Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene.

Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.

Altered Mitochondrial Dynamic in Lymphoblasts and Fibroblasts Mutated for FANCA-A Gene: The Central Role of DRP1.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and aplastic anemia. So far, 23 genes are involved in this pathology, and their mutations lead to a defect in DNA repair. In recent years, it has been observed that FA cells also display mitochondrial metabolism defects, causing an accumulation of intracellular lipids and oxidative damage. However, the molecular mechanisms involved in the metabolic alterations have not yet been elucidated. In this work, by using lymphoblasts and fibroblasts mutated for the FANC-A gene, oxidative phosphorylation (OxPhos) and mitochondria dynamics markers expression was analyzed. Results show that the metabolic defect does not depend on an altered expression of the proteins involved in OxPhos. However, FA cells are characterized by increased uncoupling protein UCP2 expression. FANC-A mutation is also associated with DRP1 overexpression that causes an imbalance in the mitochondrial dynamic toward fission and lower expression of Parkin and Beclin1. Treatment with P110, a specific inhibitor of DRP1, shows a partial mitochondrial function recovery and the decrement of DRP1 and UCP2 expression, suggesting a pivotal role of the mitochondrial dynamics in the etiopathology of Fanconi anemia.

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells.

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.

Lubeluzole Repositioning as Chemosensitizing Agent on Multidrug-Resistant Human Ovarian A2780/DX3 Cancer Cells.

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy.

The Hormetic Effect of Metformin: "Less Is More"?

Metformin (MTF) is the first-line therapy for type 2 diabetes (T2DM). The euglycemic effect of MTF is due to the inhibition of hepatic glucose production. Literature reports that the principal molecular mechanism of MTF is the activation of 5'-AMP-activated protein kinase (AMPK) due to the decrement of ATP intracellular content consequent to the inhibition of Complex I, although this effect is obtained only at millimolar concentrations. Conversely, micromolar MTF seems to activate the mitochondrial electron transport chain, increasing ATP production and limiting oxidative stress. This evidence sustains the idea that MTF exerts a hormetic effect based on its concentration in the target tissue. Therefore, in this review we describe the effects of MTF on T2DM on the principal target organs, such as liver, gut, adipose tissue, endothelium, heart, and skeletal muscle. In particular, data indicate that all organs, except the gut, accumulate MTF in the micromolar range when administered in therapeutic doses, unmasking molecular mechanisms that do not depend on Complex I inhibition.

Exploring Charged Polymeric Cyclodextrins for Biomedical Applications.

Over the years, cyclodextrin uses have been widely reviewed and their proprieties provide a very attractive approach in different biomedical applications. Cyclodextrins, due to their characteristics, are used to transport drugs and have also been studied as molecular chaperones with potential application in protein misfolding diseases. In this study, we designed cyclodextrin polymers containing different contents of ß- or γ-cyclodextrin, and a different number of guanidinium positive charges. This allowed exploration of the influence of the charge in delivering a drug and the effect in the protein anti-aggregant ability. The polymers inhibit Amiloid ß peptide aggregation; such an ability is modulated by both the type of CyD cavity and the number of charges. We also explored the effect of the new polymers as drug carriers. We tested the Doxorubicin toxicity in different cell lines, A2780, A549, MDA-MB-231 in the presence of the polymers. Data show that the polymers based on γ-cyclodextrin modified the cytotoxicity of doxorubicin in the A2780 cell line.

Cyclodextrin Polymers as Delivery Systems for Targeted Anti-Cancer Chemotherapy.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and ß-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

Advanced Analysis and Validation of a microRNA Signature for Fanconi Anemia.

Some years ago, we reported the generation of a Fanconi anemia (FA) microRNA signature. This study aims to develop an analytical strategy to select a smaller and more reliable set of molecules that could be tested for potential benefits for the FA phenotype, elucidate its biochemical and molecular mechanisms, address experimental activity, and evaluate its possible impact on FA therapy. In silico analyses of the data obtained in the original study were thoroughly processed and anenrichment analysis was employed to identify the classes of genes that are over-represented in the FA-miRNA population under study. Primary bone marrow mononuclear cells (MNCs) from sixFA patients and sixhealthy donors as control samples were employed in the study. RNAs containing the small RNA fractions were reverse-transcribed and real-time PCR was performed in triplicate using the specific primers. Experiments were performed in triplicate.The in-silico analysis reported six miRNAs as likely contributors to the complex pathological spectrum of FA. Among these, three miRNAs were validated by real-time PCR. Primary bone marrow mononuclear cells (MNCs) reported a significant reduction in the expression level of miRNA-1246 and miRNA-206 in the FA samples in comparison to controls.This study highlights several biochemical pathways as culprits in the phenotypic manifestations and the pathophysiological mechanisms acting in FA. A relatively low number of miRNAs appear involved in all these different phenotypes, demonstrating the extreme plasticity of the gene expression modulation. This study further highlights miR-206 as a pivotal player in regulatory functions and signaling in the bone marrow mesenchymal stem cell (BMSC) process in FA. Due to this evidence, the activity of miR-206 in FA deserves specific experimental scrutiny. The results, here presented, might be relevant in the management of FA.

