Báo cáo khoa học: Natural polyphenols as proteasome modulators and their role as anti-cancer compounds doc

REVIEW ARTICLE

Natural polyphenols as proteasome modulators and their
role as anti-cancer compounds
Laura Bonfili1, Valentina Cecarini1, Manila Amici1, Massimiliano Cuccioloni1, Mauro Angeletti1,
Jeffrey N. Keller2 and Anna M. Eleuteri1
1 Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy
2 Pennington Biomedical Research Center, Baton Rouge, LA, USA

Keywords
antioxidant; apoptosis; cancer prevention;
cancer therapy; chemical structure; drugs;
modulation; natural extracts; polyphenols;
proteasome
Correspondence
A. M. Eleuteri, Department of Molecular,
Cellular and Animal Biology, University of
Camerino, Via Gentile III da Varano, 62032
Camerino (MC), Italy
Fax: +39 0737 403247
Tel: +39 0737 403267
E-mail:

The purpose of this review is to discuss the effect of natural antioxidant
compounds as modulators of the 20S proteasome, a multi-enzymatic multicatalytic complex present in the cytoplasm and nucleus of eukaryotic cells
and involved in several cellular activities such as cell-cycle progression, proliferation and the degradation of oxidized and damaged proteins. From this
perspective, proteasome inhibition is a promising approach to anticancer
therapy and such natural antioxidant effectors can be considered as potential relevant adjuvants and pharmacological models in the study of new
drugs.

(Received 1 August 2008, revised

10 September 2008, accepted 22
September 2008)
doi:10.1111/j.1742-4658.2008.06696.x

Introduction
Nutritional studies have recently shown that a regular
consumption of polyphenolic antioxidants, contained
in fruits, vegetables and their related juices, has a positive effect in the treatment and prevention of a wide
range of pathologies, including cancer [1], stroke [2],
coronary heart disease [3,4] and neurodegenerative disease, such as Alzheimer’s disease [5]. These diseases
are, above all, characterized by oxidative damage to
cellular macromolecules, inflammatory processes and
iron misregulation, with a consequent induction of
toxicity and cell death [6]. Polyphenols, including those
found in green tea and wine, present a wide spectrum
of biological activities, including antioxidant action

[7,8], free radical scavenging, anti-inflammatory and
metal-chelating properties. It is therefore reasonable to
consider these bioactive compounds as potential therapeutic agents [5,9,10].
The biological properties of polyphenols are strongly
affected by their chemical structure. In fact, this is
responsible for their bioavailability [11], antioxidant
activity [12], and their specific interactions with cell
receptors and enzymes [13,14].
Recent studies have shown that natural flavonoids
can modulate the functionality of the proteasome
[15,16], a multi-enzymatic multi-catalytic complex
localized in the cytoplasm and nucleus of all
eukaryotic cells. The proteasome regulates several

cellular processes involved in cell-cycle regulation,

Abbreviations
AP-1, activator protein-1; BrAAP, branched-chain amino acids preferring; ChT-L, chymotrypsin-like; EGCG, ())-epigallocatechin-3-gallate;
PGPH, peptidylglutamyl-peptide hydrolyzing; SNAAP, small neutral amino acids preferring; T-L, trypsin-like; Ub, ubiquitin.

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apoptosis, degradation of oxidized, unfolded and
misfolded proteins and antigen presentation [17–21].
Increasingly, studies have focused their attention on
the regulation of proteasomal functionality by natural and synthetic polyphenols, especially in cancer
therapy [16,22–24].

The proteasome
The proteasome is a multi-catalytic protease complex
found in prokaryotic cells and in the cytoplasm and
nucleus of all eukaryotic cells, and is the major nonlysosomal system for protein degradation.
The 26S proteasome consists of a catalytic core, the
20S proteasome, with associated regulatory particles.
The molecular structure of the 20S proteasome is
extremely conserved from archaebacteria to higher
eukaryotes and is organized in four stacked rings, each
formed by seven subunits in an a7b7b7a7 configuration. The a subunits are localized in the outer rings
and the b subunits in the inner rings of this cylinderlike complex. Whereas the a and b subunits of the

Thermoplasma acidophilum proteasome are encoded by
two genes, 14 genes are involved in the assembly of
eukaryotic 20S proteasomes. In detail, seven distinct b
subunits, carrying the enzyme active sites, constitute
the two inner rings, whereas the outer ones are composed of seven different a subunits (a1-7 b1-7 b1-7
a1-7). The structures of the alpha and beta subunits
are similar and consist of a core of two antiparallel
b sheets flanked by a-helical layers [25–27].
The 19S regulatory particle (or PA700) regulates
substrate access through the outer rings and is responsible for the recognition, unfolding and translocation
of the selected substrates into the lumen of the catalytic core.
The covalent attachment of a polyubiquitin chain
facilitates substrate recognition and triggers 26S proteasome-mediated degradation. This conjugation reaction starts with the 76-amino acid peptide ubiquitin
(Ub) that binds to a Ub-activating enzyme (E1) with a
high-energy bond. Activated Ub is then transferred to
a Ub-conjugating enzyme (E2) that, together with a
Ub ligase (E3), catalyses conjugation of the Ub monomer to a lysine residue of the target protein. More
than one ubiquitin needs to be added to build a polyUb chain that serves as an unambiguous trigger for
proteolysis by the 26S proteasome in the presence of
ATP [28]. However, several proteins are degraded
within the cells in an ATP- and Ub-independent manner [29]. There is evidence that the 20S complex can
directly degrade protein substrates such as casein, lysozyme, insulin b-chain, histone H3, ornithine decarbox-

Antioxidants and proteasome in cancer treatment

ylase, dihydrofolate reductase and oxidatively
damaged proteins [30–33].
The 20S proteasome belongs to the N-terminal
nucleophile hydrolases (Ntn-hydrolases), because its
catalytic activities are related to Thr1 on the N-terminal amino acid residue as nucleophile [27,34]. Another

amino acid residue needed for the catalytic activity is
Lys33; it facilitates proton acceptance, lowering the
pKa of the amino group of Thr1 by its electrostatic
potential [35]. The catalytic mechanism also involves
the residues Glu ⁄ Asp17, Ser129, Asp166 and Ser169
[36].
According to inhibition and X-ray diffraction studies, in eukaryotes, the three major proteasome
activities, chymotrypsin-like (ChT-L, cleaving after
hydrophobic residues), trypsin-like (T-L, cleaving after
basic residues) and peptidylglutamyl-peptide hydrolysing (PGPH, cleaving after acidic residues), are associated with b subunits b5, b2 and b1, respectively
[37–40]. Proteasomes also possess two additional
distinct activities: one cleaving preferentially after
branched-chain amino acids (BrAAP activity) and the
other cleaving after small neutral amino acids (SNAAP
activity) [41,42].
During an acute immune response the immunomodulatory cytokines interferon (IFN)-c or tumour necrosis
factor-a induce the synthesis of three extra proteasome
subunits: the catalytic components b5, b2 and b1 are
replaced by three homologous subunits called b5i, b2i
and b1i, respectively. This substitution generates the
so-called immunoproteasome [43,44]. The distribution
of constitutive and immunoproteasome differs in organs
and tissues: whereas the brain contains predominantly
constitutive proteasomes, lymphoid organs are rich in
IFN-c-induced proteasomes [45].
Immunoproteasomes are involved in the T-cell
immune response generating 7–9 amino acids containing class I antigenic peptides, with aromatic, branched
chain or basic residues at the C-terminus [46–48].
IFN-c also stimulates the synthesis of a regulatory
particle, PA28 or 11S, which caps the ends of the 20S

immunoproteasome and activates it through a conformational change in the complex [49–52].
The proteasome is known to degrade the majority of
intracellular proteins, including p27kip1 [53,54], p21 [55],
IkB-a [56,57] and Bax [58], cyclins, metabolic enzymes,
transcription factors [59] and the tumour suppressor
protein p53 [60,61]. In addition, several of its enzymatic
activities (proteolytic, ATPase, de-ubiquitinating) demonstrate the key role played by the complex in essential
biological processes such as protein quality control,
antigen processing, signal transduction, cell-cycle
control, cell differentiation and apoptosis [17,62–64].

