naphthoquinones and juglone

Bacterial resistance development represents a serious threat to human health across the globe and has become a very serious clinical problem for many classes of antibiotics. Hence, there is a constant and urgent need for the discovery and development of new effective antibacterial agents to stem the emergence of resistant bacteria. 1,4-naphthoquinones are an important class of natural products and have been known for decades as a privileged scaffold in medicinal chemistry regarding their many biological properties. The significant biological properties of specific 1,4-naphthoquinones hydroxyderivatives have drawn the attention of researchers in order to find new derivatives with an optimized activity, mainly as antibacterial agents. Based on juglone, naphthazarin, plumbagin and lawsone moieties, structural optimization was realized with the purpose of improving the antibacterial activity. Thereupon, relevant antibacterial activities have been observed on different panels of bacterial strains including resistant ones. In this review, we highlight the interest of developing new 1,4-naphthoquinones hydroxyderivatives and some metal complexes as promising antibacterial agents alternatives. Here, we thoroughly report for the first time both the antibacterial activity and the chemical synthesis of four different 1,4-naphthoquinones (juglone, naphthazarin, plumbagin and lawsone) from 2002 to 2022 with an emphasis on the structure-activity relationship, when applicable.

Topics: Anti-Bacterial Agents; Bacteria; Coordination Complexes; Humans; Naphthoquinones

Juglone (5 - hydroxy - 1, 4 - naphthalene diketone) is a kind of natural naphthoquinone, present in the roots, leaves, nut-hulls, bark and wood of walnut trees. Recent studies have found that Juglone has special significance in the treatment of cancer, which plays a significant role in the resistance of cancer cell proliferation, induction of cancer cell apoptosis, induction of autophagy, anti-angiogenesis and inhibition of cancer cell migration and invasion, etc. Additionally, its derivatives also play a tumor suppressive effect. In conclusion, Juglone and its derivatives have been identified as effective anticancer drugs. This paper reviews action mechanisms of Juglone and its derivatives in cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Movement; Cell Proliferation; DNA-Directed DNA Polymerase; Humans; Naphthoquinones; Neoplasms; Neovascularization, Pathologic; NIMA-Interacting Peptidylprolyl Isomerase; Reactive Oxygen Species

Juglone is a metabolite produced by several species of plants, in particular Juglans nigra. Additionally, juglone is a 1,4-naphthoquinone that has several biological actions. Antimicrobial, antifungal, sedative, oxidizing, antihypertensive, and especially anti-proliferative actions have been described for juglone. This justifies that 1,4-naphthoquinone is a privileged structure for Medicinal Chemistry, and it is useful for the development of new prototypes with varied actions. In this work, we make a profound review of juglone synthesis methodology, the biological actions of juglone, and mainly the synthesis and pharmacological actions of juglone derivatives. We hope that the potent biological actions described for these derivatives in this review will stimulate the continuous design, synthesis, and pharmacological evaluation of new juglone derivatives.

Topics: Anti-Bacterial Agents; Antifungal Agents; Antihypertensive Agents; Hypnotics and Sedatives; Naphthoquinones

Quinones are plant-derived secondary metabolites that present some anti-proliferation and anti-metastasis effects in various cancer types both in vitro and in vivo. This review focuses on the anti-cancer prospects of plant-derived quinones, namely, aloe-emodin, juglone, β-lapachol, plumbagin, shikonin, and thymoquinone. We intend to summarize their anti-cancer effects and investigate the mechanism of actions to promote the research and development of anti-cancer agents from quinones.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Benzoquinones; Cell Line, Tumor; Cell Survival; Humans; Naphthoquinones; Neoplasms; Plant Extracts

Iron is a transition metal that forms chelates and complexes with various organic compounds, also with phenolic plant secondary metabolites. The ligands of iron affect the redox potential of iron. Electrons may be transferred either to hydroxyl radicals, hydrogen peroxide or molecular oxygen. In the first case, oxidative stress is decreased, in the latter two cases, oxidative stress is increased. This milieu-dependent mode of action may explain the non-linear mode of action of juglone and other secondary metabolites. Attention to this phenomenon may help to explain idiosyncratic and often nonlinear effects that result in biological assays. Current chemical assays are discussed that help to explore these aspects of redox chemistry.

