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Transcript of Natural polyphenols as proteasome modulators and their role as anti-cancer compounds
REVIEW ARTICLE
Natural polyphenols as proteasome modulators and theirrole as anti-cancer compoundsLaura 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
Introduction
Nutritional studies have recently shown that a regular
consumption of polyphenolic antioxidants, contained
in fruits, vegetables and their related juices, has a posi-
tive effect in the treatment and prevention of a wide
range of pathologies, including cancer [1], stroke [2],
coronary heart disease [3,4] and neurodegenerative dis-
ease, 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 thera-
peutic 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,
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: [email protected]
(Received 1 August 2008, revised
10 September 2008, accepted 22
September 2008)
doi:10.1111/j.1742-4658.2008.06696.x
The purpose of this review is to discuss the effect of natural antioxidant
compounds as modulators of the 20S proteasome, a multi-enzymatic multi-
catalytic complex present in the cytoplasm and nucleus of eukaryotic cells
and involved in several cellular activities such as cell-cycle progression, pro-
liferation 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 poten-
tial relevant adjuvants and pharmacological models in the study of new
drugs.
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.
5512 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 natu-
ral 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 non-
lysosomal 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 configura-
tion. The a subunits are localized in the outer rings
and the b subunits in the inner rings of this cylinder-
like 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 bsubunits, carrying the enzyme active sites, constitute
the two inner rings, whereas the outer ones are com-
posed of seven different a subunits (a1-7 b1-7 b1-7a1-7). 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 respon-
sible for the recognition, unfolding and translocation
of the selected substrates into the lumen of the cata-
lytic core.
The covalent attachment of a polyubiquitin chain
facilitates substrate recognition and triggers 26S pro-
teasome-mediated degradation. This conjugation reac-
tion 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 mono-
mer to a lysine residue of the target protein. More
than one ubiquitin needs to be added to build a poly-
Ub 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 man-
ner [29]. There is evidence that the 20S complex can
directly degrade protein substrates such as casein, lyso-
zyme, insulin b-chain, histone H3, ornithine decarbox-
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-termi-
nal 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 stud-
ies, 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 hydroly-
sing (PGPH, cleaving after acidic residues), are associ-
ated 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 immunomodu-
latory 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, b2iand 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 contain-
ing 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 confor-
mational 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) dem-
onstrate 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].
L. Bonfili et al. Antioxidants and proteasome in cancer treatment
FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5513
The 20S proteasome is also part of the intracellular
antioxidant defence system, being involved in the deg-
radation 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 demon-
strated. Further evidence supporting the role of the pro-
teasome in removing oxidized proteins is that SH-SY5Y
and mutated G93A cells present increased levels of pro-
tein 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 path-
ways, regulating cell proliferation, cell death, neuro-
pathological events and drug resistance in human
tumour cells. Therefore, it seems to be an attractive
target for a combined chemopreventative ⁄ chemothera-
peutic approach, which seems ideal for cancer therapy.
In particular, because proteasome inhibitors are con-
sidered 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-Jun-
NH2-terminal kinase induction, block of the nuclear
traslocation of NF-jB, generation of reactive oxygen
species, transmembrane mitochondrial potential gradi-
ent alteration, cytochrome c release, and activation of
caspase-mediated apoptosis [74,75]. Despite the accept-
able 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, thrombocyto-
penia, 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
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 phyto-
chemicals 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 (cat-
alase, 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 aro-
matic 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 pres-
ence of a catechol group in the B-ring and the 2,3 dou-
ble bond in conjunction with the 4-carbonyl group as
well as the 3- and 5-hydroxyl groups. Thus, the hydro-
philic ⁄ 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 mem-
branes and the way substitutive groups affect their
Fig. 1. The chemical structure of a flavonoid.
Antioxidants and proteasome in cancer treatment L. Bonfili et al.
5514 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS
structure, fluidity and permeability [95,96]. The degree
of hydroxylation also influences the intestinal absorp-
tion 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 func-
tion [100].
