Naphthoquinone-induced arylation inhibits Sirtuin 7 activity.

Natural or synthetic naphthoquinones have been identified to interfere with biological systems and, in particular, exhibit anticancer properties. As redox cyclers, they generate reactive oxygen species in cells and, as electrophiles, they react with nucleophiles, mainly thiols, and form covalent adducts. To further decipher the molecular mechanism of action of naphthoquinones in human cells, we analyzed their effects in HeLa cells. First, we demonstrated that the naphthoquinones menadione and plumbagin inhibited the nucleolar NAD+-dependent deacetylase Sirtuin 7 in vitro. As assessed by their inhibition of rDNA transcription, pre-rRNA processing and formation of etoposide-induced 53BP1 foci, menadione and plumbagin also inhibited Sirtuin 7 catalytic activity in vivo. Second, we established that when sulfhydryl arylation by menadione or plumbagin was prevented by the thiol reducing agent N-acetyl-L-cysteine, the inhibition of Sirtuin 7 catalytic activity was also blocked. Finally, we discuss how inhibition of Sirtuin 7 might be crucial in defining menadione or plumbagin as anti-tumor agents that can be used in combination with other anti-tumor strategies.

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.

In vitro evaluation of the reductive carbonyl idarubicin metabolism to evaluate inhibitors of the formation of cardiotoxic idarubicinol via carbonyl and aldo-keto reductases.

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo-keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.

Reactivation of nucleases with peroxidation damages induced by a menadione: ascorbate combination devastates human prostate carcinomas: ultrastructural aspects.

INTRODUCTION: Xenografts of androgen-independent human DU145 prostate metastatic carcinomas implanted in nu/nu male mice have revealed a significant survival after a prooxidant anticancer treatment consisting of a combination of menadione bisulfite and sodium ascorbate (VK3:VC). METHODS: Implanted samples of diaphragm carcinomas from longest survived mice from either oral, intraperitoneal (IP), or both oral and IP treatment groups were assessed with light, scanning, and transmission electron microscopy to analyze morphologic damages. RESULTS: Compared with previous fine structure data of in vitro untreated carcinomas, the changes induced by oral, IP, and oral with IP VK3:VC treatment dismantled those xenografts with autoschizis, and necrotic atrophy was accomplished by cell's oxidative stress whose injuries were consequent to reactivated deoxyribonucleases and ribonucleases. Tumor destructions resulted from irreversible damages of nucleus components, endoplasmic reticulum, and mitochondria there. Other alterations included those of the cytoskeleton that resulted in characteristic self-excisions named " autoschizis." All these injuries lead resilient cancer cells to necrotic cell death. CONCLUSION: The fine structure damages caused by VK3:VC prooxidant combination in the human DU145 prostate xenografts confirmed those shown in vitro and of other cell lines with histochemistry and biomolecular investigations. These devastations incurred without damage to normal tissues; thus, our data brought support for the above combination to assist in the treatment of prostate cancers and other cancers.

Nanoengineered lanthanum niobate nanocaviar anchored carbon nanofibers for trace level detection of menadione in environmental samples.

An innovative sensor is prepared by electrode modification through a nano-ranged electrode modifier composed of LaNbO4 nano caviars decorated on the enmeshed carbon nanofibers to identify excess vitamins in animal feed. Menadione (Vitamins K3) is a micronutrient fundamentally required in precise quantities for animal health upkeep. Still, its exploitation has recently resulted in water reservoir contamination through waste generated from animal husbandry. Sustainable prevention of water contamination makes menadione detection highly imperative and flickered the attention of researchers. Considering these aspects, a novel menadione sensing platform is designed by interdisciplinary incorporation of nanoscience and electrochemical engineering. The structural and crystallographic features and the electrode modifier's morphological insights were keenly investigated. The hierarchal arrangement of individual constituents in nanocomposite is benefited through hybrid heterojunction and quantum confinement that synchronously activate the menadione detection with a LOD of 68.5 nM and 67.49 nM for oxidation and reduction, respectively. The as-prepared sensor has a wide linear range (0.1-173.6 µM), high sensitivity, good selectivity, and stability. The application of this sensor is extended to a water sample to monitor the consistency of the proposed sensor.

