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Thioredoxin: friend or foe in human disease?
Review
TRENDS in Pharmacological Sciences
Vol.26 No.8 August 2005
Thioredoxin: friend or foe in human disease? Anne Burke-Gaffney1, Matthew E.J. Callister1 and Hajime Nakamura2 1
Unit of Critical Care, National Heart and Lung Institute Division, Imperial College Faculty of Medicine, Dovehouse Street, London SW3 6LY, UK 2 Thioredoxin Project, Department of Experimental Therapeutics, Translational Research Center, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo, Kyoto, 606-8507, Japan
Thioredoxin (Trx), a small, ubiquitous thiol [sulfydryl (-SH)] protein, is one of the most important regulators of reduction–oxidation (redox) balance and, thus, redoxcontrolled cell functions. Although Trx was discovered 40 years ago in bacteria, the number and diversity of processes that Trx influences in human cells have only been appreciated recently. Processes influenced by Trx include the control of cellular redox balance, the promotion of cell growth, the inhibition of apoptosis and the modulation of inflammation. Not surprisingly, the role of Trx in a wide range of human diseases and conditions, including cancer, viral disease, ischaemia– reperfusion injury, cardiac conditions, aging, premature birth and newborn physiology, is subject to intense investigation. However, whether Trx contributes to or prevents the pathology of a particular condition is not always clear. In this article, we review the role of Trx in human disease and relate this to its redox activity and biological properties, and discuss the development and use of therapies that either inhibit or augment Trx activity.
The dichotomy of Trx Any particular biological property of thioredoxin (Trx) is unlikely to be either ‘good’ or ‘bad’ in disease; indeed, the effect of Trx is likely to depend on the type and stage of the condition. Thus, in cancer, the anti-apoptotic properties of Trx are considered deleterious because they can impair the effectiveness of chemotherapy strategies that trigger apoptosis. However, in ischaemia–reperfusion injury, in which apoptosis contributes to the pathology, Trx might protect against injurious insults [1]. Likewise, the growthpromoting effects of Trx are detrimental in cancer and NADPH + H+
TrxR-(S-Se)
NADP+
TrxR-(SH Se–) + H+
rheumatoid arthritis [2,3] but are beneficial in neurodegenerative disease in which promoting neural-cell growth aids recovery [4]. In HIV infection, Trx blocks HIV replication [3] but at later stages of the disease it contributes to immunosuppression in some patients [5]. A clear understanding of which properties of Trx predominate in a particular situation is key to the successful therapeutic manipulation of Trx. The biochemistry and biological properties of Trx are well established (reviewed in [3,6,7]). However, the dichotomy of Trx in disease is less well documented. In this review, we summarize the redox activity and biological properties of Trx. These are essential to understanding the dichotomous role of Trx in disease, which is also discussed. Finally, we describe the pharmacological inhibition of Trx, strategies that are used to induce and augment Trx and its practical applications.
Redox activity and biological properties of Trx The biological properties of Trx rely, largely, on reduction– oxidation (redox) activity, which is the ability to transfer ‘reducing equivalents’ to disulfide groups in target proteins. The key to the redox activity of Trx is the presence of two cysteine residues (Cys32 and Cys35) separated by two amino acids (Gly-Pro) in its active site. These cysteines exist as a dithiol [-(SH)2] in the reduced form and a disulfide (-S2) in the oxidized form. Trx is oxidized when it transfers reducing equivalents to disulfide groups in target proteins and it is reduced back to the dithiol form by an NADPH-dependent flavoprotein, thioredoxin reductase (TrxR); this forms the so-called Trx system (Figure 1). The activities and properties of Trx and TrxR are reviewed extensively in [6–10]. Trx-(SH)2
Trx-S2
Sub-S2
Sub-(SH)2
Figure 1. Redox cycling of Trx and TrxR. Trx is oxidised as it transfers reducing equivalents to disulfide groups in target proteins. Trx is then reduced back to the dithiol form by an NADPH-dependent flavoprotein, TrxR. The C-terminus of TrxR contains Cys-SeCys as a catalytic centre. Abbreviations: NADPH, nicotinamide dinucleotide phosphate (reduced form); NADPC, nicotinamide dinucleotide phosphate (oxidized form); -S2, oxidized, disulfide form; SeK the selenolate anion; SH, the thiol anion; (SH)2, reduced, dithiol form; S-Se the selenylsulfide; Sub, protein substrate. Corresponding author: Burke-Gaffney, A. ([email protected]). Available online 28 June 2005 www.sciencedirect.com 0165-6147/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2005.06.005
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TRENDS in Pharmacological Sciences
To date, most work that relates to the role of Trx in human disease concerns Trx-1, which is one of a family of proteins that share the active-site sequence (Cys-X-X-Cys). Trx-1 is expressed ubiquitously in cytosol and translocates to the nucleus when cells are activated by, for example, oxidative stress. Trx-1 has no recognizable nuclear localization signal and it is assumed that it is carried into the nucleus bound to other proteins such as nuclear factor kB (NF-kB) and redox factor 1. The function of Trx-1 is tightly linked to these proteins [11,12]. Trx-1 is also secreted extracellularly where it is thought to mediate some inflammatory effects. In addition to the two cysteine residues in the catalytic site, human Trx-1 contains three other cysteine residues (Cys62, Cys69 and Cys73). Although not part of the active site, these contribute to essential protein conformation, and oxidation of these structural residues leads to loss of enzymatic activity. Active-site and structural cysteine residues are targets for potential inhibitors of Trx. Key biological activities of Trx that are applicable to human disease can be categorized as antioxidant, growth promoting, anti-apoptotic and inflammation modulating. Several of these activities result, in part, from redox regulation by Trx of signal transduction pathways and gene expression [13]. The principle intracellular antioxidant property of Trx results from its ability to act as a cofactor that maintains several thioredoxin peroxidase enzymes in a reduced, active form [13]. As their name indicates, these enzymes, which are members of the peroxiredoxin family of proteins, inactivate hydrogen peroxide (H2O2). Other antioxidant properties of Trx result from the reduction of glutathione peroxidase, the induction of manganese superoxide dismutase and, to some extent, the direct reduction of H2O2 by Trx [14]. S-nitrosylation of Cys69 of Trx is an important posttranslational modification that potentiates antioxidant activity [15]. Trx stimulates the growth of normal and cancerous cells in an atypical manner that does not appear to use a receptor. Mechanisms of Trx-induced-growth are multifaceted and include the provision of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and increasing sensitivity of cells to other cytokines and growth factors [2,13]. The anti-apoptotic affects of Trx might, in part, relate to its ability to bind to and inhibit apoptosis-signalregulating kinase 1 (ASK1) [13]. When Trx is oxidized, ASK1 dissociates and activates an apoptotic signalling pathway. Trx also regulates polyamine homeostasis, a pathway that has complex inhibitory and activator effects on apoptosis and cell survival [16]. Together with the growth-promoting effects, the ability of Trx to inhibit apoptosis contributes to the aggressive growth of many tumours. In addition, Trx has several significant effects on inflammatory pathways [17]. First, Trx has multiple regulatory effects on cytokine production. As with growth factors, Trx either directly induces the synthesis of or augments the production of some cytokines and inhibits the synthesis of others. These effects are caused, in part, by the involvement of Trx in redox signalling [18]. Trx also has dual regulatory effects on leukocyte movement. It acts www.sciencedirect.com