Topics: Antioxidants; Flavonoids; Herbicides; Iron; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Phenols; Plants; Polyphenols; Reactive Oxygen Species

Malignant tumor, one of the most refractory diseases, plays a threaten role in human health, the therapy and research on malignant tumor have taken a long way to go. The anti-tumor drugs which are the essential therapy strategies upgrade with the development of new anti-tumor targets and the research on tumor pathogenesis. Aurora kinase and Pin1, the novel anti-tumor targets, maintain the important relationship with tumor. Many new compounds designed on these targets have excellent anti-tumor effects and also enter into phase I or phase II clinical trial.

Topics: Antineoplastic Agents; Apoptosis; Aurora Kinases; Humans; Naphthoquinones; Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Piperazines; Protein Serine-Threonine Kinases

The stemness characteristics of cancer cells, such as self-renewal and tumorigenicity, are considered to be responsible, in part, for tumor metastasis. Epithelial-to-mesenchymal transition (EMT) plays an important role in promoting both stemness and tumor metastasis. Although the traditional medicine juglone is thought to play an anticancer role by affecting cell cycle arrest, induction of apoptosis, and immune regulation, a potential function of juglone in regulating cancer cell stemness characteristics remains unknown.. In the present study, tumor sphere formation assay and limiting dilution cell transplantation assays were performed to assess the function of juglone in regulating maintenance of cancer cell stemness characteristics. EMT of cancer cells was assessed by western blot and transwell assay. Data gathered indicates juglone inhibits stemness characteristics and EMT in cancer cells. Furthermore, we verified that metastasis was suppressed by juglone treatment. We also observed that these effects were, in part, achieved by inhibiting Peptidyl-prolyl. These results indicate that juglone inhibits maintenance of stemness characteristics and metastasis in cancer cells.

Topics: Apoptosis; Blotting, Western; Epithelial-Mesenchymal Transition; Naphthoquinones; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells

Hydrophilic, untethered 1,4-naphthoquinones (1,4-NQs) are plant secondary metabolites that are often excreted into the environment and play a role in various plant-microbial, plant-fungal, plant-insect and plant-plant interactions. The biological activity of 1,4-NQs is mainly related to their redox properties, i.e. the ability to undergo redox cycling in cells. These compounds may also undergo electrophilic addition to thiol-containing compounds. The aim of this study was to compare the impact of juglone, plumbagin, lawsone and 2-methoxy-1,4-naphthoquinone (2-met-NQ) on the antioxidant response of the green microalga Chlamydomonas reinhardtii. The algae were incubated with the examined compounds under low light for 6 h and the content of photosynthetic pigments, prenyllipid antioxidants, ascorbate, soluble thiols, proline, and superoxide dismutase activity was assessed. To examine the interaction between photosynthetic activity and naphthoquinone toxicity, we carried out the second experiment, in which C. reinhardtii was incubated with 1,4-NQs for 1 h under high light or in darkness. The pro-oxidant action of the examined 1,4-NQs depended on their reduction potentials, which decrease in order: juglone > plumbagin > 2-met-NQ > lawsone. Lawsone did not display pro-oxidant properties. Exposure to high light strongly enhanced the pro-oxidant effect of juglone, plumbagin, and 2-met-NQ, which is thought to result from the interception of the electrons from photosynthetic electron transfer chain. Only juglone was able to cause a fast depletion of plastoquinol, which may be an important mode of action of this allelochemical, responsible for its high toxicity to plants.

Topics: Antioxidants; Chlamydomonas reinhardtii; Naphthoquinones; Plants; Reactive Oxygen Species

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Screening Assays, Antitumor; Humans; Mice; Molecular Docking Simulation; Molecular Structure; Naphthoquinones; Structure-Activity Relationship; Thiazoles

Purinergic receptors are transmembrane proteins responsive to extracellular nucleotides and are expressed by several cell types throughout the human body. Among all identified subtypes, the P2×7 receptor has emerged as a relevant target for the treatment of inflammatory disease. Several clinical trials have been conducted to evaluate the effectiveness of P2×7R antagonists. However, to date, no selective antagonist has reached clinical use. In this work, we report the pharmacological evaluation of eleven N, S-acetal juglone derivatives as P2×7R inhibitors. Using in vitro assays and in vivo experimental models, we identified one derivative with promising inhibitory activity and low toxicity. Our in silico studies indicate that the 1,4-naphthoquinone moiety might be a valuable molecular scaffold for the development of novel P2×7R antagonists, as suggested by our previous studies.