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The 20S proteasome is also part of the intracellular
antioxidant defence system, being involved in the degradation of oxidized proteins [65]. In vitro studies have
shown that the 20S proteasome selectively recognizes
hydrophobic amino acid residues that are exposed
during oxidative rearrangement of the secondary and
tertiary protein structure, without ATP or ubiquitin
[66–69].
Increased activity of the proteasome and nNOS
downregulation in neuroblastoma cells expressing a
Cu ⁄ Zn superoxide dismutase mutant has been demonstrated. Further evidence supporting the role of the proteasome in removing oxidized proteins is that SH-SY5Y

and mutated G93A cells present increased levels of protein carbonyls after treatment with the proteasome
inhibitor lactacystin [70]. Treatment of normal cells with
proteasome pharmacological inhibitors, in addition to
repressing proteasome functionality, induced higher
levels of oxidized protein aggregates [71]. In addition, a
decrease in proteasome activity and increased levels of
protein aggregates were detected in senescent cells and
tissues from aged mice [71,72], further confirming that
strong oxidative stress and aging induce both subtle and
severe alterations in proteasome biology [73].
The proteasome is involved in multiple cellular pathways, regulating cell proliferation, cell death, neuropathological events and drug resistance in human
tumour cells. Therefore, it seems to be an attractive
target for a combined chemopreventative ⁄ chemotherapeutic approach, which seems ideal for cancer therapy.
In particular, because proteasome inhibitors are considered very effective and selective for the proteasome,
their application has been extensively documented.
Among them, bortezomib is the best described and the
first to be tested in humans, especially against multiple
myeloma and non-Hodgkin’s lymphoma. This drug
acts by binding the b5i and b1i proteasome subunits
and its pro-apoptotic activity is mediated by c-JunNH2-terminal kinase induction, block of the nuclear
traslocation of NF-jB, generation of reactive oxygen
species, transmembrane mitochondrial potential gradient alteration, cytochrome c release, and activation of
caspase-mediated apoptosis [74,75]. Despite the acceptable therapeutic index, patients treated with this drug
in phase I and phase II clinical trials manifest several
toxic side effects, including diarrhoea, fatigue, fluid
retention, hypokalemia, hyponatremia, thrombocytopenia, anaemia, anorexia, neutropenia and pyrexia
[74,75]. All these side effects suggest the need to limit
the dose, considering also that some of these adverse
events could be resolved by suspending the treatment.
From this perspective, the use of natural compounds

with the same properties, but which are less toxic
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and more easily accessible than synthetic drugs, can
create new scenarios for possible drug development
[23,76–78].

Flavonoids
Flavonoids represent a wide class of phenolic phytochemicals which constitute an important component of
the human diet. They can be found in fruit, vegetables,
flowers, seeds, sprouts and beverages, providing them
with much of their flavour and colour.
In addition to endogenous antioxidant systems (catalase, superoxide dismutase, glutathione peroxidase,
glutathione reductase), exogenous antioxidants have an
important role in protecting against damage derived
from oxidative agents. Natural antioxidants include
vitamins, carotenoids and polyphenols.
The chemical structure of flavonoids is that of
diphenylpropanes (C6-C3-C6) consisting of two aromatic rings linked through three carbons forming an
oxygenated heterocycle [79,80] (Fig. 1).
Flavonoids can be divided into various subclasses
considering three major factors: the chemical nature of
the molecule, variations in the number and distribution
of the phenolic hydroxyl groups across the molecule,
and their substitutions [81–83]. The main subclasses of
flavonoids are anthocyanins, flavanols, flavanones,
flavonols, flavones and isoflavones. Their structures
and food sources are summarized in Table 1.
The best-known biological effects of flavonoids
include cancer prevention [84,85], inhibition of bone

resorption [86], hormonal and cardioprotective action
[87]. Furthermore, they also possess antibacterial
[88,89] and antiviral properties [90,91].
Flavonoids have been shown to act as scavengers of
various oxidizing species, such as hydroxyl radical,
peroxy radicals or superoxide anions, due to the presence of a catechol group in the B-ring and the 2,3 double bond in conjunction with the 4-carbonyl group as
well as the 3- and 5-hydroxyl groups. Thus, the hydrophilic ⁄ lipophilic balance is critical for the antioxidant
properties of flavonoids [92–94].
Glycosylation and the number of hydroxyl groups
influence the affinity of flavonoids for cellular membranes and the way substitutive groups affect their

Fig. 1. The chemical structure of a flavonoid.

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Table 1. Subclasses of flavonoids.
Subclasses

Principal compounds

Anthocyanin
Pelargonidin

Structure

Food sources

Cyanidin, malvidin

Berry fruits, grape seeds, wine [171,172]
OH
O+

OH

OH
OH
OH

Flavanols

OH

Catechin, EGCG, ECG, EGC, EC

Tea [173], red wine, cocoa, grape juice

O

OH

OH
OH
O

Flavanones

Hesperetin, naringenin, narirutin,
eriodictyol, neohesperetin

Citrus fruit, grapefruit, bitter orange [174]
O

O

Flavonols

Myricetin, kaempferol,
quercetin glucosides

OH

Onions, tea, red wine, broccoli,
berries, apple [175]

O

Flavones

Pigenin, chrysin, luteolin

Chamomile, tea, honey, propolis [176]

O

O

Isoflavones

Genistein, daidzein

Soybeans, black beans, green
beans chickpeas [177,178]

O

O

structure, fluidity and permeability [95,96]. The degree
of hydroxylation also influences the intestinal absorption of these compounds.
The identification of flavonoid forms that can be
effectively absorbed by humans is of great interest and
it must be considered that the gastrointestinal tract
and the colonic microflora play a significant role in the
metabolism and conjugation of polyphenols before
their entry into the systemic circulation and liver [97–
99]. Dietary flavonoid metabolites such as glucuronide
and sulphate conjugates, O-methylated forms and
O-methylated glucuronidated adducts are of interest
with respect to their actions in vivo [100].
Thus, the cellular effects of flavonoid metabolites
depend on their ability to associate with cells, either

by interactions at the membrane or uptake into the
cytosol. Information regarding the uptake of

flavonoids and their metabolites from the circulation
into various cell types and whether they are further
modified by cell interactions has become more and
more important. This is a consequence of the extent
and nature of the substitutions that can influence the
potential function of flavonoids as modulators of
intracellular signalling cascades vital to cellular function [100].
Polyphenols administered at pharmacological doses
(hundreds of milligrams) or consumed as a polyphenol-rich diet (> 1 gỈdose)1), can readily saturate the
conjugation pathways leading to detectable, unconjugated compounds in the plasma. The utilized concen-

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trations influence not only quality and quantity of circulating species, but also tissues distribution of
polyphenols and their relative metabolites [11].
Flavonoids have the potential to bind the ATP-binding sites of a large number of proteins [14] including
mitochondrial ATPase [101], calcium plasma membrane ATPase [102], protein kinase A [103], protein
kinase C [104,105] and topoisomerase [106].
The structure of the flavonoids determines whether
they act as potent inhibitors of protein kinase C,
tyrosine kinase, and, most notably, phosphoinositol
3-kinase [104,107].
In this review, we discuss the property of flavonoids

to affect the proteasome proteolytic activities and their
selective and deleterious effect towards cancer cells by
inhibition of vital proteasome.

Dietary flavonoids in cancer
chemoprevention
Several epidemiological studies have suggested a positive association between the consumption of a diet rich
in fruit and vegetables and a lower incidence of
stomach, oesophagus, lung, oral cavity and pharynx,
endometrial, pancreas and colon cancers [108–110].
Studies conducted on cell cultures and animal models revealed the ability of several polyphenols to defend
cells against cancer. Russo [111] suggested that these
molecules can work as cancer-blocking agents, preventing initiation of the carcinogenic process and as
cancer-suppressing agents, inhibiting cancer promotion
and progression. In detail, polyphenols block cancer
either by activation of Nrf2 signalling, promoting
genes encoding antioxidant and detoxifying enzymes,
or through NF-jB- or activator protein-1 (AP-1)-mediated pathways. NF-jB is a transcription factor with a
key role in inflammation and carcinogenesis: it acts as
an antagonist of the tumour suppressor protein p53
and its activation induces transcriptional upregulation
of the genes involved in cell-cycle progression. The
AP-1 transcription factor is a protein complex principally comprising two proto-oncogene subfamilies, Jun
(c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1
and Fra-2), whose different dimeric combinations
influence the AP-1 functions [111–114]. AP-1 activity is
increased in several human tumours and its inhibition
is a recognized molecular target in chemoprevention.
The consumption of antioxidants may lead to a
decrease in intracellular reactive oxygen species levels

associated with DNA damage, and to the protection of
pre-malignant cells from cancer [115]. Therefore, from
this perspective, such phytochemicals, as proposed by
the ‘antioxidant hypothesis’, play an important role as
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chemopreventative agents, with the ability to exert both
a protective effect on normal, non-trasformed cells and
a toxic effect on pre-neoplastic cells [111]. This chemopreventative role has also been described as being independent of the antioxidant ability because they can
regulate mechanisms related to cells differentiation,
transformation and inflammation [111,116–118].
It is important to note that every antioxidant compound is a redox agent that, under particular conditions and in the presence of metal ions, can act as a
pro-oxidant inducing radical generation and oxidative
damage. Nevertheless in vivo, most transition metal
ions are protein-conjugated and therefore not available
to catalyse free radical reactions, thus minimizing the
pro-oxidant properties of dietary polyphenols. There
are several reports of a Cu-dependent oxidant action
towards DNA strands of natural phytochemicals, such
as curcumin, resveratrol and quercetin [119–122]. Interestingly, considering that copper levels are higher in
tumour cells than in normal cells, it has been hypothesized that the cytotoxic and anti-cancer effects of
plant-derived polyphenols may primarily derive from
their pro-oxidant capacities [122].