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as a chemoattractant for monocytes, neutrophils and T cells in vitro and following local administration in vivo [19] with a potency that is comparable to that of known chemokines. However, this effect is atypical of a chemokine because it is independent of G proteins and intracellular Ca2C is not increased. By contrast, intravenous administration of Trx suppresses lipopolysaccharide (LPS)-induced neutrophil chemotaxis, in part, by preventing L-selectin shedding from neutrophils, which is a prerequisite for migration [20]. Other inflammatory activities of Trx are attributed to the 10-kDa, C-terminally-truncated form of Trx (Trx80). This comprises either the 80 or the 84 N-terminal amino acids and is secreted into and is present in plasma [21]. Monocytes are the primary target of Trx80, which causes proliferation, cytokine production and increased expression of cell-surface antigens such as CD14 in these cells. Unlike Trx, Trx80 lacks reducing activity and its effects are independent of cysteine residues in the active-site motif. Can we predict how the biological activities of Trx impact on health and disease? It might be expected that antioxidant effects are beneficial, that the merits or otherwise of growth promoting and anti-apoptotic effects are likely to vary from condition to condition, and that the effects on inflammation might also vary. Trx in health and disease Early studies of the role of Trx in human disease showed that several primary tumours overexpress Trx compared with corresponding normal tissue [2,3]. In the 1990s, the development of commercially available ELISA kits enabled measurement of changes in extracellular concentrations of Trx in many diseases and conditions that are associated generally with oxidative stress and inflammation (Table 1). Here we highlight some of the key findings about the roles of Trx in cancer, viral disease, ischaemia–reperfusion injury, cardiac conditions, aging, premature birth and newborn physiology. Upregulation of Trx in malignant disease is well established [2]. At the onset, Trx counteracts oxidative stress associated with cancer-causing agents. However, ultimately, the growth-promoting effects of Trx on cancerous cells outweigh the beneficial, antioxidant properties. Indeed, elevated expression of Trx is associated with increased proliferation of tumour cells, inhibition of apoptosis, aggressive tumour growth, and decreased patient survival [22,23]. Processes that contribute to the cancerpromoting effects of Trx include: (i) promoting growthfactor expression in the tumour; (ii) increasing the concentration and activity of hypoxia-inducible factor 1a (HIF-1a), which leads to increased synthesis of vascular endothelial growth factor (VEGF) and vascular growth in the tumour [24]; (iii) inhibiting apoptosis, possibly by effects on ASK1 [2] and on the polyamine-induced apoptosis pathway [16]; and (iv) inhibiting tumour-suppressor proteins such as PTEN [phosphatase and tensin homolog (mutated in multiple advanced cancers 1)] [25]. Moreover, because many anti-cancer agents act by triggering apoptosis, the anti-apoptotic effects of Trx reduce the effectiveness of chemotherapy strategies. Several chemotherapeutic agents that are used clinically target components of the
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Table 1. Extracellular concentrations of Trx in human diseasea Condition
Sample
Mean control value (ng mlK1)b 11.7d
Mean disease value (ng mlK1)c 24.5d
HIV/AIDS
Plasma
Hepatocellular carcinoma Cardiopulmonary bypass
Serum Plasma
147.4
Severe burns
Plasma Synovial fluid Serum Synovial fluid Serum
81.8 28.5 41.1e 139 106h 11.8i 24.6h 22.7
287 117g 412 33.6 103.4 122.6j
Pulmonary sarcoidosis Pancreatic carcinoma Hepatitis C
BALF Plasma Serum
32.9d,k 24.4 24.9d
122.6d 54.8 37.3d
Heart failure
Serum
14.0
33.3
Rheumatoid arthritis
Plasma Synovial fluid
38.6 70.6h
86.8 335.3
Type II diabetes mellitus Asthma Hepatitis C (in haemodialysis patients) Acute lung injury
Serum Serum Serum
21 26.6d,l 28.0
38 38.8d,m 112.3
Plasma BALF
18.0d 16.0d
36.1d 61.6d
Coronary spastic angina Acute myocardial infarction
Serum Plasma
34 18.3n
64 38.8
Chronic heart failure
Plasma
12
3
Rheumatoid arthritis Rheumatoid arthritis
Associated findings
Refs
Higher concentrations of Trx associated with lower CD4 counts – –
[63]
Trx in the synovial fluid correlates with neutrophil infiltration Trx in synovial fluid might aggravate rheumatoid inflammation Elevated Trx correlates with increased platelet and white-cell counts Trx is produced locally by granuloma – Trx correlates with levels of serum ferritin and histological stages of hepatic fibrosis Trx correlates negatively with left ventricular ejection fraction Plasma Trx correlates with a urinary marker of oxidative DNA damage; Trx in the synovial fluid correlates with the number of infiltrating leukocytes Trx concentration might reflect insulin resistance Trx correlates inversely with FEV1 and PEFR Markers of liver damage
[66]
Related to the aetiology of lung injury and intensity of alveolar inflammation but not the severity of illness and outcome No correlation with vitamin E levels Trx is associated with platelet hyperaggregability and lower left ventricular ejection failure Elevated Trx correlates with markers of oxidative stress and disease severity
[76]
[64] [65]
f
[67] [68] [69] [70] [71] [40] [72]
[73] [74] [75]
[77] [78] [79]
a
Abbreviations: BALF, bronchoalveolar lavage fluid; FEV1, forced expiratory volume in 1 s; PEFR, peak expiratory flow rate. Healthy controls unless otherwise stated. c Significant increase in Trx unless otherwise stated. d Median value. e Pre-operative value. f 30 min post-operative value. g Increase in Trx not significant. h Osteoarthritis. i Osteoarthritis and healthy controls. j 1day post-injury. k Lung adrenocarcinoma or haemoptysis. l Asymptomatic asthma patients. m Moderate asthma attack. n Chest-pain syndrome. b
