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Seminars in Cancer Biology 16 (2006) 444–451

Review

Extracellular thioredoxin and thioredoxin-bindingprotein 2 in control of cancer

Hajime Nakamura a,∗, Hiroshi Masutani b, Junji Yodoi a,b

a Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japanb Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan

bstract

Thioredoxin-1 (TRX) is a redox-active protein with multiple intracellular and extracellular functions. Intracellular redox balance is maintainedy the TRX family and its related molecules. Extracellular TRX shows cytoprotective effects, while truncated Trx80 has more mitogenic activity.

xogenously administered TRX does not promote the growth of cancer in vivo and shows anti-chemotactic effect for neutrophils and anti-

nflammatory functions. Thioredoxin is released from cells in response to oxidative stress and TRX levels in plasma or serum are good markers forxidative stress associated with cancer. Thioredoxin-binding protein 2 (TBP-2) is an endogenous negative regulator of TRX and a tumor suppressor.

2006 Elsevier Ltd. All rights reserved.

Keywords: Thioredoxin; Redox; Thioredoxin-binding protein 2

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Redox regulation by thioredoxin family and related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Thioredoxin family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Thioredoxin reductase and peroxiredoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Thioredoxin-binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Extracellular roles of thioredoxin in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Cytokine/chemokine-like functions of TRX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Thioredoxin in plasma or serum as an oxidative stress marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. The safety of TRX therapy in cancer patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Control of cancer by thioredoxin-binding protein 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . .. . . .

equence of E. coli thioredoxin was reported in 1968 [2,3]. Mam-alian thioredoxin was first described in rat Novikoff hepatoma

ells in 1967 [4]. Human thioredoxin was originally cloned as aytokine-like factor named adult T cell leukemia (ATL)-derived

∗ Corresponding author. Tel.: +81 75 751 4751; fax: +81 75 751 4766.E-mail address: hnakamur@kuhp.kyoto-u.ac.jp (H. Nakamura).

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044-579X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2006.09.001

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

actor (ADF), which was first defined as an IL-2 receptor a-chainIL-2Ra, CD25)-inducing factor purified from the supernatant ofuman T cell leukemia virus type-I (HTLV-I)-transformed T celline ATL2 cells [5–8]. Here we use the abbreviation as TRX foruman thioredoxin that means human thioredoxin 1 as explainedater. TRX is a small (12 kDa) protein consisting of 105 aminocids. In most of the natural protein in man (>99%), the firstmino acid methionine in the N-terminus of TRX is removedy N-terminal Met excision process [9] and it therefore con-

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Thioredoxin was originally described in 1964 as a hydrogendonor for ribonucleotide reductase which is an essential enzymefor DNA synthesis in Escherichia coli [1]. The amino acid

ists of 104 amino acids from the N-terminal Val. The active siteequence: -Cys-Gly-Pro-Cys- of the protein is conserved from. coli to human. Using the two cysteine residues in the activeite, Trx acts as an oxido-reductase through the dithiol/disulfide

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xchange (Fig. 1). Reduced TRX with dithiol (-SH, -SH) reduceshe protein disulfide and becomes oxidized form with disulfide-S-S). Oxidized TRX is again reduced by thioredoxin reduc-ase and NADPH. TRX has additional three cysteines Cys62,ys69 and Cys73 in the structure (Fig. 1). When TRX is morexidized, the second disulfide is formed between Cys62 andys69 [10]. Furthermore, when it is further oxidized, a dimer is

ormed between Cys73 of the two molecules [11]. In addition,non-covalent dimer is formed involving Asp60 at low pH [12]

Fig. 1).

. Redox regulation by thioredoxin family and relatedroteins

“Oxidative stress” indicates that the antioxidant status of cellsnd tissues are altered by exposure to oxidants [13]. In 1990s,here was growing evidence that a small amount of oxidant suchs hydrogen peroxide plays a crucial role as a second messengern the signal transduction for cellular activation, differentia-ion and proliferation. “Redox regulation” originally indicatedhat the DNA binding activity of transcriptional factors suchs AP-1 and NF-kB is regulated by reduction and oxidationredox) of their key cysteine residues. “Redox response” is now

efined as the biological response to induce antioxidant sys-ems against oxidative stress to maintain the homeostasis inhe intracellular redox balance. Mammals contain glutathioneGSH)/glutaredoxin system and TRX system as the two major