Proteasome modulation by flavonoids
The regulation of proteasome functionality by natural
and synthetic polyphenols is a promising issue in cancer therapy. In fact, inhibition of the proteasome leads
to growth arrest in the G1 phase of the cell cycle and
the induction of apoptosis in cancer cells [21].
Published research findings have shown that polyphenolic compounds present in green and black tea

can reduce risk in a variety of diseases [123]. It has
been reported that green tea consumed as part of a
balanced controlled diet improves overall antioxidative
status and protects against oxidative damage in
humans [124]. Tea polyphenols contain catechin, flavones, anthocyanins and phenolic acid. Catechins are
the main components, with a content > 80% [125].
())-Epigallocatechin-3-gallate (EGCG) and other tea
polyphenols are potential chemopreventative agents,
able to modulate multiple intracellular signal transduction pathways, such as NF-kB signalling pathway,
MAPKs pathway and AP-1 activity [126,127]; EGCG
is also involved in the inhibition of epidermal growth
factor receptor-mediated signal transduction pathway
[128]. In addition, green tea polyphenols have been
shown to inhibit insulin-like growth factor I metabolism [129] and cyclooxigenase-2 expression and activity
in cancer cells [130].
Dou et al. [131] showed that ester bond-containing
tea polyphenols potently and selectively inhibit the

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proteasomal ChT-L, but not T-L activity, in vitro and
in Jurkat cells at concentrations found in the serum of
green tea drinkers.
The inhibition of proteasome activity by EGCG can
selectively control tumour cell growth, with the accumulation of proteasome protein substrates such as
p27Kip1 and IkB-a. This finding, along with the low
toxicity of EGCG, supports the potential role of tea

polyphenols in clinical therapies in combination with
current anti-cancer drugs [131–133].
The effect of several isolated natural polyphenols on
purified proteasomes was evaluated by our group. We
reported that EGCG strongly inhibited the ChT-L
activity of both constitutive and immunoproteasomes,
whereas it seemed to be a specific inhibitor of the
immunoproteasome BrAAP component. It was also
effective on the T-L activity of the two enzymes, but
with a lower IC50 for the inducible complex. EGCG
had also a clear antioxidant effect in Caco cells
exposed to oxidative stress, preventing oxidation and
deterioration of the proteasome functionality. Gallic
acid affected the ChT-L activity of both complexes
at the same extent, while its inhibitory effect on the
T-L activity is higher for the constitutive proteasome.
[15].
The effect of various fruit and vegetable extracts rich
in flavonoids on proteasome functionality was reported
by Dou et al. They showed that apple extract, which is
particularly rich in flavanols, and grape extract, rich in
catechins, quercetin and resveratrol, were more potent
than onion, tomato and celery in inhibiting proteasomal ChT-L activity in leukaemia Jurkat T-cell lysates.
This effect caused an accumulation of the polyubiquitinated proteins, activation of caspase 3 and caspase 7,
and cleavage of poly(ADP-ribose) polymerase. The
inhibition of proteasome activity by these fruit or vegetables may contribute to their cancer preventative
effects, although other molecular mechanisms may also
be involved [134].
Other natural polyphenols able to influence the
ubiquitin–proteasome pathway have been identified.

Some of them are described below.
Tannins
Tannins are plant-derived polyphenolic compounds
with varying molecular masses; they can be further
classified into two main groups, hydrolysable and condensed tannins, also known as proanthocyanidins. The
hydrolysable tannins contain gallotannins or ellagictannins. Upon hydrolysis, gallotannins yield glucose
and gallic acid, whereas the ellagictannins produce
ellagic acid as a degradation product [135].

Antioxidants and proteasome in cancer treatment

It has been reported that tannic acid, an example of
gallotannins, potently and specifically inhibits the
ChT-L activity of purified 20S proteasome, 26S proteasome of Jurkat T-cell extracts and the 26S proteasome in living Jurkat cells, resulting in the
accumulation of proteasomal substrates p27 and Bax
[135]. In addition, tannic acid was a potent inhibitor
of proteasomal ChT-L activity and delayed cell-cycle
progression in malignant cholangiocytes [136].
Quercetin
Onions, apples, tea and red wine are examples of foods
particularly rich in quercetin (3,3¢,4¢,5,7-pentahydroxyflavone). This flavonoid belongs to the flavonols subgroup. In a recent study, Dosenko et al. [137]
performed experiments on purified 20S proteasomes
showing that quercetin inhibits three of the proteasomal peptidase activities, in particular the ChT-L
component, in a dose-dependent manner, having comparable affinity with respect to a specific proteasome
inhibitor. Similarly, quercetin inhibited the activities of
the 26S proteasome in a cardiomyocytes culture.
Recent studies have shown that apigenin and quercetin are more potent than kaempferol and myricetin in
inhibiting the ChT-L activity of purified 20S proteasome and 26S proteasome in intact leukemia Jurkat T
cells, inducing an accumulation of ubiquitinated forms
of Bax and IkB-a, activation of caspase 3 and cleavage

of poly(ADP-ribose) polymerase. Furthermore, the
proteasome-inhibitory abilities of these compounds
were related to their apoptosis-inducing potencies [16].
Chrysin
This flavone, found in many plants, honey and propolis, possesses strong antiproliferative and antioxidant
activity, and exerts its growth-inhibitory effects either
by activating p38-MAPK, leading to the accumulation
of p21Waf1 ⁄ Cip1 protein, or by mediating the inhibition
of proteasome activity [138].
Comparing the effect of luteolin, apigenin, chrysin,
naringenin and eriodictyol on 20S-purified proteasome
and on apoptosis of tumour cells it is clear that dietary
flavonoids with OH groups on the B ring and ⁄ or the
double bond between C2 and C3 of the pyranosyl moiety are natural potent proteasome inhibitors and
tumour cell apoptosis inducers. Furthermore, neither
apigenin nor luteolin could inhibit the proteasome and
induce apoptosis in non-transformed human natural
killer cells. These findings provide a molecular basis
for the clinically observed cancer-preventive effects of
fruit and vegetables [16,22].

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Curcumin

Resveratrol

Curcumin is a natural polyphenolic compound
extracted from the spice turmeric, which has been
reported to have anti-inflammatory [139], antioxidant
and antiproliferative properties [140,141]. It modulates
multiple cellular machineries, such as the ubiquitin
proteasome system [142]. Jana et al. observed a dosedependent inhibition of proteasome activities in Neuro
2a cells treated with curcumin (2.5–50 lm), due to a
direct effect on the 20S core catalytic component
[142,143]. Curcumin treatment of human epidermal
keratinocytes increased the ChT-L activity at low doses
(up to 1 lm), whereas higher concentrations of curcumin (10 lm) caused a 46% decrease in proteasome
activity [144].
Si et al. demonstrated in HeLa cells treated with
30 lm curcumin a reduction of almost 30% in the
ChT-L, T-L and PGPH activities of the 20S
proteasome, accompanied by a marked accumulation
of ubiquitin–protein conjugates. A stronger effect
was observed on purified 20S proteasome: the
ChT-L, T-L and PGPH hydrolytic activities were
inhibited by > 90% in the presence of curcumin
(30 lm) [145]. Like resveratrol, curcumin was able
to attenuate the proteolysis-inducing factor-induced
increase in expression of the ubiquitin–proteasome
proteolytic pathway [146].

Examples of foods with high levels of resveratrol are

wine, grape skins and peanuts. Several in vivo studies
[149,150] have shown sustained resveratrol efficacy in
inhibiting or retarding tumour growth and ⁄ or progression in animal models inoculated with malignant
cell lines, or treated with tumorigenesis-inducing
drugs.
In vitro, resveratrol influenced numerous intracellular pathways leading to cell growth arrest through the
inhibition of ERK1 ⁄ 2-mediated signal transduction
pathways, the inhibition of 4b-phorbol 12-mysristate
13-acetate-dependent protein kinase C activation, the
downregulation of b-catenin expression, the inhibition
of Cdk1 and Cdk4 kinase activities, the induction of
apoptotic events, such as caspases, p53, Bax activation
and Bcl2 inhibition [149,151]. Interestingly, recent clinical trials performed with the intake of resveratrol
combined with chemotherapeutic treatments indicated
that low doses of resveratrol were capable of enhancing the chemotherapeutic efficacy in various human
cancers [152,153]. It is unclear, at this stage, whether
the molecular mechanisms mediated by resveratrol
against tumour progression involve proteasome inhibition directly, even though Liao et al. suggested that
resveratrol may interfere with the NF-jB proteasome
mediated degradation [154,155].
Extracts from various fruit and vegetables, such as
apple, grape and onion, have been investigated for
their antioxidant properties and their role in inducing
apoptosis in tumour cells, and the ubiquitin–proteasome pathway may be one of the mechanisms involved
[134]. For example, a natural musaceas plant extract,
rich in tannic acid, was able to inhibit proteasome
activity and selectively induce apoptosis in human
tumour and transformed cells [156]. We recently found
that wheat sprout hydroalcoholic extract, rich in catechin, epicatechin and epigallocatechin gallate, can
induce gradual inhibition of the 20S proteasome

ChT-L, T-L, PGPH and BrAAP components. Wheat
sprout extract affected proteasome functionality in a
Caco cell line and it influenced the expression of proapoptotic proteins [157]. We also demonstrated that
tumour cell line proteasomes showed a higher degree
of impairment with respect to normal cell proteasomes,
upon wheat sprout extract polyphenol and peptide
components treatment (unpublished data).

