Unveiling the cytotoxicity of a new gold(I) complex towards hepatocellular carcinoma by inhibiting TrxR activity.

Hepatocellular carcinoma (HCC), the predominant type of liver cancer, is an aggressive malignancy with limited therapeutic options. In this study, we assess a collection of newly designed gold(I) phosphine complexes. Remarkably, the compound GC002 exhibits the greatest toxicity to HCC cells and outperforms established medications, such as sorafenib and auranofin, in terms of antitumor efficacy. GC002 triggers irreversible necroptosis in HCC cells by increasing the intracellular accumulation of reactive oxygen species (ROS). Mechanistically, GC002 significantly suppresses the activity of thioredoxin reductase (TrxR), which plays a crucial role in regulating redox homeostasis and is often overexpressed in HCC by binding directly to the enzyme. Our in vivo xenograft study confirms that GC002 possesses remarkable antitumor activity against HCC without severe side effects. These findings not only highlight the novel mechanism of controlling necroptosis via TrxR and ROS but also identify GC002 as a promising candidate for the further development of antitumor agents targeting HCC.

Evaluating the amoeba thioredoxin reductase selenoprotein as potential drug target for treatment of Acanthamoeba infections.

The genus Acanthamoeba comprises facultative pathogens, causing Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). In both diseases, treatment options are limited, and drug development is challenging. This study aimed to investigate the role of the large thioredoxin reductase selenoprotein of Acanthamoeba (AcTrxR-L) as a potential drug target assessing the effects of the thioredoxin reductase inhibitors auranofin, TRi-1, and TRi-2 on AcTrxR-L activity and on the viability of Acanthamoeba trophozoites. Recombinant expression and purification of AcTrxR-L as a selenoprotein allowed assessments of its enzymatic activity, with reduction of various substrates, including different thioredoxin isoforms. Auranofin demonstrated potent inhibition towards AcTrxR-L, followed by TRi-1, and TRi-2 exhibiting lower effectiveness. The inhibitors showed variable activity against trophozoites in culture, with TRi-1 and TRi-2 resulting in strongly impaired trophozoite viability. Cytotoxicity tests with human corneal epithelial cells revealed lower susceptibility to all compounds compared to Acanthamoeba trophozoites, underscoring their potential as future amoebicidal agents. Altogether, this study highlights the druggability of AcTrxR-L and suggests it to be a promising drug target for the treatment of Acanthamoeba infections. Further research is warranted to elucidate the role of AcTrxR-L in Acanthamoeba pathogenesis and to develop effective therapeutic strategies targeting this redox enzyme.

Probing the Site of Glutathione Reduction by Thioredoxin/Glutathione Reductase from Schistosoma mansoni under Anaerobic Conditions.

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) is a multifunctional enzyme that catalyzes the reduction of glutathione (GSSG) and thioredoxin, as well as the deglutathionylation of peptide and non-peptide substrates. SmTGR structurally resembles known glutathione reductases (GR) and thioredoxin reductases (TrxR) but with an appended N-terminal domain that has a typical glutaredoxin (Grx) fold. Despite structural homology with known GRs, the site of glutathione reduction has frequently been reported as the Grx domain, based primarily on aerobic, steady-state kinetic measurements and x-ray crystallography. Here, we present an anaerobic characterization of a series of variant SmTGRs to establish the site of GSSG reduction as the cysteine pair most proximal to the FAD, Cys154/Cys159, equivalent to the site of GSSG reduction in GRs. Anaerobic steady-state analysis of U597C, U597S, U597C+C31S, and I592STOP SmTGR demonstrate that the Grx domain is not involved in the catalytic reduction of GSSG, as redox silencing of the C-terminus results in no modulation of the observed turnover number (∼0.025 s-1) and redox silencing of the Grx domain results in an increased observed turnover number (∼0.08 s-1). Transient-state single turnover analysis of these variants corroborates this, as the slowest rate observed titrates hyperbolically with GSSG concentration and approaches a limit that coincides with the respective steady-state turnover number for each variant. Numerical integration fitting of the transient state data can only account for the observed trends when competitive binding of the C-terminus is included, indicating that the partitioning of electrons to either substrate occurs at the Cys154/Cys159 disulfide rather than the previously proposed Cys596/Sec597 sulfide/selenide. Paradoxically, truncating the C-terminus at Ile592 results in a loss of GR activity, indicating a crucial non-redox role for the C-terminus.

