Measurement of Photorespiratory Cycle Enzyme Activities in Leaves Exposed to Abiotic Stress.

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included.

Mitochondrial Role on Cellular Apoptosis, Autophagy, and Senescence during Osteoarthritis Pathogenesis.

Authors have demonstrated that apoptosis activation is a pathway related to cartilage degradation characteristics of the OA process. Autophagy is an adaptive response to protect cells from various environmental changes, and defects in autophagy are linked to cell death. In this sense, decreased autophagy of chondrocytes has been observed in OA articular cartilage. The aim of this work was to study the role of OA mitochondria in apoptosis, autophagy, and senescence, using OA and Normal (N) transmitochondrial cybrids. Results: OA cybrids incubated with menadione showed a higher percentage of late apoptosis and necrosis than N cybrids. Stimulation of cybrids with staurosporine and IL-1ß showed that OA cybrids were more susceptible to undergoing apoptosis than N cybrids. An analysis of the antioxidant response using menadione on gene expression revealed a lower expression of nuclear factor erythroid 2-like 2 and superoxide dismutase 2 in OA than N cybrids. Activation of microtubule-associated protein 1A/1B-light chain 3 was reduced in OA compared to N cybrids. However, the percentage of senescent cells was higher in OA than N cybrids. Conclusion: This work suggests that mitochondria from OA patients could be involved in the apoptosis, autophagy, and senescence of chondrocytes described in OA cartilage.

A Combination of Biocatalysis and Fenton-Like Reaction Induced OH• Burst for Cascade Amplification of Cancer Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a novel antitumor strategy that employs Fenton or Fenton-like reactions to generate highly toxic hydroxyl radical (OH•) from hydrogen peroxide (H2O2) for inducing tumor cell death. However, the antitumor efficacy of the CDT strategy is harshly limited by the redox homeostasis of tumor cells; especially the OH • is easily scavenged by glutathione (GSH) and the intracellular H2O2 level is insufficient in the tumor cells. Herein, we propose the Mn2+-menadione (also known as vitamin K3, MK3) cascade biocatalysis strategy to disrupt the redox homeostasis of tumor cells and induce a OH• storm, resulting in enhanced CDT effect. A nanoliposome encapsulating Mn-MK3 (Mn-MK3@LP) was prepared for the treatment of hepatic tumors in this study. After Mn-MK3@LPs were taken up by tumor cells, menadione could facilitate the production of intracellular H2O2 via redox cycling, and further the cytotoxic OH • burst was induced by Mn2+-mediated Fenton-like reaction. Moreover, high-valent manganese ions were reduced by GSH and the depletion of GSH further disrupted the redox homeostasis of tumor cells, thus achieving synergistically enhanced CDT. Overall, both cellular and animal experiments confirmed that the Mn-MK3@LP cascade biocatalysis nanoliposome exhibited excellent biosafety and tumor suppression efficacy. This study may provide deep insights for developing novel CDT-based strategies for tumor therapy.

Flubendazole carbonyl reduction in drug-susceptible and drug-resistant strains of the parasitic nematode Haemonchus contortus: changes during the life cycle and possible inhibition.

Carbonyl-reducing enzymes (CREs) catalyse the reduction of carbonyl groups in many eobiotic and xenobiotic compounds in all organisms, including helminths. Previous studies have shown the important roles of CREs in the deactivation of several anthelmintic drugs (e.g., flubendazole and mebendazole) in adults infected with the parasitic nematode Haemonchus contortus, in which the activity of a CRE is increased in drug-resistant strains. The aim of the present study was to compare the abilities of nematodes of both a drug-susceptible strain (ISE) and a drug-resistant strain (IRE) to reduce the carbonyl group of flubendazole (FLU) in different developmental stages (eggs, L1/2 larvae, L3 larvae, and adults). In addition, the effects of selected CRE inhibitors (e.g., glycyrrhetinic acid, naringenin, silybin, luteolin, glyceraldehyde, and menadione) on the reduction of FLU were evaluated in vitro and ex vivo in H. contortus adults. The results showed that FLU was reduced by H. contortus in all developmental stages, with adult IRE females being the most metabolically active. Larvae (L1/2 and L3) and adult females of the IRE strain reduced FLU more effectively than those of the ISE strain. Data from the in vitro inhibition study (performed with cytosolic-like fractions of H. contortus adult homogenate) revealed that glycyrrhetinic acid, naringenin, mebendazole and menadione are effective inhibitors of FLU reduction. Ex vivo study data showed that menadione inhibited FLU reduction and also decreased the viability of H. contortus adults to a similar extent. Naringenin and mebendazole were not toxic at the concentrations tested, but they did not inhibit the reduction of FLU in adult worms ex vivo.

