Combination of TrxR1 inhibitor and lenvatinib triggers ROS-dependent cell death in human lung cancer cells.

Lung cancer is one of the most lethal diseases in the world. Although there has been significant progress in the treatment of lung cancer, there is still a lack of effective strategies for advanced cases. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, has achieved much attention due to its antitumor properties. Nevertheless, the use of lenvatinib is restricted by the characteristics of poor efficacy and drug resistance. In this study, we assessed the effectiveness of lenvatinib combined with thioredoxin reductase 1 (TrxR1) inhibitors in human lung cancer cells. Our results indicate that the combination therapy involving TrxR1 inhibitors and lenvatinib exhibited significant synergistic antitumor effects in human lung cancer cells. Moreover, siTrxR1 also showed significant synergy with lenvatinib in lung cancer cells. Mechanically, we demonstrated that ROS accumulation significantly contributes to the synergism between lenvatinib and TrxR1 inhibitor auranofin. Furthermore, the combination of lenvatinib and auranofin can activate endoplasmic reticulum stress and JNK signaling pathways to achieve the goal of killing lung cancer cells. Importantly, combination therapy with lenvatinib and auranofin exerted a synergistic antitumor effect in vivo. To sum up, the combination therapy involving lenvatinib and auranofin may be a potential strategy for treating lung cancer.

TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation.

Sterile inflammation, also known as 'inflammaging', is a hallmark of tissue aging. Cellular senescence contributes to tissue aging, in part, through the secretion of proinflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). The genetic variability of thioredoxin reductase 1 (TXNRD1) is associated with aging and age-associated phenotypes such as late-life survival, activity of daily living and physical performance in old age. TXNRD1's role in regulating tissue aging has been attributed to its enzymatic role in cellular redox regulation. Here, we show that TXNRD1 drives the SASP and inflammaging through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway independently of its enzymatic activity. TXNRD1 localizes to cytoplasmic chromatin fragments and interacts with cGAS in a senescence-status-dependent manner, which is necessary for the SASP. TXNRD1 enhances the enzymatic activity of cGAS. TXNRD1 is required for both the tumor-promoting and immune surveillance functions of senescent cells, which are mediated by the SASP in vivo in mouse models. Treatment of aged mice with a TXNRD1 inhibitor that disrupts its interaction with cGAS, but not with an inhibitor of its enzymatic activity alone, downregulated markers of inflammaging in several tissues. In summary, our results show that TXNRD1 promotes the SASP through the innate immune response, with implications for inflammaging. This suggests that the TXNRD1-cGAS interaction is a relevant target for selectively suppressing inflammaging.

Guiding bar motif of thioredoxin reductase 1 modulates enzymatic activity and inhibitor binding by communicating with the co-factor FAD and regulating the flexible C-terminal redox motif.

Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in TXNRD1, which influences the transfer of electrons. In this study, utilizing single amino acid substitution and Excitation-Emission Matrix (EEM) fluorescence spectrum analysis, we discovered that the guiding bar communicates with the FAD and modulates the electron flow of the enzyme. Differential Scanning Fluorimetry (DSF) analysis demonstrated that the aromatic amino acid in guiding bar is a stabilizer for TXNRD1. Kinetic analysis revealed that the guiding bar is vital for the disulfide reductase activity but hinders the selenocysteine-independent reduction activity of TXNRD1. Meanwhile, the guiding bar shields the selenocysteine residue of TXNRD1 from the attack of electrophilic reagents. We also found that the inhibition of TXNRD1 by caveolin-1 scaffolding domain (CSD) peptides and compound LCS3 did not bind to the guiding bar motif. In summary, the obtained results highlight new aspects of the guiding bar that restrict the flexibility of the C-terminal redox motif and govern the transition from antioxidant to pro-oxidant.

Deferoxamine Mitigates Ferroptosis and Inflammation in Hippocampal Neurons After Subarachnoid Hemorrhage by Activating the Nrf2/TXNRD1 Axis.

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood-brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis.