The Role of Thioredoxin System in Shank3 Mouse Model of Autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by difficulties in social interaction and communication, repetitive behaviors, and restricted interests. Unfortunately, the underlying molecular mechanism behind ASD remains unknown. It has been reported that oxidative and nitrosative stress are strongly linked to ASD. We have recently found that nitric oxide (NO•) and its products play an important role in this disorder. One of the key proteins associated with NO• is thioredoxin (Trx). We hypothesize that the Trx system is altered in the Shank3 KO mouse model of autism, which may lead to a decreased activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in oxidative stress, and thus, contributing to ASD-related phenotypes. To test this hypothesis, we conducted in vivo behavioral studies and used primary cortical neurons derived from the Shank3 KO mice and human SH-SY5Y cells with SHANK3 mutation. We showed significant changes in the levels and activity of Trx redox proteins in the Shank3 KO mice. A Trx1 inhibitor PX-12 decreased Trx1 and Nrf2 expression in wild-type mice, causing abnormal alterations in the levels of synaptic proteins and neurotransmission markers, and an elevation of nitrosative stress. Trx inhibition resulted in an ASD-like behavioral phenotype, similar to that of Shank3 KO mice. Taken together, our findings confirm the strong link between the Trx system and ASD pathology, including the increased oxidative/nitrosative stress, and synaptic and behavioral deficits. The results of this study may pave the way for identifying novel drug targets for ASD.

Design, synthesis and biological evaluation of novel curcumin-fluorouracil hybrids as potential anti-cancer agents.

The latest global cancer data statistics report shows that cancer poses a serious threat to human life and health; The number of new cancer and death cases worldwide is severe. Molecular hybridization is considered an effective strategy for developing new anti-cancer drugs. Curcumin (Cur) is a natural active compound containing Michael receptors that target thioredoxin reductase (TrxR). Fluorouracil (5-FU) is the first anti-metabolic drug synthesized based on certain assumptions for tumor treatment, acting on thymidylate synthase (TS). This study synthesized a series of novel hybrid derivatives of Cur and 5-FU, and evaluated their anti-tumor cell proliferation effects. Several compounds with good cytotoxic activity against tumor cells were discovered; and they exhibited high selectivity towards A549 cells, compared to normal THLE cells. Among them, the hybrid derivative F-4 has the best anti-proliferative activity in tumor cells. F-4 can target TrxR, increase reactive oxygen species levels in tumor cells, and lead to tumor cell apoptosis, which may be related to the Michael receptor structure in the chemical structure of F-4; F-4 can also target TS, leading to cell cycle arrest in G0/G1 phase, which may be related to the 5-FU structure in the chemical structure of F-4. Moreover, F-4 can effectively exert anti-tumor activity in mice, significantly reduce tumor volume and weight, and has low toxic side effects. These results indicate that Cur-5-FU hybrid derivative F-4 is a novel lead compound with in vivo anti-tumor activity and minimal side effects, which deserves further investigation.

Diallyl trisulfide alleviates dextran sulphate sodium-induced colitis in mice by inhibiting NLRP3 inflammasome activation via ROS/Trx-1 pathway.