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cer Biology 16 (2006) 444–451 445

ntioxidant systems. The intracellular amount of GSH is in the–10 mM level in mammalian cells, whereas intracellular con-ent of TRX is in the 0.1–2 �M level. Therefore, TRX mayppear as a minor component as an intracellular antioxidant.owever, it is acting as an enzyme supplying electrons to per-xiredoxins or methionine sulfoxide reductases and acts like aeneral protein disulfide reductase. TRX knock out mice arembryonic lethal [14] and thereby TRX system is playing anssential role for mammals. This may be explained by TRXlaying a crucial role in the interaction with specific target pro-eins such as in the inhibition of apoptosis by suppression ofpoptosis regulating kinase 1 (ASK1) activation [15] and in theegulation of DNA binding activity of transcriptional factorsuch as AP-1, NF-kB and p53 for the transcriptional control ofssential genes (Fig. 2) [16,17]. Alternatively, the role of TRXn defense against oxidative stress or to supply building blocksor DNA synthesis via ribonucleotide reductase is essential [3].

.1. Thioredoxin family

Proteins sharing the similar active site: -Cys-Xxx-Xxx-Cys-nd similar three-dimensional structure or thioredoxin foldelong to the thioredoxin family [3] In the cytosol, there areany family members including the classical cytosolic thiore-

oxin 1 (TRX) and glutaredoxin 1 (Fig. 3). In mitochon-ria, there are mitochondria-specific thioredoxin 2 (TRX2) andlutaredoxin 2. TRX2 is also essential for mammals. TRX2 defi-ient cells fall into apoptosis [18] and TRX2 knock out micere embryonically lethal [19]. Endoplasmic reticulum (ER) con-ains protein disulfide isomerase (PDI), calcium binding protein(CaBP1), ERp72, TRX-related transmembrane protein TMX,Rdj5 and so on. TMX plays an important role in the regulationf ER stress [20].

Macrophage migration inhibitory factor (MIF) is a proinflam-atory cytokine which was originally described as a soluble

actor expressed by activated T cells in delayed type hyper-ensitivity [21,22]. MIF also has a redox-active motif: -Cys-la-Leu-Cys- and exhibits a disulfide reductase activity [23].IF shows proinflammatory functions, whereas TRX has anti-

nflammatory and anti-apoptotic functions. However, TRX andIF share similar characteristics. Both have the similar molec-

lar weight of around 12 kDa and are secreted by a leaderlessxport pathway. They share the same interacting protein suchs Jun activation domain-binding protein 1 (JAB1) in the cells24,25]. Thioredoxin 1 (TRX) and MIF control their expressioneciprocally [26], which may explain their opposite functions.lycosylation inhibitory factor (GIF), which was originally

eported as a suppressive factor for IgE response, is a posttrans-ationally modified MIF with cysteinylation at Cys60 [27,28].he biological difference between MIF and GIF also may bexplained by redox-dependent modification possibly involvingRX.

.2. Thioredoxin reductase and peroxiredoxin

The thioredoxin system is composed of NADPH, thioredoxineductase (TrxR), and thioredoxin; together with peroxiredoxin

446 H. Nakamura et al. / Seminars in Cancer Biology 16 (2006) 444–451

Fig. 2. Biochemical characteristics of thioredoxin 1 (TRX) TRX is induced by oxidative stress and plays as an antioxidant together with peroxiredoxin. TRX is anendogenous inhibitor of ASK1 to inhibit the cellular apoptosis. TRX regulates the gene expressions via redox-dependent transcriptional factors such as NF-kB, AP-1and p53. Oxidized TRX as well as truncated TRX is released from cells.

Fig. 3. Subcellular localization of TRX family thioredoxin 1 (TRX) as well as glutaredoxin 1 (GRX1) is located in the cytosol, whereas TRX2 is in the mitochondria.Glutaredoxin 2 (GRX2) is in the mitochondria and in the nucleus (modified from Ref. [18]).

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Prx) it forms a thioredoxin peroxidase system to reduce per-xides by NADPH. Mammalian thioredoxin reductase (TrxR),hich reduces thioredoxin by NADPH has in addition a vari-

ty of substrates including selenite and lipoic acid [29]. Therere three isoforms of TrxR; TrxR 1 in the cytosol, mitochondrialpecific TrxR 2, and a testis specific TrxR called thioredoxin glu-athione reductase or TGR. There are several splicing variants ofrxR1. Peroxiredoxin (Prx), which is a thioredoxin-dependenteroxidase scavenges hydrogen peroxide involved in signaling.ammalian cells have six isoforms of Prx, which are divided

nto three subgroups [30].