Trx system [2,3], and the anti-cancer properties of other novel Trx inhibitors are under investigation [26,27]. The involvement of Trx in viral disease, and in HIV infection in particular, is complex, with protective and detrimental roles described. Individuals who are infected with HIV suffer from extreme alterations in redox balance and, thus, their levels of oxidative stress are high [28]. One of the earliest observations regarding Trx in patients with HIV was that tissue levels of Trx are depleted transiently in acute infection. This might be because viruses evade the immune defences of the host and escape elimination by causing massive loss of sulfur [29], which impairs the synthesis of essential cysteine-containing proteins such as Trx and TrxR. In the later stages of HIV infection, the concentration of Trx in the plasma is elevated because viruses can induce Trx expression. In turn, elevated Trx inhibits viral replication, in part, by manipulating redox changes in the disulfide bonds of CD4, a member of the immunoglobulin superfamily and a www.sciencedirect.com
primary receptor for HIV-1 [30]. However, high circulating levels of Trx in immuno-compromised patients can result, ultimately, in a worse outcome because neutrophil function is impeded and resistance to infection is low [28]. In general, studies of in vivo models of ischaemia– reperfusion in heart, kidney, lung and brain show that increasing the concentration of Trx, either by inducing endogenous Trx or intravenous administration of human recombinant Trx, reduces markers of tissue damage [3,31–34]. By contrast, a Trx inhibitor [MOL294 (see Chemical names)] has protective effects in an in vivo model of intestinal ischaemia–reperfusion injury [35], which might indicate that the role of Trx is organ dependent. The roles of Trx in several cardiac conditions including myocarditis, cardiac hypertrophy, heart failure, myocardial infarction and hypertension have also been studied. There is no consensus about the nature of its role because it varies between and within conditions. Thus, Trx is
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TRENDS in Pharmacological Sciences
protective in animal models of either spontaneous myocarditis [36] or experimental autoimmune myocarditis (EAM) [37] (in the latter study, as a result of suppression of chemokine release and leukocyte chemotaxis). The dichotomy of Trx is highlighted in cardiac hypertrophy. As a major antioxidant, Trx protects the heart against oxidative stress that contributes to hypertrophy [38]. However, Trx is also an essential mediator of cardiomyocyte growth and, thus, contributes to hypertrophy [39]. Heart failure also represents a state of oxidative stress. Serum concentrations of Trx are raised in congestive heart failure and correlate with the severity of the disease [40]: it is assumed that Trx concentrations are raised to counteract oxidative stress, but it is unclear whether Trx contributes to the pathology of this condition. By contrast, evidence indicates that redox imbalance and, in particular, impaired expression of Trx might have a crucial role in the development and pathogenesis of hypertension [41]. The role of Trx in the aging process is an area of growing interest [42]. Oxidative stress is linked with the aging process; thus, the strong antioxidant effects of Trx, together with its anti-apoptotic and growth-promoting effects, are likely to afford some protection against the cellular changes that are associated with aging. Trx also causes the reduction of oxidised methionine sulfoxide reductase, an enzyme that has a key role in maintaining the essential amino acid methionine in a reduced, active form [43]. Loss of this essential reduction process is associated with a decrease in lifespan and the development of conditions such as Alzheimer’s disease. Trx also exerts a cytoprotective effect in the nervous system by enhancing the action of nerve growth factor via the regulation of anti-apoptotic signalling and through its antioxidant activity [4,44]. The role of Trx in premature birth and newborn physiology is another burgeoning field [14]. Studies on the role of Trx on pre-implantation and foetal development indicate that Trx is important in developmental biology [45]. Indeed, mice that are homozygous for targeted disruption of the gene that encodes Trx die shortly after implantation, whereas heterozygous animals are viable, fertile and, in other respects, normal. Moreover, the oxidant environment during embryonic development provides an ideal scenario for redox signalling that involves Trx and other redox-active molecules. In summary, Trx appears to have dual roles in malignant disease, HIV and cardiac conditions. In general, Trx seems to be protective in ischaemia–reperfusion injuries and, perhaps, beneficial in aging and neurodegenerative disease. Pharmacological inhibition of Trx In the past decade, biological screening has identified several small diverse organic compounds that inhibit the Trx system. Compounds with direct effects on Trx include PX12, palmarumycin CP1, AW464 and MOL294 (Figure 2). Data regarding some aspects of these inhibitors, such as mechanism of action and selectivity for Trx, are incomplete. All data available to date are included below. PX12, an alkyl 2-imidazolyl disulfide, was identified as a Trx inhibitor from O50 000 compounds tested at the www.sciencedirect.com
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(a)
401
(c)
OH
N
O
N SSCHCHCH3 N
S CH3
HO
PX12
AW464 (d)
(b)
O
CH3 N
O
N N
O O O OH
HO
CO2CH3
MOL294 Palmarumycin CP1 Figure 2. Chemical structures of Trx inhibitors. (a) PX12 is an alkyl 2-imidazolyl disulfide that causes irreversible thioalkylation of the structural Cys73 residue in Trx. (b) Palmarumycin CP1 is a napthoquinone spiroketal compound with selectivity for Trx; the phenol group and enone functionality are important for maximizing inhibitory activity. (c) AW464 is a heteraromatic quinol that binds to the active-site thiol groups in Trx. (d) MOL294 is a small organic molecule that is designed to mimic the extended (b) strand of peptide substrates for Trx.
National Cancer Institute using the COMPARE program. PX12 causes irreversible thioalkylation of the structural Cys73 residue, has a median IC50 value of 8.1 mM for growth inhibition of several tumour-cell lines, and in vivo anti-tumour activity against human tumour xenografts in severe combined immunodeficient mice [7]. PX12 also decreases the concentration of HIF-1a and, downstream of this, the production of VEGF [27]. A rapid decrease in the permeability of tumour vascular can also occur [46]. In Phase I clinical trials, PX12 has anti-tumour activity in patients with advanced, solid tumours that are refractory to standard therapy [47], and decreases plasma Trx and VEGF concentrations in some patients [48]. Palmarumycin CP1 is one of a family of naphthoquinone spiroketal compounds that are natural fungal metabolites [49,50]. It has IC50 values of 1 mM and 2.4 mM for growth inhibition in two breast- cancer cell lines. The phenol group and the enone functionality of palmarumycin CP1 seem to be important for maximizing its inhibitory activity. Also, the presence of the naphthalenediol ketal enhances considerably (O30-fold) the selectivity for Trx over TrxR. Other palmarumycin fungal metabolites are also potent inhibitors of the Trx–TrxR system [49]. Whereas palmarumycin CP1 is degraded rapidly and is ineffective in xenograft tumour models, suitable prodrug formulations have been developed that are promising in assays in vivo (P. Wipf et al., unpublished). AW464, which is a lead structure of a family of quinols with selective actions against renal- and colon-cancer cell lines, inhibits Trx redox cycling by forming an irreversible complex with the active-site thiol groups in the reduced form of Trx [26]. Addition of the first thiol (nucleophilic) group of Trx to the electrophilic (b-carbon) site of AW464 is thought to be reversible. Subsequent addition of the second Trx thiol group is assumed to be irreversible. Experiments in vitro show that AW464 exerts antiproliferative effects on tumour cell lines and endothelial