Diallyl trisulfide (DATS), a sulphur-containing compound isolated from the medicinal food plant garlic, has been previously reported to attenuate experimental colitis induced by either dextran sodium sulphate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice; however, the underlying mechanism remains to be identified. In this study, we deciphered the key mechanism by which DATS alleviates ulcerative colitis (UC). We showed that oral administration of DATS for 10 consecutive days greatly restrained the infiltration of macrophages and the pathological changes in colonic tissues of mice with DSS-induced colitis. DATS treatment notably dampened the content of IL-1ß and IL-18 and suppressed NLRP3 inflammasome activation in colon. Mechanistically, DATS effectively diminished the generation of ROS in macrophages. The suppressive effect of DATS on the activation of NLRP3 inflammasome and downregulation of IL-18 and IL-1ß levels was blunted by xanthine oxidase. Further studies revealed that DATS inhibited NF-κB pathway activation by suppressing the expression of Trx-1, thereby inhibiting NLRP3 inflammasome activation. Trx-1 overexpression and interference in macrophages promoted and diminished NLRP3 inflammasome activation, respectively. In summary, garlic and its main active ingredient DATS have potentials to prevent and treat UC, and DATS functions by inhibiting NLRP3 inflammasome activation via Trx-1/ROS pathway.

Enhance the Antimycobacterial Activity of Streptomycin with Ebselen as an Antibiotic Adjuvant Through Disrupting Redox Homeostasis.

Purpose: Tuberculosis (TB) remains a major health threat worldwide, and the spread of drug-resistant (DR) TB impedes the reduction of the global disease burden. Ebselen (EbSe) targets bacterial thioredoxin reductase (bTrxR) and causes an imbalance in the redox status of bacteria. Previous work has shown that the synergistic action of bTrxR and sensitization to common antibiotics by EbSe is a promising strategy for the treatment of DR pathogens. Thus, we aimed to evaluate whether EbSe could enhance anti-TB drugs against Mycobacterium marinum (M. marinum) which is genetically related to Mycobacterium tuberculosis (Mtb) and resistant to many antituberculosis drugs. Methods: Minimum inhibitory concentrations (MIC) of isoniazid (INH), rifampicin (RFP), and streptomycin (SM) against M. marinum were determined by microdilution. The Bliss Independence Model was used to determine the adjuvant effects of EbSe over the anti-TB drugs. Thioredoxin reductase activity was measured using the DTNB assay, and its effects on bacterial redox homeostasis were verified by the elevation of intracellular ROS levels and intracellular GSH levels. The adjuvant efficacy of EbSe as an anti-TB drug was further evaluated in a mouse model of M. marinum infection. Cytotoxicity was observed in the macrophage cells Raw264.7 and mice model. Results: The results reveal that EbSe acts as an antibiotic adjuvant over SM on M. marinum. EbSe + SM disrupted the intracellular redox microenvironment of M. marinum by inhibiting bTrxR activity, which could rescue mice from the high bacterial load, and accelerated recovery from tail injury with low mammalian toxicity. Conclusion: The above studies suggest that EbSe significantly enhanced the anti-Mtb effect of SM, and its synergistic combination showed low mammalian toxicity in vitro and in vivo. Further efforts are required to study the underlying mechanisms of EbSe as an antibiotic adjuvant in combination with anti-TB drug MS.

Glucose fluctuations aggravate cardiomyocyte apoptosis by enhancing the interaction between Txnip and Akt.