RASSF1A promotes radiosensitivity in nasopharyngeal carcinoma by promoting FoxO3a and inhibiting the Nrf2/TXNRD1 signaling pathway.

Radiotherapy is widely used as the first-line treatment for nasopharyngeal carcinoma (NPC). However, the resistance of some patients to treatment lowers its clinical effectiveness. Compared to typical epithelial cells, NPC markedly lowers the Ras-association domain family 1A (RASSF1A) protein expression. RASSF1A overexpression sensitizes NPC cells to radiotherapy. Mechanistically, RASSF1A promotes the expression of Forkhead box O3a (FoxO3a) in the nucleus and inhibits the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway via binding to the Kelch-like ECH-associated protein 1 (Keap1) promoter. Through elevating intracellular ROS levels, RASSF1A overexpression inhibits the expression of thioredoxin reductase 1 (TXNRD1), a crucial Nrf2 target gene, and increases NPC sensitivity to radiation. Immunohistochemical staining of NPC tissue sections revealed that the expression of RASSF1A is negatively correlated with that of TXNRD1. The traditional Chinese medicine component andrographolide (AGP), which induces RASSF1A expression, increased the sensitivity of NPC cells to radiotherapy in vitro and in vivo. Our findings implied that RASSF1A increases the sensitivity of NPC to radiation by increasing FoxO3a expression in the nucleus, inhibiting the Nrf2/TXNRD1 signaling pathway, and elevating intracellular ROS levels. AGP targets RASSF1A and may be a promising adjuvant sensitizer for enhancing radiosensitivity in NPC.

Keratinocytes Exposed to Blue or Red Light: Proteomic Characterization Showed Cytoplasmic Thioredoxin Reductase 1 and Aldo-Keto Reductase Family 1 Member C3 Triggered Expression.

Several cell-signaling mechanisms are activated by visible light radiation in human keratinocytes, but the key regulatory proteins involved in this specific cellular response have not yet been identified. Human keratinocytes (HaCaT cells) were exposed to blue or red light at low or high irradiance for 3 days in cycles of 12 h of light and 12 h of dark. The cell viability, apoptotic rate and cell cycle progression were analyzed in all experimental conditions. The proteomic profile, oxidative stress and mitochondrial morphology were additionally evaluated in the HaCaT cells following exposure to high-irradiance blue or red light. Low-irradiance blue or red light exposure did not show an alteration in the cell viability, cell death or cell cycle progression. High-irradiance blue or red light reduced the cell viability, induced cell death and cell cycle G2/M arrest, increased the reactive oxygen species (ROS) and altered the mitochondrial density and morphology. The proteomic profile revealed a pivotal role of Cytoplasmic thioredoxin reductase 1 (TXNRD1) and Aldo-keto reductase family 1 member C3 (AKR1C3) in the response of the HaCaT cells to high-irradiance blue or red light exposure. Blue or red light exposure affected the viability of keratinocytes, activating a specific oxidative stress response and inducing mitochondrial dysfunction. Our results can help to address the targets for the therapeutic use of light and to develop adequate preventive strategies for skin damage. This in vitro study supports further in vivo investigations of the biological effects of light on human keratinocytes.

Exploration of 3,4-unsubstituted coumarins as thioredoxin reductase 1 inhibitors for cancer therapy.

