Evaluation of antioxidant activation by potential nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Keap1 complex inhibitors

Purpose: To investigate the binding affinities of forty-one (41) National Cancer Institute (NCI)-generated compounds, to the Nrf2 ligand, and possible activation of Nrf2 in the MCF-7 cell line.Methods: To investigate the inhibition of the Nrf2/Keap1 complex, the MCF-7 cell line was treated with each of the 41 compounds, at a working concentration of 30 μM. The extent of Nrf2 activation and corresponding Nrf2/Keap1 complex inhibition was evaluated in terms of Nrf2 expression and its antioxidant-associated enzyme gamma-glutamylcysteine synthetase (GCS), using western blotanalysis.Results: Twenty-nine compounds out of the 41 targeted compounds activated GCS, and some showed comparable or greater activation capacity than the standard Nrf2 activator tBHQ. To confirm that the activation of GCS was mediated via Nrf2 activation, cell lysates were tested for their Nrf2 protein expression, and it was found that Nrf2 was activated by the examined compounds for more than 24 h, indicating that the effect of the chosen compounds were not transient.Conclusion: These results might be useful for identifying better targets for cytoprotection, and for oxidative stress alleviation through Nrf2 pathway activation. Further studies are required on the effects of these targets on the prevention and treatment of various oxidative stress disorders, including cancer.

Protocatechuic Acid, a Simple Plant Secondary Metabolite, Induced Apoptosis by Promoting Oxidative Stress through HO-1 Downregulation and p21 Upregulation in Colon Cancer Cells

Gastrointestinal cancers, particularly colorectal cancer, are mainly influenced by the dietary factor. A diet rich in fruits and vegetables can help to reduce the incidence of colorectal cancer thanks to the phenolic compounds, which possess antimutagenic and anticarcinogenic properties. Polyphenols, alongside their well-known antioxidant properties, also show a pro-oxidative potential, which makes it possible to sensitize tumor cells to oxidative stress. HO-1 combined with antioxidant activity, when overexpressed in cancer cells, is involved in tumor progression, and its inhibition is considered a feasible therapeutic strategy in cancer treatment. In this study, the effects of protocatechuic acid (PCA) on the viability of colon cancer cells (CaCo-2), annexin V, LDH release, reactive oxygen species levels, total thiol content, HO-1, γ-glutamylcysteine synthetase, and p21 expression were evaluated. PCA induced, in a dose-dependent manner, a significantly reduced cell viability of CaCo-2 by oxidative/antioxidant imbalance. The phenolic acid induced modifications in levels of HO-1, non-proteic thiol groups, γ-glutamylcysteine synthetase, reactive oxygen species, and p21. PCA induced a pro-oxidant effect in cancer cells, and the in vitro pro-apoptotic effect on CaCo-2 cells is mediated by the modulation of redox balance and the inhibition of the HO-1 system that led to the activation of p21. Our results suggest that PCA may represent a useful tool in prevention and/or therapy of colon cancer.

Overexpression of γ-glutamylcysteine synthetase gene from Caragana korshinskii decreases stomatal density and enhances drought tolerance

Background Gamma-glutamylcysteine synthetase (γ-ECS) is a rate-limiting enzyme in glutathione biosynthesis and plays a key role in plant stress responses. In this study, the endogenous expression of the Caragana korshinskiiγ-ECS (Ckγ-ECS) gene was induced by PEG 6000-mediated drought stress in the leaves of C. korshinskii. and the Ckγ-ECS overexpressing transgenic Arabidopsis thaliana plants was constructed using the C. korshinskii. isolated γ-ECS. Results Compared with the wildtype, the Ckγ-ECS overexpressing plants enhanced the γ-ECS activity, reduced the stomatal density and aperture sizes; they also had higher relative water content, lower water loss, and lower malondialdehyde content. At the same time, the mRNA expression of stomatal development-related gene EPF1 was increased and FAMA and STOMAGEN were decreased. Besides, the expression of auxin-relative signaling genes AXR3 and ARF5 were upregulated. Conclusions These changes suggest that transgenic Arabidopsis improved drought tolerance, and Ckγ-ECS may act as a negative regulator in stomatal development by regulating the mRNA expression of EPF1 and STOMAGEN through auxin signaling.

Role of glutathione on cell adhesion and volume.

Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of 7-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to transport of GSH out, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.