Genistein
Computational docking data suggest that genistein,
one of the predominant soy isoflavones, was a
weaker proteasome inhibitor than EGCG. Like
EGCG, genistein at 1 lm was able to inhibit ChT-L
activity in purified 20S and 26S proteasomes of
LNCaP and MCF-7 cell extracts. Furthermore, inhibition of the proteasome by genistein in intact
LNCaP and MCF-7 cells was associated with the
accumulation of ubiquitinated proteins and the
proteasome target proteins p27Kip1, IkB-a and Bax.
Following genistein-mediated proteasome inhibition,
p53 protein accumulation occurred, associated with
increased levels of p53 downstream target proteins
such as p21Waf1. Finally, the proteasome-inhibitory
and apoptosis-inducing effects of genistein were
observed in SV40-transformed human fibroblasts
(VA-13), but not in their parental normal lung fibroblast counterpart (WI-38) [147]. Genistein induced
apoptosis of p815 mastocytoma cells, in part mediated by proteasome. The enzyme activity was inhibited at early time points after genistein treatment
[148].

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Oleuropein
Oleuropein, the major constituent of Olea europea leaf
extract, olive oil and olives, was reported to enhance

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proteasome activity in vitro more strongly than other
known chemical activators, possibly through conformational changes in the proteasome. Moreover, continuous treatment of early-passage human embryonic
fibroblasts with oleuropein decreased the intracellular
levels of reactive oxygen species, reduced the amount
of oxidized proteins through increased proteasomemediated degradation rates and retained proteasome
function during replicative senescence [158].

New potential drugs in cancer
treatment
Multiple lines of evidence have proposed a positive
effect of natural phytochemical compounds like flavonoids against several human malignancies.
The use of natural polyphenols in the prevention
and treatment of cancer is now well documented (see
above). Several studies have reported the anti-cancer
activity of numerous natural compounds and their
cooperative action in association with chemotherapeutic drugs (see above).

Table 2 summarizes some phytochemical compounds
that have been proposed as potential chemopreventative, chemoprotective and chemopotentiator agents

and selected for ongoing phase I–III clinical trials.
Moreover, based on the inhibitory effect of naturally
occurring flavonoids on proteasome functionality, several studies have been performed in order to design
more effective compounds in cancer treatment.
Smith et al. tried to clarify the model of interaction
of EGCG with proteasome subunits through docking
studies, demonstrating that inhibition of the 20S proteasome ChT-L activity by EGCG was time-dependent
and irreversible, and implicated the acylation of the b5
subunit’s catalytic N-terminal threonine (Thr1) [159].
This mechanism is similar to that of lactacystin-based
inhibition [160]. However, EGCG is very unstable
under neutral or alkaline conditions (i.e. physiologic
pH). Landis-Piwowar et al. synthesized novel EGCG
analogues with -OH groups eliminated from the
B- and ⁄ or D-rings. In addition, they also synthesized
putative pro-drugs in which -OH groups were protected by peracetate that can be removed by cellular

Table 2. Polyphenols in active clinical trials (data from the National Cancer Institute, ).

Polyphenols

Source

Curcumin

Turmeric
Turmeric

Clinical trial
phase

Type of cancer

Combined with

Phase
Phase
Phase
Phase
Phase

Metastatic colon cancer
Pancreatic cancer
Osteosarcoma
Colorectal cancer
Stage IV breast cancer

Gemcitabine
Gemcitabine

Phase II
Phase II

Vitamin D and
soy isoflavones
Synthetic genistein
Resveratrol

Grape skins

Green tea extract

Polyphenon E

Advanced pancreatic cancer
Adenocarcinoma of the prostate

Phase
Phase
Phase
Phase

Prostate cancer
Colon cancer
Colorectal cancer
Healthy adults at increased
risk of developing melanoma
Chronic lymphocytic leukemia
Advanced non small cell lung cancer
Human papillomavirus and low-grade
cervical intraepithelial neoplasia
Lung cancer
Bronchial dysplasia
Prostate cancer
High-grade prostatic intraepithelial
neoplasia
Breast cancer
Nonmetastatic bladder cancer
Prostate cancer

Green tea,
decaffeinated
black tea

II
I–II
I
I

Phase I–II
Phase I–II
Phase II
Phase
Phase
Phase
Phase

Tea polyphenols
and theaflavins

III
III
I–II
II
II

II
II
II
II

Phase II
Phase II
Phase II

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS

Gemcitabine
hydrochloride
and genistein
Gemcitabine

Erlotinib

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L. Bonfili et al.

cytosolic esterases. They demonstrated how decreasing
the number of -OH groups from either the B- or
D-ring leads to diminished proteasome inhibitory
activity in vitro [161].
It has been reported that acetylated synthetic tea
analogues are much more potent than natural EGCG
in inhibiting the proteasome in cultured tumour cells,
possessing the potential to be developed into novel
anticancer drugs [162]. Methylation had no effect on

the nucleophilic susceptibility of EGCG and epicatechin-3-gallate, but may disrupt the ability of these
polyphenols to interact with Thr1 of the proteasome
b5 subunit [163]. Osanai et al. have shown that
analogues of EGCG containing a para-amino group
on the D-ring were more effective than analogues with
an hydroxyl substituent in enhancing proteasome
inhibition and inducing apoptosis, demonstrating their
potential as anticancer agents [164].
In addition, recent studies reported relationships
between the molecular structures of natural polyphenols and their inhibitory effects on the proteasome
[22,165]. As mentioned previously for EGCG, the IC50
values measured for chrysin, luteolin, apigenin, naringenin and eriodictyol were strictly related to the number of OH amount on the B-ring and to the presence
of an unsaturated C-ring group on the flavonoid
molecule [22]. Furthermore, methylation of quercetin,
chrysin, luteolin and apigenin reduced their ability
to inhibit the proteasome and to induce apoptosis in
cancer cells [165].

Concluding remarks
Epidemiological studies highlight numerous health
benefits of a diet supplemented with natural flavonoids [166–169]. The proteasome is responsible for
degrading most intracellular proteins, including oxidized proteins and the proteins involved in cell-cycle
regulation and apoptosis, processes crucial to oncogenesis. Thus, the proteasome can be considered a
potential target in cancer therapy [170] and its
modulation by polyphenols may contribute to the
cancer-preventive effect. Furthermore, when combined with common cancer therapies, polyphenols
may enhance their antitumor activity in a synergistic
way. Studying natural occurring polyphenols, like the
compounds mentioned, their bioavailability, the
structure–activity relations and the way they affect,

through modulation of the proteasome, protein degradation and all the cellular pathways in which the
proteasome is involved, represents a promising starting point for designing and developing novel anticancer drugs.
5520

Acknowledgements
The authors wish to thank Dr Matteo Mozzicafreddo
for technical assistance.

References
1 Shankar S, Ganapathy S & Srivastava RK (2007)
Green tea polyphenols: biology and therapeutic implications in cancer. Front Biosci 12, 4881–4899.
2 Neto CC (2007) Cranberry and blueberry: evidence for
protective effects against cancer and vascular diseases.
Mol Nutr Food Res 51, 652–664.
3 Widlansky ME, Hamburg NM, Anter E, Holbrook M,
Kahn DF, Elliott JG, Keaney JF Jr & Vita JA (2007)
Acute EGCG supplementation reverses endothelial
dysfunction in patients with coronary artery disease.
J Am Coll Nutr 26, 95–102.
4 Khan N & Mukhtar H (2007) Tea polyphenols for
health promotion. Life Sci 81, 519–533.
5 Mandel S & Youdim MB (2004) Catechin polyphenols:
neurodegeneration and neuroprotection in neurodegenerative diseases. Free Radical Biol Med 37, 304–317.
6 Halliwell B (2001) Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18, 685–716.
7 Prior RL (2003) Fruits and vegetables in the prevention of cellular oxidative damage. Am J Clin Nutr 78,
570S–578S.
8 Urso ML & Clarkson PM (2003) Oxidative stress,
exercise, and antioxidant supplementation. Toxicology
189, 41–54.
9 Ramassamy C (2006) Emerging role of polyphenolic

compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets. Eur J
Pharmacol 545, 51–64.
10 Weinreb O, Mandel S, Amit T & Youdim MB (2004)
Neurological mechanisms of green tea polyphenols in
Alzheimer’s and Parkinson’s diseases. J Nutr Biochem
15, 506–516.
11 Scalbert A & Williamson G (2000) Dietary intake and
bioavailability of polyphenols. J Nutr 130, 2073S–2085S.
12 Rice-Evans CA, Miller NJ & Paganga G (1996) Structure–antioxidant activity relationships of flavonoids
and phenolic acids. Free Radical Biol Med 20, 933–956.
13 Williamson MP, McCormick TG, Nance CL & Shearer
WT (2006) Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4:
potential for HIV-1 therapy. J Allergy Clin Immunol
118, 1369–1374.
14 Conseil G, Baubichon-Cortay H, Dayan G, Jault JM,
Barron D & Di Pietro A (1998) Flavonoids: a class of
modulators with bifunctional interactions at vicinal
ATP- and steroid-binding sites on mouse P-glycoprotein. Proc Natl Acad Sci USA 95, 9831–9836.

