Sunflower seed-derived bioactive peptides show antioxidant and anti-inflammatory activity: From in silico simulation to the animal model.

The evolving field of food technology is increasingly dedicated to developing functional foods. This study explored bioactive peptides from sunflower protein isolate (SPI), obtained from defatted flour, a by-product of the oil processing industry. SPI underwent simulated gastrointestinal digestion and the obtained peptide-enriched fraction (PEF) showed antioxidant properties in vivo, in zebrafish. Among the peptides present in PEF identified by mass spectrometry analysis, we selected those with antioxidant properties by in silico evaluation, considering their capability to interact with Keap1, key protein in the regulation of antioxidant response. The selected peptides were synthesized and evaluated in a cellular model. As a result, DVAMPVPK, VETGVIKPG, TTHTNPPPEAE, LTHPQHQQQGPSTG and PADVTPEEKPEV activated Keap1/Nrf2 pathway leading to Antioxidant Response Element-regulated enzymes upregulation. Since the crosstalk between Nrf2 and NF-κB is well known, the potential anti-inflammatory activity of the peptides was assessed and principally PADVTPEEKPEV showed good features both as antioxidant and anti-inflammatory molecule.

Exploring the Anticancer Activity of Tamoxifen-Based Metal Complexes Targeting Mitochondria.

Two new 'hybrid' metallodrugs of Au(III) (AuTAML) and Cu(II) (CuTAML) were designed featuring a tamoxifen-derived pharmacophore to ideally synergize the anticancer activity of both the metal center and the organic ligand. The compounds have antiproliferative effects against human MCF-7 and MDA-MB 231 breast cancer cells. Molecular dynamics studies suggest that the compounds retain the binding activity to estrogen receptor (ERα). In vitro and in silico studies showed that the Au(III) derivative is an inhibitor of the seleno-enzyme thioredoxin reductase, while the Cu(II) complex may act as an oxidant of different intracellular thiols. In breast cancer cells treated with the compounds, a redox imbalance characterized by a decrease in total thiols and increased reactive oxygen species production was detected. Despite their different reactivities and cytotoxic potencies, a great capacity of the metal complexes to induce mitochondrial damage was observed as shown by their effects on mitochondrial respiration, membrane potential, and morphology.

From In Silico to a Cellular Model: Molecular Docking Approach to Evaluate Antioxidant Bioactive Peptides.

The increasing need to counteract the redox imbalance in chronic diseases leads to focusing research on compounds with antioxidant activity. Among natural molecules with health-promoting effects on many body functions, bioactive peptides are gaining interest. They are protein fragments of 2-20 amino acids that can be released by various mechanisms, such as gastrointestinal digestion, food processing and microbial fermentation. Recent studies report the effects of bioactive peptides in the cellular environment, and there is evidence that these compounds can exert their action by modulating specific pathways. This review focuses on the newest approaches to the structure-function correlation of the antioxidant bioactive peptides, considering their molecular mechanism, by evaluating the activation of specific signaling pathways that are linked to antioxidant systems. The correlation between the results of in silico molecular docking analysis and the effects in a cellular model was highlighted. This knowledge is fundamental in order to propose the use of bioactive peptides as ingredients in functional foods or nutraceuticals.

Nrf2-Activating Bioactive Peptides Exert Anti-Inflammatory Activity through Inhibition of the NF-κB Pathway.