Menadione and protocatechuic acid: A drug combination with antitumor effects in murine osteosarcoma cells.

Osteosarcoma (OS) is a primary malignant bone tumor that has an abnormal expression of oncogenesis and tumor suppressors and causes dysregulation of various signaling pathways. Thus, novel therapeutic strategies for OS are needed to overcome the resistance of traditional treatments. This study evaluated the cytotoxic and anticancer effects of the association between menadione (MEN) and protocatechuic acid (PCA) in murine OS cells (UMR-106). The concentrations were 3.12 µM of isolated MEN, 500 µM of isolated PCA, and their associations. We performed cell viability assays, morphology modification analysis, cell migration by the wound-healing method, apoptosis by flow cytometry, reactive oxygen species (ROS) production, gene expression of NOX by RT-qPCR, and degradation of MMP-2 and 9 by zymography. Our results showed that the association of MEN+PCA was more effective in OS cells than the compounds alone. The association decreased cell viability, delayed cell migration, and decreased the expression of NOX-2 and ROS. In addition, the MEN+PCA association induced a slight increase in the apoptotic process. In summary, the association can enhance the compound's antitumor effects and establish a higher selectivity for tumor cells, possibly caused by significant mitochondrial damage and antioxidant properties.

Menadione Contribution to the In Vitro Radical Scavenging Potential of Phytochemicals Naringenin and Lignin.

Vitamin K3 (menadione), classified as a pro-vitamin, is a synthetic form of the fat-soluble family of vitamin K compounds. The combination of the vitamin with other molecules sharing structural and/or functional similarities, such as naturally occurring polyphenols, vitamins, or biopolymers, could potentiate mutual improvement of their antioxidant activity. The aim of the present study was to evaluate the role and contribution of vitamin K3 to the in vitro radical scavenging capacity of double and triple combinations with the phytochemicals naringenin and lignin, as well as assess possible intermolecular interactions between the bioactive compounds. Comparative analyses of the DPPH and ABTS radical scavenging activity of the pure substances vitamin K3, naringenin, and lignin; the two-component systems lignin/vitamin K3 and vitamin K3/naringenin; and the triple combination vitamin K3/flavonoid/lignin were carried out. The experimental results demonstrated increased DPPH and ABTS activities of the vitamin in combination with lignin compared to those of the two pure substances, i.e., a synergistic effect was observed. The registered significant increases in the radical scavenging activity of the triple combination determined via both methods are indicative of a remarkable potentiation effect, i.e., higher antioxidant potential exceeding the additive activity of the three pure substances.

Synergistic and Additive Effects of Menadione in Combination with Antibiotics on Multidrug-Resistant Staphylococcus aureus: Insights from Structure-Function Analysis of Naphthoquinones.

Antimicrobial resistance (AMR) interferes with the effective treatment of infections and increases the risk of microbial spread and infection-related illness and death. The synergistic activities of combinations of antimicrobial compounds offer satisfactory approaches to some extent. Structurally diverse naphthoquinones (NQs) including menadione (-CH3 group at C2) exhibit substantial antimicrobial activities against multidrug-resistant (MDR) pathogens. We explored the combinations of menadione with antibiotic ciprofloxacin or ampicillin against Staphylococcus aureus and its biofilms. We found an additive (0.590 %) were also observed. However, preformed biofilms were not affected. Dent formation was also evident in S. aureus treated with the test compounds. The structure-function relationship (SFR) of NQs was used to determine and predict their activity pattern against pathogens. Analysis of 10 structurally distinct NQs revealed that the compounds with -Cl, -Br, -CH3 , or -OH groups displayed the lowest MICs (32-256 µg/mL). Furthermore, 1,4-NQs possessing a halogen or -CH3 moiety showed elevated ROS activity, whereas molecules with an -OH group affected cell integrity. Improved activity of antimicrobial combinations and SFR approaches are significant in antimicrobial therapies.