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS


L. Bonfili et al.

15 Pettinari A, Amici M, Cuccioloni M, Angeletti M, Fioretti E & Eleuteri AM (2006) Effect of polyphenolic
compounds on the proteolytic activities of constitutive
and immuno-proteasomes. Antioxid Redox Signal 8,
121–129.
16 Chen D, Daniel KG, Chen MS, Kuhn DJ, LandisPiwowar KR & Dou QP (2005) Dietary flavonoids as
proteasome inhibitors and apoptosis inducers in human

leukemia cells. Biochem Pharmacol 69, 1421–1432.
17 Tambyrajah WS, Bowler LD, Medina-Palazon C &
Sinclair AJ (2007) Cell cycle-dependent caspase-like
activity that cleaves p27(KIP1) is the beta(1) subunit of
the 20S proteasome. Arch Biochem Biophys 466,
186–193.
18 Jung T, Bader N & Grune T (2007) Oxidized proteins:
intracellular distribution and recognition by the proteasome. Arch Biochem Biophys 462, 231–237.
19 Poppek D & Grune T (2006) Proteasomal defense of
oxidative protein modifications. Antioxid Redox Signal
8, 173–184.
20 Chen D, Frezza M, Shakya R, Cui QC, Milacic V,
Verani CN & Dou QP (2007) Inhibition of the proteasome activity by gallium(III) complexes contributes
to their anti-prostate tumor effects. Cancer Res 67,
9258–9265.
21 Chen WJ & Lin JK (2004) Induction of G1 arrest and
apoptosis in human jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1 ⁄ WAF1, and Bax proteins.
J Biol Chem 279, 13496–13505.
22 Chen D, Chen MS, Cui QC, Yang H & Dou QP
(2007) Structure–proteasome-inhibitory activity relationships of dietary flavonoids in human cancer cells.
Front Biosci 12, 1935–1945.
23 Dou QP & Li B (1999) Proteasome inhibitors as potential novel anticancer agents. Drug Resist Update 2,
215–223.
24 Landis-Piwowar KR, Milacic V, Chen D, Yang H,
Zhao Y, Chan TH, Yan B & Dou QP (2006) The proteasome as a potential target for novel anticancer drugs
and chemosensitizers. Drug Resist Update 9, 263–273.
25 Groll M, Ditzel L, Lowe J, Stock D, Bochtler M,
Bartunik HD & Huber R (1997) Structure of 20S pro˚
teasome from yeast at 2.4 A resolution. Nature 386,
463–471.

26 Adams J (2003) The proteasome: structure, function,
and role in the cell. Cancer Treat Rev 29(Suppl. 1),
3–9.
27 Lowe J, Stock D, Jap B, Zwickl P, Baumeister W &
Huber R (1995) Crystal structure of the 20S protea˚
some from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539.
28 Pickart CM & Fushman D (2004) Polyubiquitin
chains: polymeric protein signals. Curr Opin Chem Biol
8, 610–616.

Antioxidants and proteasome in cancer treatment

29 Shringarpure R, Grune T, Mehlhase J & Davies KJ
(2003) Ubiquitin conjugation is not required for the
degradation of oxidized proteins by proteasome. J Biol
Chem 278, 311–318.
30 Liu CW, Corboy MJ, DeMartino GN & Thomas PJ
(2003) Endoproteolytic activity of the proteasome.
Science 299, 408–411.
31 Jung T & Grune T (2008) The proteasome and its role
in the degradation of oxidized proteins. IUBMB Life,
doi: 10.1002/iub.114.
32 Asher G, Bercovich Z, Tsvetkov P, Shaul Y & Kahana
C (2005) 20S proteasomal degradation of ornithine
decarboxylase is regulated by NQO1. Mol Cell 17,
645–655.
33 Amici M, Sagratini D, Pettinari A, Pucciarelli S, Angeletti M & Eleuteri AM (2004) 20S proteasome mediated degradation of DHFR: implications in
neurodegenerative disorders. Arch Biochem Biophys
422, 168–174.
34 Seemuller E, Lupas A, Stock D, Lowe J, Huber R &

Baumeister W (1995) Proteasome from Thermoplasma acidophilum: a threonine protease. Science 268,
579–582.
35 Arendt CS & Hochstrasser M (1999) Eukaryotic 20S
proteasome catalytic subunit propeptides prevent active
site inactivation by N-terminal acetylation and promote particle assembly. EMBO J 18, 3575–3585.
36 Heinemeyer W, Fischer M, Krimmer T, Stachon U &
Wolf DH (1997) The active sites of the eukaryotic 20S
proteasome and their involvement in subunit precursor
processing. J Biol Chem 272, 25200–25209.
37 Rock KL, Gramm C, Rothstein L, Clark K, Stein R,
Dick L, Hwang D & Goldberg AL (1994) Inhibitors of
the proteasome block the degradation of most cell proteins and the generation of peptides presented on
MHC class I molecules. Cell 78, 761–771.
38 Chen P & Hochstrasser M (1996) Autocatalytic subunit processing couples active site formation in the 20S
proteasome to completion of assembly. Cell 86, 961–
972.
39 Groll M, Heinemeyer W, Jager S, Ullrich T, Bochtler
M, Wolf DH & Huber R (1999) The catalytic sites of
20S proteasomes and their role in subunit maturation:
a mutational and crystallographic study. Proc Natl
Acad Sci USA 96, 10976–10983.
40 McCormack TA, Cruikshank AA, Grenier L, Melandri
FD, Nunes SL, Plamondon L, Stein RL & Dick LR
(1998) Kinetic studies of the branched chain amino
acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin
beta-lactone. Biochemistry 37, 7792–7800.
41 Borissenko L & Groll M (2007) 20S proteasome and
its inhibitors: crystallographic knowledge for drug
development. Chem Rev 107, 687–717.

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS

5521


Antioxidants and proteasome in cancer treatment

L. Bonfili et al.

42 Orlowski M, Cardozo C & Michaud C (1993) Evidence
for the presence of five distinct proteolytic components
in the pituitary multicatalytic proteinase complex.
Properties of two components cleaving bonds on the
carboxyl side of branched chain and small neutral
amino acids. Biochemistry 32, 1563–1572.
43 Groettrup M, Khan S, Schwarz K & Schmidtke G
(2001) Interferon-gamma inducible exchanges of 20S
proteasome active site subunits: why? Biochimie 83,
367–372.
44 Aki M, Shimbara N, Takashina M, Akiyama K,
Kagawa S, Tamura T, Tanahashi N, Yoshimura T,
Tanaka K & Ichihara A (1994) Interferon-gamma
induces different subunit organizations and functional
diversity of proteasomes. J Biochem (Tokyo) 115,
257–269.
45 Noda C, Tanahashi N, Shimbara N, Hendil KB &
Tanaka K (2000) Tissue distribution of constitutive
proteasomes, immunoproteasomes, and PA28 in rats.
Biochem Biophys Res Commun 277, 348–354.
46 Goldberg AL, Cascio P, Saric T & Rock KL (2002)

The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.
Mol Immunol 39, 147–164.
47 Elliott T, Smith M, Driscoll P & McMichael A (1993)
Peptide selection by class I molecules of the major histocompatibility complex. Curr Biol 3, 854–866.
48 Sharova NP (2006) Immune proteasomes and immunity. Ontogenez 37, 171–178.
49 Rechsteiner M, Realini C & Ustrell V (2000) The proteasome activator 11S REG (PA28) and class I antigen
presentation. Biochem J 345, 1–15.
50 Dubiel W, Pratt G, Ferrell K & Rechsteiner M (1992)
Purification of an 11S regulator of the multicatalytic
protease. J Biol Chem 267, 22369–22377.
51 DeMartino GN & Slaughter CA (1999) The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem 274, 22123–22126.
52 Amici M & Eleuteri AM (2007) Structure and Function
of the 20S Proteasomes. Nova Science, Sulmona, Italy.
53 Pagano M, Tam SW, Theodoras AM, Beer-Romero P,
Del Sal G, Chau V, Yew PR, Draetta GF & Rolfe M
(1995) Role of the ubiquitin–proteasome pathway in
regulating abundance of the cyclin-dependent kinase
inhibitor p27. Science 269, 682–685.
54 Sun J, Nam S, Lee CS, Li B, Coppola D, Hamilton
AD, Dou QP & Sebti SM (2001) CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and
p27KIP1 expression and apoptosis and inhibits the
growth of the human lung adenocarcinoma A-549 in
nude mice. Cancer Res 61, 1280–1284.
55 Blagosklonny MV, Wu GS, Omura S & el-Deiry WS
(1996) Proteasome-dependent regulation of
p21WAF1 ⁄ CIP1 expression. Biochem Biophys Res
Commun 227, 564–569.

