TrxR1 mRNA Research Articles

Citrus Flavanone Effects on the Nrf2-Keap1/GSK3/NF-κB/NLRP3 Regulation and Corticotroph-Stress Hormone Loop in the Old Pituitary.

Oxidative stress and inflammation are significant causes of aging. At the same time, citrus flavanones, naringenin (NAR), and hesperetin (HES) are bioactives with proven antioxidant and anti-inflammatory properties. Nevertheless, there are still no data about flavanone's influence and its potential effects on the healthy aging process and improving pituitary functioning. Thus, using qPCR, immunoblot, histological techniques, and biochemical assays, our study aimed to elucidate how citrus flavanones (15 mg/kg b.m. per os) affect antioxidant defense, inflammation, and stress hormone output in the old rat model. Our results showed that HES restores the redox environment in the pituitary by down-regulating the nuclear factor erythroid 2-related factor 2 (Nrf2) protein while increasing kelch-like ECH-associated protein 1 (Keap1), thioredoxin reductase (TrxR1), and superoxide dismutase 2 (SOD2) protein expression. Immunofluorescent analysis confirmed Nrf2 and Keap1 down- and up-regulation, respectively. Supplementation with NAR increased Keap1, Trxr1, glutathione peroxidase (Gpx), and glutathione reductase (Gr) mRNA expression. Decreased oxidative stress aligned with NLRP3 decrement after both flavanones and glycogen synthase kinase-3 (GSK3) only after HES. The signal intensity of adrenocorticotropic hormone (ACTH) cells did not change, while corticosterone levels in serum decreased after both flavanones. HES showed higher potential than NAR in affecting a redox environment without increasing the inflammatory response, while a decrease in corticosterone level has a solid link to longevity. Our findings suggest that HES could improve and facilitate redox and inflammatory dysregulation in the rat's old pituitary.

PSMD12 promotes non-small cell lung cancer progression through activating the Nrf2/TrxR1 pathway.

Non-small cell lung cancer (NSCLC) contributes to the vast majority of cancer-related deaths. Proteasome 26S subunit, non-ATPase 12 (PSMD12), a subunit of 26S proteasome complex, is known to play the tumor-promoting role in several types of cancer but its function in NSCLC remains elusive. To explore the role and underlying mechanisms of PSMD12 in NSCLC. The PSMD12 expression in human normal lung epithelial cell line (BEAS-2B) and four NSCLC cell lines (A549, NCI-H1299, NCI-H1975, Calu-1) were determined by qRT-PCR and western blot. Malignant phenotypes of NSCLC cells were detected by CCK-8, EdU staining, immunofluorescence staining for E-cadherin, flow cytometry, and Transwell assays to assess cell viability, proliferation, epithelial-mesenchymal transition (EMT), apoptosis, migration and invasion. Dual luciferase assay was used to verify the regulatory role of transcription factor on the promoter. We identified the upregulation of PSMD12 in NSCLC tissues based on the GEO datasets, which further verified in NSCLC and BEAS-2B cell lines. PSMD12 knockdown significantly suppressed malignant behaviors of NSCLC cells, including cell growth, invasion, and migration, while PSMD12 overexpression presented the opposite effects. Interestingly, we found that PSMD12 upregulated the tumor-promoting factor TrxR1 mRNA expression. For its potential mechanisms, we demonstrated that PSMD12 elevated transcription factor Nrf2 protein level and promoted Nrf2 nuclear translocation. And Nrf2 further increased TrxR1 promoter activity and enhanced TrxR1 transcription. Meanwhile, we proved that TrxR1 overexpression erased the inhibitory effect of PSMD12 knockdown. PSMD12 promotes NSCLC progression by activating the Nrf2/TrxR1 pathway, providing a novel prognostic and therapeutic target for NSCLC treatment.

Thioredoxin reductase 1 regulates hepatic inflammation and macrophage activation during acute cholestatic liver injury.

