Inducing ubiquitination and degradation of TrxR1 protein by LW-216 promotes apoptosis in non-small cell lung cancer via triggering ROS production.

Thioredoxin reductases are frequently overexpressed in various solid tumors as a protective mechanism against heightened oxidative stress. Inhibitors of this system, such as Auranofin, are effective in eradicating cancer cells. However, the clinical significance of thioredoxin reductase 1 (TrxR1) in lung cancer, as well as the potential for its antagonist as a treatment option, necessitated further experimental validation. In this study, we observed significant upregulation of TrxR1 specifically in non-small cell lung cancer (NSCLC), rather than small cell lung cancer. Moreover, TrxR1 expression exhibited associations with survival rate, tumor volume, and histological classification. We developed a novel TrxR1 inhibitor named LW-216 and assessed its antitumor efficacy in NSCLC. Our results revealed that LW-216 is effectively bound with intracellular TrxR1 at sites R371 and G442, facilitating TrxR1 ubiquitination and suppressing TrxR1 expression, while not affecting TrxR2 expression. Treatment of LW-216-induced DNA damage and cell apoptosis in NSCLC cells through the generation of reactive oxygen species (ROS). Importantly, supplementation with N-acetylcysteine (NAC) or ectopic TrxR1 expression reversed LW-216-induced apoptosis. Furthermore, LW-216 displayed potent tumor growth inhibition in NSCLC cell-implanted mice, reducing TrxR1 expression in xenografts. Remarkably, LW-216 exhibited superior antitumor activity compared to Auranofin in vivo. Collectively, our research provides compelling evidence supporting the potential of targeting TrxR1 by LW-216 as a promising therapeutic strategy for NSCLC.

Therapeutic targeting of thioredoxin reductase 1 causes ferroptosis while potentiating anti-PD-1 efficacy in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) faces low response rates to anti-PD-1 immunotherapies, highlighting the need for enhanced treatment strategies. Auranofin, which inhibits thioredoxin reductase (TrxR) through its gold-based composition, has shown potential in cancer treatment. It targets the TrxR system, essential for safeguarding cells from oxidative stress. The overproduction of TrxR in cancerous cells supports their proliferation. However, auranofin's interference with this system can upset the cellular redox equilibrium, boost levels of reactive oxygen species, and trigger the death of cancer cells. This study is the first to highlight TXNRD1 as a crucial factor contributing to resistance to anti-PD-1 treatment in HNSCC. In this study, we identified targetable regulators of resistance to immunotherapy-induced ferroptosis in HNSCC. We observed a link of thioredoxin reductase 1 (TXNRD1) with tumoral PD-L1 expression and ferroptosis suppression in HNSCC. Moreover, HNSCC tumors with aberrant TXNRD1 expression exhibited a lack of PD-1 response, NRF2 overexpression, and PD-L1 upregulation. TXNRD1 inhibition promoted ferroptosis in HNSCC cells with NRF2 activation and in organoid tumors derived from patients lacking a PD-1 response. Mechanistically, TXNRD1 regulated PD-L1 transcription and maintained the redox balance by binding to ribonucleotide reductase regulatory subunit M2 (RRM2). TXNRD1 expression disruption sensitized HNSCC cells to anti-PD-1-mediated Jurkat T-cell activation, promoting tumor killing through ferroptosis. Moreover, TXNRD1 inhibition through auranofin cotreatment synergized with anti-PD-1 therapy to potentiate immunotherapy-mediated ferroptosis by mediating CD8+ T-cell infiltration and downregulating PD-L1 expression. Our findings indicate that targeting TXNRD1 is a promising therapeutic strategy for improving immunotherapy outcomes in patients with HNSCC.

Synergistic targeting of TrxR1 and ATM/AKT pathway in human colon cancer cells.

Thioredoxin reductase 1 (TrxR1) has emerged as a promising target for cancer therapy. In our previous research, we discovered several new TrxR1 inhibitors and found that they all have excellent anti-tumor activity. At the same time, we found these TrxR1 inhibitors all lead to an increase in AKT phosphorylation in cancer cells, but the detailed role of AKT phosphorylation in TrxR1 inhibitor-mediated cell death remains unclear. In this study, we identified the combination of AKT and TrxR1 inhibitor displayed a strong synergistic effect in colon cancer cells. Furthermore, we demonstrated that the synergistic effect of auranofin (TrxR1 inhibitor) and MK-2206 (AKT inhibitor) was caused by ROS accumulation. Importantly, we found that ATM inhibitor KU-55933 can block the increase of AKT phosphorylation caused by auranofin, and exhibited a synergistic effect with auranofin. Taken together, our study demonstrated that the activation of ATM/AKT pathway is a compensatory mechanism to cope with ROS accumulation induced by TrxR1 inhibitor, and synergistic targeting of TrxR1 and ATM/AKT pathway is a promising strategy for treating colon cancer.