5522

56 Perkins ND (2000) The Rel ⁄ NF-kappa B family: friend
and foe. Trends Biochem Sci 25, 434–440.
57 Verma IM, Stevenson JK, Schwarz EM, Van Antwerp
D & Miyamoto S (1995) Rel ⁄ NF-kappa B ⁄ I kappa B
family: intimate tales of association and dissociation.
Gene Dev 9, 2723–2735.
58 Li B & Dou QP (2000) Bax degradation by the ubiquitin ⁄ proteasome-dependent pathway: involvement in
tumor survival and progression. Proc Natl Acad Sci
USA 97, 3850–3855.
59 Basbous J, Jariel-Encontre I, Gomard T, Bossis G &
Piechaczyk M (2008) Ubiquitin-independent- versus
ubiquitin-dependent proteasomal degradation of the
c-Fos and Fra-1 transcription factors: is there a unique
answer? Biochimie 90, 296–305.
60 Yang Y, Li CC & Weissman AM (2004) Regulating
the p53 system through ubiquitination. Oncogene 23,
2096–2106.
61 Maki CG, Huibregtse JM & Howley PM (1996) In vivo
ubiquitination and proteasome-mediated degradation
of p53(1). Cancer Res 56, 2649–2654.
62 Goldberg AL (2003) Protein degradation and protection against misfolded or damaged proteins. Nature
426, 895–899.
63 Cecarini V, Gee J, Fioretti E, Amici M, Angeletti M,
Eleuteri AM & Keller JN (2007) Protein oxidation and
cellular homeostasis: emphasis on metabolism. Biochim
Biophys Acta 1773, 93–104.
64 Amici M, Cecarini V, Pettinari A, Bonfili L, Angeletti
M, Barocci S, Biagetti M, Fioretti E & Maria Eleuteri A
(2007) Binding of aflatoxins to the 20S proteasome:
effects on enzyme functionality and implications for oxidative stress and apoptosis. Biol Chem 388, 107–117.

65 Bader N & Grune T (2006) Protein oxidation and proteolysis. Biol Chem 387, 1351–1355.
66 Davies KJ (1993) Protein modification by oxidants and
the role of proteolytic enzymes. Biochem Soc Trans 21,
346–353.
67 Giulivi C, Pacifici RE & Davies KJ (1994) Exposure of
hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the
multicatalytic proteinase complex, proteasome. Arch
Biochem Biophys 311, 329–341.
68 Grune T, Reinheckel T & Davies KJ (1997) Degradation of oxidized proteins in mammalian cells. FASEB J
11, 526–534.
69 Davies KJ (2001) Degradation of oxidized proteins by
the 20S proteasome. Biochimie 83, 301–310.
70 Aquilano K, Rotilio G & Ciriolo MR (2003) Proteasome activation and nNOS down-regulation in neuroblastoma cells expressing a Cu,Zn superoxide
dismutase mutant involved in familial ALS. J Neurochem 85, 1324–1335.
71 Demasi M & Davies KJ (2003) Proteasome inhibitors
induce intracellular protein aggregation and cell death

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS


L. Bonfili et al.

72

73
74

75

76

77
78

79
80
81

82

83

84

85

86

87

88

by an oxygen-dependent mechanism. FEBS Lett 542,
89–94.
Hyun DH, Lee M, Halliwell B & Jenner P (2003) Proteasomal inhibition causes the formation of protein
aggregates containing a wide range of proteins, including nitrated proteins. J Neurochem 86, 363–373.
Keller JN, Gee J & Ding Q (2002) The proteasome in
brain aging. Ageing Res Rev 1, 279–293.
Yang H, Landis-Piwowar KR, Chen D, Milacic V &
Dou QP (2008) Natural compounds with proteasome
inhibitory activity for cancer prevention and treatment.

Curr Protein Pept Sci 9, 227–239.
Orlowski RZ & Kuhn DJ (2008) Proteasome inhibitors
in cancer therapy: lessons from the first decade. Clin
Cancer Res 14, 1649–1657.
Almond JB & Cohen GM (2002) The proteasome: a novel
target for cancer chemotherapy. Leukemia 16, 433–443.
Hanna J & Finley D (2007) A proteasome for all occasions. FEBS Lett 581, 2854–2861.
Gopalan B, Shanker M, Scott A, Branch CD, Chada S
& Ramesh R (2008) MDA-7 ⁄ IL-24, a novel tumor
suppressor ⁄ cytokine is ubiquitinated and regulated by
the ubiquitin-proteasome system, and inhibition of
MDA-7 ⁄ IL-24 degradation enhances the antitumor
activity. Cancer Gene Ther 15, doi: 10.1038/sj.cgt.
7701095.
Harborne JB (1986) Nature, distribution and function
of plant flavonoids. Prog Clin Biol Res 213, 15–24.
Harborne JB (1980) Plant phenolics. Encyclopedia
Plant Physiol 8, 329–395.
Rice-Evans C (2004) Flavonoids and isoflavones:
absorption, metabolism, and bioactivity. Free Radical
Biol Med 36, 827–828.
Hollman PC & Katan MB (1999) Health effects and
bioavailability of dietary flavonols. Free Radical Res
31(Suppl.), S75–S80.
Ross JA & Kasum CM (2002) Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev
Nutr 22, 19–34.
Le Marchand L (2002) Cancer preventive effects of
flavonoids – a review. Biomed Pharmacother 56, 296–
301.
Mizunuma H, Kanazawa K, Ogura S, Otsuka S &

Nagai H (2002) Anticarcinogenic effects of isoflavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer. Oncology
62, 78–84.
Yamaguchi M & Gao YH (1998) Inhibitory effect of
genistein on bone resorption in tissue culture. Biochem
Pharmacol 55, 71–76.
Sadzuka Y, Sugiyama T, Shimoi K, Kinae N & Hirota
S (1997) Protective effect of flavonoids on doxorubicininduced cardiotoxicity. Toxicol Lett 92, 1–7.
Haraguchi H, Tanimoto K, Tamura Y, Mizutani K &
Kinoshita T (1998) Mode of antibacterial action of

Antioxidants and proteasome in cancer treatment

89

90

91

92

93

94

95

96

97

98

99

100

101

retrochalcones from Glycyrrhiza inflata. Phytochemistry
48, 125–129.
Iinuma M, Tsuchiya H, Sato M, Yokoyama J, Ohyama M, Ohkawa Y, Tanaka T, Fujiwara S & Fujii T
(1994) Flavanones with potent antibacterial activity
against methicillin-resistant Staphylococcus aureus.
J Pharm Pharmacol 46, 892–895.
Ozcelik B, Orhan I & Toker G (2006) Antiviral and
antimicrobial assessment of some selected flavonoids.
Z Naturforsch [C] 61, 632–638.
Tait S, Salvati AL, Desideri N & Fiore L (2006) Antiviral activity of substituted homoisoflavonoids on enteroviruses. Antiviral Res 72, 252–255.
Madsen HL, Andersen CM, Jorgensen LV & Skibsted
LH (2000) Radical scavenging by dietary flavonoids. A
kinetic study of antioxidant efficiencies. Eur Food Res
Technol 211, 240–246.
Tournaire C, Croux S, Maurette MT, Beck I, Hocquaux M, Braun AM & Oliveros E (1993) Antioxidant
activity of flavonoids: efficiency of singlet oxygen (1
delta g) quenching. J Photochem Photobiol B 19, 205–
215.
Modak B, Contreras ML, Gonzalez-Nilo F & Torres
R (2005) Structure–antioxidant activity relationships of
flavonoids isolated from the resinous exudate of Heliotropium sinuatum. Bioorg Med Chem Lett 15, 309–312.
Bhendrich A (2006) Flavonoid–membrane interactions:

possible consequences for biological effects of some
polyphenolic compounds. Acta Pharmacol Sin 27,
27–40.
Arora A, Byrem TM, Nair MG & Strasburg GM
(2000) Modulation of liposomal membrane fluidity by
flavonoids and isoflavonoids. Arch Biochem Biophys
373, 102–109.
Spencer JP, Schroeter H, Rechner AR & Rice-Evans C
(2001) Bioavailability of flavan-3-ols and procyanidins:
gastrointestinal tract influences and their relevance to
bioactive forms in vivo. Antioxid Redox Signal 3, 1023–
1039.
Spencer JP, Chowrimootoo G, Choudhury R, Debnam
ES, Srai SK & Rice-Evans C (1999) The small intestine
can both absorb and glucuronidate luminal flavonoids.
FEBS Lett 458, 224–230.
Donovan JL, Crespy V, Manach C, Morand C, Besson
C, Scalbert A & Remesy C (2001) Catechin is metabolized by both the small intestine and liver of rats.
J Nutr 131, 1753–1757.
Spencer JP, Abd-el-Mohsen MM & Rice-Evans C
(2004) Cellular uptake and metabolism of flavonoids
and their metabolites: implications for their bioactivity.
Arch Biochem Biophys 423, 148–161.
Di Pietro A, Godinot C, Bouillant ML & Gautheron
DC (1975) Pig heart mitochondrial ATPase: properties
of purified and membrane-bound enzyme. Effects of
flavonoids. Biochimie 57, 959–967.

