells in vitro,
also depends on the exposure time. In terms of exposure
time, the co-treatment of human colorectal tumor cells
HCT116 or prostate cancer cells PPC-1 with quercetin (≤ 50
µM) and anti-microtubule drugs taxol (0.1 µM) or nocoda-
zole (10 µM) (both drugs induce G2/M arrest), was shown to
be bimodal [116]. A short-term activity of quercetin against
chemotherapeutic drugs was cytoprotective: for example, at
72 h treatment of HCT116, the viability index of nocodazole
treated cells was 65%. However, cellular viability was com-
parable to that of vehicle treated control cells when nocoda-
zole was applied in combination with 50 µM quercetin. This
antagonistic effect of quercetin, characterized by absence of
G2/M arrest, was not related to a lack of uptake or increased
efflux of the anti-microtubule drug. A mechanistic analysis
showed that a combined treatment results in lack of proper
mobilization of B1-CDK complex to microtubule organizing
center to initiate mitosis [117]. However, long-term applica-
tions of the combined treatments were inhibitory to cancer
cell growth. When tested in colony formation assay over 8
days, the combination of quercetin and taxol synergistically
suppressed the clonogenic survival of treated cells [116].
In vivo, however, the most important effect of quercetin
given to laboratory animals observed so far, is the increasing
bioavailability of orally applied anticancer drugs. In rats,
paclitaxel bioavailability upon oral intake was shown to be
significantly enhanced if the animals had been pretreated
with quercetin [118]. This effect of quercetin is considered to
be a result of its inhibitory effects on the efflux pump MDR1
and the CYP450 isoform CYP3A4 that is responsible for
converting paclitaxel to its principal, but inactive, metabolite
6α-hydroxytaxol. A similar effect (increased bioavailability),
was also observed in rats treated with quercetin and doxoru-
bicin [119].
Even more than quercetin, EGCG may be an important
generator of ROS and, accordingly, a pro-oxidative sub-
stance [75,101]. In a dose-dependent manner, EGCG inhib-
ited the growth of human lung cancer H1299 cells in vitro
and in vivo (xenograft tumors), with IC
50 values at 20 µM
and 0.15 µM, respectively [120]. It is interesting that the
effective concentrations of EGCG in xenograft tumors are
two orders of magnitude lower than those observed against
H1299 cells in culture. EGCG was reported to induce oxida-
tive stress in H1299 cells and also in the xenograft model
[120]. This is of great importance for understanding EGCG’s
action, since it can, even at concentrations lower than 50 µM,
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 11
induce apoptosis of cancer cells through generation of intra-
cellular ROS. Its pro-oxidative activity is mediated particu-
larly by H2O2, generated through its auto-oxidation [121].
H2O2 is a strong endogenous oxidant which reacts with tran-
sition metal centers. The increase in H2O2 levels may trigger
Fe(II)-dependent formation of highly toxic •OH, which, in
turn, induces apoptotic cell death. It also reacts with seleno-
proteins, and SH groups in selected proteins such as catalase,
glutathione peroxidases, and peroxiredoxins [122]. Such a
specifically localized production of H2O2, thus, may modify
specific SH groups which may further induce changes in
specific redox-signaling pathways [27]. The cells with lower
H2O2-eliminating activity have been found to be more sensi-
tive to EGCG [120]. The cell line A549 with high NRF2
basal level, is markedly resistant to the induction of apopto-
sis by EGCG (even at 100 µM for 72 h). Similarly as in the
case of chemoresistance induced by conventional che-
motherapeutics, the resistance to EGCG in these cells is
linked to NRF2-mediated overexpression of HO-1 protein
[123]. While HO-1 has a cytoprotective function in normal
cells, the abnormally elevated expression of HO-1 in cancer
cells, contributes to developing resistance to chemotheraphy
[124]. However, a combination of EGCG (30 µM) with lute-
olin (10 µM) which is an efficient NRF2 inhibitor, synergis-
tically increased apoptosis in head and neck as well as lung
cancer cell lines, including A549 [125].
CONCLUSION AND PERSPECTIVES
Side-effects associated with the application of conven-
tional cytostatics are a major concern. Herein, we discussed
the potential use of natural polyphenols as adjuvants for the
purpose of reducing the side effects on normal cells and/ or
increasing selectivity on cancer cells.
Plant polyphenols (Fig. 1) are generally recognized and
have been studied as antioxidants. Some of them act as effi-
cient scavengers of various free rad icals and interrupters of
radical chain reactions. Some polyphenols are inhibitors of
radical producing enzymes like NADPH oxidases and xan-
thine oxidase. Many polyphenols have also been found to
induce the transcription activity of NRF2, the main regulator
of antioxidant and detoxifying enzymes. Their antioxidative
activities provide the mechanistic basis for their anti-
inflammatory and chemopreventive capacity and may b e
expected to alleviate side-effects of the cytostatics. However,
recently, NRF2 has been shown to be overexpressed in can-
cer cells of various types, participating in d eveloping their
defense machinery, and associated with developing
chemoresistance to cytostatics. Luteolin, apigenin and chry-
sin, have been reported to reduce NRF2 expression at both
mRNA and protein levels and restore sensitivity to cytostat-
ics. Their common 5,7-dihydroxy-4H-chromen-4-one frag-
ment may provide a starting point for the hit-to-lead design
of novel chemosensitizing adjuvants to conventional anti-
cancer drugs.