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples.

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10-12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase.

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone - giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

Synthesis of mitochondria-targeted menadione cation derivatives: Inhibiting mitochondrial thioredoxin reductase (TrxR2) and inducing apoptosis in MGC-803 cells.

Menadione (VK3) is used as a powerful inducer of cellular reactive oxygen species (ROS) for many years and displays the high anti-cancer activities in vivo. Recently, the development of mitochondria-targeted drugs has been more and more appreciated. Here, the thirteen derivatives of VK3 were synthesized, which could localize in mitochondria by the triphenylphosphonium (TPP) cation or the nitrogen-based cation. The results of cytotoxicity from six human cancer cell lines showed that the targeted compounds T1-T13 displayed higher activity than VK3 with the average IC50 value around 1 µM. The results of cytotoxicity indicated that the substitutes on C-2, the linear alkyl chains on C-3 and cation moiety all could affect the cytotoxicity. The mechanistic studies showed that five representative compounds (T2, T3, T5, T8 and T13) could localize in cellular mitochondria, elicit ROS burst and collapse mitochondrial membrane potential (ΔΨm), leading to cytochrome C release and apoptosis in MGC-803 cells. Particularly, they could obviously inhibit mitochondrial thioredoxin reductase TrxR2 expression, thus leading to aggravate cellular oxidative stress.

Effects of prolonged subchronic benzo(α)pyrene exposure on rat liver morphology and CYP1A expression during treatment with menadione, quercetin, or tocopherol.

Arylamines and polycyclic aromatic hydrocarbons (PAHs) are hazardous anthropogenic pollutants in the environment. The toxicity of PAHs, which include benzo(α)pyrene (BP), is mediated by the activation of Р450 cytochromes of the 1А subfamily (CYP1A1 and CYP1A2). Previously, we have demonstrated that tocopherol, quercetin, and menadione inhibit the expression and activity of CYP1A in the liver of male Wistar rats after administration of a high BP dose to the rats for 3 days. Here, we confirmed the effects of tocopherol, quercetin, and menadione on the expression and activity of CYP1A and on rat liver morphology during prolonged administration (90 days) of a low BP dose. We revealed that subchronic oral administration of a low BP dose has no influence on CYP1A expression as compared to controls but can cause pathomorphological changes in rat liver tissue. These changes are abrogated by tocopherol, attenuated by quercetin, and enhanced by menadione.

Menadione Potentiates Auranofin-Induced Glioblastoma Cell Death.

Glioblastoma (GBM) is the most aggressive primary brain tumor. Recently, agents increasing the level of oxidative stress have been proposed as anticancer drugs. However, their efficacy may be lowered by the cytoprotective activity of antioxidant enzymes, often upregulated in neoplastic cells. Here, we assessed the mRNA and protein expression of thioredoxin reductase 1 (TrxR1), a master regulator of cellular redox homeostasis, in GBM and non-tumor brain tissues. Next, we examined the influence of an inhibitor of TrxR1, auranofin (AF), alone or in combination with a prooxidant menadione (MEN), on growth of GBM cell lines, patient-derived GBM cells and normal human astrocytes. We detected considerable amount of TrxR1 in the majority of GBM tissues. Treatment with AF decreased viability of GBM cells and their potential to form colonies and neurospheres. Moreover, it increased the intracellular level of reactive oxygen species (ROS). Pre-treatment with ROS scavenger prevented the AF-induced cell death, pointing to the important role of ROS in the reduction of cell viability. The cytotoxic effect of AF was potentiated by treatment with MEN. In conclusion, our results identify TrxR1 as an attractive drug target and highlights AF as an off-patent drug candidate in GBM therapy.