In vitro and in vivo efficacy of a cosmetic product formulated with new lipid particles for the treatment of aged skin.

BACKGROUND: The cumulative oxidative damage causes an acceleration in the skin aging. OBJECTIVES: To evaluate the ability of a new patented matrix of lipid particles (SIREN CAPSULE TECHNOLOGY™) to have superior anti-aging properties due to its high sensitivity to reactive oxygen species (ROS), testing its efficacy versus free or encapsulated vitamins. METHODS: An in vitro study was conducted to evaluate the protective effects of lipid particles using menadione as an enhancer of oxidative stress. Subsequently, in vivo studies evaluated skin hydration, skin barrier function, and smoothness and wrinkle depth. For this purpose, gels containing free or encapsulated vitamins were used as controls. RESULTS: In vitro, the SIREN CAPSULE TECHNOLOGY™ gel shows inhibitory activity against ROS production through menadione induction. In fact, at both tested concentrations, ROS production is lower than in the control samples (placebo, free vitamins, encapsulated vitamins). In vivo, the net effect of SIREN CAPSULE TECHNOLOGY™ gel versus the others permitted to conclude that lipid particles exert a higher skin moisturizing effect (20.17%) and a stronger effect in reducing transepidermal water loss (-16.29%) after 4 weeks of treatment. As for surface analysis, a gel based on SIREN CAPSULE TECHNOLOGY™ improves the skin texture in a similar way than gel containing encapsulated vitamins (Ra and Rz variations in 4 weeks). CONCLUSIONS: SIREN CAPSULE TECHNOLOGY™ represents an advance and a successful strategy to develop cosmetic products for the treatment of skin conditions associated with an accumulation of ROS. SIREN CAPSULE TECHNOLOGY™ represents a result-oriented breakthrough in the effective delivery of active ingredients to the skin.

Phylloquinone is preferable over menadione as a growth factor for Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis is the etiological agent of chronic periodontitis. Menadione (vitamin K3) and phylloquinone (vitamin K1) are well-known growth factors for P. gingivalis, while menadione is widely used in growth experiments. Here we attempted to determine the differences in phylloquinone and menadione in P. gingivalis growth experiments, which have not been well studied to date. METHODS: We investigated the effects of menadione and phylloquinone on the growth of two W83 strains and seven ATCC 33277 strains of P. gingivalis. RESULTS: The ATCC 33277 strains grew well with phylloquinone at 2.9 µM in a complex medium (nutrient medium) and at 29 µM in two minimal media. In contrast, the W83 strains grew well without menadione or phylloquinone in three different culture media. Menadione at 2.9 µM, the conventionally used concentration for culturing P. gingivalis, supported the growth of most ATCC 33277 strains but inhibited the growth of some W83 and ATCC 33277 strains. Furthermore, menadione at 14.5 µM frequently inhibited cell growth, while phylloquinone at 145 µM promoted cell growth. CONCLUSIONS: These results indicate that menadione and phylloquinone act as growth factors for ATCC 33277 but that menadione also can inhibit P. gingivalis growth. Thus, we propose that phylloquinone be used instead of menadione in P. gingivalis growth experiments requiring vitamin K.

Antibacterial activity of menadione alone and in combination with oxacillin against methicillin-resistant Staphylococcus aureus and its impact on biofilms.