Topics: Acetals; Adenosine Triphosphate; Humans; Naphthoquinones; Receptors, Purinergic P2X7

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Membrane; Colorectal Neoplasms; Humans; Nanoparticles; Naphthoquinones; Oxaliplatin

To verrify the anti-tumor efficacy and toxicity between juglone (Jug) and Jug-loaded PLGA nanoparticles (Jug-PLGA-NPs).. Jug-PLGA-NPs were prepared by ultrasonic emulsification. The anti-tumor activity of Jug (2, 3, 4 µg/mL) and Jug-PLGA-NPs (Jug: 2, 3, 4 µg/mL) in vitro was measured by MTT assay and cell apoptosis analysis. The distribution, anti-tumor effect and biological safety in vivo was evaluated on A375 nude mice.. With the advantage of good penetration and targeting properties, Jug-PLGA-NPs significantly inhibited proliferation and migration of melanoma cells both in vitro and in vivo (P<0.05 or P<0.01) with acceptable biocompatibility.. Jug can inhibit the growth of melanoma but is highly toxic. With the advantage of sustained release, tumor targeting, anti-tumor activity and acceptable biological safety, Jug-PLGA-NPs provide a new pharmaceutical form for future application of Jug.

Topics: Animals; Cell Line, Tumor; Delayed-Action Preparations; Drug Carriers; Melanoma; Mice; Mice, Nude; Nanoparticles; Naphthoquinones; Particle Size; Polylactic Acid-Polyglycolic Acid Copolymer

Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value.. Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds, and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome, and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA, and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2, and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified.. Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.

Topics: Chromosomes; Genome; Juglans; Lipids; Naphthoquinones

To observe the expression of c-Myc protein in cervical cancer HeLa cells and explore the effect of juglone on the proliferation and apoptosis of HeLa cells by affecting c-Myc ubiquitination.. HeLa cells treated with different concentrations (0, 10, 20, or 50 μmol/L) of juglone or with 20 μmol/L juglone for different time lengths were examined for expression of c-Myc protein with Western blotting. The half-life of c-Myc protein was determined using cycloheximide (CHX) and c-Myc protein degradation was detected using coimmunoprecipitation. We also assessed the effects of 20 μmol/L juglone combined with 0, 1.0 or 2.0 μmol/L MG132 (a proteasome inhibitor) on c-Myc expression. The effects of 20 μmol/L juglone on the proliferation and apoptosis of HeLa cells with RNA interference-mediated knockdown of c-Myc were evaluated with MTT assay and flow cytometry.. Treatment with juglone significantly lowered c-Myc protein expression in HeLa cells in a concentration-and time-dependent manner (. Juglone inhibits proliferation and promotes apoptosis of HeLa cells by affecting the ubiquitination of c-Myc protein.

Topics: Apoptosis; Cell Proliferation; Female; HeLa Cells; Humans; Naphthoquinones; Ubiquitination; Uterine Cervical Neoplasms

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Topics: Arabidopsis; Arabidopsis Proteins; Humans; Naphthoquinones; Ribonuclease P; RNA Precursors; RNA, Transfer

Schistosomiasis remains one of the world's leading health concerns, affecting millions. The granulomatous reaction is the most significant immunopathological change associated with Schistosoma mansoni infection, resulting in significant mortality. Recent progress has been made in the search for new natural compounds to reduce schistosomiasis and its immunopathology. Walnuts contain the phenolic compound Juglone (5-hydroxy-1,4-naphthoquinone), which has antiparasitic, anti-inflammatory, immunoregulatory, and antioxidant properties. There were three groups of infected mice: untreated (IU), Juglone-treated (JUG T), and praziquantel-treated (PZ). In mice treated at 8 mg Juglone /kg body weight, a reduction of 63.1 % and 52.1 % were observed in the number of male and female worms, respectively. In addition, the number of eggs/g tissue was reduced by 65.7 % in the liver, 58.58 % in the intestine, and 62.31 % in the liver and intestine combined. In addition, Juglone decreased hepatic granuloma size by 55.1 % and collagen fiber deposition by 23.4 % compared to PZQ (41.18 % and 11.2 %, respectively). Interestingly, the JUG T group had significantly lower levels of IL-4, IL-13, IL-37, TNF-α, TGF-β, and IFN-γ than PZ mice (p < 0.05). While IL-10 and IL-17 levels rose (p < 0.01), Juglone could restore hepatic ALT, AST, GGT, and LDH activities following infection. In addition, it increased catalase, SOD, GSH, and GST while decreasing NO and LPO in comparison to the infected group. Moreover, anti-SWAP, SEA, and CAP IgG levels increased significantly. IgE levels did not change significantly, however. Juglone could be used as an antifibrotic, immunomodulatory, and schistosomicidal agent; thus, it could be used in place of PZQ.