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cells, but not fibroblasts, with an IC50 value of w0.5 mM [51]. Preliminary studies also show anti-tumour effects in vivo [52]. Further chemical syntheses and structure– activity screening has revealed a series of (arylsulfonyl) indole-substituted quinols with better growth-inhibitory properties in vitro [53]. However, despite recent studies that support the molecular interaction between heteroaromatic quinols and Trx [54], other cellular targets that contain Trx motifs, such as protein-disulfide isomerases, and, thus, other mechanisms of anti-tumour activity, cannot be ruled out. MOL294 is a small organic molecule that mimics an extended (b) strand of peptide substrates that bind Trx. It also has a potent electrophilic moiety. In vitro, MOL294 inhibits the NF-kB-mediated expression of vascular cell adhesion molecule 1 with an IC50 value of 2.5 mM [55]. In a mouse model of asthma, MOL294 reduces eosinophil infiltration of the airway, mucus secretion, interleukin 13 (IL-13) and eotaxin release and hyperactivity of the airway to methacholine [56]. In a mouse model of intestinal ischaemia–reperfusion injury, MOL294 inhibits the local increase in vascular permeability, neutrophil accumulation, haemorrhage and production of pro-inflammatory cytokines [35]. MOL294 has some selectivity for Trx compared with other oxidoreductases (e.g. glutathione reductase) but, as with the quinols, effects on other members of this superfamily cannot be ruled out (M. Kahn et al., unpublished). Strategies to induce and administer Trx Geranylgeranylacetone (GGA), which was derived originally from a natural plant constituent and used clinically as an anti-ulcer drug, induces Trx and heat shock protein 72 [57], and protects against ethanol-induced injury to hepatic cells and gastric cells [57,58]. GGA also reduces neurotoxicity in spinal cord neurons, which indicates that it might be effective in treating neurodegenerative diseases of the spinal cord [59]. Temocapril, a novel nonsulfydryl-containing inhibitor of angiotensin-converting enzyme, also increases the expression of Trx in animal studies and reduces the severity of EAM in rats [60]. Systemic administration of Trx to mice reduces LPS-induced neutrophil migration, myocardial ischaemia– reperfusion injury, cerebral ischaemia, EAM and interstitial lung disease [1,20,34,37,61]. The protective mechanisms include anti-apoptotic effects, reducing oxidative damage, and suppressing leukocyte chemotaxis and chemokine expression, which indicates that some effects of systemically administered Trx are likely to be intracellular. Indeed, studies in vitro show that exogenous Trx enters cells to exert antioxidant, anti-apoptotic and cytoprotective effects [62]. The therapeutic dose of Trx, administered intravenously to mice, to prevent leukocyte infiltration, is O1 mg mlK1, which is at least 10 times more than either serum or plasma levels in patients with oxidative-stressassociated disorders (Table 1). This indicates that, although host cells secrete Trx to combat oxidative stress, higher doses must be administered to reduce oxidative stress and inflammation. Ongoing studies to investigate the possible side-effects of Trx administration indicate that such treatment does www.sciencedirect.com
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Chemical names AW464: 4-hydroxy-4-(benzothiazol-2-yl)cyclohexadienone MOL294: methyl (4R/S)-4-hydroxy-4-[((5S,8S)/(5R,8R))-8-methyl-1,2dioxo-2-phenyl-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin5-yl]2-butynoate PX12: 1-methylhydroxypropyl 2-imidazoloyl disulfide
not alter carcinogen-induced tumour size in Trx-transgenic and control mice (Y-W. Kwon et al., unpublished). Further studies to clarify the short-term and long-term effects of the administration of Trx on tumour growth and/or chemotherapy treatment are underway. Concluding remarks Trx has been measured in many clinical conditions. Understanding how Trx affects cell function, its physiological relevance and its role in disease are crucial for the development of a rational therapeutic approach to manipulate the activity of Trx. Cancer is likely to be a promising field for treatment with Trx inhibitors, whereas conditions that are associated with cell injury might benefit from administration of Trx. However, there is a need to understand further the balance between beneficial and detrimental effects before therapeutic manipulation of Trx becomes standard clinical practice. Acknowledgements We thank the Wellcome Trust for their support (A.B-G., University Award; M.E.J.C., Clinical Training Fellowship).
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cardiac hypertrophy through regulation of thioredoxin activity. Circulation 109, 2581–2586 Kishimoto, C. et al. (2001) Serum thioredoxin (TRX) levels in patients with heart failure. Jpn. Circ. J. 65, 491–494 Tanito, M. et al. (2004) Enhanced oxidative stress and impaired thioredoxin expression in spontaneously hypertensive rats. Antioxid. Redox Signal. 6, 89–97 Yoshida, T. et al. (2003) The role of thioredoxin in the aging process: involvement of oxidative stress. Antioxid. Redox Signal. 5, 563–570 Stadtman, E.R. et al. (2003) Oxidation of methionine residues of proteins: biological consequences. Antioxid. Redox Signal. 5, 577–582 Bai, J. et al. (2003) Critical roles of thioredoxin in nerve growth factormediated signal transduction and neurite outgrowth in PC12 cells. J. Neurosci. 23, 503–509 Kobayashi-Miura, M. et al. (2002) Thioredoxin, an anti-oxidant protein, protects mouse embryos from oxidative stress-induced developmental anomalies. Free Radic. Res. 36, 949–956 Jordan, B.F. et al. (2005) The thioredoxin-1 inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) decreases vascular permeability in tumor xenografts monitored by dynamic contrast enhanced magnetic resonance imaging. Clin. Cancer Res. 11, 529–536 Kirkpatrick, L.D.T. et al. (2004) Results from a Phase I study of PX-12, a thioredoxin inhibitor in patients with advanced solid malignancies. Proc. Am. Assoc. Clin. Oncol. 22, 217 Baker, A. et al. (2004) Pharmacodynamic proteomic studies of the thioredoxin-1 inhibitor PX-12 in mice and patients. Proc Am Assoc Clin Oncol 22, 3662 Wipf, P. et al. (2001) New inhibitors of the thioredoxin-thioredoxin reductase system based on a naphthoquinone spiroketal natural product lead. Bioorg. Med. Chem. Lett. 11, 2637–2641 Wipf, P. et al. (2004) Natural product based inhibitors of the thioredoxin-thioredoxin reductase system. Org. Biomol. Chem. 2, 1651–1658 Mukherjee, A. et al. (2005) Cytotoxic and antiangiogenic activity of AW464 (NSC 706704), a novel thioredoxin inhibitor: an in vitro study. Br. J. Cancer 92, 350–358 Wells, G. et al. (2003) 4-Substituted 4-hydroxycyclohexa-2,5-dien-1ones with selective activities against colon and renal cancer cell lines. J. Med. Chem. 46, 532–541 Berry, J.M. et al. (2005) Quinols as novel therapeutic agents. 