Coumarin and its derivatives have emerged as promising candidates in drug discovery. While the activity of coumarins as anticancer agents with different biological targets has been thoroughly investigated, reports on the potential of coumarins in the inhibition of thioredoxin reductase (TrxR) are still scarce. We focus on the design and synthesis of 3,4-unsubstituted coumarin analogues with systematic incorporation of substituents at the fifth to eighth positions of coumarin, which allowed definitive structure-activity relationship analysis to be conducted. In the obtained library, the substitution at the sixth position of the coumarin core with an aromatic or a cyclopropyl group turned out to be more activity enhancing. A bulky aromatic substituent with a large CF3 group encourages ligand alignment in a manner that enables covalent bond formation with the catalytic TrxR1 residue, according to the docking results. Our observations indicate that the activity of a series of coumarin analogues towards thioredoxin reductase 1 (TrxR1) is dependent on the nature (size and electronic effect) and the position of the substituent and more importantly - the accessibility of the Michael acceptor functionality. Several compounds (with at least 90% inhibition of the rat TrxR1 enzyme at 200 µM concentration) were further examined in in vitro cell-based assays to assess the cytotoxic effects on various cancer cell lines. The analogue 6-(4-(trifluoromethyl)phenyl)-2H-chromen-2-one was selected as the lead compound for further optimization. The results presented herein pave the way for the development of the next generation of coumarin-based TrxR1 inhibitors, where modification of the Michael acceptor moiety and incorporation of different aryl substituents at the sixth position of the coumarin core are planned.

NFATc1-mediated expression of SLC7A11 drives sensitivity to TXNRD1 inhibitors in osteoclast precursors.

Excess osteoclast activity is found in many bone metabolic diseases, and inhibiting osteoclast differentiation has proven to be an effective strategy. Here, we revealed that osteoclast precursors (pre-OCs) were more susceptible to thioredoxin reductase 1 (TXNRD1) inhibitors than bone marrow-derived monocytes (BMDMs) during receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis. Mechanistically, we found that nuclear factor of activated T-cells 1 (NFATc1) upregulated solute carrier family 7 member 11 (SLC7A11) expression through transcriptional regulation during RANKL-induced osteoclastogenesis. During TXNRD1 inhibition, the rate of intracellular disulfide reduction is significantly reduced. Increased cystine transport leads to increased cystine accumulation, which leads to increased cellular disulfide stress and disulfidptosis. We further demonstrated that SLC7A11 inhibitors and treatments that prevent disulphide accumulation could rescue this type of cell death, but not the ferroptosis inhibitors (DFO, Ferro-1), the ROS scavengers (Trolox, Tempol), the apoptosis inhibitor (Z-VAD), the necroptosis inhibitor (Nec-1), or the autophagy inhibitor (CQ). An in vivo study indicated that TXNRD1 inhibitors increased bone cystine content, reduced the number of osteoclasts, and alleviated bone loss in an ovariectomized (OVX) mouse model. Together, our findings demonstrate that NFATc1-mediated upregulation of SLC7A11 induces targetable metabolic sensitivity to TXNRD1 inhibitors during osteoclast differentiation. Moreover, we innovatively suggest that TXNRD1 inhibitors, a classic drug for osteoclast-related diseases, selectively kill pre-OCs by inducing intracellular cystine accumulation and subsequent disulfidptosis.

TXNRD1 knockdown inhibits the proliferation of endothelial cells subjected to oscillatory shear stress via activation of the endothelial nitric oxide synthase/apoptosis pathway.

Atherosclerosis is the main cause of cardiovascular disease, and fluid shear stress is a key factor regulating its occurrence and development. Oscillatory shear stress (Oss) is an important pro-atherosclerosis factor. Oss mainly occurs in areas that are susceptible to atherosclerosis, but the exact mechanism of atherosclerosis induction remains unclear. Therefore, starting from the atheroprone phenotype that Oss stimulates abnormal vascular endothelial cell proliferation, this study aimed to reveal the underlying mechanism of Oss-induced atherosclerosis formation and to identify new targets for the prevention and treatment of atherosclerosis. In this study, the gene encoding thioredoxin reductase 1 (TXNRD1), which is closely related to atherosclerosis development and cell proliferation, was screened by analyzing the transcriptome sequencing data of static and Oss-treated human aortic endothelial cells (HAECs). Moreover, this study successfully verified that TXNRD1 mRNA and protein were significantly upregulated in Oss-treated HAECs. Oss significantly promoted the proliferation, migration, and tube formation of HAECs, whereas TXNRD1 knockdown impaired the proliferation, migration, and tube formation of Oss-treated HAECs, and this process was mainly achieved via activation of the apoptosis pathway. To further clarify whether Oss-sensitive TXNRD1 affects the apoptosis rate and proliferative ability of HAECs by regulating the endothelial nitric oxide synthase (eNOS) pathway, we used NG-nitro-L-arginine methyl ester (L-NAME) to inhibit eNOS activity and nitric oxide (NO) production. L-NAME significantly reversed the promoting effect of TXNRD1 knockdown on Oss-treated HAEC apoptosis, and it also abolished the inhibitory effect of TXNRD1 knockdown on the proliferation and S + G2 phase cell mass of Oss-treated HAECs. In conclusion, this study showed that TXNRD1 knockdown inhibited the proliferation of HAECs exposed to Oss by activating the eNOS/apoptosis pathway, revealing that TXNRD1 is involved in the dysregulation of Oss-induced endothelial cell proliferation. These findings provide new directions and insights into the prevention and treatment of atherosclerosis.