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS

5523


Antioxidants and proteasome in cancer treatment

L. Bonfili et al.

102 Barzilai A & Rahamimoff H (1983) Inhibition of
Ca2+-transport ATPase from synaptosomal vesicles by
flavonoids. Biochim Biophys Acta 730, 245–254.
103 Revuelta MP, Cantabrana B & Hidalgo A (1997)
Depolarization-dependent effect of flavonoids in rat
uterine smooth muscle contraction elicited by CaCl2.
Gen Pharmacol 29, 847–857.
104 Gamet-Payrastre L, Manenti S, Gratacap MP, Tulliez
J, Chap H & Payrastre B (1999) Flavonoids and the
inhibition of PKC and PI 3-kinase. Gen Pharmacol 32,
279–286.
105 Lee SF & Lin JK (1997) Inhibitory effects of phytopolyphenols on TPA-induced transformation, PKC
activation, and c-jun expression in mouse fibroblast
cells. Nutr Cancer 28, 177–183.
106 Boege F, Straub T, Kehr A, Boesenberg C, Christiansen K, Andersen A, Jakob F & Kohrle J (1996)
Selected novel flavones inhibit the DNA binding or the
DNA religation step of eukaryotic topoisomerase I.
J Biol Chem 271, 2262–2270.
107 Ferriola PC, Cody V & Middleton E Jr (1989) Protein
kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure–activity relationships. Biochem
Pharmacol 38, 1617–1624.
108 Reddy L, Odhav B & Bhoola KD (2003) Natural
products for cancer prevention: a global perspective.

Pharmacol Ther 99, 1–13.
109 Block G, Patterson B & Subar A (1992) Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 18, 1–29.
110 Mathew A, Peters U, Chatterjee N, Kulldorff M &
Sinha R (2004) Fat, fiber, fruits, vegetables, and risk of
colorectal adenomas. Int J Cancer 108, 287–292.
111 Russo GL (2007) Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochem Pharmacol
74, 533–544.
112 Greenwald P (2004) Cancer chemoprevention. BMJ
324, 714–718.
113 Surh YJ (2003) Cancer chemoprevention with dietary
phytochemicals. Nat Rev Cancer 3, 768–780.
114 Surh YJ, Kundu JK, Na HK & Lee JS (2005) Redoxsensitive transcription factors as prime targets for chemoprevention with anti-inflammatory and antioxidative
phytochemicals. J Nutr 135, 2993S–3001S.
115 Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a
dawn for evolutionary medicine. Annu Rev Genet 39,
359–407.
116 Hayes JD, Kelleher MO & Eggleston IM (2008) The
cancer chemopreventive actions of phytochemicals
derived from glucosinolates. Eur J Nutr 47(Suppl. 2),
73–88.
117 Baumeister P, Reiter M, Zieger S, Matthias C & Harreus U (2008) DNA-protective potential of polyphenols
in human mucosa cell cultures. HNO 56, 795–798.

5524

118 Trosko JE (2006) Dietary modulation of the multistage, multimechanisms of human carcinogenesis:
effects on initiated stem cells and cell–cell communication. Nutr Cancer 54, 102–110.
119 de la Lastra CA & Villegas I (2007) Resveratrol as an
antioxidant and pro-oxidant agent: mechanisms and
clinical implications. Biochem Soc Trans 35, 1156–1160.

120 Ahsan H & Hadi SM (1998) Strand scission in DNA
induced by curcumin in the presence of Cu(II). Cancer
Lett 124, 23–30.
121 Rahman A, Shahabuddin, Hadi SM & Parish JH
(1990) Complexes involving quercetin, DNA and
Cu(II). Carcinogenesis 11, 2001–2003.
122 Zheng LF, Wei QY, Cai YJ, Fang JG, Zhou B, Yang
L & Liu ZL (2006) DNA damage induced by resveratrol and its synthetic analogues in the presence of
Cu(II) ions: mechanism and structure-activity relationship. Free Radical Biol Med 41, 1807–1816.
123 Mukhtar H & Ahmad N (2000) Tea polyphenols: prevention of cancer and optimizing health. Am J Clin
Nutr 71 (6 Suppl.), 1698S–1702S; discussion 1703S–
1704S.
124 Erba D, Riso P, Bordoni A, Foti P, Biagi PL & Testolin G (2005) Effectiveness of moderate green tea consumption on antioxidative status and plasma lipid
profile in humans. J Nutr Biochem 16, 144–149.
125 Dalluge JJ, Nelson BC, Thomas JB & Sander LC
(1998) Selection of column and gradient elution system
for the separation of catechins in green tea using highperformance liquid chromatography. J Chromatogr A
793, 265–274.
126 Shimizu M, Deguchi A, Lim JT, Moriwaki H, Kopelovich L & Weinstein IB (2005) ())-Epigallocatechin gallate and polyphenon E inhibit growth and activation
of the epidermal growth factor receptor and human
epidermal growth factor receptor-2 signaling pathways
in human colon cancer cells. Clin Cancer Res 11, 2735–
2746.
127 Dong Z, Ma W, Huang C & Yang CS (1997) Inhibition of tumor promoter-induced activator protein 1
activation and cell transformation by tea polyphenols,
())-epigallocatechin gallate, and theaflavins. Cancer
Res 57, 4414–4419.
128 Givant-Horwitz V, Davidson B & Reich R (2004)
Laminin-induced signaling in tumor cells: the role of
the Mr 67,000 laminin receptor. Cancer Res 64, 3572–

3579.
129 Ahmed S, Rahman A, Hasnain A, Lalonde M, Goldberg VM & Haqqi TM (2002) Green tea polyphenol
epigallocatechin-3-gallate inhibits the IL-1 beta-induced
activity and expression of cyclooxygenase-2 and nitric
oxide synthase-2 in human chondrocytes. Free Radical
Biol Med 33, 1097–1105.
130 Hussain T, Gupta S, Adhami VM & Mukhtar H
(2005) Green tea constituent epigallocatechin-3-gallate

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS


L. Bonfili et al.

131

132

133

134

135

136

137

138

139

140

141

142

143

selectively inhibits COX-2 without affecting COX-1
expression in human prostate carcinoma cells. Int J
Cancer 113, 660–669.
Nam S, Smith DM & Dou QP (2001) Ester bond-containing tea polyphenols potently inhibit proteasome activity
in vitro and in vivo. J Biol Chem 276, 13322–13330.
Smith DM, Wang Z, Kazi A, Li LH, Chan TH & Dou
QP (2002) Synthetic analogs of green tea polyphenols
as proteasome inhibitors. Mol Med 8, 382–392.
Wan SB, Chen D, Dou QP & Chan TH (2004) Study
of the green tea polyphenols catechin-3-gallate (CG)
and epicatechin-3-gallate (ECG) as proteasome inhibitors. Bioorg Med Chem 12, 3521–3527.
Chen MS, Chen D & Dou QP (2004) Inhibition of
proteasome activity by various fruits and vegetables is
associated with cancer cell death. In Vivo 18, 73–80.
Nam S, Smith DM & Dou QP (2001) Tannic acid
potently inhibits tumor cell proteasome activity,
increases p27 and Bax expression, and induces G1
arrest and apoptosis. Cancer Epidemiol Biomarkers
Prev 10, 1083–1088.
Marienfeld C, Tadlock L, Yamagiwa Y & Patel T

(2003) Inhibition of cholangiocarcinoma growth by
tannic acid. Hepatology 37, 1097–1104.
Dosenko VE, Nagibin VS, Tumanovskaia LV, Zagorii
V & Moibenko AA (2006) The influence of quercetin
on the activity of purified 20S, 26S proteasome and
proteasomal activity in isolated cardiomyocytes.
Biomed Khim 52, 138–145.
Weng MS, Ho YS & Lin JK (2005) Chrysin induces
G1 phase cell cycle arrest in C6 glioma cells through
inducing p21Waf1 ⁄ Cip1 expression: involvement of
p38 mitogen-activated protein kinase. Biochem
Pharmacol 69, 1815–1827.
Jin CY, Lee JD, Park C, Choi YH & Kim GY (2007)
Curcumin attenuates the release of pro-inflammatory
cytokines in lipopolysaccharide-stimulated BV2
microglia. Acta Pharmacol Sin 28, 1645–1651.
Mehta K, Pantazis P, McQueen T & Aggarwal BB
(1997) Antiproliferative effect of curcumin (diferuloylmethane) against human breast tumor cell lines.
Anticancer Drugs 8, 470–481.
Iqbal M, Sharma SD, Okazaki Y, Fujisawa M &
Okada S (2003) Dietary supplementation of curcumin
enhances antioxidant and phase II metabolizing
enzymes in ddY male mice: possible role in protection
against chemical carcinogenesis and toxicity. Pharmacol
Toxicol 92, 33–38.
Dikshit P, Goswami A, Mishra A, Chatterjee M &
Jana NR (2006) Curcumin induces stress response,
neurite outgrowth and prevent NF-kappaB activation
by inhibiting the proteasome function. Neurotox Res 9,
29–37.