Polyphenols administered at pharmacological doses
(hundreds of milligrams) or consumed as a polyphe-
nol-rich diet (> 1 gÆdose)1), can readily saturate the
conjugation pathways leading to detectable, unconju-
gated compounds in the plasma. The utilized concen-
Table 1. Subclasses of flavonoids.
Subclasses Principal compounds Structure Food sources
Anthocyanin
Pelargonidin
Cyanidin, malvidin
OH
OH
OH
OHO+
Berry fruits, grape seeds, wine [171,172]
Flavanols Catechin, EGCG, ECG, EGC, EC
OH
OH
OH
OH
OH
O
Tea [173], red wine, cocoa, grape juice
Flavanones Hesperetin, naringenin, narirutin,
eriodictyol, neohesperetin
O
O
Citrus fruit, grapefruit, bitter orange [174]
Flavonols Myricetin, kaempferol,
quercetin glucosides
OH
O
O
Onions, tea, red wine, broccoli,
berries, apple [175]
Flavones Pigenin, chrysin, luteolin O
O
Chamomile, tea, honey, propolis [176]
Isoflavones Genistein, daidzein O
O
Soybeans, black beans, green
beans chickpeas [177,178]
L. Bonfili et al. Antioxidants and proteasome in cancer treatment
FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5515
trations influence not only quality and quantity of cir-
culating species, but also tissues distribution of
polyphenols and their relative metabolites [11].
Flavonoids have the potential to bind the ATP-bind-
ing sites of a large number of proteins [14] including
mitochondrial ATPase [101], calcium plasma mem-
brane 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 cancerchemoprevention
Several epidemiological studies have suggested a posi-
tive 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 mod-
els revealed the ability of several polyphenols to defend
cells against cancer. Russo [111] suggested that these
molecules can work as cancer-blocking agents, prevent-
ing 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)-medi-
ated 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 princi-
pally 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
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 chemo-
preventative role has also been described as being inde-
pendent 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 com-
pound is a redox agent that, under particular condi-
tions 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]. Inter-
estingly, considering that copper levels are higher in
tumour cells than in normal cells, it has been hypothe-
sized 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 can-
cer 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 poly-
phenolic 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, flav-
ones, 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 transduc-
tion 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 metabo-
lism [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
Antioxidants and proteasome in cancer treatment L. Bonfili et al.
5516 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 accu-
mulation 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 proteaso-
mal ChT-L activity in leukaemia Jurkat T-cell lysates.
This effect caused an accumulation of the polyubiquiti-
nated proteins, activation of caspase 3 and caspase 7,
and cleavage of poly(ADP-ribose) polymerase. The
inhibition of proteasome activity by these fruit or vege-
tables 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 con-
densed tannins, also known as proanthocyanidins. The
hydrolysable tannins contain gallotannins or ellagic-
tannins. Upon hydrolysis, gallotannins yield glucose
and gallic acid, whereas the ellagictannins produce
ellagic acid as a degradation product [135].
It has been reported that tannic acid, an example of
gallotannins, potently and specifically inhibits the
ChT-L activity of purified 20S proteasome, 26S pro-
teasome of Jurkat T-cell extracts and the 26S protea-
some 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-pentahydroxyf-lavone). This flavonoid belongs to the flavonols sub-
group. In a recent study, Dosenko et al. [137]
performed experiments on purified 20S proteasomes
showing that quercetin inhibits three of the prot-
easomal peptidase activities, in particular the ChT-L
component, in a dose-dependent manner, having com-
parable 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 querce-
tin are more potent than kaempferol and myricetin in
inhibiting the ChT-L activity of purified 20S protea-
some 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 propo-
lis, 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 moi-
ety 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].
L. Bonfili et al. Antioxidants and proteasome in cancer treatment
FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5517
Curcumin
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 dose-
dependent 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 curcu-
min (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].