Thimerosal, a competitive thioredoxin reductase 1 (TrxR1) inhibitor discovered via high-throughput screening.

Thioredoxin reductase 1 (TrxR1) is considered as an important anti-cancer drug target, inhibition of which can induce reactive oxygen species (ROS)-mediated apoptosis of human cancer cells. Here, we developed and optimized a high-throughput screening (HTS) assay based on enzyme kinetics for the discovery of TrxR1 inhibitors. By utilizing this assay, we performed a HTS for 2500 compounds from an in-house library against TrxR1. We found that a vaccine preservative, thimerosal, strongly inhibited TrxR1 in a competitive and reversible manner with an IC50 of 24.08 ± 0.86 nM. In addition, we determined that thiomersal has an inhibitory effect on the proliferation of A549 lung cancer cell line, with a GI50 of 6.81 ± 0.09 µM, slightly more potent than auranofin (GI50 = 11.85 ± 0.56 µM). Furthermore, we showed by flow cytometer that thimerosal effectively increased the content of ROS in A549 cells. Therefore, our work provided a high-throughput screening assay to quickly and effectively discover TrxR1 inhibitors, identifying thiomersal as a novel TrxR1 inhibitor and chemical probe.

Selenite Ameliorates Cadmium-induced Cytotoxicity Through Downregulation of ROS Levels and Upregulation of Selenoprotein Thioredoxin Reductase 1 in SH-SY5Y Cells.

Cadmium (Cd) as a ubiquitous toxic heavy metal in the environment, causes severe hazards to human health, such as cellular stress and organ injury. Selenium (Se) was reported to reduce Cd toxicity and the mechanisms have been intensively studied so far. However, it is not yet crystal clear whether the protective effect of Se against Cd-induced cytotoxicity is related to selenoproteins in nerve cells or not. In this study, we found that Cd inhibited selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1) and decreased the expression level of TrxR1, resulting in cellular oxidative stress, and Se supplements ameliorated Cd-induced cytotoxicity in SH-SY5Y cells. Mechanistically, the detoxification of Se against Cd is attributed to the increase of the cellular TrxR activity and upregulated TrxR1 protein level, culminating in strengthened antioxidant capacity. Results showed that Se supplements attenuated the ROS production and apoptosis in SH-SY5Y cells, and significantly mitigated Cd-induced SH-SY5Y cell death. This study may be a valuable reference for shedding light on the mechanism of Cd-induced cytotoxicity and the role of TrxR1 in Se-mitigated cytotoxicity of Cd in neuroblast cells, which may be helpful for understanding the therapeutic potential of Cd and Se in treating or preventing neurodegenerative diseases, like Alzheimer's disease (AD) and Parkinson's disease (PD).

The molecular evaluation of thioredoxin (TXN1 & TXN2), thioredoxin reductase 1 (TXNRd1), and oxidative stress markers in a binary rat model of hypo- and hyperthyroidism after treatment with gallic acid.