Redox status and inflammation are related to the pathogenesis of the majority of diseases. Therefore, understanding the role of specific food-derived molecules in the regulation of their specific pathways is a relevant issue. Our previous studies indicated that K-8-K and S-10-S, milk and soy-derived bioactive peptides, respectively, exert antioxidant effects through activation of the Keap1/Nrf2 pathway. A crosstalk between Nrf2 and NF-κB, mediated by the action of heme oxygenase (HO-1), is well known. On this basis, we studied if these peptides, in addition to their antioxidant activity, could exert anti-inflammatory effects in human cells. First, we observed an increase of HO-1 expression in Caco-2 cells treated with K-8-K and S-10-S, following the activation of the Keap1/Nrf2 pathway. Moreover, when cells are treated with the two peptides and stimulated by TNF-α, the levels of NF-κB in the nucleus decreased in comparison with TNF-α alone. In the same conditions, we observed the downregulation of the gene expression of proinflammatory cytokines (IL1B, IL6, and TNF), while the anti-inflammatory cytokine gene, IL1RN, was upregulated in Caco-2 cells processed as reported above. Then, when the cells were pretreated with the two peptides and stimulated with LPS, a different proinflammatory factor, (TNF-α) was estimated to have a lower secretion in the supernatant of cells. In conclusion, these observations confirmed that Nrf2-activating bioactive peptides, K-8-K and S-10-S, exerted anti-inflammatory effects by inhibiting the NF-κB pathway.

New Platinum(II) Complexes Affecting Different Biomolecular Targets in Resistant Ovarian Carcinoma Cells.

Resistance to platinum-based anticancer drugs represents an important limit for their clinical effectiveness and one of the most important field of investigation in the context of platinum compounds. From our previous studies, PtII complexes containing the triphenylphosphino moiety have been emerging as promising agents, showing significant cytotoxicity to resistant ovarian carcinoma cells. Two brominated triphenylphosphino trans-platinum derivatives were prepared and evaluated on human tumor cell lines, sensitive and resistant to cisplatin. The new complexes exert a notable antiproliferative effect on resistant ovarian carcinoma cells, showing a remarkable intracellular accumulation and the ability to interact with different intracellular targets. The interaction with DNA, the collapse of mitochondrial transmembrane potential, and the impairment of intracellular redox state were demonstrated. Moreover, a selectivity towards the selenocysteine of thioredoxin reductase was observed. The mechanism of action is discussed with regard to the resistance phenomenon in ovarian carcinoma cells.

Fermented Soy-Derived Bioactive Peptides Selected by a Molecular Docking Approach Show Antioxidant Properties Involving the Keap1/Nrf2 Pathway.

Bioactive peptides are a group of molecules with health beneficial properties, deriving from food matrices. They are protein fragments consisting of 2-20 amino acids that can be released by microbial fermentation, food processing and gastrointestinal digestion. Once hydrolyzed from their native proteins, they can have different functions including antioxidant activity, which is important for cell protection by oxidant agents. In this work, fermented soy products were digested in vitro in order to improve the release of bioactive peptides. These were extracted, purified and analyzed in vitro and in a cellular model to assess their antioxidant activity. Peptide sequences were identified by LC-MS/MS analysis and a molecular docking approach was used to predict their ability to interact with Keap1, one of the key proteins of the Keap1/Nrf2 pathway, the major system involved in redox regulation. Peptides showing a high score of interaction were selected and tested for their antioxidant properties in a cellular environment using the Caco-2 cell line and examined for their capability to defend cells against oxidative stress. Our results indicate that several of the selected peptides were indeed able to activate the Keap1/Nrf2 pathway with the consequent overexpression of antioxidant and phase II enzymes.

Platinum(II) Complexes Bearing Triphenylphosphine and Chelating Oximes: Antiproliferative Effect and Biological Profile in Resistant Cells.

Platinum(II) complexes of the type [Pt(Cl)(PPh3 ){(κ2 -N,O)-(1{C(R)=N(OH)-2(O)C6 H4 })}] with R=Me, H, (1 and 2) were synthesized and characterized. Single-crystal X-ray diffraction confirmed the proposed (SP4-3) configuration for 1. Study of the antiproliferative activity, performed on a panel of human tumor cell lines and on mesothelial cells, highlighted complex 2 as the more effective. In particular, it showed a remarkable cytotoxicity in ovarian carcinoma cells (A2780) and interestingly, a significant antiproliferative effect on cisplatin resistant cells (A2780cis). Investigation into the intracellular mechanism of action demonstrated that 2 had a lower ability to platinate DNA than did cisplatin, which was taken as reference, and a notably higher uptake in resistant cells. A significant accumulation in mitochondria, along with the ability to induce concentration-dependent mitochondrial membrane depolarization and intracellular reactive oxygen species production, allowed us to propose a mitochondrion-mediated pathway as responsible for the interesting cytotoxic profile of complex 2.

