Stimulation of mitochondrial hydrogen sulfide and glutathione production improves Frank-Starling response of the rat heart via NO-dependent pathway

The Frank-Starling response of the heart is known to be mediated by NO signaling, which is regulated by GSH and H2S. We hypothesized that stimulation of endogenous H2S or GSH synthesis improve Frank-Starling response . Wistar male rats were injected with propargylglycine (PAG, 11.3mg/kg-1, 40min,n=12), an inhibitor of H2S producing enzyme (cystationine-γ-lyase) and L-cysteine (121mg/kg-1, 30min,n=20), a precursor of H2S and GSH. Pretreatment with PAG or L-cysteine separately slightly improved P-V dependence of the isolated rat heart, but combination of PAG and L-cysteine (n=12) improved heart contractile activity. H2S content, cNOS activity, nitrate reductase activity and nitrite content increased by 2, 3.83, 2.5 and 1.3 times in cardiac mitochondria as well as GSH and GSSG levels increased by 2.24 and 1.86 times in heart homogenates of PAG+L-cysteine group comparing to control (all P<0.05). Inhibition of glutathione with DL-buthionine-sulfoximine (22.2mg/kg-1, 40min, n=6) drastically decreased Frank-Starling response of the heart and prevented PAG+L-cysteine induced increase of GSH and GSSG levels (BSO+PAG+L-cysteine,n=9). Inhibitor of NOS, N-nitro-L-arginine-methylester hydrochloride (40min, 27mg/kg-1), abolished positive inotropy induced by PAG+L-cysteine pretreatment (L-NAME+PAG+L-cysteine,n=7). Thus, PAG+L-cysteine administration improves Frank-Starling response with up-regulation of mitochondrial H2S, glutathione and NO synthesis that might be promising approach in treatment of myocardial dysfunction.

Hollow Gold Nanoparticles Loaded with L-Buthionine-Sulfoximine as a Novel Nanomedicine for In Vitro Cancer Cell Therapy

A novel nanomedicine, constructed by simultaneously binding L-buthionine-sulfoximine (BSO) and thiolated polyethylene glycol (PEG) to the surface of 100 nm hollow gold nanoparticles (HAuNS), was expected to be used in effective therapeutics of cancer. The current study is aimed at evaluating in vitro the antitumor efficacy of newly synthesized BSO-loaded PEG-SH-HAuNS (BSO@HAuNS) with strong resonances in near-infrared (NIR) as a chemotherapy agents against a line of human lung cancer cells (A549). Here, we conducted cytotoxicity assays and found BSO@HAuNS to efficiently kill human lung cancer cells by ROS generation, indicating that BSO facilitated an increased susceptibility of cancer cells to PEG-SH-HAuNS. Based on flow cytometry analysis, BSO@HAuNS can induce apoptosis and necrosis in mitochondrial-dependent pathway in A549 cells. Our results revealed a novel class of nanomedicine with high potential to be implemented as effective chemotherapy agents for patients diagnosed with unresectable lung cancer.

GSH and Zinx Supplementation Attenuate Cadmium-Induced Cellular Stress and Stimulation of Choline Uptake in Cultured Neonatal Rat Choroid Plexus Epithelia

Choroid plexus (CP) sequesters cadmium and other metals, protecting the brain from these neurotoxins. These metals can induce cellular stress and modulate homeostatic functions of CP, such as solute transport. We previously showed in primary cultured neonatal rat CP epithelial cells (CPECs) that cadmium induced cellular stress and stimulated choline uptake at the apical membrane, which interfaces with cerebrospinal fluid in situ. Here, in CPECs, we characterized the roles of glutathione (GSH) and Zinx supplementation in the adaptive stress response to cadmium. Cadmium increased GSH and decreased the reduced GSH-to-oxidized GSH (GSSG) ratio. Heat shock protein-70 (Hsp70), heme oxygenase (HO-1), and metallothionein (Mt-1) were induced along with the catalytic and modifier subunits of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. Inhibition of GCL by l-buthionine sulfoximine (BSO) enhanced stress protein induction and stimulation of choline uptake by cadmium. Zinx alone did not induce Hsp70, HO-1, or GCL subunits, or modulate choline uptake. Zinx supplementation during cadmium exposure attenuated stress protein induction and stimulation of choline uptake; this effect persisted despite inhibition of GSH synthesis. These data indicated up-regulation of GSH synthesis promotes adaptation to cadmium-induced cellular stress in CP, but Zinx may confer cytoprotection independent of GSH.