Cholestatic liver diseases, including primary sclerosing cholangitis, are characterized by periportal inflammation with progression to hepatic fibrosis and ultimately cirrhosis. We recently reported that the thioredoxin antioxidant response is dysregulated during primary sclerosing cholangitis. The objective of this study was to examine the impact of genetic and pharmacological targeting of thioredoxin reductase 1 (TrxR1) on hepatic inflammation and liver injury during acute cholestatic injury. Primary mouse hepatocytes and intrahepatic macrophages were isolated from 3-day bile duct ligated (BDL) mice and controls. Using wildtype and mice with a liver-specific deletion of TrxR1 (TrxR1LKO), we analyzed the effect of inhibition or ablation of TrxR1 signaling on liver injury and inflammation. Immunohistochemical analysis of livers from BDL mice and human cholestatic patients revealed increased TrxR1 staining in periportal macrophages and hepatocytes surrounding fibrosis. qPCR analysis of primary hepatocytes and intrahepatic macrophages revealed increased TrxR1 mRNA expression following BDL. Compared with sham controls, BDL mice exhibited increased inflammation, necrosis, and increased mRNA expression of pro-inflammatory cytokines, fibrogenesis, the NLRP3 inflammatory complex, and increased activation of NFkB, all of which were ameliorated in TrxR1LKO mice. Importantly, following BDL, TrxR1LKO induced periportal hepatocyte expression of Nrf2-dependent antioxidant proteins and increased mRNA expression of basolateral bile acid transporters with reduced expression of bile acid synthesis genes. In the acute BDL model, the TrxR1 inhibitor auranofin (10mg/kg/1d preincubation, 3d BDL) ameliorated BDL-dependent increases in Nlrp3, GsdmD, Il1β, and TNFα mRNA expression despite increasing serum alanine aminotransferase, aspartate aminotransferase, bile acids, and bilirubin. These data implicate TrxR1-signaling as an important regulator of inflammation and bile acid homeostasis in cholestatic liver injury.

Expression of thioredoxin reductase 1 (TrxR1) in gastric cancer and its effect on the growth of gastric cancer cells

To investigate the relationship between the expression of thioredoxin reductase 1 (TrxR1) in gastric cancer tissues and the survival time of patients with gastric cancer and its effect on the growth of gastric cancer cells. The expression level of TrxR1 mRNA in gastric cancer tissues and adjacent tissues of 76 patients was determined by real-time PCR assays, and the relationship between the mRNA expression level of TrxR1 and the clinicopathological characteristics and prognosis of gastric cancer patients was analyzed. Three gastric cancer tissues and adjacent tissues were randomly selected, and the expression of TrxR1 protein was detected by immunohistochemistry and Western blot. The expression levels of TrxR1 in human gastric cancer cells line and gastric mucosa epithelial cells were determined by Western blot and quantitative real-time PCR assays. Then, AGS cells were transfected with siRNA sequences to silence the expression of TrxR1. AGS cells were divided into 3 groups according to different processing negative control group: transfected with NC-siRNA, TRXR1 siRNA interference group 1: transfected with TRXR1-siRNA1, TRXR1 siRNA interference 2 group: transfected with TRXR1-siRNA2. The expression of TrxR1 mRNA in AGS cells of each group was detected by Real-time PCR. The proliferation of AGS cells was determined by MTT and colony formation assays following TrxR1 silencing. TrxR1 mRNA and protein expression levels in gastric cancer tissues were significantly up-regulated compared with adjacent tissues, and were mainly located in the cytoplasm. High expression of TrxR1 was associated with TNM stage and lymph node metastasis, and the overall survival time of patients with high expression of TrxR1 was shorter than those with low expression level. TrxR1 mRNA and protein in AGS cells of TRXR1-siRNA1 group and AGS cells of TRXR1-siRNA2 group were significantly reduced compared with NC-siRNA group (P＜0.05). And AGS cell clone formation and proliferation ability were decreased. The high expression of TrxR1 in gastric cancer tissues indicates poor prognosis of patients, and the silencing of TrxR1 can inhibit the proliferation of gastric cancer cells.