Introduction. Antibiotic resistance is a major threat to public health, particularly with methicillin-resistant Staphylococcus aureus (MRSA) being a leading cause of antimicrobial resistance. To combat this problem, drug repurposing offers a promising solution for the discovery of new antibacterial agents.Hypothesis. Menadione exhibits antibacterial activity against methicillin-sensitive and methicillin-resistant S. aureus strains, both alone and in combination with oxacillin. Its primary mechanism of action involves inducing oxidative stress.Methodology. Sensitivity assays were performed using broth microdilution. The interaction between menadione, oxacillin, and antioxidants was assessed using checkerboard technique. Mechanism of action was evaluated using flow cytometry, fluorescence microscopy, and in silico analysis.Aim. The aim of this study was to evaluate the in vitro antibacterial potential of menadione against planktonic and biofilm forms of methicillin-sensitive and resistant S. aureus strains. It also examined its role as a modulator of oxacillin activity and investigated the mechanism of action involved in its activity.Results. Menadione showed antibacterial activity against planktonic cells at concentrations ranging from 2 to 32 µg ml-1, with bacteriostatic action. When combined with oxacillin, it exhibited an additive and synergistic effect against the tested strains. Menadione also demonstrated antibiofilm activity at subinhibitory concentrations and effectively combated biofilms with reduced sensitivity to oxacillin alone. Its mechanism of action involves the production of reactive oxygen species (ROS) and DNA damage. It also showed interactions with important targets, such as DNA gyrase and dehydroesqualene synthase. The presence of ascorbic acid reversed its effects.Conclusion. Menadione exhibited antibacterial and antibiofilm activity against MRSA strains, suggesting its potential as an adjunct in the treatment of S. aureus infections. The main mechanism of action involves the production of ROS, which subsequently leads to DNA damage. Additionally, the activity of menadione can be complemented by its interaction with important virulence targets.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids.

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

Vitamin k3-Loaded Magnetic Nanoparticle-Mediated Synergistic Magnetothermodynamic Therapy Evokes Massive ROS and Immune Modulation for Augmented Antitumor Potential.

Magnetic nanoparticle (MNP)-mediated magnetic hyperthermia (MHT) under an alternating magnetic field (AMF) causes tumor regression via reactive oxygen species (ROS) generation. However, less therapeutic efficacy has been reported due to the generation of low levels of ROS in a hypoxic tumor microenvironment. Therefore, improved treatments are required to generate relatively high levels of ROS to promote irreversible oxidative damage to the tumor cells. Herein, we report a magnetothermodynamic (MTD) therapy, as a robust and versatile approach for cancer treatment, by combining the magnetothermodynamic-related ROS and heat-related immunological effect in order to overcome the aforementioned obstacle. The synergistic therapy was achieved by the development of vitamin k3 (Vk3)-loaded copper zinc ferrite nanoparticles (Vk3@Si@CuZnIONPs) as an efficient MTD agent. The in vitro results unveiled that enhanced ROS production under the influence of AMF is a predominant aspect in yielding an assertive anticancer response. The in vivo antitumor response was assessed in an ectopic tumor model of A549 lung adenocarcinoma by MTD. The tumor inhibition rate of 69% was achieved within 20 days of MTD treatment, exhibiting complete tumor eradication within 30 days. The validation of antitumor response was marked by severe apoptosis (TUNEL, Caspase-3) in the Vk3@Si@CuZnIONPs + AMF-treated group. The higher expression level of heat shock proteins and proinflammatory cytokines (IL-6, TNF-α, IL-1α, IL-1ß) was speculated to play a role in the activation of immune response for faster tumor regression in the MTD-treated group. Therefore, by implementing a dual ROS and heat-mediated immunogenic effect, the antitumor efficiency of future cancer magnetotherapies will be greatly enhanced.

The conditional mitochondrial protein complexome in the Arabidopsis thaliana root and shoot.

Protein complexes are important for almost all biological processes. Hence, to fully understand how cells work, it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues. Moreover, the dynamics of protein interaction play crucial roles in regulating the (dis)association of protein complexes and, in turn, regulating biological processes such as metabolism. Here, mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic (dis)associations. Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment. These included changes in enzymatic protein complexes involving γ-amino butyric acid transaminase (GABA-T), Δ-ornithine aminotransferase (Δ-OAT), or proline dehydrogenase 1 (POX1) that are expected to affect proline metabolism. Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid (TCA) cycle and the abundance of complexes of the oxidative phosphorylation pathway. In addition, we compared the mitochondrial complexes of roots and shoots. Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus, the formation of super-complexes in the oxidative phosphorylation pathway, and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.

Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes.

OBJECTIVE: The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. DESIGN: Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. RESULTS: Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p = 0.034) and OA chondrocytes (∼0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. CONCLUSIONS: Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.

Vitamin K3 (Menadione) is a multifunctional microbicide acting as a photosensitizer and synergizing with blue light to kill drug-resistant bacteria in biofilms.