Considering possible use of polyphenols as anticancer
therapy adjuvants, their pro-oxidant activity should not be
neglected. Polyphenols with catechol and/ or pyrogallol
fragments which are able to form complexes with transition
metal ions like Cu (II) and/or form quinone forms and un-
dergo redox cycling, and thus cause ROS production, may,
synergistically with conventional anticancer drugs, produce
cytostatic effects selectively on cancer cells.
The biological activity of polyphenols greatly depends
upon the dose applied, the type of cell, exposure time and
environmental conditions. Their oral bioavailability is often
restricted, as well. Therefore, we strongly recommend exten-
sive clinical studies of the application of combination thera-
pies to specific cancer types along with developing novel
polyphenol formulations to be included in such studies.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Science,
Education and Sport, Republic of Croatia. Authors are thank-
ful to Mr. Aaron Etra for a careful revision of the text.
REFERENCES
[1] Centers for Disease Control and Prevention.
http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm (Ac-
cessed July 4, 2014).
[2] http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted
(Accessed December 15, 2014)
[3] Pignatti, F.; Jonsson, B.; Blumenthal, G.; Justice R. Assessment of
benefits and risks in development of targeted therapies for cancer -
The view of regulatory authorities. Mo l. Oncol., 2014, DOI: .
[4] Rang, H.P.; Dale M.M.; Ritter J.M.; Flower R.J. Rang & Dale's
Pharmacology, 6th Ed.; Elsevier: Edinburgh, 2007.
[5] Lawenda, B.D.; Kelly, K.M.; Ladas, E.J.; Sagar, S.M.; Vickers, A.;
Blumberg J.B. Should supplemental antioxidant administration be
avoided during chemotherapy and radiation therapy? J. Natl. Can-
cer Inst., 2008, 100(11), 773-783.
[6] Ramanathan, B.; Jan, K.Y.; Chen, C.H.; Hour, T.C.; Yu, H.J.; Pu,
Y.S. Resistance to paclitaxel is proportional to cellular total anti-
oxidant capacity. Cancer Res., 2005, 65(18), 8455-8460.
[7] Benedict, W.F.; Banerjee, A.; Venkatesan, N. Cyclophosphamide-
induced oncogenic transformation, chromosomal breakage, and sis-
ter chromatid exchange following microsomal activation. Cancer
Res., 1978, 38(9), 2922-2924.
[8] Zheng, J.; Lee, H.C.; B in Sattar, M.M.; Huang, Y.; B ian, J.S. Car-
dioprotective effects of epigallocatechin-3-gallate against doxoru-
bicin-induced cardiomyocyte injury. Eur. J. Pharmacol., 2011,
652(1-3), 82-88.
[9] Domitrović, R.; Cvijanović, O.; Pugel, E.P.; Zagorac, G.B.;
Mahmutefendić, H.; Škoda, M. Luteolin ameliorates cisplatin-
induced nephrotoxicity in mice through inhibition of platinum ac-
cumulation, inflammation and apoptosis in the kidney. Toxicology,
2013, 310, 115-123.
[10] Khurana, S.; Piche, M.; Hollingsworth, A.; Venkataraman, K.; Tai,
T.C. Oxidative stress and cardiovascular health: therapeutic
potential of polyphenols. Can. J. Physiol. Pharmacol., 2013, 91(3),
198-212.
[11] Gupta, S.C.; Tyagi, A.K.; Deshmukh-Taskar, P.; Hinojosa, M.;
Prasad, S.; Aggarwal, B.B. Downregulation of tumor necrosis
factor and other proinflammatory biomarkers by polyphenols.
Arch. Biochem. Biophys., 2014, 559, 91-99.
[12] Stepanić V.; Novak Kujundžić R.; Gall Trošelj K. In: Epigenetics
and Epigenomics; Payne, C., Ed.; InTech: Rijeka, 2014; pp. 173-
209.
[13] Bentires-Alj, M.; Barbu, V.; Fillet, M.; Chariot, A.; Relic, B.;
Jacobs, N.; Gielen, J.; Merville, M.P.; Bour, V. NF-κB transcrip-
tion factor induces drug resistance through MDR1 expression in
cancer cells. Oncogene, 2003, 22(1), 90-97.
[14] Duan, J.; Mansour, H.M.; Zhang, Y.; Deng, X.; Chen, Y.; Wang, J.;
Pan, Y.; Zhao, J. Reversion of multidrug resistance by co-
encapsulation of doxorubicin and curcumin in chitosan/poly(butyl
12 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić e t al.
cyanoacrylate) nanoparticles. Int. J. Pharm., 2012, 426(1-2), 193-
201.
[15] Sreekanth, C.N.; Bava, S.V.; Sreekumar, E.; Anto, R.J. Molecular
evidences for the chemosensitizing efficacy of liposomal curcumin
in paclitaxel chemotherapy in mouse models of cervical cancer.
Oncogene, 2011, 30(28), 3139-3152.