Antioxidant effects of Lycium barbarum polysaccharides on photoreceptor degeneration in the light-exposed mouse retina

We assessed the neuroprotective effects of Lycium barbarum Polysaccharides (LBP) on photoreceptor degeneration and the mechanisms involved in oxidative stress in light-exposed mouse retinas. Mice were given a gavage of LBP (150 mg/kg or 300 mg/kg) or phosphate buffered saline (PBS) for 7 days before exposure to light (5000 lx for 24 h). We found that LBP significantly improved the electroretinography (ERG) amplitudes of the a- and b-waves that had been attenuated by light exposure. In addition, changes caused by light exposure including photoreceptor cell loss, nuclear condensation, an increased number of mitochondria vacuoles, outer membrane disc swelling and cristae fractures were distinctly ameliorated by LBP. LBP treatment also significantly prevented the generation of reactive oxygen species (ROS) compared with PBS treatment. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and thioredoxin reductase (TrxR1) mRNA were decreased in PBS-treated mice compared with controls but increased remarkably in LBP-treated mice. The mRNA levels of the DNA repair gene Poly (ADP-ribose) polymerase (PARP14) was increased in PBS-treated mice but decreased significantly in the LBP-treated mice. Our findings indicate that pretreatment with LBP effectively protected photoreceptor cells against light-induced retinal damage probably through the up-regulation of the antioxidative genes Nrf2 and TrxR1, the elimination of oxygen free radicals, and the subsequent reduction in the mitochondrial reaction to oxidative stress and enhancement in antioxidant capacity. In addition, the decreased level of PARP14 mRNA in LBP-treated mice also indicated a protective effect of LBP on delaying photoreceptor in the light-damaged retina.

MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3′ UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23.

The Protective Effects of Different Sources of Maternal Selenium on Oxidative Stressed Chick Embryo Liver.

The experiment was conducted to investigate the protective effects of different sources of maternal selenium (Se) on oxidative stressed chick embryo. A total of 270 Lingnan Yellow broiler breeders were randomly allocated into three treatments with five replicates for 18 birds each. Breeders were fed with basal diet (BD) including 0.04mg/kg Se or BD supplemented with sodium selenite (SS) or selenomethionine (SM) at a level of 0.15mg Se/kg. The rearing experiment lasted for 8weeks after an 8-week pre-test. Twenty eggs were collected from each replicate during the last 10-day, then incubated in a commercial incubator. On embryonic 17th, fertile eggs were transferred into 39.5°C temperature stimulation for 6h. Afterward, five eggs were randomly selected from each replicate for collecting chick embryo sample. The results showed that Se supplementation in the diet of breeders resulted in lower reactive oxygen species (ROS), heat shock protein 70 (HSP70), malondialdehyde (MDA), carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) concentrations and higher glutathione peroxidase (GPx), total superoxide dismutase (T-SOD), and catalase (CAT) activities in heat stress treated chick embryo (P < 0.05), and ROS, MDA, carbonyl, 8-OHdG concentrations in SM treatment were lower than those in SS treatment (P < 0.05). Se supplementation elevated cellular glutathione peroxidase (GPx1) mRNA level and activity, cytoplasmic thioredoxin reductase (TrxR1) activity and selenoprotein P (SelP) mRNA and protein level (P < 0.05), and maternal SM showed a higher value than maternal SS in upregulating GPx1, TrxR1, and SelP mRNA levels as well as GPx1 and TrxR1 activities or SelP protein level (P < 0.05). This study indicated that maternal Se can enhance antioxidative capacity and reduce ROS concentration and oxidative damage by upregulating the expression of antioxidative selenoprotein, and maternal SM is superior to SS in heat stress treated chick embryo.

Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50% of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.