Cutaneous bacterial wound infections typically involve gram-positive cocci such as Staphylococcus aureus (SA) and usually become biofilm infections. Bacteria in biofilms may be 100-1000-fold more resistant to an antibiotic than the clinical laboratory minimal inhibitory concentration (MIC) for that antibiotic, contributing to antimicrobial resistance (AMR). AMR is a growing global threat to humanity. One pathogen-antibiotic resistant combination, methicillin-resistant SA (MRSA) caused more deaths globally than any other such combination in a recent worldwide statistical review. Many wound infections are accessible to light. Antimicrobial phototherapy, and particularly antimicrobial blue light therapy (aBL) is an innovative non-antibiotic approach often overlooked as a possible alternative or adjunctive therapy to reduce antibiotic use. We therefore focused on aBL treatment of biofilm infections, especially MRSA, focusing on in vitro and ex vivo porcine skin models of bacterial biofilm infections. Since aBL is microbicidal through the generation of reactive oxygen species (ROS), we hypothesized that menadione (Vitamin K3), a multifunctional ROS generator, might enhance aBL. Our studies suggest that menadione can synergize with aBL to increase both ROS and microbicidal effects, acting as a photosensitizer as well as an ROS recycler in the treatment of biofilm infections. Vitamin K3/menadione has been given orally and intravenously worldwide to thousands of patients. We conclude that menadione/Vitamin K3 can be used as an adjunct to antimicrobial blue light therapy, increasing the effectiveness of this modality in the treatment of biofilm infections, thereby presenting a potential alternative to antibiotic therapy, to which biofilm infections are so resistant.

Effect of the combination of Lactobacillus acidophilus (probiotic) with vitamin K3 and vitamin E on Escherichia coli and Staphylococcus aureus: An in vitro pathogen model.

The gut microbiota plays a key role in maintaining health and regulating the host's immune response. The use of probiotics and concomitant vitamins can increase mucus secretion by improving the intestinal microbial population and prevent the breakdown of tight junction proteins by reducing lipopolysaccharide concentration. Changes in the intestinal microbiome mass affect multiple metabolic and physiological functions. Studies on how this microbiome mass and the regulation in the gastrointestinal tract are affected by probiotic supplements and vitamin combinations have attracted attention. The current study evaluated vitamins K and E and probiotic combinations effects on Escherichia coli and Staphylococcus aureus. Minimal inhibition concentrations of vitamins and probiotics were determined. In addition, inhibition zone diameters, antioxidant activities and immunohistochemical evaluation of the cell for DNA damage were performed to evaluate the effects of vitamins and probiotics. At the specified dose intervals, L. acidophilus and vitamin combinations inhibit the growth of Escherichia coli and Staphylococcus aureus. It could thus contribute positively to biological functions by exerting immune system­strengthening activities.

Retinoic acid and evernyl-based menadione-triazole hybrid cooperate to induce differentiation of neuroblastoma cells.

Neuroblastoma arises when immature neural precursor cells do not mature into specialized cells. Although retinoic acid (RA), a pro-differentiation agent, improves the survival of low-grade neuroblastoma, resistance to retinoic acid is found in high-grade neuroblastoma patients. Histone deacetylases (HDAC) inhibitors induce differentiation and arrest the growth of cancer cells; however, HDAC inhibitors are FDA-approved mostly for liquid tumors. Therefore, combining histone deacetylase (HDAC) inhibitors and retinoic acid can be explored as a strategy to trigger the differentiation of neuroblastoma cells and to overcome resistance to retinoic acid. Based on this rationale, in this study, we linked evernyl group and menadione-triazole motifs to synthesize evernyl-based menadione-triazole hybrids and asked if the hybrids cooperate with retinoic acid to trigger the differentiation of neuroblastoma cells. To answer this question, we treated neuroblastoma cells using evernyl-based menadione-triazole hybrids (6a-6i) or RA or both and examined the differentiation of neuroblastoma cells. Among the hybrids, we found that compound 6b inhibits class-I HDAC activity, induces differentiation, and RA co-treatments increase 6b-induced differentiation of neuroblastoma cells. In addition, 6b reduces cell proliferation, induces expression of differentiation-specific microRNAs leading to N-Myc downregulation, and RA co-treatments enhance the 6b-induced effects. We observed that 6b and RA trigger a switch from glycolysis to oxidative phosphorylation, maintain mitochondrial polarization, and increase oxygen consumption rate. We conclude that in evernyl-based menadione-triazole hybrid, 6b cooperates with RA to induce differentiation of neuroblastoma cells. Based on our results, we suggest that combining RA and 6b can be pursued as therapy for neuroblastoma. Schematic representation of RA and 6b in inducing differentiation of neuroblastoma cells.