2.(1) 4-(1-Arylsulfonylindol-2-yl)-4-hydroxycyclohexa-2,5-dien-1-ones and related agents as potent and selective antitumor agents. J. Med. Chem. 48, 639–644 Bradshaw, T.D. et al. (2005) Elucidation of thioredoxin as a molecular target for antitumor quinols. Cancer Res. 65, 3911–3919 Misra-Press, A. et al. (2002) Identification of a novel inhibitor of the NF kappa B pathway. Curr. Med. Chem. 1, 29 Henderson, W.R., Jr. et al. (2002) A small molecule inhibitor of redoxregulated NF-kappa B and activator protein-1 transcription blocks allergic airway inflammation in a mouse asthma model. J. Immunol. 169, 5294–5299 Hirota, K. et al. (2000) Geranylgeranylacetone enhances expression of thioredoxin and suppresses ethanol-induced cytotoxicity in cultured hepatocytes. Biochem. Biophys. Res. Commun. 275, 825–830 Bai, J. et al. (2002) Thioredoxin suppresses 1-methyl-4-phenylpyridinium-induced neurotoxicity in rat PC12 cells. Neurosci. Lett. 321, 81–84 Kikuchi, S. et al. (2002) Effect of geranylgeranylaceton on cellular damage induced by proteasome inhibition in cultured spinal neurons. J. Neurosci. Res. 69, 373–381 Yuan, Z. et al. (2003) Temocapril treatment ameliorates autoimmune myocarditis associated with enhanced cardiomyocyte thioredoxin expression. Mol. Cell. Biochem. 248, 185–192 Hoshino, T. et al. (2003) Redox-active protein thioredoxin prevents proinflammatory cytokine- or bleomycin-induced lung injury. Am. J. Respir. Crit. Care Med. 168, 1075–1083 Kondo, N. et al. (2004) Redox-sensing release of human thioredoxin from T lymphocytes with negative feedback loops. J. Immunol. 172, 442–448 Nakamura, H. et al. (1996) Elevation of plasma thioredoxin levels in HIV-infected individuals. Int. Immunol. 8, 603–611 Miyazaki, K. et al. (1998) Elevated serum level of thioredoxin in patients with hepatocellular carcinoma. Biotherapy 11, 277–288
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65 Nakamura, H. et al. (1998) Measurements of plasma glutaredoxin and thioredoxin in healthy volunteers and during open-heart surgery. Free Radic. Biol. Med. 24, 1176–1186 66 Maurice, M.M. et al. (1999) Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis. Arthritis Rheum. 42, 2430–2439 67 Yoshida, S. et al. (1999) Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-alpha-induced production of IL-6 and IL-8 from cultured synovial fibroblasts. J. Immunol. 163, 351–358 68 Abdiu, A. et al. (2000) Thioredoxin blood level increases after severe burn injury. Antioxid. Redox Signal. 2, 707–716 69 Koura, T. et al. (2000) Expression of thioredoxin in granulomas of sarcoidosis: possible role in the development of T lymphocyte activation. Thorax 55, 755–761 70 Nakamura, H. et al. (2000) Expression of thioredoxin and glutaredoxin, redox-regulating proteins, in pancreatic cancer. Cancer Detect. Prev. 24, 53–60 71 Sumida, Y. et al. (2000) Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection. J. Hepatol. 33, 616–622
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72 Jikimoto, T. et al. (2002) Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. Mol. Immunol. 38, 765–772 73 Kakisaka, Y. et al. (2002) Elevation of serum thioredoxin levels in patients with type 2 diabetes. Horm. Metab. Res. 34, 160–164 74 Yamada, Y. et al. (2003) Elevated serum levels of thioredoxin in patients with acute exacerbation of asthma. Immunol. Lett. 86, 199–205 75 Kato, A. et al. (2003) Elevation of blood thioredoxin in hemodialysis patients with hepatitis C virus infection. Kidney Int. 63, 2262–2268 76 Callister, M.E.J. et al. (2003) Thioredoxin concentrations are elevated in plasma and bronchoalveolar lavage fluid from patients with acute lung injury. Am. J. Respir. Crit. Care Med. 167, A662 77 Miwa, K. et al. (2003) Increased oxidative stress with elevated serum thioredoxin level in patients with coronary spastic angina. Clin. Cardiol. 26, 177–181 78 Miyamoto, S. et al. (2003) Plasma thioredoxin levels and platelet aggregability in patients with acute myocardial infarction. Am. Heart J. 146, 465–471 79 Jekell, A. et al. (2004) Elevated circulating levels of thioredoxin and stress in chronic heart failure. Eur. J. Heart Fail. 6, 883–890
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Vol.26 No.8 August 2005
Thioredoxin: friend or foe in human disease? Anne Burke-Gaffney1, Matthew E.J. Callister1 and Hajime Nakamura2 1
Unit of Critical Care, National Heart and Lung Institute Division, Imperial College Faculty of Medicine, Dovehouse Street, London SW3 6LY, UK 2 Thioredoxin Project, Department of Experimental Therapeutics, Translational Research Center, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo, Kyoto, 606-8507, Japan
Thioredoxin (Trx), a small, ubiquitous thiol [sulfydryl (-SH)] protein, is one of the most important regulators of reduction–oxidation (redox) balance and, thus, redoxcontrolled cell functions. Although Trx was discovered 40 years ago in bacteria, the number and diversity of processes that Trx influences in human cells have only been appreciated recently. Processes influenced by Trx include the control of cellular redox balance, the promotion of cell growth, the inhibition of apoptosis and the modulation of inflammation. Not surprisingly, the role of Trx in a wide range of human diseases and conditions, including cancer, viral disease, ischaemia– reperfusion injury, cardiac conditions, aging, premature birth and newborn physiology, is subject to intense investigation. However, whether Trx contributes to or prevents the pathology of a particular condition is not always clear. In this article, we review the role of Trx in human disease and relate this to its redox activity and biological properties, and discuss the development and use of therapies that either inhibit or augment Trx activity.
The dichotomy of Trx Any particular biological property of thioredoxin (Trx) is unlikely to be either ‘good’ or ‘bad’ in disease; indeed, the effect of Trx is likely to depend on the type and stage of the condition. Thus, in cancer, the anti-apoptotic properties of Trx are considered deleterious because they can impair the effectiveness of chemotherapy strategies that trigger apoptosis. However, in ischaemia–reperfusion injury, in which apoptosis contributes to the pathology, Trx might protect against injurious insults [1]. Likewise, the growthpromoting effects of Trx are detrimental in cancer and NADPH + H+
TrxR-(S-Se)
NADP+
TrxR-(SH Se–) + H+
rheumatoid arthritis [2,3] but are beneficial in neurodegenerative disease in which promoting neural-cell growth aids recovery [4]. In HIV infection, Trx blocks HIV replication [3] but at later stages of the disease it contributes to immunosuppression in some patients [5]. A clear understanding of which properties of Trx predominate in a particular situation is key to the successful therapeutic manipulation of Trx. The biochemistry and biological properties of Trx are well established (reviewed in [3,6,7]). However, the dichotomy of Trx in disease is less well documented. In this review, we summarize the redox activity and biological properties of Trx. These are essential to understanding the dichotomous role of Trx in disease, which is also discussed. Finally, we describe the pharmacological inhibition of Trx, strategies that are used to induce and augment Trx and its practical applications.