.3. Thioredoxin-binding proteins

Yeast two hybrid screening revealed that TRX binds to intra-ellular proteins. One of them is ASK1 as mentioned above [15].educed TRX binds to ASK1 in the cytosol under the normalondition. When TRX is oxidized by oxidative stress, ASK1s dissociated from TRX and becomes a homodimer to tran-uce the apoptosis signal. The other binding protein for TRXs thioredoxin-binding protein 2 (TBP-2), which is identical toitamin D3 upregulated protein 1 (VDUP1) [31].

. Extracellular roles of thioredoxin in cancer

As previously described, TRX was originally cloned as aytokine-like factor named ADF [6]. Independently, TRX waslso identified as an autocrine growth factor named 3B6-IL-produced by Epstein-Barr virus-transformed B cells [32,33]

r as a B cell growth factor named MP6-BCGF produced bycell hybridoma MP6 [34]. Moreover, eosinophil cytotoxic-

ty enhancing factor (ECEF) was found as a truncated form ofRX which is the N-terminal 1–80 (or 1–84) residues of TRX

Trx80) [35] and a component of “early pregnancy factor” whichas an immunosuppressive factor in pregnant female serumas also identified as TRX [36]. These historical reports collec-

ively have shown that TRX has various important extracellularunctions.

.1. Cytokine/chemokine-like functions of TRX

Initially TRX was reported to show mitogenic activity oneukemic cells [37,38]. However, the mitogenic activity wasnly significant in the presence of a reducing agent such as-mercaptoethanol (2-ME). In the medium of cell culture or inlasma, TRX is easily oxidized in the absence of 2-ME or dithio-hreitol (DTT) [39,40] and present in the dimer form which haso growth promoting effect on cancer cells [41]. Actually, C73s reported to be essential for the activity of “early pregnancyactor”, suggesting that the dimer of TRX in plasma functionss early pregnancy factor [42]. Interestingly, C32S/C35S mutantRX in which two cysteines Cys32 and Cys35 in the active sitere replaced with serines has no reducing activity and no growth

romoting activity even in the presence of DTT [43], suggestinghat the intact active site interacting with the target protein on theell membrane may be necessary for the growth promoting activ-ty of TRX. In contrast, the truncated form of TRX (Trx80) which

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cer Biology 16 (2006) 444–451 447

as originally reported as ECEF [35] shows different functionsike a cytokine generated from TRX. It has mitogenic activityn peripheral blood mononuclear cells (PBMC) [44]. Moreover,rx80 which is also present in the non-covalent dimer form inlasma increases the expressions of CD14, CD40, CD54 andD86 on monocytes [45] from PBMC and induces the secretionf IL-12 in CD40+ monocytes. Truncated thioredoxin Trx80 ispotent cytokine for monocytes directing the immune system

o a Th1 response [46]. To complicate things, the C32S/C35Sutant Trx80 still has mitogenic activity and IL-12 inducing

ctivity similar to that of Trx80 [47]. Further studies are neces-ary for understanding the signal transduction via Trx80.

Extracellular TRX shows cytoprotective effects. For exam-le, extracellular TRX suppresses TNF-a induced apoptosis [48]nd hydrogen-peroxide-induced endothelial cell damage [49].ince extracellular TRX becomes oxidized, it is rather hard toxplain these cytoprotective effects by the antioxidative functionf TRX. Extracellular TRX suppresses the secretion of the intra-ellular endogenous TRX in response to oxidative stress [50].he cytoprotective effect may be explained by the interaction ofxtracellular TRX with membrane-associated target moleculesncluding membrane-bound TRX. The study is still in progress tolarify the molecular mechanisms of the cytoprotective effects.s for the cytokine stimulation, it was reported previously thatRX treated with DTT stimulates the productions of IL-1, IL-, and IL-8 together with tetradecanoyl phorbolacetate [51].owever, more recently it was also shown that extracellularRX becomes oxidized and inhibits lipopolysaccharide (LPS)-

nduced IL-1� expression and secretion in macrophages [40],uggesting that in normal condition without reducing agent,RX suppresses the production of inflammatory cytokines.

Chemotactic effect of TRX was first observed for theosinophils from the patients with hypereosinophilia [52]. TRXhowed enhancing effects on the chemotactic and chemokineticigration of eosinophils. Later, the chemotactic effect of TRXas shown for T lymphocytes, monocytes and neutrophils fromealthy volunteers by a Boyden-chamber assay [53]. C32S/C35Sutant TRX has no chemotactic activity for neutrophils andonocytes. The chemotactic effect of TRX was also observed

y a mouse air pouch model. Truncated thioredoxin Trx80 hashemotactic effect for monocytes [54].