Simvastatin inhibits the core promoter of the TXNRD1 gene and lowers cellular TrxR activity in HepG2 cells

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing redox-active enzyme that is thought to be important during carcinogenesis. We have recently shown that treatment with statins, HMGCoA reductase inhibitors, reduces the levels of TrxR1 in liver of both rat and human. The reduced TrxR1 levels were correlated with inhibited hepatocarcinogenesis in a rat model. The aim of the present study was to investigate if statins affect the activity of the human TXNRD1 core promoter, which guides expression of TrxR1, and if the effects by statins on TrxR1 expression in liver could be reproduced in a cellular model system. We found that simvastatin and fluvastatin decreased cellular TrxR activity in cultured human liver-derived HepG2 cells with approximately 40% (p<0.05). Simvastatin, but not fluvastatin or atorvastatin, also reduced the TXNRD1 promoter activity in HepG2 cells by 20% (p<0.01). In line with this result, TrxR1 mRNA levels decreased with about 25% in non-transfected HepG2 cells upon treatment with simvastatin (p<0.01). Concomitant treatment with mevalonate could not reverse these effects of simvastatin, indicating that other mechanisms than HMGCoA reductase inhibition was involved. Also, simvastatin did not inhibit sulforaphane-derived stimulation of the TXNRD1 core promoter activity, suggesting that the inhibition by simvastatin was specific for basal and not Nrf2-activated TrxR1 expression. In contrast to simvastatin, the two other statins tested, atorvastatin or fluvastatin, did not influence the TrxR1 mRNA levels. Thus, our results reveal a simvastatin-specific reduction of cellular TrxR1 levels that at least in part involves direct inhibitory effects on the basal activity of the core promoter guiding TrxR1 expression.

Effect of selenium sources on the expression of cellular glutathione peroxidase and cytoplasmic thioredoxin reductase in the liver and kidney of broiler breeders and their offspring

In total, 180 Lingnan Yellow broiler breeders were used to investigate the effect of different sources of selenium (Se) on the expression of cellular glutathione peroxidase (GPx1) and cytoplasmic thioredoxin reductase (TrxR1) in the liver and kidney of broiler breeders and their offspring by quantitative real-time PCR. There were 6 replicates of 3 dietary treatments. Broiler breeders were fed corn-soy-based diets supplemented with 0.15 mg/kg of Se from sodium selenite, Se-enriched yeast (SY), or selenomethionine (SM). At the end of the feeding trial, liver and kidney mRNA levels of GPx1 and TrxR1 were determined by quantitative real-time PCR, as well as the activity of GPx1 and TrxR1 in liver and kidney of breeders and their offspring. The results showed that, compared with sodium selenite, SY or SM significantly increased (P < 0.05) the activity of TrxR1 in the liver and kidney of broiler breeders and their offspring but not the GPx1 activity. The liver GPx1 and TrxR1 mRNA levels in SY or SM groups were higher (P < 0.05) than that in the sodium selenite group. And the kidney TrxR1 mRNA levels were also significantly increased (P < 0.05) by using SY or SM, whereas there was no significant difference in the kidney GPx1 mRNA levels between the organic or inorganic sources of Se used.

Synergy between sulforaphane and selenium in the up-regulation of thioredoxin reductase and protection against hydrogen peroxide-induced cell death in human hepatocytes