Docosahexaenoic Acid Potentiates the Anticancer Effect of the Menadione/Ascorbate Redox Couple by Increasing Mitochondrial Superoxide and Accelerating ATP Depletion.

BACKGROUND/AIM: Mitochondria-targeted anticancer drugs ("mitocans") of natural origin are attractive candidates as adjuvants in cancer therapy. The redox couple menadione/ascorbate (M/A), which belongs to the "mitocans" family, induces selective oxidative stress in cancerous mitochondria and cells, respectively. DHA has also been found to regulate the mevalonate pathway, which is closely related to the prenylation of the cytotoxic menadione to the non-cytotoxic menaquinone. The aim of this study was to elucidate the ability of docosahexaenoic acid (DHA) to potentiate the anticancer effect of M/A by increasing ROS production, as well as affecting steady-state ATP levels in cancer cells. MATERIALS AND METHODS: The experiments were performed on leukemic lymphocyte Jurkat. Cells were treated with DHA, M/A, and their combination (M/A/DHA) and four parameters were examined using the following assays: cell viability and proliferation, steady-state ATP, mitochondrial superoxide, intracellular hydroperoxides. Three independent experiments with two or six parallel measurements were performed for each parameter. RESULTS: The triple combination M/A/DHA was characterized by much higher antiproliferative activity and cytotoxicity than M/A and DHA administered alone. DHA significantly accelerated M/A-induced ATP depletion in cells, which was accompanied by an additional increase in mitochondrial superoxide compared to cells treated with M/A or DHA alone. CONCLUSION: DHA significantly enhanced M/A-induced cytotoxicity in leukemic lymphocytes by inducing severe mitochondrial oxidative stress and accelerated ATP depletion. Selective DHA-mediated suppression of cholesterol synthesis in cancer cells (involved in the prenylation of cytotoxic menadione to the less cytotoxic phylloquinone), as well as DHA-mediated inhibition of superoxide dismutase are suggested to underlie the potentiation of the anticancer effect of M/A.

Genome-Wide Gene Expression Analyses of the AtfA/AtfB-Mediated Menadione Stress Response in Aspergillus nidulans.

The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of control, ΔatfA, ΔatfB, and ΔatfAΔatfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and ΔatfA (ΔatfB) strains and between the ΔatfB (ΔatfA) and the ΔatfAΔatfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans, the number of differentially expressed genes for ΔatfA was significantly higher than for ΔatfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of ΔatfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (ΔatfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase (agdB), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.

Vitamin K3 Suppresses Pyroptosis in THP-1 Cells through Inhibition of NF-κB and JNK Signaling Pathways.

Vitamin K, a necessary nutritional supplement for human, has been found to exhibit anti-inflammatory activity. In the present study, we investigated the effects of vitamin K family on lipopolysaccharide (LPS) plus nigericin induced pyroptosis and explored the underlying mechanism of its action in THP-1 monocytes. Results showed that vitamin K3 treatment significantly suppressed THP-1 pyroptosis, but not vitamin K1 or K2, as evidenced by increased cell viability, reduced cellular lactate dehydrogenase (LDH) release and improved cell morphology. Vitamin K3 inhibited NLRP3 expression, caspase-1 activation, GSDMD cleavage and interleukin (IL)-1ß secretion in pyrophoric THP-1 cells. In addition, vitamin K3 inhibited the pro-inflammatory signaling pathways including nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK). Vitamin K3 treatment also attenuated tissue damage and reduced serum LDH, IL-1ß and IL-6 levels in LPS-induced systemic inflammation of mice. The reduced myeloperoxidase (MPO) activityand F4/80 expression indicated that vitamin K3 effectively reduced the infiltration of neutrophils and macrophages. Moreover, NLRP3 expression in monocytes/macrophages were also decreased in vitamin K3-treatedmice after LPS challenge. These findings suggest that vitamin K3 potently alleviates systemic inflammation and organ injury via inhibition of pyroptosis in monocytes and may serve as a novel therapeutic strategy for patients with inflammatory diseases.