Jolkinolide B synergistically potentiates the antitumor activity of GPX4 inhibitors via inhibiting TrxR1 in cisplatin-resistant bladder cancer cells.

Glutathione peroxidase 4 (GPX4) is a promising anticancer therapeutic target; however, the application of GPX4 inhibitors (GPX4i) is limited owing to intrinsic or acquired drug resistance. Hence, understanding the mechanisms underlying drug resistance and discovering molecules that can overcome drug resistance are crucial. Herein, we demonstrated that GPX4i killed bladder cancer cells by inducing lipid reactive oxygen species-mediated ferroptosis and apoptosis, and cisplatin-resistant bladder cancer cells were also resistant to GPX4i, representing a higher half-maximal inhibitory concentration value than that of parent bladder cancer cells. In addition, thioredoxin reductase 1 (TrxR1) overexpression was responsible for GPX4i resistance in cisplatin-resistant bladder cancer cells, and inhibiting TrxR1 restored the sensitivity of these cells to GPX4i. In vitro and in vivo studies revealed that Jolkinolide B (JB), a natural diterpenoid and previously identified as a TrxR1 inhibitor, potentiated the antiproliferative efficacy of GPX4i (RSL3 and ML162) against cisplatin-resistant bladder cancer cells. Furthermore, GPX4 knockdown and inhibition could augment JB-induced paraptosis and apoptosis. Our results suggest that inhibiting TrxR1 can effectively improve GPX4 inhibition-based anticancer therapy. A combination of JB and GPX4i, which is well-tolerated and has several anticancer mechanisms, may serve as a promising therapy for treating bladder cancer.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. METHODS: Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. RESULTS: Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. CONCLUSION: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

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.

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.

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.

Antiproliferative, antimigratory, and apoptotic effects of diffractaic and vulpinic acids as thioredoxin reductase 1 inhibitors on cervical cancer.

Cervical cancer is among the most frequently observed cancer types in females. New therapeutic targets are needed because of the side impacts of existing cancer drugs and the inadequacy of treatment methods. Thioredoxin reductase 1 (TrxR1) is often overexpressed in many cancer cells, and targeting TrxR1 has become an attractive target for cancer therapy. This study investigated the anticancer impacts of diffractaic and vulpinic acids, lichen secondary metabolites, on the cervical cancer HeLa cell line. XTT findings demonstrated showed that diffractaic and vulpinic acids suppressed the proliferation of HeLa cells in a dose- and time-dependent manner and IC50 values were 22.52 µg/ml and 66.53 µg/ml at 48 h, respectively. Each of these lichen metabolites significantly suppressed migration. Diffractaic acid showed an increase in both the BAX/BCL2 ratio by qPCR analysis and the apoptotic cell population via flow cytometry analysis on HeLa cells. Concerning vulpinic acid, although it decreased the BAX/BCL2 ratio in this cells, it increased apoptotic cells according to the flow cytometry analysis results. Diffractaic and vulpinic acids significantly suppressed TrxR1 enzyme activity rather than the gene and protein expression levels in HeLa cells. This research demonstrated for the first time, that targeting TrxR1 with diffractaic and vulpinic acids was an effective therapeutic strategy for treating cervical cancer.

Elovanoid-N34 modulates TXNRD1 key in protection against oxidative stress-related diseases.

The thioredoxin (TXN) system is an NADPH + H+/FAD redox-triggered effector that sustains homeostasis, bioenergetics, detoxifying drug networks, and cell survival in oxidative stress-related diseases. Elovanoid (ELV)-N34 is an endogenously formed lipid mediator in neural cells from omega-3 fatty acid precursors that modulate neuroinflammation and senescence gene programming when reduction-oxidation (redox) homeostasis is disrupted, enhancing cell survival. Limited proteolysis (LiP) screening of human retinal pigment epithelial (RPE) cells identified TXNRD1 isoforms 2, 3, or 5, the reductase of the TXN system, as an intracellular target of ELV-N34. TXNRD1 silencing confirmed that the ELV-N34 target was isoform 2 or 3. This lipid mediator induces TXNRD1 structure changes that modify the FAD interface domain, leading to its activity modulation. The addition of ELV-N34 decreased membrane and cytosolic TXNRD1 activity, suggesting localizations for the targeted reductase. These results show for the first time that the lipid mediator ELV-N34 directly modulates TXNRD1 activity, underling its protection in several pathologies when uncompensated oxidative stress (UOS) evolves.

Protective effect of tretinoin derivative and TXNRD1 protein on streptozotocin induced gestational diabetes via an age-rage signaling-pathway.