Dietary isothiocyanates and selenium are chemopreventive agents and potent inducers of antioxidant enzymes. It has been previously shown that sulforaphane and selenium have a synergistic effect on the upregulation of thioredoxin reductase-1 (TrxR-1) in human hepatoma HepG2 cells. In this paper, further evidence is presented to show that sulforaphane and selenium synergistically induce TrxR-1 expression in immortalised human hepatocytes. Sulforaphane was found to be more toxic toward hepatocytes than HepG2 cells with IC50=25.1 and 56.4μM, respectively. Sulforaphane can protect against hydrogen peroxide-induced cell death and this protection was enhanced by co-treatment with selenium. Using siRNA to knock down TrxR-1 or Nrf2, sulforaphane (5μM)-protected cell viability was reduced from 73% to 46% and 34%, respectively, suggesting that TrxR-1 is an important enzyme in protection against hydrogen peroxide-induced cell death. Sulforaphane-induced TrxR-1 expression was positively associated with significant levels of Nrf2 translocation into the nucleus, but co-treatment with selenium showed no significant increase in Nrf2 translocation. Moreover, MAPK (ERK, JNK and p38) and PI3K/Akt signalling pathways were found to play no significant role in sulforaphane-induced Nrf2 translocation into the nucleus. However, blocking ERK and JNK signalling pathways decreased sulforaphane-induced TrxR-1 mRNA by about 20%; whereas blocking p38 and PI3K/AKT increased TrxR-1 transcription. In summary, a combination of sulforaphane and selenium resulted in a synergistic upregulation of TrxR-1 that contributed to the enhanced protection against free radical-mediated oxidative damage in human hepatocytes.

Selenium homeostasis and induction of thioredoxin reductase during long term selenite supplementation in the rat

Selenium is a candidate treatment for liver tumour prevention in chronic liver disease. In this study, we have studied selenium uptake, distribution and accumulation in rats provided with water containing tumour-preventive doses of sodium selenite for 10 weeks. Male Fischer 344 rats were given drinking water containing 1 μg/mL or 5 μg/mL sodium selenite. Selenium levels were monitored in serum and liver tissue over the 10-week period, and the kinetics of induction of the redox-active cytosolic selenoenzyme thioredoxin reductase were followed. Selenite exposure via drinking water caused a dose-dependent increase in blood and liver selenium levels, with plateaus at 6 and 8 weeks, respectively. These plateaus were reached at the same level of selenium regardless of dose, and no further accumulation was observed. A selenium-dependent increase in the activity of TrxR1 in parallel with the increase in liver selenium levels was also seen, and the induction of TrxR1 mRNA was seen only during the first three days of treatment, when the levels of selenium in the liver were increasing. Sodium selenite at 1 and 5 μg/mL did not affect body weight or relative liver mass. We concluded that long-term treatment with selenite did not cause accumulation of selenium and that the activity of TrxR1 in the liver rose with the selenium levels. We therefore suggest that sodium selenite at doses up to 5 μg/mL could be used for long-term tumour prevention.

Abstract 5360: Differential induction of the anti-oxidant response by arsenic trioxide and darinaparsin in leukemic cells

Abstract Darinaparsin (DAR) is a significantly more potent arsenical than arsenic trioxide (ATO) at mediating apoptosis in various malignant cell lines and is highly active against Acute Promyelocytic Leukemia (APL) cells (NB4). The mechanisms behind DAR's increased efficacy versus ATO remain to be elucidated. We previously reported that DAR triggers apoptosis by inducing cellular responses that do not completely overlap with ATO (Diaz et al. Leukemia). Oxidative damage has been postulated to be a key mechanism by which arsenicals initiate the apoptotic process. Consistent with its increased cytotoxicity, we observed that DAR induces superoxide (O2·−) and hydrogen peroxide (H2O2), as shown respectively by hydorethidine and dichloro-dihydro-fluorescein staining quantified by flow cytometry. In response to oxidative stress, cells induce an arsenal of protective proteins of which HO-1 (heme oxygenase-1), NQO1 (NAD(P)H:quinone oxidoreductase), TrxR1 (thioredoxin-reductase-1) and γGCS (glutamyl cysteine synthetase) are well recognized members. We therefore studied the expression of these stress response proteins after ATO and DAR treatment. As expected, we observed a significantly higher increase in NQO1, TrxR1 and γGCS mRNA and protein levels as assessed by Western Blot and quantitative real PCR, with DAR than with ATO. However, HO-1 expression was not induced by DAR. Nrf2 is a transcription factor that mediates the expression of key protective enzymes through antioxidant response elements (AREs). AREs have been identified in the regulatory regions of genes encoding detoxification enzymes and other cytoprotective proteins, including NQO1, TrxR1, γGCS, and HO-1. Nucleo-cytoplasmic fractionation followed by Western Blot show that both compounds induce Nrf2 translocation to the nucleus. Transcriptional regulation of these detoxifying enzymes involves a competition between the activator Nrf2 and the repressor Bach1 for interaction with their AREs. Accordingly, ChIP assays demonstrate that Bach1 is released and Nrf2 is recruited to the HO-1 ARE in ATO treated cells but not in cells treated with DAR. HO-1's widely reported cytoprotective effects in vitro and in vivo have been related to the generation of the antioxidants biliverdin and bilirubin. In order to elucidate whether the lack of HO-1 expression contributes to the increased efficacy of DAR, we examined the response to ATO and DAR in co-treatment with OB-24, a recently developed HO-1 inhibitor. As indicated by Propidium Iodide staining followed by flow cytometry analysis, pharmacological inhibition of HO-1 increased ATO toxicity but has no significant effect on DAR induced apoptosis. We therefore hypothesize that the increased efficacy of DAR versus ATO might be partially due to the absence of HO-1 up-regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2011-5360