In contrast, TRX in higher concentration (>1 �g/ml) in circu-ation shows anti-chemotactic effects. It blocks the activation of38 mitogen activated protein kinase in neutrophils, l-selectinhedding on the cell surface of neutrophils, the adhesion of neu-rophils on endothelial cells and the extravasation of neutrophilsnto the inflammatory site as shown in Fig. 4 [55,56]. Thisnti-chemotactic effect of TRX was also observed for MCP--induced monocyte chemotaxis [57]. Oxidized TRX as wells reduced TRX shows the same anti-chemotactic effect foreutrophil, since reduced TRX is converted to oxidized formmmediately in plasma. The effect of the C32S/C35S mutationn TRX on the anti-chemotactic effect is at present controversial

56,58]. C32S/C35S mutant TRX has no anti-chemotactic effectn LPS-induced neutrophil extravasation in the mouse air pouch

odel [56], whereas it is effective to suppress MCP-1-inducedigration of monocytes [58].

448 H. Nakamura et al. / Seminars in Cancer Biology 16 (2006) 444–451

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ig. 4. Anti-inflammatory functions of thioredoxin 1 (TRX) TRX suppresses pndothelial cells and transmigration of neutrophils into the inflammatory site.

.2. Thioredoxin in plasma or serum as an oxidative stressarker

Thioredoxin 1 (TRX) is released by cells in response toxidative stress [50]. Plasma or serum levels of TRX are mea-urable by a sensitive sandwich enzyme immunosorbent assayELISA) [59]. Serum/plasma levels of TRX are good markersor oxidative stress in a variety of disorders [60]. For exam-le, plasma levels of TRX are elevated in patients with acquiredmmunodeficiency syndrome (AIDS) and negatively correlatedith the intracellular levels of GSH, suggesting that the HIV

nfected individuals with AIDS are suffering from systemicxidative stress [55,59]. In patients with type C chronic hep-titis, serum levels of TRX and ferritin are good markers for thefficacy of interferon therapy [61]. In the case of cancer, serumevels of TRX are elevated in patients with hepatocellular carci-oma [62] and pancreatic cancer [63]. The serum levels of TRXecrease after the removal of the main tumor, suggesting thatancer tissues are the main source of the elevated TRX in serum62].

.3. The safety of TRX therapy in cancer patients

Since TRX shows anti-inflammatory effect in circulation, thelinical application of TRX therapy is now planned. EspeciallyRX blocks the neutrophil infiltration into the inflammatory site.

or example, the administration of recombinant human TRXrhTRX) inhibits bleomycin or inflammatory cytokine-inducednterstitial pneumonia [64] and LPS-induced neutrophil infiltra-ion in bronchoalveolar space [65]. Therefore, acute respiratory

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APK activation in neutrophils. TRX inhibits l-selectin shedding, adhesion on

istress syndrome (ARDS)/acute lung injury (ALI) is one disor-er which is a good target for TRX therapy. ARDS/ALI is causedy various etiologies including anti-cancer agents such as gefi-inib, a molecular-targeted agent that inhibits epidermal growthactor receptor (EGFR) tyrosine kinase. Then, how about theafety of TRX therapy in cancer-bearing patients? Although thentracellular expression of TRX in cancer tissues is associatedith resistance to anti-cancer agents [66,67], there is no evi-ence showing that exogenously administered rhTRX promoteshe growth of cancer. Actually, there is no promoting effect ofdministered rhTRX on the growth of the tumor planted in nudeice [65]. In addition, administered rhTRX has no inhibitory

ffect on the anti-cancer agent to suppress the tumor growthn nude mice [65]. It may be explained by that the cellularptake of exogenous TRX is quite limited and administeredRX in plasma immediately becomes the oxidized form whichas no tumor growth stimulatory activity as previously men-ioned. Thioredoxin 1 (TRX) expression is enhanced in cancerissues and now inhibitors for TRX and/or TrxR are studied asew anti-cancer agents [68]. From this aspect, TRX gene ther-py may be dangerous in cancer-bearing patients. In contrast,he administration of rhTRX may be safe and applicable even inancer-bearing patients to attenuate the inflammatory disordersssociated with the leukocyte infiltration.