[16] Gall Trošelj, K.; Kujundžić Novak, R. Curcumin in combined
cancer therapy. Curr. Pharm. Des., 2014, 20(42) 6682-6696 .
[17] Kim, T.H.; Shin, Y.J.; Won, A.J.; Lee, B.M.; Choi, W.S.; Jung,
J.H.; Chung, H.Y.; Kim, H.S. Resveratrol enhances chemosensitiv-
ity of dox orubicin in multidrug-resistant human breast cancer cells
via increased cellular influx of doxorubicin. Biochim. Biophys.
Acta., 2014, 1840(1) 615-625.
[18] Wang. X.J.; Sun, Z.; Villeneuve, N.F.; Zhang, S.; Zhao, F.; Li, Y.;
Chen, W.; Yi, X.; Zheng, W.; Wondrak, G.T.; Wong, P.K.; Zhang,
D.D. Nrf2 enhances resistance of cancer cells to chemotherapeutic
drugs, the dark side of Nrf2. Carcinogenesis, 2008, 29(6), 1235-
1243.
[19] Del Rio, D.; Rodriguez-Mateo s, A.; Spencer, J.P.; Tognolini, M.;
Borges, G.; Crozier, A. Dietary (poly)phenolics in human health:
structures, bioavailability, and evidence of protective effects
against chronic diseases. Antioxid. Redox Signal., 2013, 18(14),
1818-1892.
[20] Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.; Mazur, M.;
Telser, J. Free radicals and antioxidants in nor mal ph ysiological
functions and human disease. Int. J. Biochem. Cell Biol., 2007,
39(1) 44-84.
[21] Paschos, A.; Pandy a, R.; Duivenvoo rden, W.C.M.; Pinthus, J.H.
Oxidative stress in prostate cancer: changing research concepts to-
wards a novel paradigm for prevention and therapeutics. Prost.
Cancer Prost. Dis., 2013, 16(3), 217-225.
[22] Halliwell, B.; Clement, M.V.; Long, L.H. Hydrogen peroxide in the
human body. FEBS Lett., 2000, 486(1), 10-13.
[23] Dröge, W. Free radicals in the physiological control of cell func-
tion. Physiol. Rev., 2002, 82(1), 47-95.
[24] Park, J.E.; Yang, J.H.; Yoon, S.J.; Lee, J.H.; Yang, E.S.; Park, J.W.
Lipid peroxidation-mediated cytotoxicity and DNA damage in
U937 cells. Biochimie, 2002, 84(12), 1199-1205.
[25] Wallace, S.S. Biological consequences of free radical-damaged
DNA bases. Free Radic. Biol. Med., 2002, 33(1), 1-14.
[26] Jung, T.; Höhn, A.; Grune, T. The proteasome and the degradation
of oxidized proteins: Part III-Redox regulation of the proteasomal
system. Redox Biol., 2014, 2, 388-394.
[27] Jones, D.P. Redox sensing: orthogonal control in cell cycle and
apoptosis signalling. J. Intern. Med., 2010, 268(5), 432-448.
[28] Jones, D.P. Radical-free biology of oxidative stress. Am. J. Physiol.
Cell Physiol., 2008, 295(4), C849-C868.
[29] von Montfort, C.; Sharov, VS.; Metzger, S.; Schöneich ,C.; Sies,
H.; Klotz, L.O. Singlet oxygen inactivates protein tyrosine phos-
phatase-1B by oxidation of the active site cysteine. Biol. Chem.
2006, 387(10-11), 13 99-1404.
[30] Čipak Gašparović, A.; Lovaković, T.; Žarković, N. Oxidative stress
and antioxidants: biological response modifiers of oxidative ho-
meostasis in cancer. Period. Biol., 2010, 112(4), 433-439.
[31] Trachootham, D.; Lu, W.; Ogasawara, M.A.; Nilsa, R.-D.V.;
Huang, P. Redox regulation of cell survival. Antioxid. Redox Sig-
nal., 2008, 10(8), 1343-1374.
[32] Jung, K.-A.; Kwak, M.-K. The Nrf2 system as a potential target for
the development of indirect antioxidants. Molecules, 2010, 15(10),
7266-7291.
[33] Augustyniak, A.; Bartosz, G.; Čipak, A.; Duburs, G.; Horáková, L.;
Luczaj, W.; Majekova, M.; Odyssoes, A.D.; Rackova, L.; Skrzy-
dlewska, E.; Stefek, M.; Štrosová, M.; Tirzitis, G.; Venskutonis,
P.R.; Viskupicova, J.; Vraka, P.S.; Žarković, N. Natural and syn-
thetic antioxidants: an updated overview. Free Radic. Res., 2010,
44(10), 1216-1262.
[34] Adachi, T.; Nakagawa, H.; Chung, I.; Hagiya, Y.; Hoshijima, K.;
Noguchi, N.; Kuo, M.T.; Ishikawa, T. Nrf2-dependent and -
independent induction of ABC transporters ABCC1, ABCC2, and
ABCG2 in HepG2 cells under oxidative stress. J. Exp. Ther. On-
col., 2007, 6(4), 335-348.