Small Structural Differences between Two Ferrocenyl Diphenols Determine Large Discrepancies of Reactivity and Biological Effects.

The ferrocenyl diphenol complexes 1,1-bis(4'-hydroxyphenyl)-2-ferrocenyl-but-1-ene (1) and 1,2-bis(4'-hydroxyphenyl)-1-ferrocenyl-but-1-ene [(Z)-2], which differ by the relative position of the two phenolic substituents, display dramatically different antiproliferative activities on cancer cells (1 is far more cytotoxic than 2). In this study, our goal was to discover the origin of this difference by comparing their reactivity and biological behaviour. In terms of common behaviour, we found that 1 and 2 are both efficient inhibitors of thioredoxin reductase (TrxR) in vitro after oxidation by a horseradish peroxidase/H2 O2 system. However, as 1 is only a moderate inhibitor of TrxR in MDA-MB-231 cells, TrxR is probably not the major target responsible for the cytotoxicity of 1. In terms of differences, we noted that 1 induced a significant redox imbalance characterised by lipid peroxidation and thiol oxidation, and a moderate decrease of the mitochondrial membrane potential in breast cancer cells, whereas 2 has almost no effect. These results underline the importance of the trans configuration in the ferrocenyl-double bond-phenol motif, which is present in 1 but is cis in (Z)-2.

Dimers of glutaredoxin 2 as mitochondrial redox sensors in selenite-induced oxidative stress.

Glutaredoxin 2 (Grx2) has been previously shown to link thioredoxin and glutathione systems receiving reducing equivalents by both thioredoxin reductase and glutathione. Grx2 catalyzes protein glutathionylation/de-glutathionylation and can coordinate an iron-sulfur cluster, forming inactive dimers stabilized by two molecules of glutathione. This protein is mainly located in the mitochondrial matrix, though other isoforms have been found in the cytosolic and nuclear cell compartments. In the present study, we have analyzed the monomeric and dimeric states of Grx2 under different redox conditions in HeLa cells, and sodium selenite was utilized as the principal oxidizing agent. After selenite treatment, an increased glutathione oxidation was associated to Grx2 monomerization and activation, specifically in the mitochondrial compartment. Interestingly, in mitochondria, a large decline of thioredoxin reductase activity was also observed concomitantly to Grx2 activity stimulation. In addition, Grx2 monomerization led to an increase free iron ions concentration in the mitochondrial matrix, induction of lipid peroxidation and decrease of the mitochondrial membrane potential, indicating that the disassembly of Grx2 dimer involved the release of the iron-sulfur cluster in the mitochondrial matrix. Moreover, sodium selenite-triggered lipid and protein oxidation was partially prevented by deferiprone, an iron chelator with mitochondriotropic properties, suggesting a role of the iron-sulfur cluster release in the observed impairment of mitochondrial functions. Thus, by sensing the overall cellular redox conditions, mitochondrial Grx2 dimers become active monomers upon oxidative stress induced by sodium selenite with the consequent release of the iron-sulfur cluster, leading to activation of the intrinsic apoptotic pathway.

Antioxidant Properties of Fermented Soy during Shelf Life.

Glycine max (soybean) is a fundamental food in human nutrition, largely utilized by the consumers, and in particular, fermented soy is mainly used. However, health benefits of the products can change during the shelf life as oxidation processes occur determining alterations of protein and lipid constituents leading to a decrease of nutritional quality. Therefore, the oxidative stability of the fermented soy during the shelf life was studied. The antioxidant potential of this product was evaluated by estimating total phenols, free radical scavenger activity using DPPH and ABTS tests, and the degree of lipid peroxidation, from I up to IX weeks. The antioxidant capacity after an initial decrease, increased again at VII-IX weeks. Lipid peroxidation was evaluated by comparing non fermented and fermented soy. The results disclosed a low amount of peroxides in the fermented soy, suggesting that fermentation brings to an improvement of the product associated to a decreased lipid peroxidation at longer times. Fractions of aqueous extract, obtained at the end of the shelf life from fermented soy, showed an enrichment in antioxidant peptides.