Jana NR, Dikshit P, Goswami A & Nukina N (2004)
Inhibition of proteasomal function by curcumin

Antioxidants and proteasome in cancer treatment

144

145

146

147

148

149

150

151

152

153

154

155

156

induces apoptosis through mitochondrial pathway.
J Biol Chem 279, 11680–11685.
Ali RE & Rattan SI (2006) Curcumin’s biphasic hormetic response on proteasome activity and heat-shock
protein synthesis in human keratinocytes. Ann NY
Acad Sci 1067, 394–399.
Si X, Wang Y, Wong J, Zhang J, McManus BM &
Luo H (2007) Dysregulation of the ubiquitin–proteasome system by curcumin suppresses coxsackievirus B3
replication. J Virol 81, 3142–3150.
Wyke SM, Russell ST & Tisdale MJ (2004) Induction
of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-kappaB activation. Br J
Cancer 91, 1742–1750.
Kazi A, Daniel KG, Smith DM, Kumar NB & Dou
QP (2003) Inhibition of the proteasome activity, a
novel mechanism associated with the tumor cell apoptosis-inducing ability of genistein. Biochem Pharmacol
66, 965–976.
Park BS, Baek SJ, Song KH, Kim KH, Jeong SJ,
Jeong MH, Seo SY, Lee SW, Yoo KS & Yoo YH
(2002) Genistein-induced apoptosis of p815 mastocytoma cells is mediated by Bax and augmented by a
proteasome inhibitor, lactacystin. Nutr Cancer 42, 248–
255.
Ulrich S, Wolter F & Stein JM (2005) Molecular mechanisms of the chemopreventive effects of resveratrol
and its analogs in carcinogenesis. Mol Nutr Food Res
49, 452–461.
Delmas D, Lancon A, Colin D, Jannin B & Latruffe N
(2006) Resveratrol as a chemopreventive agent: a
promising molecule for fighting cancer. Curr Drug
Targets 7, 423–442.
Aziz MH, Kumar R & Ahmad N (2003) Cancer chemoprevention by resveratrol: in vitro and in vivo studies and the underlying mechanisms (review). Int J
Oncol 23, 17–28.

Fulda S & Debatin KM (2004) Sensitization for anticancer drug-induced apoptosis by the chemopreventive
agent resveratrol. Oncogene 23, 6702–6711.
Pervaiz S (2004) Chemotherapeutic potential of the
chemopreventive phytoalexin resveratrol. Drug Resist
Update 7, 333–344.
Liao HF, Kuo CD, Yang YC, Lin CP, Tai HC, Chen
YY & Chen YJ (2005) Resveratrol enhances radiosensitivity of human non-small cell lung cancer NCI-H838
cells accompanied by inhibition of nuclear factorkappa B activation. J Radiat Res (Tokyo) 46, 387–
393.
Kundu JK & Surh YJ (2008) Cancer chemopreventive
and therapeutic potential of resveratrol: mechanistic
perspectives. Cancer Lett 269, 243–261.
Kazi A, Urbizu DA, Kuhn DJ, Acebo AL, Jackson
ER, Greenfelder GP, Kumar NB & Dou QP (2003) A
natural musaceas plant extract inhibits proteasome

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS

5525


Antioxidants and proteasome in cancer treatment

157

158

159

160

161

162

163

164

165

L. Bonfili et al.

activity and induces apoptosis selectively in human
tumor and transformed, but not normal and non-transformed, cells. Int J Mol Med 12, 879–887.
Amici M, Bonfili L, Spina M, Cecarini V, Calzuola I,
Marsili V, Angeletti M, Fioretti E, Tacconi R,
Gianfranceschi GL et al. (2008) Wheat sprout extract
induces changes on 20S proteasomes functionality.
Biochimie 90, 790–801.
Katsiki M, Chondrogianni N, Chinou I, Rivett AJ &
Gonos ES (2007) The olive constituent oleuropein
exhibits proteasome stimulatory properties in vitro and
confers life span extension of human embryonic fibroblasts. Rejuvenation Res 10, 157–172.
Smith DM, Daniel KG, Wang Z, Guida WC, Chan
TH & Dou QP (2004) Docking studies and model
development of tea polyphenol proteasome inhibitors:
applications to rational drug design. Proteins 54,
58–70.
Kisselev AF & Goldberg AL (2001) Proteasome inhibitors: from research tools to drug candidates. Chem Biol

8, 739–758.
Landis-Piwowar KR, Kuhn DJ, Wan SB, Chen D,
Chan TH & Dou QP (2005) Evaluation of proteasomeinhibitory and apoptosis-inducing potencies of novel
(-)-EGCG analogs and their prodrugs. Int J Mol Med
15, 735–742.
Kuhn D, Lam WH, Kazi A, Daniel KG, Song S,
Chow LM, Chan TH & Dou QP (2005) Synthetic peracetate tea polyphenols as potent proteasome inhibitors
and apoptosis inducers in human cancer cells. Front
Biosci 10, 1010–1023.
Daniel KG, Landis-Piwowar KR, Chen D, Wan SB,
Chan TH & Dou QP (2006) Methylation of green tea
polyphenols affects their binding to and inhibitory
poses of the proteasome beta5 subunit. Int J Mol Med
18, 625–632.
Osanai K, Landis-Piwowar KR, Dou QP & Chan TH
(2007) A para-amino substituent on the D-ring of
green tea polyphenol epigallocatechin-3-gallate as a
novel proteasome inhibitor and cancer cell apoptosis
inducer. Bioorg Med Chem 15, 5076–5082.
Landis-Piwowar KR, Milacic V & Dou QP (2008)
Relationship between the methylation status of dietary
flavonoids and their growth-inhibitory and apoptosis-

5526

166

167

168

169
170

171

172

173

174

175

176
177

178

inducing activities in human cancer cells. J Cell Biochem 105, 514–523.
Proteggente AR, Pannala AS, Paganga G, Van Buren
L, Wagner E, Wiseman S, Van De Put F, Dacombe C
& Rice-Evans CA (2002) The antioxidant activity of
regularly consumed fruit and vegetables reflects their
phenolic and vitamin C composition. Free Radical Res
36, 217–233.
Kuroda Y & Hara Y (1999) Antimutagenic and anticarcinogenic activity of tea polyphenols. Mutat Res
436, 69–97.
Park OJ & Surh YJ (2004) Chemopreventive potential
of epigallocatechin gallate and genistein: evidence from

epidemiological and laboratory studies. Toxicol Lett
150, 43–56.
Pietta PG (2000) Flavonoids as antioxidants. J Nat
Prod 63, 1035–1042.
Voorhees PM, Dees EC, O’Neil B & Orlowski RZ
(2003) The proteasome as a target for cancer therapy.
Clin Cancer Res 9, 6316–6325.
McGhie TK & Walton MC (2007) The bioavailability
and absorption of anthocyanins: towards a better
understanding. Mol Nutr Food Res 51, 702–713.
Duthie SJ (2007) Berry phytochemicals, genomic stability and cancer: evidence for chemoprotection at several
stages in the carcinogenic process. Mol Nutr Food Res
51, 665–674.
Yang CS, Sang S, Lambert JD, Hou Z, Ju J & Lu G
(2006) Possible mechanisms of the cancer-preventive
activities of green tea. Mol Nutr Food Res 50, 170–175.
Tomas-Barberan FA & Clifford MN (2000)
Flavanones, chalcones and dihydrochalcones – nature,
occurrence and dietary burden. J Sci Food Agric 80,
1073–1080.
Woodman OL & Chan E (2004) Vascular and anti-oxidant actions of flavonols and flavones. Clin Exp Pharmacol Physiol 31, 786–790.
Martens S & Mithofer A (2005) Flavones and flavone
synthases. Phytochemistry 66, 2399–2407.
Nielsen IL & Williamson G (2007) Review of the factors affecting bioavailability of soy isoflavones in
humans. Nutr Cancer 57, 1–10.
Sarkar FH & Li Y (2004) The role of isoflavones in
cancer chemoprevention. Front Biosci 9, 2714–2724.

FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS