Due to their beneficial properties, fermented foods are considered important constituents of the human diet. They also contain bioactive peptides, health-promoting compounds studied for a wide range of effects. In this work, several antioxidant peptides extracted from fermented milk proteins were investigated. First, enriched peptide fractions were purified and analysed for their antioxidant capacity in vitro and in a cellular model. Subsequently, from the most active fractions, 23 peptides were identified by mass spectrometry MS/MS), synthesized and tested. Peptides N-15-M, E-11-F, Q-14-R and A-17-E were selected for their antioxidant effects on Caco-2 cells both in the protection against oxidative stress and inhibition of ROS production. To define their action mechanism, the activation of the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2(Keap1/Nrf2) pathway was studied evaluating the translocation of Nrf2 from cytosol to nucleus. In cells treated with N-15-M, Q-14-R and A-17-E, a higher amount of Nrf2 was found in the nucleus with respect to the control. In addition, the three active peptides, through the activation of Keap1/Nrf2 pathway, led to overexpression and increased activity of antioxidant enzymes. Molecular docking analysis confirmed the potential ability of N-15-M, Q-14-R and A-17-E to bind Keap1, showing their destabilizing effect on Keap1/Nrf2 interaction.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. While the exact causes of ALS are still unclear, the discovery that familial cases of ALS are related to mutations in the Cu/Zn superoxide dismutase (SOD1), a key antioxidant enzyme protecting cells from the deleterious effects of superoxide radicals, suggested that alterations in SOD1 functionality and/or aberrant SOD1 aggregation strongly contribute to ALS pathogenesis. A new scenario was opened in which, thanks to the generation of SOD1 related models, different mechanisms crucial for ALS progression were identified. These include excitotoxicity, oxidative stress, mitochondrial dysfunctions, and non-cell autonomous toxicity, also implicating altered Ca²⁺ metabolism. While most of the literature considers motor neurons as primary target of SOD1-mediated effects, here we mainly discuss the effects of SOD1 mutations in non-neuronal cells, such as glial and skeletal muscle cells, in ALS. Attention is given to the altered redox balance and Ca²⁺ homeostasis, two processes that are strictly related with each other. We also provide original data obtained in primary myocytes derived from hSOD1(G93A) transgenic mice, showing perturbed expression of Ca²⁺ transporters that may be responsible for altered mitochondrial Ca²⁺ fluxes. ALS-related SOD1 mutants are also responsible for early alterations of fundamental biological processes in skeletal myocytes that may impinge on skeletal muscle functions and the cross-talk between muscle cells and motor neurons during disease progression.

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.

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.

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.