The Anti-Inflammatory Effect of Lactose-Modified Hyaluronic Acid Molecules on Primary Bronchial Fibroblasts of Smokers.

The progression of smoking-related diseases is characterized by macrophage-mediated inflammation, which is responsible for an increased expression of proinflammatory cytokines and galectins, molecules that bind specifically to ß-galactoside sugars. This study aimed to assess the anti-inflammatory and antioxidant effects of a broad selection of differently lactose-modified hyaluronic acids (HA) named HYLACH®, which are able to bind proinflammatory galectins. The best HYLACH ligands for Gal-3 were selected in silico and their activities were tested in vitro on primary human bronchial fibroblasts obtained from smokers and inflamed with the conditioned medium of activated U937 monocytes. Changes in cell viability, ROS generation, proinflammatory mediators, and MMP expression, at both gene and protein levels, were analyzed. The in silico results show that HYLACH with a percentage of lactosylation of 10-40% are the best ligands for Gal-3. The in vitro study revealed that HYLACH compounds with 10, 20, and 40% lactosylation (HYLACH-1-2-3) administrated to inflamed cell cultures counteracted the oxidative damage and restored gene and protein expression for IL-1ß, TNF-α, IL-6, Gal-1, Gal-3, and MMP-3 to near baseline values. The evidence that HYLACH attenuated macrophage-induced inflammation, inhibited MMP expression, and exhibited antioxidative effects provide an initial step toward the development of a therapeutic treatment suitable for smoking-related diseases.

PTC596-Induced BMI-1 Inhibition Fights Neuroblastoma Multidrug Resistance by Inducing Ferroptosis.

Neuroblastoma (NB) is a paediatric cancer with noteworthy heterogeneity ranging from spontaneous regression to high-risk forms that are characterised by cancer relapse and the acquisition of drug resistance. The most-used anticancer drugs exert their cytotoxic effect by inducing oxidative stress, and long-term therapy has been demonstrated to cause chemoresistance by enhancing the antioxidant response of NB cells. Taking advantage of an in vitro model of multidrug-resistant (MDR) NB cells, characterised by high levels of glutathione (GSH), the overexpression of the oncoprotein BMI-1, and the presence of a mutant P53 protein, we investigated a new potential strategy to fight chemoresistance. Our results show that PTC596, an inhibitor of BMI-1, exerted a high cytotoxic effect on MDR NB cells, while PRIMA-1MET, a compound able to reactivate mutant P53, had no effect on the viability of MDR cells. Furthermore, both PTC596 and PRIMA-1MET markedly reduced the expression of epithelial-mesenchymal transition proteins and limited the clonogenic potential and the cancer stemness of MDR cells. Of particular interest is the observation that PTC596, alone or in combination with PRIMA-1MET and etoposide, significantly reduced GSH levels, increased peroxide production, stimulated lipid peroxidation, and induced ferroptosis. Therefore, these findings suggest that PTC596, by inhibiting BMI-1 and triggering ferroptosis, could be a promising approach to fight chemoresistance.

Anti-Inflammatory and Pro-Regenerative Effects of Hyaluronan-Chitlac Mixture in Human Dermal Fibroblasts: A Skin Ageing Perspective.

Inflammation and the accumulation of reactive oxygen species (ROS) play an important role in the structural and functional modifications leading to skin ageing. The reduction of inflammation, cellular oxidation and dermal extracellular matrix (ECM) alterations may prevent the ageing process. The aim of this study is to investigate the expression of pro-inflammatory markers and ECM molecules in human dermal fibroblasts derived from young and middle-aged women and the effects of lactose-modified chitosan (Chitlac®, CTL), alone or in combination with mid-MW hyaluronan (HA), using an in vitro model of inflammation. To assess the response of macrophage-induced inflamed dermal fibroblasts to HA and CTL, changes in cell viability, pro-inflammatory mediators, MMPs and ECM molecules expression and intracellular ROS generation are analysed at gene and protein levels. The expression of pro-inflammatory markers, galectins, MMP-3 and ECM molecules is age-related. CTL, HA and their combination counteracted the oxidative damage, stimulating the expression of ECM molecules, and, when added to inflamed cells, restored the baseline levels of IL-1ß, TNF-α, GAL-1, GAL-3 and MMP-3. In conclusion, HA and CTL mixture attenuated the macrophage-induced inflammation, inhibited the MMP-3 expression, exhibited the anti-oxidative effects and exerted a pro-regenerative effect on ECM.

In Vitro Effects of Low Doses of ß-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation.

ß-caryophyllene (BCP), a plant-derived sesquiterpene, has been reported to have anti-inflammatory and antioxidant effects. The purpose of this study is to evaluate the effects of BCP in combination with ascorbic acid (AA) and d-glucosamine (GlcN) against macrophage-mediated inflammation on in vitro primary human chondrocytes. Changes in cell viability, intracellular ROS generation, gene expression of pro-inflammatory mediators, metalloproteinases (MMPs), collagen type II and aggrecan were analyzed in primary human chondrocytes exposed to the conditioned medium (CM) of activated U937 monocytes and subsequently treated with BCP alone or in combination with AA and GlcN. The CM-induced chondrocyte cytotoxicity was reduced by the presence of low doses of BCP alone or in combination with AA and GlcN. The exposure of cells to CM significantly increased IL-1ß, NF-κB1 and MMP-13 expression, but when BCP was added to the inflamed cells, alone or in combination with AA and GlcN, gene transcription for all these molecules was restored to near baseline values. Moreover, chondrocytes increased the expression of collagen type II and aggrecan when stimulated with AA and GlcN alone or in combination with BCP. This study showed the synergistic anti-inflammatory and antioxidative effects of BCP, AA and GlcN at low doses on human chondrocyte cultures treated with the CM of activated U937 cells. Moreover, the combination of the three molecules was able to promote the expression of collagen type II and aggrecan. All together, these data could suggest that BCP, AA and GlcN exert a chondro-protective action.

Aerobic pyruvate metabolism sensitizes cells to ferroptosis primed by GSH depletion.

Ferroptosis is a non-accidental, regulated form of cell death operated by lipid peroxidation under strict control of GPx4 activity. This is consistent with the notion that lipid peroxidation is initiated by radicals produced from decomposition of traces of pre-existing lipid hydroperoxides. The question, therefore, emerges about the formation of these traces of lipid hydroperoxides interacting with Fe2+. In the most realistic option, they are produced by oxygen activated species generated during aerobic metabolism. Screening for metabolic sources of superoxide supporting ferroptosis induced by GSH depletion, we failed to detect, in our cell model, a role of respiratory chain. We observed instead that the pyruvate dehydrogenase complex -as other α keto acid dehydrogenases already known as a major source of superoxide in mitochondria- supports ferroptosis. The opposite effect on ferroptosis by silencing either the E1 or the E3 subunit of the pyruvate dehydrogenase complex pointed out the autoxidation of dihydrolipoamide as the source of superoxide. We finally observed that GSH depletion activates superoxide production, seemingly through the inhibition of the specific kinase that inhibits pyruvate dehydrogenase. In summary, this set of data is compatible with a scenario where the more electrophilic status produced by GSH depletion not only activates ferroptosis by preventing GPx4 activity, but also favors the formation of lipid hydroperoxides. In an attractive perspective of tissue homeostasis, it is the activation of energetic metabolism associated to a decreased nucleophilic tone that, besides supporting energy demanding proliferation, also sensitizes cells to a regulated form of death.

Anti-Inflammatory Performance of Lactose-Modified Chitosan and Hyaluronic Acid Mixtures in an In Vitro Macrophage-Mediated Inflammation Osteoarthritis Model.

The development and progression of osteoarthritis (OA) is associated with macrophage-mediated inflammation that generates a broad spectrum of cytokines and reactive oxygen species (ROS). This study investigates the effects of mid-MW hyaluronic acid (HA) in combination with a lactose-modified chitosan (CTL), on pro-inflammatory molecules and metalloproteinases (MMPs) expression, using an in vitro model of macrophage-mediated inflammation. METHODS: To assess chondrocyte response to HA and CTL in the presence of macrophage derived inflammatory mediators, cells were exposed to the conditioned medium (CM) of U937 activated monocytes and changes in cell viability, pro-inflammatory mediators and MMPs expression or ROS generation were analysed. RESULTS: CTL induced changes in chondrocyte viability that are reduced by the presence of HA. The CM of activated U937 monocytes (macrophages) significantly increased gene expression of pro-inflammatory molecules and MMPs and intracellular ROS generation in human chondrocyte cultures. HA, CTL and their combinations counteracted the oxidative damage and restored gene transcription for IL-1ß, TNF-α, Gal-1, MMP-3 and MMP-13 to near baseline values. CONCLUSIONS: This study suggests that HA-CTL mixture attenuated macrophage-induced inflammation, inhibited MMPs expression and exhibited anti-oxidative effects. This evidence provides an initial step toward the development of an early stage OA therapeutic treatment.

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3ε.

Ras-selective lethal small molecule 3 (RSL3), a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating the glutathione peroxidase glutathione peroxidase 4 (GPx4). Here, we report the purification of the protein indispensable for GPx4 inactivation by RSL3. Mass spectrometric analysis identified 14-3-3 isoforms as candidates, and recombinant human 14-3-3ε confirms the identification. The function of 14-3-3ε is redox-regulated. Moreover, overexpression or silencing of the gene coding for 14-3-3ε consistently controls the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein operating in cell signaling further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives.

