Targeting Thioredoxin Reductase Research Articles

Targeting thioredoxin reductase by eupalinilide B promotes apoptosis of colorectal cancer cells in vitro and in vivo

Aberrant activation of thioredoxin reductase (TrxR) is correlated with tumor occurrence and progression, suggesting that TrxR inhibitors can be used as antitumor agents. In this study, we evaluated the anticancer efficacy of eupalinilides B on colorectal cancer cells. Eupalinilides B primarily targeted the conserved selenocysteine 498 residues in TrxR. Besides, it inhibited the enzyme activity in an irreversible manner. After eupalinilides B was used to pharmacologically inhibit TrxR, reactive oxygen species accumulated, and the intracellular redox balance was broken, finally causing oxidative stress-induced tumor cell apoptosis. Significantly, eupalinilides B treatment inhibited in vivo tumor growth. Targeting TrxR by eupalinilides B reveals the new mechanism underlying eupalinilides B and provides insight in developing eupalinilides B as the candidate antitumor chemotherapeutic agent for the treatment of cancer.

Abstract 2910: Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma progression

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. Poor outcomes for traditional treatments demand targeted therapies based on identified mechanisms that drive tumor development and sustain its malignancy. Molecular pathology studies have classified GBM into subtypes differing in treatment responses and survival rates. Among these subtypes, the mesenchymal (MES) group is associated with the worst prognosis. The Hippo pathway transcriptional co-activator with PDZ-binding motif (TAZ) is one of the three transcriptional regulators that drive the GBM MES gene expression program. Studies from our lab and others have previously shown that TAZ activation is able to promote GBM MES differentiation and progression. Therefore, finding vulnerabilities of TAZ-driven tumors may help to develop targeted therapeutic methods for MES GBM. In an orthotopic GBM mouse model, we found that TAZ-driven tumors show increased expression of thioredoxin reductase 1 (TrxR1), which is responsible for catalyzing the reduction of thioredoxin and maintaining the cellular redox balance. In searching for the cause of increased expression of TrxR1, we found that glucose deprivation can induce TrxR1 expression, suggesting TrxR1 may be an adaptive mechanism for tumor cells to survive in glucose deprivation conditions. Indeed, either depleting TrxR1 genetically or inhibiting TrxR1 pharmacologically can promote tumor cell death under glucose deprivation. Furthermore, knockout TrxR1 in a TAZ-driven mouse GBM model significantly decelerated tumor progression. Collectively, our studies revealed that inhibition of the Trx redox system under glucose deprivation conditions leads to a synergistic cell death induction in GBM cells, therefore making targeting TrxR1 a potential therapeutic strategy for TAZ-driven MES GBM. Citation Format: Miaolu Tang, Kaitlyn Dirks, Soo Yeon Kim, Jessica Thorpe, Wei Li. Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2910.

Diffractaic acid exerts anti-cancer effects on hepatocellular carcinoma HepG2 cells by inducing apoptosis and suppressing migration through targeting thioredoxin reductase 1

Hepatocellular carcinoma (HCC) represents one of the most common malignant tumors worldwide. Due to the limited number of available drugs and their side effects, the development of new chemotherapeutic strategies for HCC treatment has become increasingly important. This study is aimed at investigating whether diffractaic acid (DA), one of the secondary metabolites of lichen, exhibits a potential anticancer effect on HepG2 cells and whether its anticancer effect is mediated by inhibition of thioredoxin reductase 1 (TRXR1), which is a target of chemotherapeutic strategies due to overexpression in tumor cells including HCC. XTT assay results showed that DA exhibited strong cytotoxicity on HepG2 cells with an IC50 value of 78.07 µg/mL at 48 h. Flow cytometric analysis results revealed that DA displayed late apoptotic and necrotic effects on HepG2 cells. Consistent with these findings, real-time PCR results showed that DA did not alter the BAX/BCL2 ratio in HepG2 cells but upregulated the P53 gene. Moreover, the wound healing assay results revealed a strong anti-migratory effect of DA in HepG2 cells. Real-time PCR and Western blot analyses demonstrated that DA increased TRXR1 gene and protein expression levels, whereas enzyme activity studies disclosed that DA inhibited TRXR1. These findings suggest that DA has an anticancer effect on HepG2 cells by targeting the enzymatic inhibition of TRXR1. In conclusion, DA as a TRXR1 inhibitor can be considered an effective chemotherapeutic agent which may be a useful lead compound for the treatment of HCC.

Benzophenanthridine Alkaloid Chelerythrine Elicits Necroptosis of Gastric Cancer Cells via Selective Conjugation at the Redox Hyperreactive C-Terminal Sec498 Residue of Cytosolic Selenoprotein Thioredoxin Reductase.

Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec498 residue of the enzyme, but the neighboring Cys497 and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec498. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of N-acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec498 and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis.