. Control of cancer by thioredoxin-binding protein 2

We isolated TBP-2/Vitamin D3 upregulated protein 1VDUP1), which was originally reported as the product of aene whose expression was up-regulated in HL-60 cells stimu-

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ated with 1a, 25-dihydroxyvitamin D3 [31,69]. The interactionf TBP-2/VDUP1 with TRX was observed in vitro and in vivo.nterestingly, TBP-2/VDUP1 only binds to the reduced form ofRX and acts as an apparent negative regulator of TRX [31].ater, other groups also reported the interaction between TRXnd this protein [70,71]. Although the mechanism is unknown,reciprocal expression pattern of TRX and TBP-2 was often

eported upon various stimulation [72,73].TBP-2 has a growth suppressive activity. Overexpression of

BP-2 was shown to resulted in growth suppression [74–77].BP-2 expression is upregulated by Vitamin D3 treatment,erum or IL-2 deprivation, leading to growth arrest. TBP-2as found predominantly in the nucleus [76]. TBP-2 mRNA

xpression is down-regulated in several tumors [72,78] andymphoma [79], suggesting a close association between theeduction and tumorigenesis. TBP-2 expression is also down-egulated in melanoma metastasis [80].

Loss of TBP-2 seems to be an important step of HTLV-I trans-ormation. In an in vitro model, HTLV-I-infected T-cells requireL-2 to proliferate in the early phase of transformation, but sub-equently lost cell cycle control in the late phase, as indicated by

heir continuous proliferative state in the absence of IL-2. Thehange of cell growth phenotype has been suggested to be one ofhe oncogenic transformation processes [81]. The expression ofBP-2 is lost in HTLV-I-positive IL-2-independent T-cell lines

Tati

ig. 5. Thioredoxin binding protein 2 (TBP-2) is a regulator of lipid metabolism anethylated in IL-2 independent ATL-2 cells and histones H3 and H4 are deacetylated.

nd a histone acetylase inhibitor (suberoylanilide hydroxamic acid: SAHA) recovere

cer Biology 16 (2006) 444–451 449

ue to the DNA methylation and histone deacetylation, but main-ained in HTLV-I-positive IL-2-dependent T-cell lines as well asTLV-I-negative T-cell lines (Fig. 5) [82]. We generated miceith targeted inactivation of TBP-2 (TBP-2−/− mice). In theseice, plasma free fatty acids levels are higher, whereas glucose

evels are lower than those of wild type mice. TBP-2−/− miceere predisposed to death with bleeding tendency, and hepatic

nd renal dysfunction as a result of 48 h fasting. Inability of fattycid utilization plays an important role in the anomaly of TBP-−/− mice, displaying a characteristically metabolic feature ofeye (-like) syndrome [83]. The tumor incidence of the knockut mice is under investigation. The HcB-19 strain, which has apontaneous mutation in TBP-2/Txnip/VDUP1 gene, has beeneported to have an increased incidence of hepatocellular car-inoma (HCC), showing that TBP-2/Txnip/VDUP1 is a candi-ate tumor suppressor gene in vivo [84]. TBP-2/Txnip/VDUP1eficient mice also exhibited decreased NK cells and reducedumor rejection [85]. Considering that TBP-2 is one of importantenes which are upregulated by a histone deacetylase (HDAC)nhibitor [72], induction of TBP-2 by HDAC inhibitors and othergents seems to be a good therapeutic approach against cancers.

BP-2 was reported to interact with various cellular target suchs JAB1 and FAZF and may be a component of a transcrip-ional repressor complex [74], although precise mechanism ofts molecular action remains to be elucidated.

d a suppressor of cancer. In the promoter region of the TBP-2 gene, CpG areSequential treatment with a demethylase (5-aza-2′-deoxycytidine: 5-aza-CdR)

d the expression of TBP-2 (modified from Ref. [83]).

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There are three highly homologous genes of TBP-2/VDUP1.e characterized a TBP-2 homologue, TLIMP (TBP-2 like

nducible membrane protein). TLIMP is a novel VD3-or PPAR-�igand-inducible membrane-associated protein and plays a reg-latory role in cell proliferation and PPAR-� activation [86].nother TBP-2 homologous gene, DRH1 is reported to beown-regulated in hepatocellular carcinoma [87]. These resultsndicate that the familial members of TBP-2 also play a role inancer suppression.

. Conclusion

Thioredoxin plays crucial roles intracellularly and extracel-ularly in cancer. TBP-2 is an endogenous suppressor of cancerrowth. Studies are in progress to understand the molecularechanisms of the thioredoxin system in cancer and to develop

ew strategies for cancer therapy.

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