Insight into antioxidant properties of milk-derived bioactive peptides in vitro and in a cellular model.

Milk is a nutritionally important source of bioactive peptides with anti-inflammatory, immunomodulatory, anticancer, and antioxidant properties. These compounds can be useful as ingredients of functional food. For this reason, in the last decades, bioactive peptides attracted the interest of researchers and food companies. In this work, the results obtained with six milk-derived bioactive peptides (Y-4-R, V-6-R, V-7-K, A-10-F, R-10-M, and H-9-M) synthesized and studied for their antioxidant properties in vitro and in a cellular model, are reported. These molecules correspond to peptide fragments derived from parent compounds able to cross the apical membrane of Caco-2 cell layer and released in the basolateral compartment. In vitro, antioxidant tests such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and crocin bleaching showed antioxidant activity mainly for peptides Y-4-R and V-6-R, respectively. In Caco-2 cells, peptides V-6-R, H-9-R, Y-4-R, and particularly R-10-M and V-7-K are able to prevent the decrease of viability due to oxidative stress. The latter peptide is also the most effective in protecting cells from lipid peroxidation. In conclusion, the reported hydrolyzed peptides are shown to exert the antioxidant properties both in vitro and in a cellular model.

Significance of the mitochondrial thioredoxin reductase in cancer cells: An update on role, targets and inhibitors.

Thioredoxin reductase 2 (TrxR2) is a key component of the mitochondrial thioredoxin system able to transfer electrons to peroxiredoxin 3 (Prx3) in a reaction mediated by thioredoxin 2 (Trx2). In this way, both the level of hydrogen peroxide and thiol redox state are modulated. TrxR2 is often overexpressed in cancer cells conferring apoptosis resistance. Due to their exposed flexible arm containing selenocysteine, both cytosolic and mitochondrial TrxRs are inhibited by a large number of molecules. The various classes of inhibitors are listed and the molecules acting specifically on TrxR2 are extensively described. Particular emphasis is given to gold(I/III) complexes with phosphine, carbene or other ligands and to tamoxifen-like metallocifens. Also chemically unrelated organic molecules, including natural compounds and their derivatives, are taken into account. An important feature of many TrxR2 inhibitors is provided by their nature of delocalized lipophilic cations that allows their accumulation in mitochondria exploiting the organelle membrane potential. The consequences of TrxR2 inhibition are presented focusing especially on the impact on mitochondrial pathophysiology. Inhibition of TrxR2, by hindering the activity of Trx2 and Prx3, increases the mitochondrial concentration of reactive oxygen species and shifts the thiol redox state toward a more oxidized condition. This is reflected by alterations of specific targets involved in the release of pro-apoptotic factors such as cyclophilin D which acts as a regulator of the mitochondrial permeability transition pore. Therefore, the selective inhibition of TrxR2 could be utilized to induce cancer cell apoptosis.

Oxidative changes in lipids, proteins, and antioxidants in yogurt during the shelf life.

Oxidation processes in milk and yogurt during the shelf life can result in an alteration of protein and lipid constituents. Therefore, the antioxidant properties of yogurt in standard conditions of preservation were evaluated. Total phenols, free radical scavenger activity, degree of lipid peroxidation, and protein oxidation were determined in plain and skim yogurts with or without fruit puree. After production, plain, skim, plain berries, and skim berries yogurts were compared during the shelf life up to 9 weeks. All types of yogurts revealed a basal antioxidant activity that was higher when a fruit puree was present but gradually decreased during the shelf life. However, after 5-8 weeks, antioxidant activity increased again. Both in plain and berries yogurts lipid peroxidation increased until the seventh week of shelf life and after decreased, whereas protein oxidation of all yogurts was similar either in the absence or presence of berries and increased during shelf life. During the shelf life, a different behavior between lipid and protein oxidation takes place and the presence of berries determines a protection only against lipid peroxidation.