Synthesis of thia-Michael-Type Adducts between Naphthoquinones and N-Acetyl-L-Cysteine and Their Biological Activity.

A series of naphthoquinones, namely, 1,4-naphthoquinone, menadione, plumbagin, juglone, naphthazarin, and lawsone, were reacted with N-acetyl-L-cysteine, and except for lawsone, which did not react, the related adducts were obtained. After the tuning of the solvent and reaction conditions, the reaction products were isolated as almost pure from the complex reaction mixture via simple filtration and were fully characterized. Therefore, the aim of this work was to evaluate whether the antitumor activity of new compounds of 1,4-naphthoquinone derivatives leads to an increase in ROS in tumor cell lines of cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), and osteosarcoma (SaOS2, U2OS) and in normal dermal fibroblast (HDFa). The MTT assay was used to assay cell viability, the DCF-DA fluorescent probe to evaluate ROS induction, and cell-cycle analysis to measure the antiproliferative effect. Compounds 8, 9, and 12 showed a certain degree of cytotoxicity towards all the malignant cell lines tested, while compound 11 showed biological activity at higher IC50 values. Compounds 8 and 11 induced increases in ROS generation after 1 h of exposure, while after 48 h of treatment, only 8 induced an increase in ROS formation in HeLa cells. Cell-cycle analysis showed that compound 8 caused an increase in the number of G0/G1-phase cells in the HeLa experiment, while for the U2OS and SH-SY5Y cell lines, it led to an accumulation of S-phase cells. Therefore, these novel 1,4-naphthoquinone derivatives may be useful as antitumoral agents in the treatment of different cancers.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

Nitroreductase Increases Menadione-Mediated Oxidative Stress in Aspergillus nidulans.

Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O2•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O2•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O2•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O2•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O2•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O2•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O2•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

Low- or high-white light irradiance induces similar conidial stress tolerance in Metarhizium robertsii.

White light during mycelial growth influences high conidial stress tolerance of the insect-pathogenic fungus Metarhizium robertsii, but little is known if low- or high-white light irradiances induce different stress tolerances. The fungus was grown either in the dark using two culture media: on minimal medium (Czapek medium without sucrose = MM) or on potato dextrose agar (PDA) or PDA medium under five different continuous white light irradiances. The stress tolerances of conidia produced on all treatments were evaluated by conidial germination on PDA supplemented with KCl for osmotic stress or on PDA supplemented with menadione for oxidative stress. Conidia produced on MM in the dark were more tolerant to osmotic and oxidative stress than conidia produced on PDA in the dark or under the light. For osmotic stress, growth under the lower to higher irradiances produced conidia with similar tolerances but more tolerant than conidia produced in the dark. For oxidative stress, conidia produced under the white light irradiances were generally more tolerant to menadione than conidia produced in the dark. Moreover, conidia produced in the dark germinated at the same speed when incubated in the dark or under lower irradiance treatment. However, at higher irradiance, conidial germination was delayed compared to germination in the dark, which germinated faster. Therefore, growth under light from low to high irradiances induces similar conidial higher stress tolerances; however, higher white light irradiances cause a delay in germination speed.

The ubiquitin-proteasome system and its crosstalk with mitochondria as therapeutic targets in medicine.

The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.

Anharmonicity and Spectra-Structure Correlations in MIR and NIR Spectra of Crystalline Menadione (Vitamin K3).