α-Chaconine Affects the Apoptosis, Mechanical Barrier Function, and Antioxidant Ability of Mouse Small Intestinal Epithelial Cells

α-Chaconine is the most abundant glycoalkaloid in potato and toxic to the animal digestive system, but the mechanisms underlying the toxicity are unclear. In this study, mouse small intestinal epithelial cells were incubated with α-chaconine at 0, 0.4, and 0.8 μg/mL for 24, 48, and 72 h to examine apoptosis, mechanical barrier function, and antioxidant ability of the cells using a cell metabolic activity assay, flow cytometry, Western blot, immunofluorescence, and fluorescence quantitative PCR. The results showed that α-chaconine significantly decreased cell proliferation rate, increased apoptosis rate, decreased transepithelial electrical resistance (TEER) value, and increased alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) activities, and there were interactions between α-chaconine concentration and incubation time. α-Chaconine significantly reduced the relative and mRNA expressions of genes coding tight junction proteins zonula occludens-1 (ZO-1) and occludin, increased malondialdehyde (MDA) content, decreased total glutathione (T-GSH) content, reduced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and γ-glutamylcysteine synthetase (γ-GCS) and the mRNA expressions of SOD, CAT, GSH-Px, and γ-GCS genes. In conclusion, α-chaconine disrupts the cell cycle, destroys the mechanical barrier and permeability of mucosal epithelium, inhibits cell proliferation, and accelerates cell apoptosis.

The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in Arabidopsis

In Arabidopsis thaliana there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of nad1-493, nad4-403, nad7-698 and ccmFN2-356 sites in Arabidopsis mitochondria. The absence of editing in mef100 leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.

Mutation of SlSBPASE Aggravates Chilling-Induced Oxidative Stress by Impairing Glutathione Biosynthesis and Suppressing Ascorbate-Glutathione Recycling in Tomato Plants

Sedoheptulose-1,7-bisphosphatase (SBPase) is a crucial enzyme for photosynthetic carbon assimilation in the Calvin-Benson cycle. Previous studies have shown that overexpression of SBPase is advantageous to chilling tolerance in plants; however, the mechanisms of SBPase acting in the improvement of chilling tolerance remain largely unknown. In the present study, we aimed to uncover the essential role of SBPase in the response of tomato plants to oxidative stress induced by low temperature. To fulfill that, we performed an array of comparative studies between slsbpase mutant plants that we previously generated using CRISPR/Cas9 genome editing system and their wild-type counterparts under chilling stress. It was observed that following a 24 h chilling treatment, slsbpase mutant plants accumulated higher levels of reactive oxygen species (ROS) than wild-type plants and consequently, more severe lipid peroxidation occurred in slsbpase plants. Activity assay of antioxidant enzymes showed that mutation in SlSBPASE significantly decreased activities of peroxidase (POD) and ascorbate peroxidase (APX), but surprisingly did not significantly alter activities of superoxide dismutase (SOD) and catalase (CAT) under the chilling condition. Notably, mutation in SlSBPASE reduced the contents of total ascorbate (AsA) and total glutathione (GSH) and suppressed the recycling of AsA and GSH in chilling-stressed tomato plants. In addition, activities of two GSH biosynthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) and transcript abundance of their coding genes (GSH1 and GSH2) were markedly reduced in slsbpase mutant plants in comparison with those in wild-type plants under chilling stress. Furthermore, exogenous GSH remarkably mitigated chilling damage in slsbpase plants. Collectively, these results support that mutation in SlSBPASE aggravates chilling-induced oxidative stress by suppressing GSH biosynthesis and AsA-GSH recycling and suggest that SBPase is required for optimal response to chilling stress in tomato plants. The findings also shed light on the idea to mitigate chilling-induced damages by genetically manipulating a photosynthetic enzyme in plants.

The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.

The Sulfilimine Analogue of Allicin, S-Allyl-S-(S-allyl)-N-Cyanosulfilimine, Is Antimicrobial and Reacts with Glutathione

When cells of garlic (Allium sativum) are disrupted by wounding, they produce the defense substance allicin (diallylthiosulfinate). Allicin is an efficient thiol trap and readily passes through cell membranes into the cytosol, where it behaves as a redox toxin by oxidizing the cellular glutathione (GSH) pool and producing S-allylmercaptoglutathione (GSSA). An N-cyanosulfilimine analogue of allicin (CSA), which was predicted to have similar reactivity towards thiol groups but be more stable in storage, was synthesized and its properties investigated. Similarly to allicin, CSA was shown to inhibit the growth of various bacteria, a fungus (baker’s yeast), and Arabidopsis roots. A chemogenetic screen showed that yeast mutants with compromised GSH levels and metabolism were hypersensitive to CSA. GSH reacted with CSA to produce allyltrisulfanylglutathione (GS3A), which was a white solid virtually insoluble in water. Yeast Δgsh1 mutants are unable to synthesize GSH because they lack the γ-glutamylcysteine synthetase (GSH1) gene, and they are unable to grow without GSH supplementation in the medium. GS3A in the growth medium supported the auxotrophic requirement for GSH in Δgsh1 mutants. This result suggests that GS3A is being reduced to GSH in vivo, possibly by the enzyme glutathione reductase (GR), which has been shown to accept GSSA as a substrate. The results suggest that CSA has a mode of action similar to allicin and is effective at similar concentrations.