The Glucose-Glutamine Metabolic Interplay in MCF-7 Cells, a Hormone-Sensitive Breast Cancer Model.

BACKGROUND: Selective deprivation of glutamine has been shown to accelerate the generation of reactive oxygen species (ROS) and to impair the activity of a specific pentose phosphate pathway (PPP) located within the endoplasmic reticulum (ER). The consequent oxidative damage suggests that glucose flux through this reticular pathway might contribute to the redox stress of breast cancer cells. We thus evaluated whether this response is reproduced when the glutamine shortage is coupled with the glucose deprivation. METHODS: Cancer growth, metabolic plasticity and redox status were evaluated under saturating conditions and after 48 h starvation (glucose 2.5 mM, glutamine 0.5 mM). The Seahorse technology was used to estimate adenosine triphosphate (ATP)-linked and ATP-independent oxygen consumption rate (OCR) as well as proton efflux rate (PER). 18F-fluoro-deoxy-glucose (FDG) uptake was evaluated through the LigandTracer device. Proliferation rate was estimated by the carboxyfluorescein-diacetate-succinimidyl ester (CFSE) staining, while cell viability by the propidium iodide exclusion assay. RESULTS: Starvation reduced the proliferation rate of MCF-7 cells without affecting their viability. It also decreased lactate release and PER. Overall OCR was left unchanged although ATP-synthase dependent fraction was increased under nutrient shortage. Glutaminolysis inhibition selectively impaired the ATP-independent and the oligomycin-sensitive OCR in control and starved cultures, respectively. The combined nutrient shortage decreased the cytosolic and mitochondrial markers of redox stress. It also left unchanged the expression of the reticular unfolded protein marker GRP78. By contrast, starvation decreased the expression of hexose-6P-dehydrogenase (H6PD) thus decreasing the glucose flux through the ER-PPP as documented by the profound impairment in the uptake rate of FDG. CONCLUSIONS: When combined with glucose deprivation, glutamine shortage does not elicit the expected enhancement of ROS generation in the studied breast cancer cell line. Combined with the decreased activity of ER-PPP, this observation suggests that glutamine interferes with the reticular glucose metabolism to regulate the cell redox balance.

Terpyridine Functionalized Cyclodextrin Nanoparticles as Carriers for Doxorubicin: Analysis of In Vitro Antiproliferative Activity.

BACKGROUND/AIM: We have recently described the development of cyclodextrin-based nanoparticles (NPs) functionalized with terpyridine and decorated with biotin-terpyridine ligands via Cu(II) and Fe(II) coordination. In the present study, we report the performance of these novel NPs as a delivery system for anticancer drugs. In particular, we analyzed the feasibility of loading these new NPs with the topoisomerase II inhibitor Doxorubicin (Doxo), still administered to patients to treat different forms of cancers. We developed Doxo-encapsulated polymeric NPS to generate nanoformulations with higher efficacy than free Doxo. MATERIALS AND METHODS: We investigated the inhibition of cell proliferation in A2780, A549, SKHep1, and MDA-MB-453 cancer cell lines using the MTT assay. RESULTS: NPs loaded with Doxo displayed higher antiproliferative activity than free Doxo. CONCLUSION: The NPs generated in this study inhibited the proliferation of cancer cells and were able to entrap the classic anticancer drug Doxo. The Doxo-loaded NP showed increased cytotoxicity in comparison to free Doxo.

Crosstalk between the Rod Outer Segments and Retinal Pigmented Epithelium in the Generation of Oxidative Stress in an In Vitro Model.

Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE.

Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by a dysfunctional DNA repair and an oxidative stress accumulation due to defective mitochondrial energy metabolism, not counteracted by endogenous antioxidant defenses, which appear down-expressed compared to the control. Since the antioxidant response lack could depend on the hypoacetylation of genes coding for detoxifying enzymes, we treated lymphoblasts and fibroblasts mutated for the FANC-A gene with some histone deacetylase inhibitors (HDACi), namely, valproic acid (VPA), beta-hydroxybutyrate (OHB), and EX527 (a Sirt1 inhibitor), under basal conditions and after hydrogen peroxide addition. The results show that VPA increased catalase and glutathione reductase expression and activity, corrected the metabolic defect, lowered lipid peroxidation, restored the mitochondrial fusion and fission balance, and improved mitomycin survival. In contrast, OHB, despite a slight increase in antioxidant enzyme expressions, exacerbated the metabolic defect, increasing oxidative stress production, probably because it also acts as an oxidative phosphorylation metabolite, while EX527 showed no effect. In conclusion, the data suggest that VPA could be a promising drug to modulate the gene expression in FA cells, confirming that the antioxidant response modulation plays a pivotal in FA pathogenesis as it acts on both oxidative stress levels and the mitochondrial metabolism and dynamics quality.