[35] Bryan, H.K.; Olayanju, A.; Goldring, C.E.; Park, B.K.The Nrf2 cell
defence pathway: Keap1-dependent and -independent mechanisms
of regulation. Biochem Pharmacol. 2013, 85(6), 705-717.
[36] Sporn, M.B; Liby, K.T. NRF2 and cancer: the good, the bad and
the importance of co ntext. Nat. Rev. Cancer., 2012, 12(8), 564-571.
[37] Bao, L.J.; Jaramillo, M.C.; Zhang, Z.B.; Zheng, Y.X.; Yao, M.;
Zhang, D.D.; Yi, X.F. Nrf2 induces cisplatin resistan ce through ac-
tivation of autophagy in ovarian carcinoma. Int. J. Clin. Exp.
Pathol., 2014, 7(4), 1502-1513.
[38] Jaramillo, M.C.; Zhang, D.D. The emerging role of the Nrf2-Keap1
signaling pathway in cancer. Genes Dev., 2013, 27(20) , 2179-2191.
[39] Fang, J.; Lu , J.; Holmgren, A. Thioredoxin reductase is irreversibly
modified by curcumin: a novel molecular mechanism for its anti-
cancer activity. J. Biol. Chem., 2005, 280(26), 25284-25290.
[40] Dangles, O. Antioxidant activity of plant phenols: chemical
mechanisms and biological significance. Curr. Org. Chem., 2012,
16(6), 692-714.
[41] Sosa, V.; Moliné, T.; Somoza, R.; Paciucci, R.; Kondoh, H.;
LLeonart, M.E. Oxidative stress and cancer: an overview. Ageing
Res. Rev., 2013, 12(1), 376-390 .
[42] Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: the next gen-
eration. Cell, 2011, 144(5) 646-674.
[43] Mahalingaiah , P.K.; Singh, K.P. Chronic o xidativ e stress increases
growth and tumorigenic potential of MCF-7 breast cancer cells.
PLoS One, 2014, 9(1), e87371.
[44] Toyokuni, S.; Okamoto, K.; Yodoi, J.; Hiai , H. Persistent oxidative
stress in cancer. FEBS Lett., 1995, 358(1), 1-3.
[45] Reuter, S.; Gupta, S.C.; Chaturvedi, M.M.; Aggarwal, B.B. Oxida-
tive stress, inflammation, and cancer: how are they linked? Free
Radic. Biol. Med., 2010, 49(11), 1603-1616.
[46] Maraldi T. Natural compounds as modulators of NADPH oxidases.
Oxid. Med. Cell. Longev., 2013, 2013, ID 271602.
[47] Federico, A.; Morgillo, F.; Tuccillo, C.; Ciardiello, F.; Loguercio,
C. Chronic inflammation and oxidative stress in human carcino-
genesis. Int. J. Cancer, 2007, 121(11), 2381-2386.
[48] Kujundžić Novak, R.; Žarković N.; Gall Trošelj K. Pyridine nu-
cleotides in regulation of cell death and survival by redox and non-
redox reactions. Crit. Rev. Eukaryot. Gene Expr., 2014, 24 (4), 287-
309.
[49] Kamp, D.W.; Shacter, E.; Weitzman, S.A. Chronic inflammation
and cancer: the role of the mitochondria. Oncology (New York),
2011, 25(5), 400-413.
[50] Cai, Y.Z.; Sun. M.; Xing, J.; Luo, Q.; Corke, H. Structure-radical
scavenging activity relationships of phenolic compounds from tra-
ditional Chinese medicinal plants. Life Sci., 2006, 78(25), 2872-
2888.
[51] Stepanić, V.; Gall Trošelj, K.; Lučić, B.; Marković, Z.; Amić, D.
Bond dissociation free energy as a general parameter for flavonoid
radical scavenging activity. Food Chem., 2013, 141(2) , 1562 -1570.
[52] Lamoral-Theys, D.; Pottier, L.; Dufrasne, F.; Nève, J.; Dubois, J.;
Kornienko, A.; Kiss, R.; Ingrassia, L. Natural polyphenols that dis-
play anticancer properties through inhibition of kinase activity.
Curr. Med. Chem., 2010, 17(9), 812-825.
[53] Boly, R.; Gras, T.; Lamkami, T.; Guissou, P.; Serteyn, D.; Kiss, R.;
Dubois, J. Quercetin inhibits a large panel of kinases implicated in
cancer cell biology. Int. J. Oncol., 2011, 38(3), 833-842.
[54] Wang, Y.J.; Pan, M.H.; Cheng, A.L.; Lin, L.I.; Ho, Y.S.; Hsieh,
C.Y.; Lin, J.K. Stability of curcumin in buffer solutions and charac-
terization of its degradation products. J. Pharm. Biomed. Anal.,
1997, 15(12) , 1867-1876.
[55] de Boer, V.C.J.; de Goffau, M.C.; Arts, I.C.W.; Hollman, P.C.H.;
Keijer, J. SIRT1 stimulation by polyphenols is affected by their
stability and metabolism. Mech . Ageing Dev., 2006, 127(7), 618-
627.
[56] Chiou, Y.S.; Wu, J.C.; Huang, Q.; Shahidi, F.; Wang, Y.J.; Ho,
C.T.; Pan, M.H, Metabolic and colonic microbiota transformation
may enhance the bioactivities of dietary polyphenols, J. Funct.