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1.

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

Lack of glutathione peroxidase-8 in the ER impacts on lipid composition of HeLa cells microsomal membranes.

GPx8 is a glutathione peroxidase homolog inserted in the membranes of endoplasmic reticulum (ER), where it seemingly plays a role in controlling redox status by preventing the spill of H2O2. We addressed the impact of GPx8 silencing on the lipidome of microsomal membranes, using stably GPx8-silenced HeLa cells. The two cell lines were clearly separated by Principal Component Analysis (PCA) and Partial Least Square Discriminant analysis (PLS-DA) of lipidome. Considering in detail the individual lipid classes, we observed that unsaturated glycerophospholipids (GPL) decreased, while only in phosphatidylinositols (PI) a substitution of monounsaturated fatty acids (MUFA) for polyunsaturated fatty acids (PUFA) was observed. Among sphingolipids (SL), ceramides (CER) decreased while sphingomyelins (SM) and neutral glycophingolipids (nGSL) increased. Here, in addition, longer chains than in controls in the amide fatty acid were present. The increase up to four folds of the CER (d18:1; c24:0) containing three hexose units, was the most remarkable species increasing in the differential lipidome of siGPx8 cells. Quantitative RT-PCR complied with lipidomic analysis specifically showing an increased expression of: i) acyl-CoA synthetase 5 (ACSL5); ii) CER synthase 2 and 4; iii) CER transporter (CERT); iv) UDP-glucosyl transferase (UDP-GlcT), associated to a decreased expression of UDP-galactosyl transferase (UDP-GalT). A role of the unfolded protein response (UPR) and the spliced form of the transcription factor XBP1 on the transcriptional changes of GPx8 silenced cells was ruled-out. Similarly, also the involvement of Nrf2 and NF-κB. Altogether our results indicate that GPx8-silencing of HeLa yields a membrane depleted by about 24% of polyunsaturated GPL and a corresponding increase of saturated or monounsaturated SM and specific nGSL. This is tentatively interpreted as an adaptive mechanism leading to an increased resistance to radical oxidations. Moreover, the marked shift of fatty acid composition of PI emerges as a possibly relevant issue in respect to the impact of GPx8 on signaling pathways.

Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase.

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1-9) or methoctramine (10-14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 µM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

Redox status in a model of cancer stem cells.

Reversible oxidation of Cys residues is a crucial element of redox homeostasis and signaling. According to a popular concept in oxidative stress signaling, the oxidation of targets of signals can only take place following an overwhelming of the cellular antioxidant capacity. This concept, however, ignores the activation of feedback mechanisms possibly leading to a paradoxical effect. In a model of cancer stem cells (CSC), stably overexpressing the TAZ oncogene, we observed that the increased formation of oxidants is associated with a globally more reduced state of proteins. Redox proteomics revealed that several proteins, capable of undergoing reversible redox transitions, are indeed more reduced while just few are more oxidized. Among the proteins more oxidized, G6PDH emerges as both more expressed and activated by oxidation. This accounts for the observed more reduced state of the NADPH/NADP+ couple. The dynamic redox flux generating this apparently paradoxical effect is rationalized in a computational system biology model highlighting the crucial role of G6PDH activity on the rate of redox transitions eventually leading to the reduction of reversible redox switches.

Ternary Hybrid γ-Fe2 O3 /Cr(VI) /Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue.

Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts.

Glutathione peroxidase 8 is transcriptionally regulated by HIFα and modulates growth factor signaling in HeLa cells.

GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both the functionality of two hypoxia-response elements (HREs) within the promoter, GPx8 HRE1 and GPx8 HRE2, and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2'-bipyridyl (BP), induces GPx8 expression at both mRNA and protein level. Luciferase activity of pGL3(GPx8wt), containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8 HRE1 within pGL3(GPx8wt) resulted in a significantly higher inhibition of luciferase activity than mutating GPx8 HRE2. Electrophoretic mobility-shift assay showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8 HRE1. In DES-treated cells transfected with pGL3(GPx8wt) or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8 HREs, with GPx8 HRE1 playing the major role. Fibroblast growth factor (FGF) treatment increases GPx8 mRNA expression, and reporter gene experiments indicate that induction occurs via HIF. Comparing the effects of depleting GPx8 on the downstream effectors of FGF or insulin signaling revealed that absence of GPx8 results in a 16- or 12-fold increase in phosphorylated ERK1/2 by FGF or insulin treatment, respectively. Furthermore, in GPx8-depleted cells, phosphorylation of AKT by insulin treatment increases 2.5-fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.

Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern.

RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.