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, inhi-
bition 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 fibro-
blast counterpart (WI-38) [147]. Genistein induced
apoptosis of p815 mastocytoma cells, in part medi-
ated by proteasome. The enzyme activity was inhib-
ited at early time points after genistein treatment
[148].
Resveratrol
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 pro-
gression in animal models inoculated with malignant
cell lines, or treated with tumorigenesis-inducing
drugs.
In vitro, resveratrol influenced numerous intracellu-
lar 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 clin-
ical trials performed with the intake of resveratrol
combined with chemotherapeutic treatments indicated
that low doses of resveratrol were capable of enhanc-
ing 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 inhibi-
tion 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–protea-
some 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 cate-
chin, 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 pro-
apoptotic 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).
Oleuropein
Oleuropein, the major constituent of Olea europea leaf
extract, olive oil and olives, was reported to enhance
Antioxidants and proteasome in cancer treatment L. Bonfili et al.
5518 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS
proteasome activity in vitro more strongly than other
known chemical activators, possibly through confor-
mational changes in the proteasome. Moreover, con-
tinuous 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 proteasome-
mediated degradation rates and retained proteasome
function during replicative senescence [158].
New potential drugs in cancertreatment
Multiple lines of evidence have proposed a positive
effect of natural phytochemical compounds like flavo-
noids 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 chemotherapeu-
tic drugs (see above).
Table 2 summarizes some phytochemical compounds
that have been proposed as potential chemopreventa-
tive, 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, sev-
eral 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 pro-
teasome ChT-L activity by EGCG was time-dependent
and irreversible, and implicated the acylation of the b5subunit’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 pro-
tected by peracetate that can be removed by cellular
Table 2. Polyphenols in active clinical trials (data from the National Cancer Institute, http://www.cancer.gov).
Polyphenols Source
Clinical trial
phase Type of cancer Combined with
Curcumin Turmeric Phase III Metastatic colon cancer Gemcitabine
Turmeric Phase III Pancreatic cancer Gemcitabine
Phase I–II Osteosarcoma
Phase II Colorectal cancer
Phase II Stage IV breast cancer Gemcitabine
hydrochloride
and genistein
Phase II Advanced pancreatic cancer Gemcitabine
Vitamin D and
soy isoflavones
Phase II Adenocarcinoma of the prostate
Synthetic genistein Phase II Prostate cancer
Resveratrol Grape skins Phase I–II Colon cancer
Phase I Colorectal cancer
Phase I Healthy adults at increased
risk of developing melanoma
Green tea extract Polyphenon E Phase I–II Chronic lymphocytic leukemia
Phase I–II Advanced non small cell lung cancer Erlotinib
Phase II Human papillomavirus and low-grade
cervical intraepithelial neoplasia
Phase II Lung cancer
Phase II Bronchial dysplasia
Phase II Prostate cancer
Phase II High-grade prostatic intraepithelial
neoplasia
Phase II Breast cancer
Phase II Nonmetastatic bladder cancer
Tea polyphenols
and theaflavins
Green tea,
decaffeinated
black tea
Phase II Prostate cancer
L. Bonfili et al. Antioxidants and proteasome in cancer treatment
FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5519
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 epicate-
chin-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 polyphe-
nols and their inhibitory effects on the proteasome
[22,165]. As mentioned previously for EGCG, the IC50
values measured for chrysin, luteolin, apigenin, narin-
genin and eriodictyol were strictly related to the num-
ber 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 flavo-
noids [166–169]. The proteasome is responsible for
degrading most intracellular proteins, including oxi-
dized proteins and the proteins involved in cell-cycle
regulation and apoptosis, processes crucial to onco-
genesis. 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 com-
bined 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 deg-
radation and all the cellular pathways in which the
proteasome is involved, represents a promising start-
ing point for designing and developing novel anti-
cancer drugs.
Acknowledgements
The authors wish to thank Dr Matteo Mozzicafreddo
for technical assistance.
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