Oxidative stress plays an important role in the pathology of thyroid disorders. This study examined the effect of gallic acid (GA) on the oxidative status and expression of liver antioxidant genes including thioredoxin (TXN1 & TXN2) and thioredoxin reductase1 (TXNRd1) in hypo- and hyperthyroid rat models. Forty-nine male Wistar rats were randomly assigned into seven groups as follows: control group, hypothyroid and hyperthyroid groups respectively induced by propylthiouracil and levothyroxine, hypo- and hyper thyroid-treated groups (where the groups were separately treated with 50 and 100 mg/kg of GA daily, orally). The levels of thyroid hormones and serum oxidative stress markers were evaluated after 5 weeks. The relative expression of TXN1,2 and TXNRd1 genes was measured via real-time qRT-PCR. The mean level of total antioxidant capacity (TAC), malondialdehyde, and uric acid index diminished in the hypothyroid group. Increased TAC reached almost the level of control in hypothyroid groups treated with GA. Elevation of thiol index in the hypothyroid group was observed (p < 0.01), which diminished to the control level after GA treatment. The relative expression of TXN1, TXNRd1, and TXN2 genes in the hypothyroid and hyperthyroid groups significantly increased compared to the control group (p ≥ 0.05), but in the groups treated with GA, the expression of these genes declined significantly (p ≥ 0.05). Our results indicated GA can affect the expression of TXN system genes in the rat liver. Also, the results suggest GA has a more positive effect on modulating serum oxidative parameters in hypothyroid rat models than in hyperthyroid.

Metabolism-Related Gene TXNRD1 Regulates Inflammation and Oxidative Stress Induced by Cigarette Smoke through the Nrf2/HO-1 Pathway in the Small Airway Epithelium.

Chronic obstructive pulmonary disease (COPD), a small airway disease, is regarded as a metabolic disorder. To further uncover the metabolic profile of COPD patients, it is necessary to identify metabolism-related differential genes in small airway epithelium (SAE) of COPD. Metabolism-related differential genes in SAE between COPD patients and nonsmokers were screened from GSE128708 and GSE20257 datasets. KEGG, GO, and PPI analyses were performed to evaluate the pathway enrichment, term enrichment, and protein interaction of candidate metabolism-related differential genes, respectively. RT-PCR was used to verify the mRNA expression of the top ten differential genes. Western blotting was used to evaluate the protein expression of TXNRD1. TXNRD1 inhibitor auranofin (AUR) was used to assess the impact of TXNRD1 on oxidative stress and inflammation induced by cigarette smoke extraction (CSE). Twenty-four metabolism-related differential genes were selected. ALDH3A1, AKR1C3, CYP1A1, AKC1C1, CPY1B1, and TXNRD1 in the top ten genes were significantly upregulated after CSE simulation for 24 h in human bronchial epithelial (16HBE) cells. Among them, CYP1A1 and TXNRD1 also have a significant upregulation in primary SAE after simulation of CSE for 24 h. The overexpression of protein TXNRD1 has also been detected in 16HBE cells, primary SAE stimulated with CSE, and mouse lung exposed to cigarette smoke (CS). Additionally, inhibition of TXNRD1 with 0.1 µM AUR alleviated the expression of IL-6 and reactive oxygen species (ROS) induced by CSE by activating the Nrf2/HO-1 pathway in 16HBE cells. This study identified twenty-four metabolism-related differential genes associated with COPD. TXNRD1 might participate in the oxidative stress and inflammation induced by CS by regulating the activation of the Nrf2/HO-1 pathway.

Menadione Potentiates Auranofin-Induced Glioblastoma Cell Death.