Prevention of Teratogenesis in Pregnancies of Obese Rats by Vitamin E Supplementation

Congenital malformations are a common adverse outcome in pregnancies complicated by pregestational obesity, although the underlying mechanisms are still unrevealed. Our aim was to study the effect of oxidative stress in obesity-induced teratogenesis. Wistar rats were fed a high-fat diet for 13 weeks, with (OE group) or without (O group) vitamin E supplementation. Then, rats were mated and sacrificed at day 11.5 of gestation. Embryos from O dams presented a 25.9 ± 3.5% rate of malformations (vs. 8.7 ± 3.4% in C rats), which was reduced in the OE group (11.5 ± 2.3%). Pregestational obesity induced hepatic protein and DNA oxidation and a decline in antioxidant enzymes. Importantly, glutathione content was also decreased, limiting the availability of this antioxidant in the embryos. Vitamin E supplementation efficiently maintained glutathione levels in the obese mothers, which could be used in their embryos to prevent oxidation-induced malformations. To test the effect of decreasing glutathione levels alone in a cell culture model of neuroepithelium, murine embryonic stem cells (ESC) were induced to form neuronal precursors and glutathione synthesis was inhibited with the gamma–glutamylcysteine synthesis inhibitor, buthionine sulfoximine (BSO). BSO inhibited the expression of Pax3, a gene required for neural tube closure that is also inhibited by oxidative stress. Taken together, our data indicate that obesity causes malformations through the depletion of maternal glutathione, thereby decreasing glutathione-dependent free radical scavenging in embryos, which can be prevented by vitamin E supplementation.

Resistance to different anthracycline chemotherapeutics elicits distinct and actionable primary metabolic dependencies in breast cancer

Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.

RNAi-mediated gene knockdown of progesterone 5β-reductases in Digitalis lanata reduces 5β-cardenolide content

Key message Studying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation. Abstract Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Ethylene reduces glucose sensitivity and reverses photosynthetic repression through optimization of glutathione production in salt-stressed wheat (Triticum aestivum L.)

Ethylene plays a crucial role throughout the life cycle of plants under optimal and stressful environments. The present study reports the involvement of exogenously sourced ethylene (as ethephon; 2-chloroethyl phosphonic acid) in the protection of the photosynthetic activity from glucose (Glu) sensitivity through its influence on the antioxidant system for adaptation of wheat (Triticum aestivum L.) plants under salt stress. Ten-day-old plants were subjected to control and 100 mM NaCl and treated with 200 µl L−1 ethephon on foliage at 20 days after seed sowing individually or in combination with 6% Glu. Plants receiving ethylene exhibited higher growth and photosynthesis through reduced Glu sensitivity in the presence of salt stress. Moreover, ethylene-induced reduced glutathione (GSH) production resulted in increased psbA and psbB expression to protect PSII activity and photosynthesis under salt stress. The use of buthionine sulfoximine (BSO), GSH biosynthesis inhibitor, substantiated the involvement of ethylene-induced GSH in the reversal of Glu-mediated photosynthetic repression in salt-stressed plants. It was suggested that ethylene increased the utilization of Glu under salt stress through its influence on photosynthetic potential and sink strength and reduced the Glu-mediated repression of photosynthesis.

GSH-Independent Induction of ER Stress during Hypoglycaemia in the Retinal Cells of Mice

Glucose is one of the most important metabolic substrates of the retina, and glycaemic imbalances can lead to serious side effects, including retinopathy. We previously showed that hypoglycaemia induces retinal cell death in mice, as well as the implication of glutathione (GSH) in this process. This study aimed to analyse the role of low glucose-induced decrease in GSH levels in endoplasmic reticulum (ER) stress. We cultured 661W photoreceptor-like cells under various glucose conditions and analysed ER stress markers at the mRNA and protein levels. We used the ERAI (“ER stress-activated indicator”) mouse model to test ER stress in both ex vivo, on retinal explants, or in vivo, in mice subjected to hypoglycaemia. Moreover, we used buthionine sulfoximine (BSO) and glutamate cysteine ligase (Gclm)-KO mice as models of low GSH to test its effects on ER stress. We show that the unfolded protein response (UPR) is triggered in 661W cells and in ERAI mice under hypoglycaemic conditions. Low GSH levels promote cell death, but have no impact on ER stress. We concluded that low glucose levels induce ER stress independently of GSH levels. Inhibition of ER stress could prevent neurodegeneration, which seems to be an early event in the pathogenesis of diabetic retinopathy.

Positive and Negative Regulation of Ferroptosis and Its Role in Maintaining Metabolic and Redox Homeostasis

Ferroptosis is a recently recognized regulated form of cell death characterized by accumulation of lipid-based reactive oxygen species (ROS), particularly lipid hydroperoxides and loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4). This iron-dependent form of cell death is morphologically, biochemically, and also genetically discrete from other regulated cell death processes, which include autophagy, apoptosis, necrosis, and necroptosis. Ferroptosis is defined by three hallmarks, defined as the loss of lipid peroxide repair capacity by GPX4, the bioavailability of redox-active iron, and oxidation of polyunsaturated fatty acid- (PUFA-) containing phospholipids. Experimentally, it can be induced by many compounds (e.g., erastin, Ras-selective lethal small-molecule 3, and buthionine sulfoximine) and also can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and deferoxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin and liproxstatin). The sensitivity of a cell towards ferroptotic cell death is tightly associated with the metabolism of amino acid, iron, and polyunsaturated fatty acid metabolism, and also with the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis sensitivity is also governed by many regulatory proteins, which also link ferroptosis to the function of key tumour suppressor pathways. In this review, we highlight the discovery of ferroptosis, the mechanism of ferroptosis regulation, and its association with other cellular metabolic processes.