Effects of hyperoxia on cytoplasmic thioredoxin system in alveolar type epithelial cells of premature rats

This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 (Trx1) and thioredoxin reductase-1 (TrxR1) in alveolar type II epithelial cells (AECII) of premature rats. Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation. AECII were isolated and purified from the lungs of premature rats. When cultured to 80% confluence, in vitro cells were randomly divided into air group and hyperoxia group. Cells in the hyperoxia group were continuously exposed to 95% O(2)/5% CO(2) and those in the air group to 95% air/5% CO(2). After 12, 24 and 48 h, cells in the two groups were harvested to detect their reactive oxygen species (ROS), apoptosis, TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols, respectively. The results showed that AEC II exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group (P<0.001). Moreover, TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group (P<0.001). RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AECII exposed to hyperoxia for 12 and 24 h (P<0.01), respectively. At 48 h, the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group (P>0.05). Western blotting showed the changes of Trx1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR. It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AECII in a certain period, however, also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity, which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.

Novel mechanism of ethaselen in poorly differentiated colorectal RKO cell growth inhibition: Simultaneous regulation of TrxR transcription, expression and enzyme activity

Thioredoxin reductase (TrxR) is a ubiquitous intracellular redox enzyme that regulates tumor growth and proliferation in various cancer cells. Ethaselen (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane), a novel anticancer agent, is designed to target mammalian TrxR1 with the aims of cancer growth inhibition and TrxR inactivation. In this study, we demonstrated that ethaselen significantly inhibits cell growth in the poorly differentiated colorectal RKO cell line, and simultaneously downregulates mammalian TrxR1 mRNA transcript levels, protein expression and enzyme activity, which differs from its actions in moderately differentiated colorectal LoVo cells. Ethaselen’s significant abatement of the Wnt/beta-catenin cell differentiation-related signaling pathway was also observed in RKO cells; this apparently leads to its strong inhibitory effect on cell growth and TrxR1 activity in this cell line. These results suggest that ethaselen has a novel mechanism affecting cell growth in the poorly differentiated RKO colorectal cell line.