Glioblastoma (GBM) is the most aggressive primary brain tumor. Recently, agents increasing the level of oxidative stress have been proposed as anticancer drugs. However, their efficacy may be lowered by the cytoprotective activity of antioxidant enzymes, often upregulated in neoplastic cells. Here, we assessed the mRNA and protein expression of thioredoxin reductase 1 (TrxR1), a master regulator of cellular redox homeostasis, in GBM and non-tumor brain tissues. Next, we examined the influence of an inhibitor of TrxR1, auranofin (AF), alone or in combination with a prooxidant menadione (MEN), on growth of GBM cell lines, patient-derived GBM cells and normal human astrocytes. We detected considerable amount of TrxR1 in the majority of GBM tissues. Treatment with AF decreased viability of GBM cells and their potential to form colonies and neurospheres. Moreover, it increased the intracellular level of reactive oxygen species (ROS). Pre-treatment with ROS scavenger prevented the AF-induced cell death, pointing to the important role of ROS in the reduction of cell viability. The cytotoxic effect of AF was potentiated by treatment with MEN. In conclusion, our results identify TrxR1 as an attractive drug target and highlights AF as an off-patent drug candidate in GBM therapy.

LDHA-mediated metabolic reprogramming promoted cardiomyocyte proliferation by alleviating ROS and inducing M2 macrophage polarization.

AIMS: Metabolic switching during heart development contributes to postnatal cardiomyocyte (CM) cell cycle exit and loss of regenerative capacity in the mammalian heart. Metabolic control has potential for developing effective CM proliferation strategies. We sought to determine whether lactate dehydrogenase A (LDHA) regulated CM proliferation by inducing metabolic reprogramming. METHODS AND RESULTS: LDHA expression was high in P1 hearts and significantly decreased during postnatal heart development. CM-specific LDHA knockout mice were generated using CRISPR/Cas9 technology. CM-specific LDHA knockout inhibited CM proliferation, leading to worse cardiac function and a lower survival rate in the neonatal apical resection model. In contrast, CM-specific overexpression of LDHA promoted CM proliferation and cardiac repair post-MI. The α-MHC-H2B-mCh/CAG-eGFP-anillin system was used to confirm the proliferative effect triggered by LDHA on P7 CMs and adult hearts. Metabolomics, proteomics and Co-IP experiments indicated that LDHA-mediated succinyl coenzyme A reduction inhibited succinylation-dependent ubiquitination of thioredoxin reductase 1 (Txnrd1), which alleviated ROS and thereby promoted CM proliferation. In addition, flow cytometry and western blotting showed that LDHA-driven lactate production created a beneficial cardiac regenerative microenvironment by inducing M2 macrophage polarization. CONCLUSIONS: LDHA-mediated metabolic reprogramming promoted CM proliferation by alleviating ROS and inducing M2 macrophage polarization, indicating that LDHA might be an effective target for promoting cardiac repair post-MI.

Biochemical and molecular assessment of selenium forms for the alleviation of oxidative stress in senescent human fibroblasts.

Selenium enhances the cellular antioxidant capacity and alleviates oxidative stress. We investigated the transcriptional and enzymatic activities of selenium-dependent glutathione peroxidase 1 and thioredoxin reductase 1 (TrxR1), and levels of glutathione, hydrogen peroxide, lipid peroxides, and protein carbonyls in primary passage 5 (P5) and senescent passage 25 (P25) and 30 (P30) fibroblasts. Cells were incubated in either standard Dulbecco growth medium (CM1) containing normal plasma selenium levels (0.8 µmol/l), or in CM2, CM3, and CM4 containing 3 µmol/l (5 µmol/l for TrxR1) sodium selenite, L-hydroxyselenomethionine, or Se-methylselenocysteine, respectively. Gene transcripts and activities of both investigated enzymes as well as the levels of reduced glutathione were significantly increased in CM2-, CM3-, and CM4-incubated senescent P25 and P35 cells compared against those incubated in CM1. In congruence, although all oxidative stress parameters including oxidized glutathione were significantly lower in CM2-, CM3-, and CM4-incubated senescent cells compared against those incubated in CM1, such reductions were of significantly higher magnitude in CM3 and CM4 cells compared against those in CM2. In conclusion, organic L-hydroxyselenomethionine and Se-methylselenocysteine are equally more potent at alleviating oxidative stress in senescent cells than inorganic sodium selenite, and thus could be beneficial for use in elderly subjects and those with oxidative stress-associated disease.

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm.

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

Selenocysteine Machinery Primarily Supports TXNRD1 and GPX4 Functions and Together They Are Functionally Linked with SCD and PRDX6.