Foods, 2014, 7(1), 3-25.
[57] Day, A.J.; Bao, Y.; Morgan , M.R.; Williamson, G. Conjug ation
position of quercetin glucuronides and effect on biological activity.
Free Radic. Biol. Med., 2000, 29(12), 1234-1243.
[58] Kawai, Y.; Nishikawa, T.; Shiba, Y.; Saito, S.; Murota, K.; Shibata,
N.; Kobayashi, M.; Kanayama, M.; Uchida, K.; Terao, J. Macro-
phage as a target of quercetin glucuronides in human atheroscle-
rotic arteries: implication in the anti-atherosclerotic mechanism of
dietary flavonoids. J. Biol. Chem., 2008, 283(14), 9424-9434.
[59] Kawai Y. β-Glucuronidase activity and mitochondrial dysfunction:
the sites where flavonoid glucuronides act as anti-inflammatory
agents. J. Clin. Biochem. Nutr., 2014, 54(3), 145-150.
[60] Ishisaka, A.; Kawabata K.; Miki S.; Shiba, Y.; Minekawa, S.;
Nishikawa, T.; Mukai, R.; Terao, J.; Kawai, Y. M itochondrial dys-
Selected Attributes of Polyphenols in Targeting Oxidative Stress in Cancer Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 13
function leads to deconjugation of quercetin glucuronides in in-
flammatory macrophages. PLoS One, 2013, 8(11), e80843.
[61] Son, T.G.; Camandola, S.; Mattson, M.P. Hormetic dietary phyto-
chemicals. Neuromolecular Med., 2008, 10(4), 236-246.
[62] Vargas, A.J.; Burd, R. Hormesis and synergy: pathways and
mechanisms of quercetin in cancer prevention and management.
Nutr. Rev., 2010, 68(7), 418-428.
[63] de Moura, C.F.G.; Noguti J.; de Jesus, G.P.P.; Ribeiro, F.A.P;
Garcia, F.A.; Gollucke, A.P.B.; Aguiar, O.; Ribeiro, D.A. Polyphe-
nols as a chemopreventive agent in oral carcinogenesis: putative
mechanisms of action using in-vitro and in-vivo test systems. Eur.
J. Cancer Prev., 2013, 22(5), 467-472.
[64] Amorati, R.; Foti, M.C.; Valgimigli, L. Antioxidant activity of
essential oils. J. Agric. Food Chem., 2013, 61(46), 10835-10847.
[65] Pietta, P.-G., Flavonoids as antioxidants. J. Nat. Prod., 2000, 63(7),
1035-1042.
[66] Wright, J.S.; Johnson, E.R.; DiLabio, G.A. Predicting the activity
of phenolic antioxidants: theoretical method, analysis of substituent
effects, and application to major families of antioxidants. J. Am.
Chem. Soc., 2001, 123(6), 1173-1183.
[67] Litwinienko, G.; Ingold, K. U. Solvent effects on the rates and
mechanisms of reaction of phenols with free radicals. Acc. Chem.
Res. 2007, 40(3), 222-230 .
[68] Weinberg, D.R.; Gagliardi, C.J.; Hull, J.F.; Murphy, C.F.; Kent,
C.A.; Westlake, B.C.; Paul, A.; Ess, D.H.; McCafferty, D.G.;
Meyer, T.J. Proton-coupled electron transfer. Chem. Rev., 2012,
112(7), 4016-4093.
[69] Alberto, M.E.; Russo, N.; Grand, A.; Galano, A. A physicochemi-
cal examination of the free radical scavenging activity of Trolox:
mechanism, kinetics and influence of the environment. Phys.
Chem. Chem. Phys., 2013, 15(13), 4642-4650.
[70] Li, D.D.; Han, R.M.; Liang, R.; Chen, C.H.; Lai, W.; Zhang J.P.;
Skibsted, L.H. Hydroxyl radical reaction with trans-resveratrol: ini-
tial carbon radical adduct formation followed by rearrangement to
phenoxyl radical. J. Phys. Chem. B., 2012, 116(24), 7154-7161.
[71] Amić, A.; Marković, Z.; Dimitrić Marković, J.M.; Stepanić, V.;
Lučić, B.; Amić, D. Towards an improved prediction of the free
radical scavenging potency of flavonoids: the significance of dou-
ble PCET mechanisms. Food Chem., 2014, 152, 578-585.
[72] de Souza, R.F.V.; De Giovani, W.F. Antioxidant properties of
complexes of flavonoids with metal ions. Redox Rep., 2004, 9(2),
97-104.
[73] Moridani, M.Y.; Pourahmad, J.; Bui, H.; Siraki, A.; O'Brien, P.J.
Dietary flavonoid iron complexes as cytoprotective superoxide
radical scavengers. Free Radic. Biol. Med. , 2003, 34(2), 243-253.
[74] Kostyuk, V.A.; Potapovich, A.I.; Strigunova, E.N.; Kostyuk, T.V.;
Afanasev, I.B. Experimental evidence that flavonoid metal com-
plexes may act as mimics of superoxide dismutase. Arch. Bioch em.