Characterization of Hydrophilic Gold(I) N-Heterocyclic Carbene (NHC) Complexes as Potent TrxR Inhibitors Using Biochemical and Mass Spectrometric Approaches.

We report here on the synthesis of a series of mono- and dinuclear gold(I) complexes exhibiting sulfonated bis(NHC) ligands and novel hydroxylated mono(NHC) Au(I) compounds, which were also examined for their biological activities. Initial cell viability assays show strong antiproliferative activities of the hydroxylated mono(NHC) gold compounds (8 > 9 > 10) against 2008 human ovarian cancer cells even after 1 h incubation. In order to gain insight into the mechanism of biological action of the gold compounds, their effect on the pivotal cellular target seleno-enzyme thioredoxin reductase (TrxR), involved in the maintenance of intracellular redox balance, was investigated in depth. The compounds' inhibitory effects on TrxR and glutathione reductase (GR) were studied comparatively, using either the pure proteins or cancer cell extracts. The results show a strong and selective inhibitory effect of TrxR, specifically for the hydroxyl-functionalized NHC gold(I) complexes (8-10). Valuable information on the gold compounds' molecular reactivity with TrxR was gained using the BIAM (biotin-conjugated iodoacetamide) assay and performing competition experiments by mass spectrometry (MS). In good agreement, both techniques suggest the binding affinity of the mono(NHC) Au(I) complexes toward selenols and thiols. Notably, for the first time, bis-carbene formation from mono-carbenes in buffered solution could be observed by MS, which may provide new insights into the speciation mechanisms of bioactive Au(I) NHC complexes. Furthermore, the compounds' interactions with another relevant in cellulo target, namely telomeric G-quadruplex DNA-a higher-order DNA structure playing key roles in telomere function-was investigated by means of FRET melting assays. The lack of interactions with this type of nucleic acid secondary structure support the idea of selective targeting of the hydrophilic Au(I) NHC compounds toward proteins such as TrxR.

Exploring the C

A family of cyclometalated Au(III) complexes featuring a tridentate C^N^C scaffold has been synthesized and characterized. Microwave assisted synthesis of the ligands has also been exploited and optimized. The biological properties of the thus formed compounds have been studied in cancer cells and demonstrate generally moderate antiproliferative effects. Initial mechanistic insights have also been gained on the gold complex [Au(C^N^C)(GluS)] (3), and support the idea that the thioredoxin system may be a target for this family of compounds together with other relevant intracellular thiol-containing molecules.

Tamoxifen-like metallocifens target the thioredoxin system determining mitochondrial impairment leading to apoptosis in Jurkat cells.

Tamoxifen-like metallocifens (TLMs) of the group-8 metals (Fe, Ru, and Os) show strong anti-proliferative activity on cancer cell lines resistant to apoptosis, owing to their unique redox properties. In contrast, the thioredoxin system, which is involved in cellular redox balance, is often overexpressed in cancer cells, especially in tumour types resistant to standard chemotherapies. Therefore, we investigated the effect of these three TLMs on the thioredoxin system and evaluated the input of the metallocene unit in comparison with structurally related organic tamoxifens. In vitro, all three TLMs became strong inhibitors of the cytosolic (TrxR1) and mitochondrial (TrxR2) isoforms of thioredoxin reductase after enzymatic oxidation with HRP/H2O2 while none of the organic analogues was effective. In Jurkat cells, TLMs inhibited mainly TrxR2, resulting in the accumulation of oxidized thioredoxin 2 and cell redox imbalance. Overproduction of ROS resulted in a strong decrease in the mitochondrial membrane potential, translocation of cytochrome c to the cytosol and activation of caspase 3, thus leading to apoptosis. None of these events occurred with organic tamoxifens. The mitochondrial fraction of cells exposed to TLMs contained a high amount of the corresponding metal, as quantified by ICP-OES. The lipophilic and cationic character associated with the singular redox properties of the TLMs could explain why they alter the mitochondrial function. These results provide new insights into the mechanism of action of tamoxifen-like metallocifens, underlying their prodrug behaviour and the pivotal role played by the metallocenic entity in their cytotoxic activity associated with the induction of apoptosis.