Hyperbaric oxygen preconditioning ameliorates anxiety-like behavior and cognitive impairments via upregulation of thioredoxin reductases in stressed rats

The present study examined the protective effect of hyperbaric oxygen preconditioning (HBO-PC) and the role of thioredoxin reductase (TrxR) in a post-traumatic stress disorder (PTSD)-induced rat model by using single prolonged stress (SPS). Rats were randomly divided into Sham, HBO, SPS and HBO + SPS groups. HBO-PC was conducted by exposing rats to 100% oxygen at 2.5 atm absolute for 1 h each day for 5 consecutive days. SPS was performed 24 h after the last HBO-PC conditioning event. At 1 h, 6 h, 12 h, 24 h and 72 h after SPS, TrxR mRNA expression was analyzed in the hippocampus; Nissl and TUNEL staining were performed at 72 h after SPS. The results indicated that HBO-PC was able to significantly preserve viable neurons in the CA1 subfield of hippocampus following SPS exposure, as evidenced by reduced amounts of CA1 neuronal apoptosis. Furthermore, HBO-PC upregulate the expression of TrxR-1 and TrxR-2 mRNA in the hippocampus at 6 h and 12 h after SPS exposure and ameliorated anxiety-like behavior and cognitive impairments normally induced by SPS. Taken together, these findings suggest that HBO-PC is beneficial for the improvement of anxiety-like behavior and cognitive impairments induced by SPS exposure, and this effect might be associated with inhibition of neuronal apoptosis via upregulation of TrxR in stressed rats.

Abstract 1896: Selenium and isothiocyanates increases TrxR1 and GI-GPx expression but DNA methylation of LINE-1 remains unchanged in Caco-2 cells

Abstract Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression through epigenetic events to modify cancer susceptibility. Evidence from epidemiological, in vivo and in vitro studies have suggested that the chemopreventive effect of cruciferous vegetables is attributed to the glucosinolate break-down products, isothiocyanates (ITCs). Similarly, selenium (Se) is an integral part of many cellular antioxidant enzymes and like isothiocyanates has been inversely associated with the incidence of cancer as a result of influencing multiple pathways in the development of cancer. However, their efficacy might depend on the chemical form in which they are administered. We therefore examined the synergistic effect of two forms of selenium, selenium-methylselenocysteine (Se-MSC) and selenite (200nM) with ITCs (6µM) such as iberin and sulforaphane on the expression of antioxidant selenoenzymes such as thioredoxin-reductase (TrxR1) and gastrointestinal glutathione peroxidise (GI-GPx) in an in vitro model using Caco-2 cells. Our preliminary results suggest that the simultaneous addition of Se-MSC and iberin significantly induced TrxR1 and GI-GPx mRNA and protein expression more than either compound alone by ∼6 fold (p&amp;lt;0.001). In addition to the antioxidant protection provided by these selenoproteins, DNA methylation is essential for normal development, but aberrant DNA methylation patterns are a hallmark of most cancers and include global DNA hypomethylation, region-specific hypermethylation and increased DNA methyltransferases (DNMTs) activity. We therefore also examined the impact of Se and ITCs on DNA methylation, specifically examining factors modulating DNMT1, 3A, 3B expression and methylation of the LINE-1 retrotransposon, a surrogate marker of global DNA methylation. Caco-2 cells were treated with Se-MSC and selenite using 0.2, 0.5, 1, 2 and 5µM, in addition to the ITCs (6µM) after 5 and 10 days of treatment. Our initial data suggests that methylation of LINE-1 in the control group was indeed low (55%) and treatment of cells with DNMT inhibitor 5-aza-2 deoxycytidine resulted in a 45% reduction in LINE-1 methylation as expected. Unexpectedly, none of the food compounds or dose used in this study modified significantly the methylation status of LINE-1. Interestingly, although the methylation levels of this marker remain unchanged, the mRNA expression of the DNMTs showed the tendency to decrease, following the same pattern in the different treatments and time courses albeit with small differences, which suggests that both de novo and maintenance DNMTs work in concert to create and propagate methylation patterns. However, only DNMT3B showed a significant reduction of 35 and 38% after 10 days of treatment with Se-MSC (500nM) (p=0.04) and selenite (1µM) (p=0.035) respectively. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1896.