Biophys., 2004, 428(2), 20 4-208.
[75] Barik, A.; Mishra, B.; Shen, L.; Mohan, H.; Kadam, R.M.; Dutta,
S.; Zhan g, H.-Y.; Priyadarsini, K.I. Evaluation of a new copper(II)-
curcumin complex as superoxide dismutase mimic and its free
radical reactions. Free Radic. Biol. Med., 2005, 39(6), 811-822.
[76] Lambert, J.D.; Elias, R.J. The antioxidant and pro-oxidant activities
of green tea polyphenols: a role in cancer prevention. Arch. Bio-
chem. Biophys., 2010, 501(1), 65 -72.
[77] Lin, J.K.; Chen, P.C.; Ho, C.T.; Lin-Shian, S.Y. In: Free Radicals
in Food; Morello, M.; Shahidi, F.; Ho, V.T., Eds.; ACS Sympo-
sium Series; American Chemical Society: Washington, DC, 2002;
pp. 264-281.
[78] Hunyadi, A.; Martins, A.; Danko, B.; Chuang, D.-W.; Trouillas , P.;
Chang, F.-R.; Wu, Y.-C.; Falkay G. Discovery of the first non-
planar flavonoid that can strongly inhibit xanthine oxidase: pro-
toapigenone 1'-O-propargyl ether. Tetrahedron Lett., 2013, 54(48),
6529-6532.
[79] Block, K.I.; Koch, A.C.; Mead, M.N.; Tothy, P.K.; Newman, R.A.;
Gyllenhaal, C. Impact of antioxidant supplementation on che-
motherapeutic toxicity: A systematic review of the evidence from
randomized controlled trials. Int. J. Cancer, 2008, 123(6), 12 27-
1239.
[80] Heaney, M.L.; Gardner, J.R.; Karasavvas, N.; Golde, D.W.;
Scheinberg, D.A.; Smith, E.A.; O'Connor, O.A. V itamin C antago-
nizes the cytotoxic effects of antineoplastic drugs. Cancer Res.,
2008, 68(19) , 8031-8038.
[81] Fukui, M.; Yamabe, N.; Zhu, B.T. Resvera trol attenuates the anti-
cancer efficacy of paclitaxel in human breast cancer cells in vitro
and in vivo. Eur. J. Cancer, 2010, 46(10), 1882-1891.
[82] Alexandre, J.; Hu, Y.; Lu, W.; Pelicano, H.; Huang, P. Novel ac-
tion of paclitaxel against cancer cells: bystander effect mediated by
reactive oxygen species. Cancer Res., 2007, 67(8), 3512-3517.
[83] Wang, G.-Q.; Dawsey, S.M.; Li, J.-Y.; Taylor, P.R.; Li, B.; Blot,
W.J.; Weinstein, W.M.; Liu, F.S.; Lewin, K.J.; Wang, H.; Wiggett,
S.; Gail, M.H.; Yang C.S. Effects of vitamin/mineral supplementa-
tion on the prevalence of histological dysplasia and early cancer of
the esophagus and stomach: results from the General Population
Trial in Linxian, China. Cancer Epidemiol. Biomarkers Prev.,
1994, 3(2), 161-166.
[84] Taylor, P.R.; Li, B .; Dawsey, S.M.; Li, J.Y.; Yang, C.S.; Guo, W.;
Blot, W.J. Prevention of esophageal cancer: the nutrition interven-
tion trials in Linxian, China. Lin xian Nutrition Intervention Trials
Study Group. Cancer Res., 1994, 54(7 Suppl), 2029s-2031s.
[85] Limburg, P.; Qiao, Y.; Mark, S.; Wang, G.; Perez-Perez, G.; Blase r,
M.; Wu, Y.; Zou, X.; Dong, Z.; Taylor, P.; Dawsey, S. Helicobacter
pylori seropositivity and subsite-specific gastric cancer risks in
Linxian, China. J. Natl. Cancer Inst., 2001, 93(3), 226-233.
[86] Rieke, C.; Papendieck, A.; Sokolova, O.; Naumann, M. Helicobac-
ter pylori-induced tyrosine phosphorylation of IKKβ contributes to
NF-κB activation. Biol. Chem., 2011, 392(4), 387-393.
[87] Huang, F.Y.; Chan, A.O.; Rashid, A.; Wong, D.K.; Cho, C.H.;
Yuen, M.F. Helicobacter pylori induces promoter methylation of
E-cadherin via interleukin-1β activation of nitric oxide production
in gastric cancer cells. Cancer, 2012, 118(20 ), 4969 -4980.
[88] Speciale, A.; Ch irafisi, J.; Saija, A.; Cimino, F. Nutritional antioxi-
dants and adaptive cell responses: an update. Curr. Mol. Med.,
2011, 11(9), 770-789.
[89] Tanigawa, S.; Fujii, M.; Hou, D.X. Action of Nrf2 and Keap1 in
ARE-mediated NQO1 expression by quercetin. Free Radic. Biol.
Med., 2007, 42(11), 1690-1703.
[90] Granado-Serrano, A.B.; Martín, M.A.; Bravo, L.; Goya, L.; Ramos,
S. Quercetin modulates Nrf2 and glutathione-related defenses in
HepG2 cells: Involvement of p38. Chem. Biol. Interact., 2012,
195(2), 154-164.