Redox activity and biological properties of Trx The biological properties of Trx rely, largely, on reduction– oxidation (redox) activity, which is the ability to transfer ‘reducing equivalents’ to disulfide groups in target proteins. The key to the redox activity of Trx is the presence of two cysteine residues (Cys32 and Cys35) separated by two amino acids (Gly-Pro) in its active site. These cysteines exist as a dithiol [-(SH)2] in the reduced form and a disulfide (-S2) in the oxidized form. Trx is oxidized when it transfers reducing equivalents to disulfide groups in target proteins and it is reduced back to the dithiol form by an NADPH-dependent flavoprotein, thioredoxin reductase (TrxR); this forms the so-called Trx system (Figure 1). The activities and properties of Trx and TrxR are reviewed extensively in [6–10]. Trx-(SH)2
Trx-S2
Sub-S2
Sub-(SH)2
Figure 1. Redox cycling of Trx and TrxR. Trx is oxidised as it transfers reducing equivalents to disulfide groups in target proteins. Trx is then reduced back to the dithiol form by an NADPH-dependent flavoprotein, TrxR. The C-terminus of TrxR contains Cys-SeCys as a catalytic centre. Abbreviations: NADPH, nicotinamide dinucleotide phosphate (reduced form); NADPC, nicotinamide dinucleotide phosphate (oxidized form); -S2, oxidized, disulfide form; SeK the selenolate anion; SH, the thiol anion; (SH)2, reduced, dithiol form; S-Se the selenylsulfide; Sub, protein substrate. Corresponding author: Burke-Gaffney, A. ([email protected]). Available online 28 June 2005 www.sciencedirect.com 0165-6147/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2005.06.005
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To date, most work that relates to the role of Trx in human disease concerns Trx-1, which is one of a family of proteins that share the active-site sequence (Cys-X-X-Cys). Trx-1 is expressed ubiquitously in cytosol and translocates to the nucleus when cells are activated by, for example, oxidative stress. Trx-1 has no recognizable nuclear localization signal and it is assumed that it is carried into the nucleus bound to other proteins such as nuclear factor kB (NF-kB) and redox factor 1. The function of Trx-1 is tightly linked to these proteins [11,12]. Trx-1 is also secreted extracellularly where it is thought to mediate some inflammatory effects. In addition to the two cysteine residues in the catalytic site, human Trx-1 contains three other cysteine residues (Cys62, Cys69 and Cys73). Although not part of the active site, these contribute to essential protein conformation, and oxidation of these structural residues leads to loss of enzymatic activity. Active-site and structural cysteine residues are targets for potential inhibitors of Trx. Key biological activities of Trx that are applicable to human disease can be categorized as antioxidant, growth promoting, anti-apoptotic and inflammation modulating. Several of these activities result, in part, from redox regulation by Trx of signal transduction pathways and gene expression [13]. The principle intracellular antioxidant property of Trx results from its ability to act as a cofactor that maintains several thioredoxin peroxidase enzymes in a reduced, active form [13]. As their name indicates, these enzymes, which are members of the peroxiredoxin family of proteins, inactivate hydrogen peroxide (H2O2). Other antioxidant properties of Trx result from the reduction of glutathione peroxidase, the induction of manganese superoxide dismutase and, to some extent, the direct reduction of H2O2 by Trx [14]. S-nitrosylation of Cys69 of Trx is an important posttranslational modification that potentiates antioxidant activity [15]. Trx stimulates the growth of normal and cancerous cells in an atypical manner that does not appear to use a receptor. Mechanisms of Trx-induced-growth are multifaceted and include the provision of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and increasing sensitivity of cells to other cytokines and growth factors [2,13]. The anti-apoptotic affects of Trx might, in part, relate to its ability to bind to and inhibit apoptosis-signalregulating kinase 1 (ASK1) [13]. When Trx is oxidized, ASK1 dissociates and activates an apoptotic signalling pathway. Trx also regulates polyamine homeostasis, a pathway that has complex inhibitory and activator effects on apoptosis and cell survival [16]. Together with the growth-promoting effects, the ability of Trx to inhibit apoptosis contributes to the aggressive growth of many tumours. In addition, Trx has several significant effects on inflammatory pathways [17]. First, Trx has multiple regulatory effects on cytokine production. As with growth factors, Trx either directly induces the synthesis of or augments the production of some cytokines and inhibits the synthesis of others. These effects are caused, in part, by the involvement of Trx in redox signalling [18]. Trx also has dual regulatory effects on leukocyte movement. It acts www.sciencedirect.com
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as a chemoattractant for monocytes, neutrophils and T cells in vitro and following local administration in vivo [19] with a potency that is comparable to that of known chemokines. However, this effect is atypical of a chemokine because it is independent of G proteins and intracellular Ca2C is not increased. By contrast, intravenous administration of Trx suppresses lipopolysaccharide (LPS)-induced neutrophil chemotaxis, in part, by preventing L-selectin shedding from neutrophils, which is a prerequisite for migration [20]. Other inflammatory activities of Trx are attributed to the 10-kDa, C-terminally-truncated form of Trx (Trx80). This comprises either the 80 or the 84 N-terminal amino acids and is secreted into and is present in plasma [21]. Monocytes are the primary target of Trx80, which causes proliferation, cytokine production and increased expression of cell-surface antigens such as CD14 in these cells. Unlike Trx, Trx80 lacks reducing activity and its effects are independent of cysteine residues in the active-site motif. Can we predict how the biological activities of Trx impact on health and disease? It might be expected that antioxidant effects are beneficial, that the merits or otherwise of growth promoting and anti-apoptotic effects are likely to vary from condition to condition, and that the effects on inflammation might also vary. Trx in health and disease Early studies of the role of Trx in human disease showed that several primary tumours overexpress Trx compared with corresponding normal tissue [2,3]. In the 1990s, the development of commercially available ELISA kits enabled measurement of changes in extracellular concentrations of Trx in many diseases and conditions that are associated generally with oxidative stress and inflammation (Table 1). Here we highlight some of the key findings about the roles of Trx in cancer, viral disease, ischaemia–reperfusion injury, cardiac conditions, aging, premature birth and newborn physiology. Upregulation of Trx in malignant disease is well established [2]. At the onset, Trx counteracts oxidative stress associated with cancer-causing agents. However, ultimately, the growth-promoting effects of Trx on cancerous cells outweigh the beneficial, antioxidant properties. Indeed, elevated expression of Trx is associated with increased proliferation of tumour cells, inhibition of apoptosis, aggressive tumour growth, and decreased patient survival [22,23]. Processes that contribute to the cancerpromoting effects of Trx include: (i) promoting growthfactor expression in the tumour; (ii) increasing the concentration and activity of hypoxia-inducible factor 1a (HIF-1a), which leads to increased synthesis of vascular endothelial growth factor (VEGF) and vascular growth in the tumour [24]; (iii) inhibiting apoptosis, possibly by effects on ASK1 [2] and on the polyamine-induced apoptosis pathway [16]; and (iv) inhibiting tumour-suppressor proteins such as PTEN [phosphatase and tensin homolog (mutated in multiple advanced cancers 1)] [25]. Moreover, because many anti-cancer agents act by triggering apoptosis, the anti-apoptotic effects of Trx reduce the effectiveness of chemotherapy strategies. Several chemotherapeutic agents that are used clinically target components of the
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Table 1. Extracellular concentrations of Trx in human diseasea Condition
Sample
Mean control value (ng mlK1)b 11.7d
Mean disease value (ng mlK1)c 24.5d
HIV/AIDS
Plasma
