Targeting Thioredoxin Reductase Research Articles

Targeting Thioredoxin Reductase: Anticancer Agents and Chemopreventive Compounds

Thioredoxin reductase plays a critical role in the regulation of cancer cell apoptosis, making it an attractive target for the design of new anticancer drugs. Several classes of compounds have been considered as potential antitumor or chemopreventive agents; most of them apparently interact with thioredoxin reductase's C-terminal redox center.

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, ‘auranofin-like’ gold(I) complexes all containing the [Au(PEt3)]+ synthon and the ligands: Cl−, Br−, cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

Augmented antitumor effects of combination therapy of cisplatin with ethaselen as a novel thioredoxin reductase inhibitor on human A549 cell in vivo

Ethaselen (1, 2-[bis (1, 2-Benzisoselenazolone-3 (2H) -ketone)] ethane, BBSKE), as a novel organoselenium compound targeting thioredoxin reductase (TrxR), has been reported to inhibit tumor growth and TrxR activity in several human tumor cell lines. It has now entered Phase I clinical trails. Here we report the effects of ethaselen and cisplatin (cis-diamminedichloroplatinum II, DDP) combination therapy (ethaselen 36 mg/kg, i.g., o.d. x 10 d and cisplatin 1 mg/kg, i.p., single at day 0) on human A549-grafted nude mouse model (female, BALB/c nude mouse, n = 5, treatment after tumor volume reached 100 mm(3)). Compared to single drug administration (either ethaselen: 36 mg/kg, i.g., o.d. x 10 d or cisplatin: 1.0 mg/kg, i.p., single at day 0), the combination therapy showed significantly reduced tumor size (presumably due to a synergistic effect) and no obvious toxic damage (both in terms of body weight maintenance and liver/kidney damage). These results will be significant in the development of novel anti-tumoral therapeutic strategies directed to non-small cell lung cancer (NSCLC).

Targeting Thioredoxin Reductase 1 Reduction in Cancer Cells Inhibits Self-Sufficient Growth and DNA Replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide

Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia and has potential anticancer activity against a wide range of solid tumors. ATO exerts its effect mainly through elevated oxidative stress, but the exact molecular mechanism remains elusive. The thioredoxin (Trx) system comprising NADPH, thioredoxin reductase (TrxR), and Trx and the glutathione (GSH) system composed of NADPH, glutathione reductase, and GSH supported by glutaredoxin are the two electron donor systems that control cellular proliferation, viability, and apoptosis. Recently, the selenocysteine-dependent TrxR enzyme has emerged as an important molecular target for anticancer drug development. Here, we have discovered that ATO irreversibly inhibits mammalian TrxR with an IC(50) of 0.25 microM. Both the N-terminal redox-active dithiol and the C-terminal selenothiol-active site of reduced TrxR may participate in the reaction with ATO. The inhibition of MCF-7 cell growth by ATO was correlated with irreversible inactivation of TrxR, which subsequently led to Trx oxidation. Furthermore, the inhibition of TrxR by ATO was attenuated by GSH, and GSH depletion by buthionine sulfoximine enhanced ATO-induced cell death. These results strongly suggest that the ATO anticancer activity is by means of a Trx system-mediated apoptosis. Blocking cancer cell DNA replication and repair and induction of oxidative stress by the inhibition of both Trx and GSH systems are suggested as cancer chemotherapeutic strategies.

Examination of anticancer mechanisms in ras‐induced cancer cells by targeting thioredoxin reductase 1 knockdown

Thioredoxin reductase 1 (TR1) is a major antioxidant and redox regulator in mammals. This selenium-containing oxidoreductase is enriched in many malignant cells and has been proposed as a target for cancer therapy. To assess the role of TR1 in the malignancy process, we used RNAi technology to knockdown its expression in a lung cancer cell line whereby reversing many of the malignant phenotypes (Yoo et al. JBC, 281: , 2006). To further elucidate the role of TR1 in cancer therapy, we targeted the removal of TR1 expression in DT cells that overexpress oncogenic ras and manifest numerous malignant phenotypes. We found that DT cells overexpress TR1 compared with its normal control, NIH3T3 cells. Knockdown of TR1 resulted in a significant reduction in its anchorage-independent growth properties. We examined gene expression in normal (NIH3T3), cancer (DT) and TR1 knockdown (DT/siTR1) cells by microarray analysis and found several mRNAs that are characteristically elevated in cancer cells were down-regulated in the DT/siTR cells. We are expanding these findings and examining additional properties of DT/siTR1 cells to better evaluate TR1 as a target for cancer therapy. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

215 Targeting thioredoxin reductase ‐ a possible new treatment for malignant mesothelioma

215 Targeting thioredoxin reductase ‐ a possible new treatment for malignant mesothelioma

Motexafin Gadolinium, a Tumor-selective Drug Targeting Thioredoxin Reductase and Ribonucleotide Reductase

Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 microM (kcat/Km of 4.86 x 10(4) M(-1) s(-1)). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 microM) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 x 10(5) M(-1) s(-1)) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 microM reduced human Trx (IC50 2 microM) or 4 mM dithiothreitol (IC50 6 microM) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair.

SiRNA knockdown‐mRNA knock‐in as a means of assessing elenoprotein function

A knockdown/knock-in strategy was designed for studying selenoprotein (SP) function. The technique entails the initial knockdown of gene expression using RNAi technology wherein the siRNA target is directed to the SP mRNA 3′-untranslated region (3′-UTR). Gene expression is then knocked-in circumventing the siRNA by mutations within the target region of the knock-in construct. We tested this strategy by targeting thioredoxin reductase 1 (TR1), which is an essential Sec-containing oxidoreductase that controls intracellular redox state in mammals. TR1 knockdown cells grew more slowly, were more sensitive to UV irradiation and had increased apoptosis in response to UV than control cells. Cells transfected with a construct encoding the wild type TR1 gene and having mutations in the sequences targeted by siRNA, restored TR1 expression and catalytic activity. Replacement of TR1 reversed the sensitivity of TR1 knockdown cells by UV irradiation. The data demonstrate that the RNAi-based knock-in technology provides a means of studying SP function and this approach provides an alternative means for examining many of the subtleties of protein function not available using RNAi technology alone. In addition, by targeting the 3′-UTR, the coding sequence is not altered and can be changed in subsequent knock-in experiments. This research was supported by the Intramural Research Program of the NIH, NCI, CCR, and by NIH GM065204.