Gold complex compounds that inhibit drug-resistant Staphylococcus aureus by targeting thioredoxin reductase

IntroductionThere is a significant need for new antimicrobial compounds that are effective against drug-resistant microbes. Thioredoxin reductase (TrxR) is critical in redox homeostasis and was identified as a potential drug target and confirmed through inhibition by compounds auranofin and Bay11-7085.MethodsAdditional TrxR inhibitors were designed and found to exhibit antimicrobial activity against Gram-positive (Enterococcus faecium and Staphylococcus aureus) and glutathione-deficient bacteria (Helicobacter pylori). Investigational compounds were tested for antimicrobial activity, anti-biofilm efficacy, target impact, and cytotoxicity.ResultsThe first-generation molecules AU1 and AU5 inhibited TrxR activity and inhibited methicillin-resistant S. aureus strain MW2 with minimal inhibitory concentrations (MIC) of 0.125 and 0.5 μg/mL, respectively. In an S. aureus enzymatic assay, AU1 inhibited TrxR enzymatic activity in a dose-dependent manner causing a decrease in intracellular free thiols. In addition, biofilm studies demonstrated that AU1 and AU5 reduced biofilm formation at 1X MIC and disrupted mature biofilms at 4X MIC. Cytotoxicity profiles were created using human cell lines and primary cells with LD50 exceeding MICs by at least 12X.DiscussionThus, AU1 and AU5 were TrxR inhibitors that yielded low-concentration antimicrobial activity impacting S. aureus in planktonic and biofilm forms with limited toxic liability.

Structure-based drug design of multi-targeting inhibitors of human pathogen fungi

The development of multi-targeting inhibitors is proposed to improve the antifungal effect. In a single chemical entity it is possible to inhibit two different targets. Thioredoxin reductase and glutathione reductase belong to the two main systems responsible for the maintenance of cell homeostasis. Herein, we describe the discovery and selection of dual-inhibitors targeting thioredoxin reductase and glutathione reductase proteins of Aspergillus fumigatus. Both proteins were modeled and molecular dynamics were performed to obtain different conformational frames to ensemble docking. After the independent virtual screening of each protein, a ranking containing molecules selected for both proteins were generated. Fourteen molecules were tested in vitro against A. fumigatus, Fusarium oxysporum, Paracoccidioides brasiliensis, Candida albicans and Cryptococcus neoformans by broth microdilution assay. Octenidine (OCT) was the most promising molecule which exhibited MIC < 0.5 µg/mL for all fungi tested. The literature describes the action of OCT in the cell membrane of gram-negative bacteria. Thus, we assume that the mechanism in which OCT acts on fungal cells is an association of disrupting the cell membrane and also the dual-inhibition of thioredoxin reductase and glutathione reductase which implies the increase of ROS and further fungal cell death.

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.

Selenium speciation determines the angiogenesis effect through regulating selenoproteins to trigger ROS-mediated cell apoptosis and cell cycle arrest

Tumor angiogenesis is closely related to tumor development, immune escape, and drug resistance. Therefore, the development of effective anti-tumor angiogenesis drugs is of great research significance. Although the current clinical angiogenesis inhibitors have achieved certain efficacy, they also pose the problems of limited and short duration of efficacy, drug resistance, and intrinsic toxicity. Anti-tumor angiogenesis strategies targeting endothelial cells (ECs) have attracted widespread attention in the development of highly effective and low toxicity anti-angiogenesis inhibitors. Studies have verified that the trace element selenium (Se) can inhibit tumor growth by inhibiting tumor angiogenesis through different mechanisms. Nevertheless, it is unclear whether Se speciation has different effects on anti-tumor angiogenesis. Herein, we found that Se exhibited effective anti-angiogenic activity, and its mechanisms of activity were determined by its chemical speciation. Organic Se can significantly inhibit tumor angiogenesis by targeting thioredoxin reductase (TrxR) to trigger cell apoptosis and cell cycle arrest and by increasing reactive oxygen species (ROS) production in ECs. Inorganic Se can induce cell cycle arrest and increase ROS production in ECs, showing promising anti-angiogenic effects. Se nanoparticles (SeNPs) slightly inhibit tumor angiogenesis by inducing apoptosis and cell cycle arrest and by increasing the production of ROS. In summary, this study elucidates the anti-angiogenic activity of Se speciation control with a view to providing a scientific reference for the design and development of novel Se-based highly effective and low toxicity angiogenesis inhibitors.

QSAR, molecular docking, and molecular dynamics simulation–based design of novel anti-cancer drugs targeting thioredoxin reductase enzyme

QSAR, molecular docking, and molecular dynamics simulation–based design of novel anti-cancer drugs targeting thioredoxin reductase enzyme

Oridonin Induces Oxidative Stress-mediated Cancer Cells Apoptosis via Targeting Thioredoxin Reductase.

