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 Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy 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: annamaria.eleuteri@unicam.it 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 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 5512 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS L Bonfili et al 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] FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5513 Antioxidants and proteasome in cancer treatment L Bonfili et al 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 5514 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 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 The chemical structure of a flavonoid FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS L Bonfili et al Antioxidants and proteasome in cancer treatment Table 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 (> gỈdose)1), can readily saturate the conjugation pathways leading to detectable, unconjugated compounds in the plasma The utilized concen- FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5515 Antioxidants and proteasome in cancer treatment L Bonfili et al 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 5516 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 FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS L Bonfili et al 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 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 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] FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS 5517 Antioxidants and proteasome in cancer treatment L Bonfili et al 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 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 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] 5518 Oleuropein Oleuropein, the major constituent of Olea europea leaf extract, olive oil and olives, was reported to enhance FEBS Journal 275 (2008) 5512–5526 ª 2008 The Authors Journal compilation ª 2008 FEBS L Bonfili et al Antioxidants and proteasome in cancer treatment 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 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 Polyphenols in active clinical trials (data from the National Cancer Institute, http://www.cancer.gov) 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 5519 Antioxidants and proteasome in cancer treatment 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 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