BACKGROUND: Glucose fluctuations may be involved in the pathophysiological process of cardiomyocyte apoptosis, but the exact mechanism remains elusive. This study focused on exploring the mechanisms related to glucose fluctuation-induced cardiomyocyte apoptosis. METHODS: Diabetic rats established via an injection of streptozotocin were randomized to five groups: the controlled diabetic (CD) group, the uncontrolled diabetic (UD) group, the glucose fluctuated diabetic (GFD) group, the GFD group rats with the injection of 0.9% sodium chloride (NaCl) (GFD + NaCl) and the GFD group rats with the injection of N-acetyl-L-cysteine (NAC) (GFD + NAC). Twelve weeks later, cardiac function and apoptosis related protein expressions were tested. Proteomic analysis was performed to further analyze the differential protein expression pattern of CD and GFD. RESULTS: The left ventricular ejection fraction levels and fractional shortening levels were decreased in the GFD group, compared with those in the CD and UD groups. Positive cells tested by DAB-TUNEL were increased in the GFD group, compared with those in the CD group. The expression of Bcl-2 was decreased, but the expressions of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in response to glucose fluctuations. Compared with CD, there were 527 upregulated and 152 downregulated proteins in GFD group. Txnip was one of the differentially expressed proteins related to oxidative stress response. The Txnip expression was increased in the GFD group, while the Akt phosphorylation level was decreased. The interaction between Txnip and Akt was enhanced when blood glucose fluctuated. Moreover, the application of NAC partially reversed glucose fluctuations-induced cardiomyocyte apoptosis. CONCLUSIONS: Glucose fluctuations lead to cardiomyocyte apoptosis by up-regulating Txnip expression and enhancing Txnip-Akt interaction.

Endogenous electrophiles and peroxymonocarbonate can link tyrosine phosphorylation cascades with the cytosolic TXNRD1 selenoprotein and the KEAP1/NRF2 system.

Endogenously formed reactive molecules, such as lipid peroxides, 4-hydroxynonenal, methylglyoxal and other reactive oxygen species, can have major effects on cells. Accumulation of these molecules is counteracted by antioxidant enzymes, including the glutathione (GSH) and thioredoxin (Trx) systems, in turn regulated by the KEAP1/NRF2 system. Receptor tyrosine kinases (RTK) and their counteracting protein tyrosine phosphatases (PTP) are also modulated through redox regulation of PTP activities. The cytosolic selenoprotein thioredoxin reductase (TXNRD1) is particularly prone to attack at its easily accessible catalytic selenocysteine (Sec) residue by reactive electrophilic compounds. Therefore, we here discuss how endogenously formed electrophiles can modulate RTK/PTP signaling in a concentration- and time dependent manner by reactions either directly or indirectly linking TXNRD1 with the KEAP1/NRF2 system. Moreover, recent findings suggest that endogenous formation of peroxymonocarbonate can efficiently inhibit PTP activities and stimulate RTK signaling, seemingly bypassing PTP reduction as otherwise supported by the GSH/Trx systems.

Thiamine disulfide derivatives in thiol redox regulation: Role of thioredoxin and glutathione systems.

Thiamine (vitamin B1), under the proper conditions, is able to reversibly open the thiazole ring, forming a thiol-bearing molecule that can be further oxidized to the corresponding disulfide. To improve the bioavailability of the vitamin, several derivatives of thiamine in the thioester or disulfide form were developed and extensively studied over time, as apparent from the literature. We have examined three thiamine-derived disulfides: thiamine disulfide, sulbutiamine, and fursultiamine with reference to their intervention in modulating the thiol redox state. First, we observed that both glutathione and thioredoxin (Trx) systems were able to reduce the three disulfides. In particular, thioredoxin reductase (TrxR) reduced these disulfides either directly or in the presence of Trx. In Caco-2 cells, the thiamine disulfide derivatives did not modify the total thiol content, which, however, was significantly decreased by the concomitant inhibition of TrxR. When oxidative stress was induced by tert-butyl hydroperoxide, the thiamine disulfides exerted a protective effect, indicating that the thiol form deriving from the reduction of the disulfides might be the active species. Further, the thiamine disulfides examined were shown to increase the nuclear levels of the transcription factor nuclear factor erythroid 2 related factor 2 and to stimulate both expression and activity of NAD(P)H quinone dehydrogenase 1 and TrxR. However, other enzymes of the glutathione and Trx systems were scarcely affected. As the thiol redox balance plays a critical role in oxidative stress and inflammation, the information presented can be of interest for further research, considering the potential favorable effect exerted in the cell by many sulfur compounds, including the thiamine-derived disulfides.