Activated macrophages upregulate inducible nitric oxide synthase (iNOS) leading to the profuse production of nitric oxide (NO) and, eventually, tissue damage. Using macrophage NO production as a biochemical marker of inflammation, we tested different parts (flower, leaf, and stem) of the medicinal plant, Spilanthes acmella. We found that extracts prepared from all three parts, especially the flowers, suppressed NO production in RAW macrophages in response to interferon-γ and lipopolysaccharide. Follow up experiments with selected bioactive molecules from the plant (α-amyrin, β-caryophylline, scopoletin, vanillic acid, trans-ferulic acid, and spilanthol) indicated that the N-alkamide, spilanthol, is responsible for the NO-suppressive effects and provides protection from NO-dependent cell death. Spilanthol reduced the expression of iNOS mRNA and protein and, as a possible underlying mechanism, inhibited the activation of several transcription factors (NFκB, ATF4, FOXO1, IRF1, ETS, and AP1) and sensitized cells to downregulation of Smad (TF array experiments). The iNOS inhibitory effect translated into an anti-inflammatory effect, as demonstrated in a phorbol 12-myristate 13-acetate-induced dermatitis and, to a smaller extent, in cerulein-induced pancreatitis. In summary, we demonstrate that spilanthol inhibits iNOS expression, NO production and suppresses inflammatory TFs. These events likely contribute to the observed anti-inflammatory actions of spilanthol in dermatitis and pancreatitis.

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1^−/− cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD⁺ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1^−/− cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1^−/− cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.

Diabetes is accompanied by fundamental rearrangements in redox homeostasis. Hyperglycemia triggers the production of reactive oxygen and nitrogen species which contributes to tissue damage in various target organs. Proliferative diabetic retinopathy (PDR) is a common manifestation of diabetic complications but information on the possible role of reactive intermediates in this condition with special regard to the involvement of the vitreous in PDR-associated redox alterations is scarce. The aim of the study was to determine key parameters of redox homeostasis [advanced glycation endproducts (AGE); protein carbonyl and glutathione (GSH)] content in the vitreous in PDR patients. The study population involved 10 diabetic patients undergoing surgery for complications of proliferative diabetic retinopathy and 8 control (non-diabetic) patients who were undergoing surgery for epiretinal membranes. Vitreal fluids were assayed for the above biochemical parameters. We found elevated levels of AGE in the vitreous of PDR patients (812.10 vs 491.69ng AGE/mg protein). Extent of protein carbonylation was also higher in the samples of diabetic patients (2.08 vs 0.67A/100μg protein). The GSH content also increased in the vitreous of PDR patients as compared to the control group (4.54 vs 2.35μmol/μg protein), respectively. The study demonstrates that diabetes-associated redox alterations also reach the vitreous with the most prominent changes being increased protein carbonylation and increased antioxidant levels.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 μM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H₂O₂-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1β, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 μM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1β (IL1β), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

Background and Purpose: Excessive oxidative stress can induce PARP1‐mediated programmed necrotic cell death, termed parthanatos. Inhibition of parthanatos may be therapeutically beneficial in a wide array of diseases associated with tissue injury and inflammation. Our goal was to identify novel molecules inhibiting parthanatos.Experimental Approach: A small library of 774 pharmacologically active compounds was screened in a Sytox Green uptake assay, which identified 20 hits that reduced hydrogen‐peroxide‐induced parthanatos with an efficiency comparable to the benchmark PARP inhibitor, PJ34.Key Results: Of these hits, two compounds, antifungal ciclopirox and dopamine receptor agonist apomorphine, inhibited PAR polymer synthesis. These two compounds prevented the binding of PARP1 to oxidatively damaged DNA but did not directly interfere with the interaction between DNA and PARP1. Both compounds inhibited mitochondrial superoxide and H₂O₂ production and suppressed DNA breakage. Since H₂O₂‐induced damage is dependent on Fe²⁺‐catalysed hydroxyl radical production (Fenton chemistry), we determined the iron chelation activity of the two test compounds and found that ciclopirox and, to a lesser extent, apomorphine act as iron chelators. We also show that the Fe²⁺ chelation and indirect PARP inhibitory effects of ciclopirox translate to anti‐inflammatory actions as demonstrated in a mouse dermatitis model, where ciclopirox reduced ear swelling, inflammatory cell recruitment and poly(ADP‐ribosyl)ation.Conclusion and Implications: Our findings indicate that the antimycotic drug, ciclopirox, acts as an iron chelator and thus targets an early event in hydrogen‐peroxide‐induced parthanatos. Ciclopirox has the potential to be repurposed as a cytoprotective and anti‐inflammatory agent.

Breast cancer patients are characterized by the oncobiotic transformation of multiple microbiome communities, including the gut microbiome. Oncobiotic transformation of the gut microbiome impairs the production of antineoplastic bacterial metabolites. The goal of this study was to identify bacterial metabolites with antineoplastic properties. We constructed a 30-member bacterial metabolite library and screened the library compounds for effects on cell proliferation and epithelial-mesenchymal transition. The metabolites were applied to 4T1 murine breast cancer cells in concentrations corresponding to the reference serum concentrations. However, yric acid, glycolic acid, d-mannitol, 2,3-butanediol, and trans-ferulic acid exerted cytostatic effects, and 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, and vanillic acid exerted hyperproliferative effects. Furthermore, 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 2,3-butanediol, and hydrocinnamic acid inhibited epithelial-to-mesenchymal (EMT) transition. We identified redox sets among the metabolites (d-mannitol—d-mannose, 1-butanol—butyric acid, ethylene glycol—glycolic acid—oxalic acid), wherein only one partner within the set (d-mannitol, butyric acid, glycolic acid) possessed bioactivity in our system, suggesting that changes to the local redox potential may affect the bacterial secretome. Of the nine bioactive metabolites, 2,3-butanediol was the only compound with both cytostatic and anti-EMT properties.