Thioredoxin reductase (TrxR) plays vital role in regulating cellular redox balance as well as redox-mediated signal transduction. Accumulating evidence supports that overactivation of TrxR is closely related to tumorigenesis and that targeting TrxR ablation reverses the growth of numerous malignant tumors, making TrxR a promising target for cancer chemotherapy. Thus, the discovery and development of molecules as promising anticancer agents that target TrxR is of great significance. Oridonin was shown to inhibit TrxR activity, but the detailed cellular mechanism is largely unknown. The study investigated the mechanism of action and underlying inhibitory properties of oridonin on TrxR in HeLa cells. A covalent docking was performed to reveal the possible interaction between oridonin and TrxR by Schrödinger Software Suite. TrxR activity was determined by 5,5'-dithiobis-2- nitrobenzoic acid reduction assay and endpoint insulin reduction assay. Sulforhodamine B and colony formation assay were employed to assess the viability and growth of cells. Reactive oxygen species level was measured by probe 2', 7'-dichlorfluorescein diacetate, and dihydroethidium. Hoechst 33342 staining, caspase 3 activation, and fluorescein-5-isothiocyanate-conjugated Annexin V and propidium iodide double staining were used to evaluate apoptosis. Here, we reported the oridonin as a potent inhibitor of TrxR. Inhibition of TrxR results in a decrease of thiols content and total glutathione, elevates reactive oxygen species levels, and finally promotes oxidative stress-mediated apoptosis of cancer cells. Targeting TrxR by oridonin discloses a novel molecular mechanism underlying the biological action of oridonin and sheds light on developing oridonin as a potential tumor therapeutic agent.

Nanoparticle delivery of miR-21-3p sensitizes melanoma to anti-PD-1 immunotherapy by promoting ferroptosis

BackgroundAlthough anti-programmed cell death protein 1 (PD-1) immunotherapy is greatly effective in melanoma treatment, low response rate and treatment resistance significantly hinder its efficacy. Tumor cell ferroptosis triggered by interferon...

Targeting thioredoxin reductase by micheliolide contributes to radiosensitizing and inducing apoptosis of HeLa cells

Inhibition of thioredoxin reductase (TrxR) is a crucial strategy for the discovery of antineoplastic drugs and radiosensitizers. As an anticancer candidate derived from Michelia, micheliolide (MCL) is converted readily from parthenolide (PTL), and has better stability and solubility than PTL. However, the anticancer mechanism of MCL has not been fully dissected. We present here for the first time that MCL-targeted inhibition of TrxR not only promotes oxidative stress-mediated HeLa cell apoptosis but also sensitizes ionizing radiation (IR) treatment. Further mechanistic studies demonstrate that MCL covalently binds to Sec at position 498 of TrxR to restrain the biological function of TrxR. It exhibits the inhibition of TrxR activity, enhancement of oxidized Trx, and sensitization of IR in the cellular environment, accompanied by the accumulation of reactive oxygen species (ROS) and the collapse of the intracellular redox balance. In addition, HeLa-shTrxR1 cells with knockdown of TrxR were more sensitive than the HeLa-shNT cells to either MCL-treated or IR-induced cytotoxicity, ROS, and apoptosis, suggesting that inhibition of TrxR by MCL is likely responsible for increased cytotoxicity and enhanced radiation response. These findings further establish the mechanistic understanding and preclinical data to support the further investigation of MCL's potential as a prospective radiosensitizer and cancer chemotherapeutic agent.

Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.

Rheumatoid arthritis drug sinomenine induces apoptosis of cervical tumor cells by targeting thioredoxin reductase in vitro and in vivo

Overexpression of thioredoxin reductase (TrxR) has been linked to tumorigenesis and phenotypic maintenance of malignant tumors. Thus, targeting TrxR with natural molecules is a promising strategy for developing anticancer drugs. Sinomenine is a naturally occurring alkaloid isolated from Sinomenium acutum. The drug, Zhengqing Fengtongning made from sinomenine, has been universally applied in rheumatoid arthritis treatment in China as well as other Asian countries for decades. Recently, increasing evidence indicates that sinomenine appears to be a promising therapeutic agent against various cancer cells. However, the exact mechanism underlying the anticancer activity of sinomenine remains unclear. In this study, we identified sinomenine as a kind of new inhibitor for TrxR. Pharmacological inhibition of TrxR by sinomenine results in the decrease of thiols content, increases the levels of reactive oxygen species, and finally facilitates oxidative stress-mediated cancer cell apoptosis. It is vital that knockdown in TrxR1 by shRNA can increase cell sensitivity to sinomenine. Treatment with sinomenine in vivo leads to a decrease in TrxR activity and tumor growth, and an increase in apoptosis. Our findings provide a novel action mechanism related to sinomenine and presents an insight on how to develop sinomenine as a chemotherapeutic agent for cancer therapy.