Enzymatic oxidation of ansa-ferrocifen leads to strong and selective thioredoxin reductase inhibition in vitro.

This paper reports the inhibitory effect on the cytosolic thioredoxin reductase (TrxR1) in vitro by the ansa-ferrocifen derivative (ansa-FcdiOH, 1). We found that 1 decreased only slightly enzyme activity (IC50=8µM), while 1*, the species generated by enzymatic oxidation by the HRP (horseradish peroxidase)/H2O2 mixture, strongly inhibited TrxR1 (IC50=0.15µM). At the same concentrations, neither 1 nor 1* had effect on glutathione reductase (GR). The most potent TrxR1 inhibitor did not appear to be the corresponding quinone methide as it was the case for ferrocifens of the acyclic series, or the stabilized carbocation as in the osmocifen series, but rather the quinone methide radical. This hypothesis was confirmed by ab-initio calculations of the species generated by oxidation of 1 and by EPR spectroscopy. BIAM (biotin-conjugated iodoacetamide) assay showed that 1* targeted both cysteine and selenocysteine of the C-terminal redox center of TrxR1.

Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells.

The synthesis and the biological effects of two ferrocifen analogs in the osmium series, namely the monophenolic complex 1, the tamoxifen-like complex 2 and their oxidized quinone methide (QM) derivatives, 1-QM and 2-QM, are reported. Inhibition of purified thioredoxin reductase (TrxR) is observed with 1 and 2 only after their enzymatic oxidation by the hydrogen peroxide/horseradish peroxidase (H2O2/HRP) system with IC50 of 2.4 and 1.2µM respectively. However, this inhibition is larger than that obtained with the corresponding quinone methides (IC50=5.4µM for 1-QM and 3.6µM for 2-QM). The UV-Vis spectra of 1 or 2 incubated in the presence of H2O2/HRP show that the species generated is not a quinone methide, but probably the corresponding cation. In Jurkat cells, 2 shows high toxicity (IC50=7.4µM), while 1 is less effective (IC50=42µM). Interestingly, a significant inhibition of TrxR activity is observed in cells incubated with 2 (about 70% inhibition with 15µM) while the inhibition induced by 1 is much weaker (about 30% inhibition with 50µM). This strong inhibition of TrxR by 2 leads to accumulation of thioredoxin and peroxiredoxin 3 in oxidized form and to a decrease of the mitochondrial membrane potential (MMP). These results show that cytotoxicity of the osmocifens depends on their oxidation within the cell and that inhibition of thioredoxin reductase by oxidized species is a key factor in rationalizing the cytotoxicity of these complexes on Jurkat cells.

Comparative effects of selenate and selenite on selenium accumulation, morphophysiology, and glutathione synthesis in Ulva australis.

The capacity of Ulva australis Areschoug to tolerate and accumulate selenium (Se) supplied in the form of selenate or selenite was investigated. The macroalga was provided for 3 and 7 days with concentrations of selenate (Na2SeO4) or selenite (Na2SeO3) ranging from 0 to 400 µM. U. australis exhibited the highest ability to accumulate selenium when fed with 100 µM selenate and 200 µM selenite after 7 days, and accumulation values were respectively 25 and 36 ppm Se. At the same concentrations, stimulation of the synthesis of chlorophylls and carotenoids was observed. Elevated doses of selenate or selenite decreased Se accumulation inside algal cells, perhaps through repression of membrane transporters. This effect was more pronounced in thalli cultivated with selenate. There were no morphological and ultrastructural alterations in thalli exposed to Se. However, selenite induced the increase of the oxidized fraction of glutathione (GSSG), perhaps because of its capacity to bind the thiol group of reduced glutathione (GSH). In conclusion, this study highlights the capacity of U. australis to resist to very high concentrations of selenite and selenate, which are normally toxic to other organisms. Also, the lack of bioconcentration in U. australis indicates that this alga does not facilitate delivery of Se in the food chain and remains safe for consumption when it grows in water bodies contaminated with Se. Its potential for the removal of excess Se from water bodies appears limited.

Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State.

The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.