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD.

A thioredoxin reductase 1 inhibitor pyrano [3,2-a] phenazine inhibits A549 cells proliferation and migration through the induction of reactive oxygen species production.

BACKGROUND: Thioredoxin reductase 1 (TrxR1) inhibitor, pyrano [3,2-a] phenazine, named CPUL-1, was synthesized with potential anticancer activity. The aim of the present work was to explore the potential anti-proliferative and anti-metastatic ability of CPUL-1 against A549 cancer cell lines in vitro. METHODS AND RESULTS: First, Cell Counting Kit-8 (CCK8) assay was used to assess cell proliferation. The A549 cell migration was evaluated by wound healing assay and transwell assay. Second, the epithelial-mesenchymal transition (EMT)-related proteins in A549 cells treated with CPUL-1 were analyzed by western blot methods. Then, TrxR1 enzyme activity assay and reactive oxygen species (ROS) assay were conducted to evaluate the effect of CPUL-1 on TrxR1 inhibition and ROS levels. Finally, western blotting was used to explore the mechanism of CPUL-1. The study results revealed that the ability of cell proliferation and migration was decreased under CPUL-1 treatment. CPUL-1 could distinctly restrain the migration and invasion of A549 cells through inhibiting EMT process. The results of TrxR1 enzyme activity assay, ROS assay and western blotting showed that CPUL-1 influenced EMT via inducing ROS-mediated ERK/JNK signaling by inhibiting TrxR1 enzyme activity. CONCLUSIONS: Together, proliferation suppression and anti-metastasis activity of CPUL-1 in A549 cells were demonstrated by all the evidence. Our findings highlight the great potential of phenazine compound CPUL-1 to suppress A549 cells proliferation and metastasis.

Exosomal circRNA FNDC3B promotes the progression of esophageal squamous cell carcinoma by sponging miR-490-5p and regulating thioredoxin reductase 1 expression.

Exosomal circular RNAs (circRNAs) have been reported to play critical roles in esophageal squamous cell carcinoma (ESCC). We aimed to investigate the function of exosomal circRNA FNDC3B (circFNDC3B). The RNA levels and protein levels were examined using RT-qPCR and western blot (WB) assays. Colony formation and EdU assays were used to assess cell proliferative ability. Cell migratory and invasive abilities were detected by wound healing and transwell assays. Cell apoptosis was measured by flow cytometry. Glycolysis was measured using commercial kits. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were applied to examine the morphology and size of exosomes. Dual-luciferase reporter, RIP and RNA pull-down assays assessed the interaction of miR-490-5p with circFNDC3B or thioredoxin reductase 1 (TXNRD1). Xenograft tumor model determined the role of exosomal circFNDC3B in vivo. We observed that circFNDC3B was upregulated in ESCC samples and cells, as well as ESCC-derived exosomes. CircFNDC3B could be delivered via exosomes in tumor cells, and the colony formation, proliferation, migration, invasion, glycolysis, and in vivo growth ability of recipient cells were weakened after co-incubation with exosomal circFNDC3B-knockdown donor cells. CircFNDC3B was a miR-490-5p sponge, and miR-490-5p inhibition reversed the role of exosomal circFNDC3B-downregulating in ESCC cells. TXNRD1 was a miR-490-5p target, and TXNRD1 elevation weakened the anti-cancer function of miR-490-5p upregulation in ESCC cells. CircFNDC3B mediated TXNRD1 expression by interacting with miR-490-5p. In conclusion, exosomal circFNDC3B drove ESCC progression via regulating the miR-490-5p/TXNRD1 axis.AbbreviationsEC: esophageal cancer; ESCC: esophageal squamous cell carcinoma; circRNA: circular RNA; WB: western blot; TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis; TXNRD1: thioredoxin reductase 1; IHC: immunohistochemistry; RT-qPCR: reverse transcription-polymerase quantitative chain reaction; GLUT1: glucose transport protein type 1; LDHA: lactate dehydrogenase A.