[91] Chian, S.; Thapa, R.; Chi, Z.; Wang, X.J.; Tang, X. Luteolin inhib-
its the Nrf2 signaling pathway and tumor growth in vivo. Biochem.
Biophys. Res. Commun., 2014, 447(4), 602-608.
[92] DeNicola, G.M.; Karreth, F.A.; Humpton, T.J.; Gopinath an, A.;
Wei, C.; Frese, K.; Mangal, D.; Yu, K.H.; Yeo, C.J.; Calhoun, E.S.;
Scrimieri, F.; Winter, J.M.; Hruban, R.H. Iacobuzio-Donahue C,
Kern S.E, Blair I.A, Tuveson D.A. Oncogene-induced Nrf2
transcription promotes ROS detoxification and tumorigenesis.
Nature, 2011, 475(73 54), 10 6-109.
[93] Tang, X.; Wang, H.; Fan, L.; Wu, X.; Xin, A.; Ren, H.; Wang, X.J.
Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE
pathway and sensitization of human lung carcinoma A549 cells to
therapeutic drugs. Free Radic. Biol. Med., 2011, 50(11), 1599 -16 09.
[94] Chian, S.; Li, Y.Y.; Wang, X.J.; Tang, X.W. Luteolin sensitizes
two oxaliplatin-resistant colorectal cancer cell lines to chemothera-
peutic drugs via inhibition of the Nrf2 pathway. Asian Pac. J. Can-
cer Prev., 2014, 15(6), 2911-2916.
[95] Gao, A.M.; Ke, Z.P.; Wang, J.N.; Yang, J.Y.; Chen, S.Y.; Chen, H.
Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma
BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2
pathway. Carcinogenesis, 2013, 34(8), 1806-1814.
[96] Gao, A.M.; Ke, Z.P.; Shi, F.; Sun, G.C.; Chen, H. Chrysin en-
hances sensitivity of BEL-7402/ADM cells to doxorubicin by sup-
pressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway. Chem. Biol. In-
teract., 2013, 206(1), 100-108.
[97] Hu, L.; Miao, W.; Loignon, M.; Kandouz, M.; Batist, G. Putative
chemopreventive molecules can increase Nrf2-regulated cell de-
fense in some human cancer cell lines, resulting in resistance to
common cytotoxic therapies. Cancer Chemother. Pharmacol.,
2010, 66(3), 467-474.
[98] Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L. Plant
polyphenols: chemical properties, biological activities, and synthe-
sis. Angew. Chem. Int. Ed., 2011, 50(3), 586-621.
[99] Chu, K.O.; Chan, S.-O.; Pang, C.P.; Wang, C.C. Pro-oxidative and
antioxidative controls and signaling modification of polyphenolic
phytochemicals: contribution to health promotion and disease pre-
vention? J. Agric. Food Chem., 2014, 62(18), 4026-4038.
14 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 6 Stepanić e t al.
[100] Fukumoto, L.R.; Mazza, G. Assessing antioxidant and prooxidant
activities of phenolic compounds. J. Agric. Food Chem., 2000,
48(8), 3597-3604.
[101] Kim, H.S.; Quon, M.J.; Kim, J.A. New insights into the mecha-
nisms of polyphenols beyond antioxidant properties; lessons from
the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol.,
2014, 2, 187-195.
[102] Hadi, S.M.; Bhat, S.H.; Azmi, A.S.; Hanif, S.; Shamim, U.; Ullah,
M.F. Oxidative breakage of cellular DNA by p lant polyphenols: a
putative mechanism for anticancer properties. Semin. Cancer Biol.,
2007, 17(5), 370-376.
[103] Chang, Y.F.; Chi, C. W.; Wang, J.J. Reactive oxygen species pro-
duction is involved in quercetin-induced apoptosis in human hepa-
toma cells. Nutr. Cancer, 2006, 55(2), 201-209.
[104] Hussain, H.H.; Babic, G..; Durst, T.; Wright, J.S.; Flueraru, M.;
Chichirau, A.; Chepelev, L.L. Development of novel antioxidants:
design, synthesis, and reactivity. J. Org. Chem., 2003, 68(18),
7023-7032.
[105] Robaszkiewicz, A.; Balcerczyk, A.; Bartosz, G. Antioxidative and
prooxidative effects of quercetin on A549 cells. Cell Biol. Int.,
2007, 31(10) , 1245-1250.
[106] Smith, N.A.; Byl, J.A.; Mercer, S.L.; Deweese, J.E.; Osheroff, N.
Etoposide quinone is a covalent poison of human topoisomerase
IIβ. Biochemistry, 2014, 53(19), 3229-3236.
[107] Bolton, J.L.; Thatcher, G.R. Potential mechanisms of estrogen
quinone carcinogenesis. Chem. Res. Toxicol., 2008, 21(1), 93-101.
[108] Doroshow J.H. Reductive activation of mitomycin C: a delicate
balance. J. Natl. Cancer Inst., 1992, 84(15), 1138-1139.