Hepatocellular carcinoma Cardiopulmonary bypass
Serum Plasma
147.4
Severe burns
Plasma Synovial fluid Serum Synovial fluid Serum
81.8 28.5 41.1e 139 106h 11.8i 24.6h 22.7
287 117g 412 33.6 103.4 122.6j
Pulmonary sarcoidosis Pancreatic carcinoma Hepatitis C
BALF Plasma Serum
32.9d,k 24.4 24.9d
122.6d 54.8 37.3d
Heart failure
Serum
14.0
33.3
Rheumatoid arthritis
Plasma Synovial fluid
38.6 70.6h
86.8 335.3
Type II diabetes mellitus Asthma Hepatitis C (in haemodialysis patients) Acute lung injury
Serum Serum Serum
21 26.6d,l 28.0
38 38.8d,m 112.3
Plasma BALF
18.0d 16.0d
36.1d 61.6d
Coronary spastic angina Acute myocardial infarction
Serum Plasma
34 18.3n
64 38.8
Chronic heart failure
Plasma
12
3
Rheumatoid arthritis Rheumatoid arthritis
Associated findings
Refs
Higher concentrations of Trx associated with lower CD4 counts – –
[63]
Trx in the synovial fluid correlates with neutrophil infiltration Trx in synovial fluid might aggravate rheumatoid inflammation Elevated Trx correlates with increased platelet and white-cell counts Trx is produced locally by granuloma – Trx correlates with levels of serum ferritin and histological stages of hepatic fibrosis Trx correlates negatively with left ventricular ejection fraction Plasma Trx correlates with a urinary marker of oxidative DNA damage; Trx in the synovial fluid correlates with the number of infiltrating leukocytes Trx concentration might reflect insulin resistance Trx correlates inversely with FEV1 and PEFR Markers of liver damage
[66]
Related to the aetiology of lung injury and intensity of alveolar inflammation but not the severity of illness and outcome No correlation with vitamin E levels Trx is associated with platelet hyperaggregability and lower left ventricular ejection failure Elevated Trx correlates with markers of oxidative stress and disease severity
[76]
[64] [65]
f
[67] [68] [69] [70] [71] [40] [72]
[73] [74] [75]
[77] [78] [79]
a
Abbreviations: BALF, bronchoalveolar lavage fluid; FEV1, forced expiratory volume in 1 s; PEFR, peak expiratory flow rate. Healthy controls unless otherwise stated. c Significant increase in Trx unless otherwise stated. d Median value. e Pre-operative value. f 30 min post-operative value. g Increase in Trx not significant. h Osteoarthritis. i Osteoarthritis and healthy controls. j 1day post-injury. k Lung adrenocarcinoma or haemoptysis. l Asymptomatic asthma patients. m Moderate asthma attack. n Chest-pain syndrome. b
Trx system [2,3], and the anti-cancer properties of other novel Trx inhibitors are under investigation [26,27]. The involvement of Trx in viral disease, and in HIV infection in particular, is complex, with protective and detrimental roles described. Individuals who are infected with HIV suffer from extreme alterations in redox balance and, thus, their levels of oxidative stress are high [28]. One of the earliest observations regarding Trx in patients with HIV was that tissue levels of Trx are depleted transiently in acute infection. This might be because viruses evade the immune defences of the host and escape elimination by causing massive loss of sulfur [29], which impairs the synthesis of essential cysteine-containing proteins such as Trx and TrxR. In the later stages of HIV infection, the concentration of Trx in the plasma is elevated because viruses can induce Trx expression. In turn, elevated Trx inhibits viral replication, in part, by manipulating redox changes in the disulfide bonds of CD4, a member of the immunoglobulin superfamily and a www.sciencedirect.com
primary receptor for HIV-1 [30]. However, high circulating levels of Trx in immuno-compromised patients can result, ultimately, in a worse outcome because neutrophil function is impeded and resistance to infection is low [28]. In general, studies of in vivo models of ischaemia– reperfusion in heart, kidney, lung and brain show that increasing the concentration of Trx, either by inducing endogenous Trx or intravenous administration of human recombinant Trx, reduces markers of tissue damage [3,31–34]. By contrast, a Trx inhibitor [MOL294 (see Chemical names)] has protective effects in an in vivo model of intestinal ischaemia–reperfusion injury [35], which might indicate that the role of Trx is organ dependent. The roles of Trx in several cardiac conditions including myocarditis, cardiac hypertrophy, heart failure, myocardial infarction and hypertension have also been studied. There is no consensus about the nature of its role because it varies between and within conditions. Thus, Trx is
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protective in animal models of either spontaneous myocarditis [36] or experimental autoimmune myocarditis (EAM) [37] (in the latter study, as a result of suppression of chemokine release and leukocyte chemotaxis). The dichotomy of Trx is highlighted in cardiac hypertrophy. As a major antioxidant, Trx protects the heart against oxidative stress that contributes to hypertrophy [38]. However, Trx is also an essential mediator of cardiomyocyte growth and, thus, contributes to hypertrophy [39]. Heart failure also represents a state of oxidative stress. Serum concentrations of Trx are raised in congestive heart failure and correlate with the severity of the disease [40]: it is assumed that Trx concentrations are raised to counteract oxidative stress, but it is unclear whether Trx contributes to the pathology of this condition. By contrast, evidence indicates that redox imbalance and, in particular, impaired expression of Trx might have a crucial role in the development and pathogenesis of hypertension [41]. The role of Trx in the aging process is an area of growing interest [42]. Oxidative stress is linked with the aging process; thus, the strong antioxidant effects of Trx, together with its anti-apoptotic and growth-promoting effects, are likely to afford some protection against the cellular changes that are associated with aging. Trx also causes the reduction of oxidised methionine sulfoxide reductase, an enzyme that has a key role in maintaining the essential amino acid methionine in a reduced, active form [43]. Loss of this essential reduction process is associated with a decrease in lifespan and the development of conditions such as Alzheimer’s disease. Trx also exerts a cytoprotective effect in the nervous system by enhancing the action of nerve growth factor via the regulation of anti-apoptotic signalling and through its antioxidant activity [4,44]. The role of Trx in premature birth and newborn physiology is another burgeoning field [14]. Studies on the role of Trx on pre-implantation and foetal development indicate that Trx is important in developmental biology [45]. Indeed, mice that are homozygous for targeted disruption of the gene that encodes Trx die shortly after implantation, whereas heterozygous animals are viable, fertile and, in other respects, normal. Moreover, the oxidant environment during embryonic development provides an ideal scenario for redox signalling that involves Trx and other redox-active molecules. In summary, Trx appears to have dual roles in malignant disease, HIV and cardiac conditions. In general, Trx seems to be protective in ischaemia–reperfusion injuries and, perhaps, beneficial in aging and neurodegenerative disease. Pharmacological inhibition of Trx In the past decade, biological screening has identified several small diverse organic compounds that inhibit the Trx system. Compounds with direct effects on Trx include PX12, palmarumycin CP1, AW464 and MOL294 (Figure 2). Data regarding some aspects of these inhibitors, such as mechanism of action and selectivity for Trx, are incomplete. All data available to date are included below. PX12, an alkyl 2-imidazolyl disulfide, was identified as a Trx inhibitor from O50 000 compounds tested at the www.sciencedirect.com
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(a)
401
(c)
OH
N
O
N SSCHCHCH3 N
S CH3
HO
PX12
AW464 (d)
(b)
O
CH3 N
O
N N
O O O OH
HO
CO2CH3
MOL294 Palmarumycin CP1 Figure 2. Chemical structures of Trx inhibitors. (a) PX12 is an alkyl 2-imidazolyl disulfide that causes irreversible thioalkylation of the structural Cys73 residue in Trx. (b) Palmarumycin CP1 is a napthoquinone spiroketal compound with selectivity for Trx; the phenol group and enone functionality are important for maximizing inhibitory activity. (c) AW464 is a heteraromatic quinol that binds to the active-site thiol groups in Trx. (d) MOL294 is a small organic molecule that is designed to mimic the extended (b) strand of peptide substrates for Trx.