Treatment of lung cancer cells with cytotoxic levels of sodium selenite: Effects on the thioredoxin system

Selenium at subtoxic doses has been shown to have tumor specific cytotoxic effects. In this work, viability measurements in different lung cancer cell lines showed that selenite was more effective compared to three different conventional cytotoxic drugs. In addition, the cell line most sensitive to selenite toxicity comprised the highest level of thioredoxin reductase 1 (TrxR1). The human selenoenzyme TrxR1 is a central enzyme for cell growth, differentiation, and the protection against oxidative stress. TrxR1, which in several studies has been shown to be up-regulated in various tumor cells, is also a target for many anticancer drugs. In this study, inhibition of TrxR resulted in enhanced selenite cytotoxicity, clearly connecting the thioredoxin system to the toxic effects mediated by selenite. The complex regulation of TrxR1, involving the expression of many different transcript forms of mRNA, was investigated by real-time qPCR in lung cancer cell lines following treatment with toxic doses (2.5–10μM) of sodium selenite. Selenium treatment resulted in increased expression of almost all TrxR1 mRNA variants with increasing concentrations of selenite. On the contrary, the TrxR protein level and activity, increased at low to moderate doses followed by a decrease at higher doses, indicating impairment of protein synthesis by selenite.

6539 POSTER Malignant mesothelioma of pleura: potentialities of immunocytochemistry

The human selenoenzyme thioredoxin reductase 1 (TrxR1) is a very important enzyme for cell growth, differentiation, and the defense against oxidative stress. Several studies have shown that TrxR1 is up-regulated in tumor cells. The regulation of TrxR1 is very complex and involves the expression of different transcript forms of mRNA. We have, by quantitative polymerase chain reaction, investigated the total expression of TrxR1 mRNA and quantified the expression of alternative mRNA forms (α1/2, α6, α7/8, α10/11, α13, γ2–4, and β1) in six different human malignant mesothelioma cell lines of epithelioid, sarcomatoid, or mixed phenotype. The most abundant α-form was surprisingly α1/2 and not the expected α7/8. Selenium treatment resulted in increased expression of all α-variants, except the α10/11, where the levels were unaffected. The expression of protein isoforms was studied and the less abundant forms TrxR1v.2, TrxR1v.3, and TrxR1v.5 were detected in cell lysates and in human tumor tissue, using specific peptide antibodies. Furthermore, TrxR1v.3 and TrxR1v.5, previously not identified in human cells, were detected by mass spectrometry. Our data show differential expression of TrxR1 mRNA forms in malignant mesothelioma of different phenotype, and investigation of alternative transcript variants of TrxR1 could be a valuable tool in the diagnostics and characterization of tumors.

Quantification of alternative mRNA species and identification of thioredoxin reductase 1 isoforms in human tumor cells

The human selenoenzyme thioredoxin reductase 1 (TrxR1) is a very important enzyme for cell growth, differentiation, and the defense against oxidative stress. Several studies have shown that TrxR1 is up-regulated in tumor cells. The regulation of TrxR1 is very complex and involves the expression of different transcript forms of mRNA. We have, by quantitative polymerase chain reaction, investigated the total expression of TrxR1 mRNA and quantified the expression of alternative mRNA forms (α1/2, α6, α7/8, α10/11, α13, γ2–4, and β1) in six different human malignant mesothelioma cell lines of epithelioid, sarcomatoid, or mixed phenotype. The most abundant α-form was surprisingly α1/2 and not the expected α7/8. Selenium treatment resulted in increased expression of all α-variants, except the α10/11, where the levels were unaffected. The expression of protein isoforms was studied and the less abundant forms TrxR1v.2, TrxR1v.3, and TrxR1v.5 were detected in cell lysates and in human tumor tissue, using specific peptide antibodies. Furthermore, TrxR1v.3 and TrxR1v.5, previously not identified in human cells, were detected by mass spectrometry. Our data show differential expression of TrxR1 mRNA forms in malignant mesothelioma of different phenotype, and investigation of alternative transcript variants of TrxR1 could be a valuable tool in the diagnostics and characterization of tumors.