[109] Metodiewa, D.; Jaiswal, A.; Cenas, N.; Dickancaite, E.; Segura-
Aguilar, J. Quercetin may act as a cytotoxic prooxidant after its
metabolic activation to semiquinone and quinoidal product. Free
Radic. Biol. Med., 1999, 26(1-2), 107-116.
[110] Martin, K.R.; Appel, C.L. Polyphenols as dietary supplements: A
double-edged s word , Nutr. Diet. Suppl., 2010, 2, 1-12.
[111] Villaño, D.; Fernández-Pachón, M.S.; Moyá, M.L.; Troncoso,
A.M.; García-Parrilla, M. C. Radical scavenging ability of polyphe-
nolic compounds towards DPPH free radical. Talanta, 2007, 71(1),
230-235.
[112] Bolton, J.L.; Pisha, E.; Shen, L.; Krol, E.S.; Iverson, S.L.; Huang,
Z.; van Breemen, R .B.; Pezzuto, J.M. The reactivity of o-quinones
which do not isomerize to quinone methides correlates with alkyl-
catechol-induced toxicity in human melanoma cells. Chem. Biol.
Interact., 1997, 106(2), 133-148.
[113] Tu, T.; Giblin, D.; Gross, M.L. Structural determinant of chemical
reactivity and potential health effects of quinones from natural
products. Chem. Res. Toxicol., 2011, 24(9), 1527-1539.
[114] Yamashita, N.; Kawanishi, S. Distinct mechanisms of DNA dam-
age in apoptosis induced by quercetin and luteolin. Free Radic.
Res., 2000, 33(5), 623-633.
[115] Wätjen, W.; M ichels, G.; Steffan, B.; Niering, P.; Chovolou, Y.;
Kampkötter, A.; Tran-Thi, Q.-H.; Proksch, P.; Kahl, R. Low con-
centrations of flavonoids are protective in rat HAIIE cells whereas
high concentrations cause DNA damage and apoptosis. J. Nutr.,
2005, 135(3), 525-531.
[116] Samuel, T.; Fadlalla, K.; Turner, T.; Yehualaeshet, T.E. The fla-
vonoid quercetin transiently inhibits the activity of taxol and noco-
dazole through interference with the cell cycle. Nutr. Cancer.,
2010, 62(8), 1025-1035.
[117] Jackman, M.; Lindon, C.; Nigg, EA.; Pines, J. Active cyclin B1-
Cdk1 first appears on centrosomes in prophase. Nat. Cell. Biol.,
2003, 5(2), 143-148.
[118] Choi, J.S.; Jo, B.W.; Kim, Y.C. Enhanced paclitaxel bioavailability
after oral administration of paclitaxel or prodrug to rats pretreated
with quercetin. Eur. J. Pharm. Biopharm., 2004, 57(2), 313-318.
[119] Choi, J.S.; Piao, Y.J.; Kang, K.W. Effects of quercetin on the
bioavailability of doxorubicin in rats: role of CYP3A4 and P-gp in-
hibition by quercetin. Arch. Pharm. Res., 2011, 34(4), 607-613.
[120] Li, G.X.; Chen, Y.K.; Hou, Z.; Xiao, H.; Jin, H.; Lu, G.; Lee, M.J.;
Liu, B.; Guan, F.; Yang, Z.; Yu, A .; Yang, C.S. Pro-oxidative ac-
tivities and dose-response relationship of (-)-epigallocatechin-3-
gallate in the inhibition of lung cancer cell growth: a comparative
study in vivo and in vitro. Carcinogenesis, 2010, 31(5), 90 2-910.
[121] Nakagawa, H.; Hasumi, K.; Woo, J.T.; Nagai, K .; Wachi, M. Gen-
eration of hydrogen peroxide primarily contributes to the induction
of Fe(II)-dependent apoptosis in Jurkat cells by (-)-epigallocatechin
gallate. Carcinogenesis, 2004, 25(9), 1567-1574.
[122] Winterbourn, C.C. The biological chemistry of hydrogen peroxide.
In: Methods in Enzymolog y; Cadenas, E.; Packer, L., Eds.; Aca-
demic Press: Burlington, 2013; Vol. 528, pp. 3-25.
[123] Kweon, M.H.; Adhami, V.M.; Lee, J.S.; Mukhtar, H. Constitutive
overexpression of Nrf2 dependent heme oxygenase-1 in A549 cells
contributes to resistance to apoptosis induced by epigallocatechin
3-gallate, J. Biol. Chem., 2006, 281(44), 3361-3372.
[124] Na, H.K; Surh, Y.J. Modulation of Nrf2-mediated antioxidant and
detoxifying enzyme induction by the green tea polyphenol EGCG.
Food Chem. Toxicol., 2008, 46(4), 1271-1278.
[125] Amin, A.R.; Wang, D.; Zhang, H.; Peng, S.; Shin, H.J.; Brandes,
J.C.; Tighiouart, M.; Khuri, F.R.; Chen, Z.G.; Shin, D.M. Enhanced
anti-tumor activity by the combination of the natural compounds (-
)-epigallocatechin-3-gallate and luteolin: potential role of p53. J.
Biol. Chem., 2010, 285(45), 34557-34565.
Received: ???????????????? Revised: ???????????????? Accepted: ??? ?????????????