Targeting thioredoxin reductase by deoxyelephantopin from Elephantopus scaber triggers cancer cell apoptosis

Elevated expression of thioredoxin reductase (TrxR) is associated with the tumorigenesis and resistance to cancer chemoradiotherapy, highlighting the potential of TrxR inhibitors as anticancer drugs. Deoxyelephantopin (DET) is the major active ingredient of Elephantopus scaber and reveals potent anticancer activity. However, the potential mechanism of action and the cellular target of DET are still unknown. Here, we found that DET primarily targets the Sec residue of TrxR and irreversibly prohibits enzyme activity. Suppression of TrxR by DET leads to accumulation of reactive oxygen species and dysregulation in intracellular redox balance, eventually inducing cancer cell apoptosis mediated by oxidative stress. Noticeably, down-regulation of TrxR1 by shRNA increases cell sensitivity to DET. Collectively, targeting of TrxR1 by DET uncovers a novel mechanism of action in DET and deepens the understanding of developing DET as a potential chemotherapeutic agent for treating cancers.

Targeting CXCR4 and Thioredoxin Reductase in High Grade Neuroendocrine Tumors

Targeting CXCR4 and Thioredoxin Reductase in High Grade Neuroendocrine Tumors

Correction to: Understanding the survival mechanisms of Deinococcus radiodurans against oxidative stress by targeting thioredoxin reductase redox system.

The correction does not affect the discussion or conclusions of the article. The correct image is given below.

Cancer Therapy by Targeting Thioredoxin Reductase Based on Selenium-Containing Dynamic Covalent Bond

Thioredoxin Reductase (TrxR) plays a pivotal role in defending cells against reactive oxygen species (ROS) and maintaining a reduced intracellular environment. It has been discovered that TrxR is e...

New cyclometalated gold (III) complex targeting thioredoxin reductase: exploring as cytotoxic agents and mechanistic insights.

A new cyclometalated Au(III) complex highlighting a naphthoquinone-C^N scaffold with formula (NQ-N^C)AuIII(SAd)Cl, 1, in which NQ-N^C: 2-(5-amino-benzo[h]quinolone)-3-(3-methyl-2-butenyl)-1,4-naphthoquinone, HSAd: 1-adamantanethiol, was synthesized and characterized. The interaction of complex 1 with cysteine (CysH) was experimentally and theoretically studied. Complex 1 was more active against MCF-7 and A549 cancer cell lines and less active in a healthy cell (non-tumorigenic cells, MRC-5) than cisplatin. The DNA binding and inhibition of thioredoxin reductase of complex 1 were studied and compared with the molecular docking results. The generation of reactive oxygen species (ROS) and apoptosis of this new complex were also investigated.

EXTH-50. THIOREDOXIN REDUCTASE1 AND MGMT SYNTHETIC LETHALITY ENHANCES CYTOTOXICITY OF PRIMA-1MET(APR-246) AND AURANOFIN IN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM), the most common and advanced primary brain malignancy in adults remains an incurable disease, despite aggressive treatment with surgery, radiation therapy and chemoradiation using the alkylating agent, Temozolomide (TMZ). PRIMA-1MET (APR-246), a small molecule designed to restore mutant (mut)p53 function has been shown to affect cellular redox status through targeting thioredoxin reductase 1 (TrxR1) in wild-type (wt)p53 cancer cells. We have recently shown that PRIMA-1MET exerts cytotoxic effects status preferentially in GBM cell lines expressing low levels of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), known for its role in resistance to TMZ. We hypothesized that PRIMA-1MET mediates its growth inhibitory effects by modulating the redox balance and investigated the potential relationship between MGMT, redox balance and TrxR1. We show that PRIMA-1MET decreased TrxR1 expression levels preferentially in MGMT-low expressing isogenic GBM cell lines. Using pharmacological agents that modulate reactive oxygen species (ROS) levels i.e., ROS scavenger, N-acetylcysteine and ROS inducer, L-Buthionine-Sulfoximine, we show that PRIMA-1MET exerts its growth-inhibitory effects through increased ROS. Strikingly, we identified a novel positive relationship between MGMT and TrxR1, wherein high MGMT expression is associated with high expression of TrxR1 and low levels of ROS. Treatment with the MGMT inhibitor, O6-Benzylguanine, or the TrxR1-targeting FDA-approved drug Auranofin validated our findings. Interestingly, the latter exerted significantly more pronounced cytotoxic effects compared to PRIMA-1MET in GBM cell lines. Additional studies are warranted to assess PRIMA-1MET in combination with TrxR1-targeting therapies and propose repurposing of Auranofin as a novel strategy to improve the dismal outcome of patients with GBM.