National Cancer Institute using the COMPARE program. PX12 causes irreversible thioalkylation of the structural Cys73 residue, has a median IC50 value of 8.1 mM for growth inhibition of several tumour-cell lines, and in vivo anti-tumour activity against human tumour xenografts in severe combined immunodeficient mice [7]. PX12 also decreases the concentration of HIF-1a and, downstream of this, the production of VEGF [27]. A rapid decrease in the permeability of tumour vascular can also occur [46]. In Phase I clinical trials, PX12 has anti-tumour activity in patients with advanced, solid tumours that are refractory to standard therapy [47], and decreases plasma Trx and VEGF concentrations in some patients [48]. Palmarumycin CP1 is one of a family of naphthoquinone spiroketal compounds that are natural fungal metabolites [49,50]. It has IC50 values of 1 mM and 2.4 mM for growth inhibition in two breast- cancer cell lines. The phenol group and the enone functionality of palmarumycin CP1 seem to be important for maximizing its inhibitory activity. Also, the presence of the naphthalenediol ketal enhances considerably (O30-fold) the selectivity for Trx over TrxR. Other palmarumycin fungal metabolites are also potent inhibitors of the Trx–TrxR system [49]. Whereas palmarumycin CP1 is degraded rapidly and is ineffective in xenograft tumour models, suitable prodrug formulations have been developed that are promising in assays in vivo (P. Wipf et al., unpublished). AW464, which is a lead structure of a family of quinols with selective actions against renal- and colon-cancer cell lines, inhibits Trx redox cycling by forming an irreversible complex with the active-site thiol groups in the reduced form of Trx [26]. Addition of the first thiol (nucleophilic) group of Trx to the electrophilic (b-carbon) site of AW464 is thought to be reversible. Subsequent addition of the second Trx thiol group is assumed to be irreversible. Experiments in vitro show that AW464 exerts antiproliferative effects on tumour cell lines and endothelial
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cells, but not fibroblasts, with an IC50 value of w0.5 mM [51]. Preliminary studies also show anti-tumour effects in vivo [52]. Further chemical syntheses and structure– activity screening has revealed a series of (arylsulfonyl) indole-substituted quinols with better growth-inhibitory properties in vitro [53]. However, despite recent studies that support the molecular interaction between heteroaromatic quinols and Trx [54], other cellular targets that contain Trx motifs, such as protein-disulfide isomerases, and, thus, other mechanisms of anti-tumour activity, cannot be ruled out. MOL294 is a small organic molecule that mimics an extended (b) strand of peptide substrates that bind Trx. It also has a potent electrophilic moiety. In vitro, MOL294 inhibits the NF-kB-mediated expression of vascular cell adhesion molecule 1 with an IC50 value of 2.5 mM [55]. In a mouse model of asthma, MOL294 reduces eosinophil infiltration of the airway, mucus secretion, interleukin 13 (IL-13) and eotaxin release and hyperactivity of the airway to methacholine [56]. In a mouse model of intestinal ischaemia–reperfusion injury, MOL294 inhibits the local increase in vascular permeability, neutrophil accumulation, haemorrhage and production of pro-inflammatory cytokines [35]. MOL294 has some selectivity for Trx compared with other oxidoreductases (e.g. glutathione reductase) but, as with the quinols, effects on other members of this superfamily cannot be ruled out (M. Kahn et al., unpublished). Strategies to induce and administer Trx Geranylgeranylacetone (GGA), which was derived originally from a natural plant constituent and used clinically as an anti-ulcer drug, induces Trx and heat shock protein 72 [57], and protects against ethanol-induced injury to hepatic cells and gastric cells [57,58]. GGA also reduces neurotoxicity in spinal cord neurons, which indicates that it might be effective in treating neurodegenerative diseases of the spinal cord [59]. Temocapril, a novel nonsulfydryl-containing inhibitor of angiotensin-converting enzyme, also increases the expression of Trx in animal studies and reduces the severity of EAM in rats [60]. Systemic administration of Trx to mice reduces LPS-induced neutrophil migration, myocardial ischaemia– reperfusion injury, cerebral ischaemia, EAM and interstitial lung disease [1,20,34,37,61]. The protective mechanisms include anti-apoptotic effects, reducing oxidative damage, and suppressing leukocyte chemotaxis and chemokine expression, which indicates that some effects of systemically administered Trx are likely to be intracellular. Indeed, studies in vitro show that exogenous Trx enters cells to exert antioxidant, anti-apoptotic and cytoprotective effects [62]. The therapeutic dose of Trx, administered intravenously to mice, to prevent leukocyte infiltration, is O1 mg mlK1, which is at least 10 times more than either serum or plasma levels in patients with oxidative-stressassociated disorders (Table 1). This indicates that, although host cells secrete Trx to combat oxidative stress, higher doses must be administered to reduce oxidative stress and inflammation. Ongoing studies to investigate the possible side-effects of Trx administration indicate that such treatment does www.sciencedirect.com
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Chemical names AW464: 4-hydroxy-4-(benzothiazol-2-yl)cyclohexadienone MOL294: methyl (4R/S)-4-hydroxy-4-[((5S,8S)/(5R,8R))-8-methyl-1,2dioxo-2-phenyl-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin5-yl]2-butynoate PX12: 1-methylhydroxypropyl 2-imidazoloyl disulfide
not alter carcinogen-induced tumour size in Trx-transgenic and control mice (Y-W. Kwon et al., unpublished). Further studies to clarify the short-term and long-term effects of the administration of Trx on tumour growth and/or chemotherapy treatment are underway. Concluding remarks Trx has been measured in many clinical conditions. Understanding how Trx affects cell function, its physiological relevance and its role in disease are crucial for the development of a rational therapeutic approach to manipulate the activity of Trx. Cancer is likely to be a promising field for treatment with Trx inhibitors, whereas conditions that are associated with cell injury might benefit from administration of Trx. However, there is a need to understand further the balance between beneficial and detrimental effects before therapeutic manipulation of Trx becomes standard clinical practice. Acknowledgements We thank the Wellcome Trust for their support (A.B-G., University Award; M.E.J.C., Clinical Training Fellowship).
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