Plasma-derived extracellular vesicles released after endurance exercise exert cardioprotective activity through the activation of antioxidant pathways.

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H2O2) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.

Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.

Cyclic fasting bolsters cholesterol biosynthesis inhibitors' anticancer activity.

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology.

Mutated FANCA Gene Role in the Modulation of Energy Metabolism and Mitochondrial Dynamics in Head and Neck Squamous Cell Carcinoma.

Fanconi Anaemia (FA) is a rare recessive genetic disorder characterized by a defective DNA repair mechanism. Although aplastic anaemia is the principal clinical sign in FA, patients develop a head and neck squamous cell carcinoma (HNSCC) with a frequency 500-700 folds higher than the general population, which appears more aggressive, with survival of under two years. Since FA gene mutations are also associated with a defect in the aerobic metabolism and an increased oxidative stress accumulation, this work aims to evaluate the effect of FANCA mutation on the energy metabolism and the relative mitochondrial quality control pathways in an HNSCC cellular model. Energy metabolism and cellular antioxidant capacities were evaluated by oximetric, luminometric, and spectrophotometric assays. The dynamics of the mitochondrial network, the quality of mitophagy and autophagy, and DNA double-strand damage were analysed by Western blot analysis. Data show that the HNSCC cellular model carrying the FANCA gene mutation displays an altered electron transport between respiratory Complexes I and III that does not depend on the OxPhos protein expression. Moreover, FANCA HNSCC cells show an imbalance between fusion and fission processes and alterations in autophagy and mitophagy pathways. Together, all these alterations associated with the FANCA gene mutation cause cellular energy depletion and a metabolic switch to glycolysis, exacerbating the Warburg effect in HNSCC cells and increasing the growth rate. In addition, the altered DNA repair due to the FANCA mutation causes a higher accumulation of DNA damage in the HNSCC cellular model. In conclusion, changes in energy metabolism and mitochondrial dynamics could explain the strict correlation between HNSCC and FA genes, helping to identify new therapeutic targets.

Terpyridine functionalized cyclodextrin nanoparticles: metal coordination for tuning anticancer activity.

Multi-metal and multi-cavity systems based on the coordination properties of terpyridine functionalized cyclodextrin polymers were synthesized and characterized. Nanoparticles decorated with terpyridine derivatives via metal coordination showed high antiproliferative activity in tumor cells.

Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging.

Inflammaging is one of the evolutionarily conserved mechanisms underlying aging and is defined as the long-term consequence of the chronic stimulation of the innate immune system. As macrophages are intimately involved in initiating and regulating the inflammatory process, their dysregulation plays major roles in inflammaging. The paracrine factors, and in particular extracellular vesicles (EVs), released by mesenchymal stromal cells (MSCs) retain immunoregulatory effects on innate and adaptive immune responses. In this paper, we demonstrate that EVs derived from MSCs preconditioned with hypoxia inflammatory cytokines exerted an anti-inflammatory role in the context of inflammaging. In this study, macrophages isolated from aged mice presented elevated pro-inflammatory factor levels already in basal conditions compared to the young counterpart, and this pre-activation status increased when cells were challenged with IFN-γ. EVs were able to attenuate the age-associated inflammation, inducing a decrease in the expression of TNF-α, iNOS, and the NADase CD38. Moreover, we demonstrate that EVs counteracted the mitochondrial dysfunction that affected the macrophages, reducing lipid peroxidation and hindering the age-associated impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis. These results indicate that preconditioned MSC-derived EVs might be exploited as new anti-aging therapies in a variety of age-related diseases.

Identification of NAPRT Inhibitors with Anti-Cancer Properties by In Silico Drug Discovery.

Depriving cancer cells of sufficient NAD levels, mainly through interfering with their NAD-producing capacity, has been conceived as a promising anti-cancer strategy. Numerous inhibitors of the NAD-producing enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been developed over the past two decades. However, their limited anti-cancer activity in clinical trials raised the possibility that cancer cells may also exploit alternative NAD-producing enzymes. Recent studies show the relevance of nicotinic acid phosphoribosyltransferase (NAPRT), the rate-limiting enzyme of the Preiss-Handler NAD-production pathway for a large group of human cancers. We demonstrated that the NAPRT inhibitor 2-hydroxynicotinic acid (2-HNA) cooperates with the NAMPT inhibitor FK866 in killing NAPRT-proficient cancer cells that were otherwise insensitive to FK866 alone. Despite this emerging relevance of NAPRT as a potential target in cancer therapy, very few NAPRT inhibitors exist. Starting from a high-throughput virtual screening approach, we were able to identify and annotate two additional chemical scaffolds that function as NAPRT inhibitors. These compounds show comparable anti-cancer activity to 2-HNA and improved predicted aqueous solubility, in addition to demonstrating favorable drug-like profiles.