Mid-infrared (MIR) and near-infrared (NIR) spectra of crystalline menadione (vitamin K3) were measured and analyzed with aid of quantum chemical calculations. The calculations were carried out using the harmonic approach for the periodic model of crystal lattice and the anharmonic DVPT2 calculations applied for the single molecule model. The theoretical spectra accurately reconstructed the experimental ones permitting for reliable assignment of the MIR and NIR bands. For the first time, a detailed analysis of the NIR spectrum of a molecular system based on a naphthoquinone moiety was performed to elucidate the relationship between the chemical structure of menadione and the origin of the overtones and combination bands. In addition, the importance of these bands during interpretation of the MIR spectrum was demonstrated. The overtones and combination bands contribute to 46.4% of the total intensity of menadione in the range of 3600-2600 cm-1. Evidently, these bands play a key role in shaping of the C-H stretching region of MIR spectrum. We have shown also that the spectral regions without fundamentals may provide valuable structural information. For example, the theoretical calculations reliably reconstructed numerous overtones and combination bands in the 4000-3600 and 2800-1800 cm-1 ranges. These results, provide a comprehensive origin of the fundamentals, overtones and combination bands in the NIR and MIR spectra of menadione, and the relationship of these spectral features with the molecular structure.

Proteomic analysis of Sporothrix schenckii cell wall reveals proteins involved in oxidative stress response induced by menadione.

Sporotrichosis is an emergent subcutaneous mycosis that is a threat to both humans and other animals. Sporotrichosis is acquired by the traumatic implantation of species of the Sporothrix genus. Added to the detoxification systems, pathogenic fungi possess different mechanisms that allow them to survive within the phagocytic cells of their human host during the oxidative burst. These mechanisms greatly depend from the cell wall (CW) since phagocytic cells recognize pathogens through specific receptors associated to the structure. To date, there are no studies addressing the modulation of the expression of S. schenckii CW proteins (CWP) in response to reactive oxygen species (ROS). Therefore, in this work, a proteomic analysis of the CW of S. schenckii in response to the oxidative agent menadione (O2•-) was performed. Proteins that modulate their expression were identified which can be related to the fungal survival mechanisms within the phagocyte. Among the up-regulated CWP in response to the oxidative agent, 13 proteins that could be involved in the mechanisms of oxidative stress response in S. schenckii were identified. The proteins identified were thioredoxin1 (Trx1), superoxide dismutase (Sod), GPI-anchored cell wall protein, ß-1,3-endoglucanase EglC, glycoside hydrolase (Gh), chitinase, CFEM domain protein, glycosidase crf1, covalently-linked cell wall protein (Ccw), 30 kDa heat shock protein (Hsp30), lipase, trehalase (Treh), fructose-bisphosphate aldolase (Fba1) and citrate synthase (Cs). The identification of CWP that modulates their expression in response to superoxide ion (O2•-) in S. schenckii is a useful approach to understand how the fungus defends itself against ROS, in order to evade the phagocytic cells from the host and cause the infection.

The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2',7'-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

Combined treatment of menadione and calcitriol increases the antiproliferative effect by promoting oxidative/nitrosative stress, mitochondrial dysfunction, and autophagy in breast cancer MCF-7 cells.

The aim of this study was to determine new insights into the molecular mechanisms involved in the antiproliferative action of menadione + calcitriol (MEN+D) on MCF-7 cells. After 24 h, MEN+D inhibited the cell growth but was not observed with each single treatment. The combined drugs reduced the mitochondrial respiration at that time, as judged by an increase in the proton leak and a decrease in the ATP generation and coupling efficiency. At longer times, 48 or 96 h, either D or MEN reduced the proliferation, but the effect was higher when both drugs were used together. The combined treatment increased the superoxide anion ([Formula: see text]) and nitric oxide (NO•) contents as well as acidic vesicular organelles (AVOs) formation. The percentage of cells showing the lower mitochondrial membrane potential (ΔΨm) was highly increased by the combined therapy. LC3-II protein expression was enhanced by any treatment. In conclusion, the antiproliferative action of MEN+D involves oxidative/nitrosative stress, mitochondrial alteration, and autophagy. This combined therapy could be useful to treat breast cancer cells because it inhibits multiple oncogenic pathways more effectively than each single agent.