BACKGROUND: In the present study effect of tretinoin derivative was investigated on the pathogenesis of gestational diabetes mellitus (GDM) in mice model in vivo. MATERIALS AND METHODS: Diabetes was induced in mice by injecting Streptozotocin (STZ) for 5consecutive days at a dose of 65 mg/kg body weight through the intraperitoneal route. Tretinoin derivative was given to the mice at 0.12 and 0.25 mg/kg doses through gavage in normal saline alternately for one week after STZ injection. RESULTS: The results demonstrated that tretinoin derivative administration to the diabetic mice significantly (P<0.05) alleviated the blood FBG and FINS levels. Administration of tretinoin derivative to the diabetic mice significantly (P<0.05) promoted the blood HDL level and alleviated TC and TG levels. The administration of tretinoin derivative to the diabetic mice significantly (P<0.05) alleviated the CRP, IL-6and TNF-α production in pancreatic tissues. Tretinoin derivative administration to the diabetic mice significantly (P<0.05) elevated the SOD activity, and CAT level and lowered the MDA level in pancreatic tissues. The TXNRD1 expression in diabetic mice was comparable to that in the normal group after administration of tretinoin derivativeat the dose of 0.25 mg/kg dose. In silico data demonstrated that tretinoin derivativeinteracts with TXNRD1 protein with the binding affinity ranging from -10 to 9.4 kcal/ mol. CONCLUSION: In conclusion, tretinoin derivative administration effectively regulated streptozotocin-induced changes in fasting blood glucose, insulin level, high-density lipid level and triglyceride level in diabetic mice in vivo. The streptozotocin-induced excessive production of C-reactive protein and inflammatory cytokines was also down-regulated in diabetic mice on administration of tretinoin derivative. Therefore, tretinoin derivative can be investigated further as a therapeutic agent for the treatment of gestational diabetes mellitus.

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.

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.

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.

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.

A wide scan of plasma proteins demonstrates thioredoxin reductase 1 as a potential new diagnostic biomarker for hepatocellular carcinoma.

BACKGROUND: Patients with liver cirrhosis are recommended ultrasonography screening for early detection of hepatocellular carcinoma to increase the chances of curative treatment. However, ultrasonography alone lacks in sensitivity. Adding plasma biomarkers may increase the detection rate. We performed a broad exploratory analysis to find new plasma proteins with potential applicability for HCC screening in patients with cirrhosis. METHODS: In a protein discovery cohort of 172 patients with cirrhosis or HCC, we screened for 481 proteins with suspension bead array or proximity extension assay. From these, 24 proteins were selected for further analysis in a protein verification cohort (n = 160), using ELISA, Luminex or an electrochemiluminescence platform. A cut-off model and a stepwise logistic regression model were used to find combinations of proteins with the best discriminatory performance between HCC and cirrhosis. RESULTS: Stepwise logistic regression revealed alpha-fetoprotein (AFP), decarboxy-prothrombin (DCP), thioredoxin reductase 1 (TXNRD1), and fibroblast growth factor 21 (FGF21) as the proteins with the best discriminatory performance between HCC and cirrhosis. Adding TXNRD1 to DCP and AFP increased the AUC from 0.844 to 0.878, and combining AFP, DCP and TXNRD1 with age and sex resulted in an AUC of 0.920. FGF21, however, did not further increase the performance when including age and sex. CONCLUSION: In the present study, TXNRD1 improves the sensitivity and specificity of AFP and DCP as HCC screening tools in patients with cirrhosis. We suggest that TXNRD1 should be validated in prospective settings as a new complementary HCC biomarker together with AFP and DCP.

Nitrovin (difurazone), an antibacterial growth promoter, induces ROS-mediated paraptosis-like cell death by targeting thioredoxin reductase 1 (TrxR1).

Glioblastoma multiforme (GBM) is one of the most lethal malignant tumors in the human brain, with only a few chemotherapeutic drugs available after surgery. Nitrovin (difurazone) is widely used as an antibacterial growth promoter in livestock. Here, we reported that nitrovin might be a potential anticancer lead. Nitrovin showed significant cytotoxicity to a panel of cancer cell lines. Nitrovin induced cytoplasmic vacuolation, reactive oxygen species (ROS) generation, MAPK activation, and Alix inhibition but had no effect on caspase-3 cleavage and activity, suggesting paraptosis activation. Nitrovin-induced cell death of GBM cells was significantly reversed by cycloheximide (CHX), N-acetyl-l-cysteine (NAC), glutathione (GSH), and thioredoxin reductase 1 (TrxR1) overexpression. Vitamins C and E, inhibitors of pan-caspase, MAPKs, and endoplasmic reticulum (ER) stress failed to do so. Nitrovin-triggered cytoplasmic vacuolation was reversed by CHX, NAC, GSH, and TrxR1 overexpression but not by Alix overexpression. Furthermore, nitrovin interacted with TrxR1 and significantly inhibited its activity. In addition, nitrovin showed a significant anticancer effect in a zebrafish xenograft model, which was reversed by NAC. In conclusion, our results showed that nitrovin induced non-apoptotic and paraptosis-like cell death mediated by ROS through targeting TrxR1. Nitrovin might be a promising anticancer lead for further development.