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

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 nucleu

Natural polyphenols as proteasome modulators and their role as anti-cancer compounds

Laura Bonfili1, Valentina Cecarini1, Manila Amici1, Massimiliano Cuccioloni1, Mauro Angeletti1, Jeffrey N Keller2and 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: annamaria.eleuteri@unicam.it

(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 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.

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 acidophilumproteasome 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

com-posed 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

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, 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 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]

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.

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.

Anthocyanin

Pelargonidin

Cyanidin, malvidin

OH

OH

OH

OH

O+

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]

O

Chamomile, tea, honey, propolis [176]

O

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

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] includATP-bind-ing

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 cancer

chemoprevention

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

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 myricequerce-tin in inhibiting the ChT-L activity of purified 20S protea-some and 26S proteaprotea-some 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]

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

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 cancer

treatment

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 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 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).

Clinical trial

hydrochloride and genistein

Vitamin D and

soy isoflavones

Phase II Adenocarcinoma of the prostate

Phase I Healthy adults at increased

risk of developing melanoma

Phase I–II Advanced non small cell lung cancer Erlotinib Phase II Human papillomavirus and low-grade

cervical intraepithelial neoplasia

Phase II Bronchial dysplasia

Phase II High-grade prostatic intraepithelial

neoplasia

Phase II Nonmetastatic bladder cancer Tea polyphenols

and theaflavins

Green tea, decaffeinated black tea

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 designstart-ing and developstart-ing novel

anti-cancer drugs

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

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