Topics: Animals; Female; Liver; Male; Mice; Naphthoquinones; Schistosomiasis mansoni; Schistosomicides

7-Methyl juglone as a naturally occurring naphthoquinone showed striking antibacterial, antifungal, antivirus and anticancer activity. Its derivatives had also been characterized as key intermediates in the preparation of natural naphthoquinones and anthraquinones. Herein, we reported a regioselective synthesis of 7-methyl juglone

Topics: Antifungal Agents; Cyclization; Naphthoquinones; Oxidation-Reduction

Indomethacin [IND] is reported to treat colon cancer. However, continuous exposure to IND causes gastric ulceration, an adverse side effect in humans. This study implies the therapeutic effect of IND and juglone [JUG] against colon carcinogenesis, without gastric ulceration - an adverse side effect of IND.. Adult male Balb/C mice were divided into six groups randomly: control, AOM/DSS-induced, IND-treated, JUG-treated, IND + JUG-treated and drug-control. Levels of serum markers, haematoxylin & eosin staining to observe tissue architecture, toluidine blue staining to detect mast cells expression, Masson's trichrome and sirius-red staining were used to detect the collagen deposition. RT-PCR and western blot analysis were carried out to detect inflammation and apoptosis.. IND + JUG effectively decreased the levels of serum markers: CEA, AFP, LDH, AST and ALT. Although, IND restored colonic architecture by regulating the accumulation of mast cell and collagen content, it causes gastric ulceration. To address this adverse effect of IND, JUG was given along with IND and was shown to alleviate IND-induced gastric ulceration. AOM/DSS induced animals showed increased expression of inflammatory molecules - TNFα, NFκB and Cox-2, apoptosis regulator - Bcl-2 and decreased expression of pro-apoptotic molecules - Bad, Bax and caspase3; whereas, IND and JUG treated groups showed decreased inflammatory expression with increased expression of pro-apoptotic molecules.. IND and JUG reduce the inflammatory activity and induce apoptotic cell death, while JUG effectively prevents IND induced gastric ulceration. These findings establish that a combination of IND + JUG may serve as a promising treatment regimen for colon cancer.

Topics: Animals; Apoptosis; Azoxymethane; Carcinogenesis; Cell Count; Cell Line, Tumor; Collagen; Colonic Neoplasms; Dextran Sulfate; Indomethacin; Inflammation; Male; Mast Cells; Mice, Inbred BALB C; Naphthoquinones

Juglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time.. Human platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay.. Juglone (1 - 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca. Juglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

Topics: Blood Platelets; Humans; Naphthoquinones; Platelet Activation; Platelet Aggregation Inhibitors; Protein Disulfide-Isomerases; Proto-Oncogene Proteins c-akt; Signal Transduction; Thrombosis

We aimed to investigate the effects of juglone on angiogenesis, metastasis and cell proliferation processes in pancreatic cancer  (PC)  and to understand whether its possible effects occur via the Wnt signaling pathway by analyzing the expression levels of target genes of Wnt signaling.. PC is a silent and lethal cancer type which can only be detectable after metastasis and angiogenesis processes occured. The Wnt signaling pathway is one of the pathways that plays an active role in many biological processes in the cell. Mutations in the genes of this signaling pathway are related to the development of many cancers. Juglone, a natural compound, is shown to have cytotoxic and apoptotic effects on various cancer cells.. PANC-1 and BxPC-3 pancreatic cancer cells were treated with juglone at

Topics: beta Catenin; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Naphthoquinones; Neovascularization, Pathologic; Pancreatic Neoplasms; Wnt Signaling Pathway

Depression and anxiety disorders contribute to the global disease burden. Ursolic acid (UA), a natural compound present in many vegetables, fruits and medicinal plants, was tested in vivo for its effect on (1) enhancing resistance to stress and (2) its effect on life span.. The compound was tested for its antioxidant activity in C. elegans. Stress resistance was tested in the heat and osmotic stress assay. Additionally, the influence on normal life span was examined. RT-PCR was used to assess possible serotonin targets.. UA prolonged the life span of C. elegans. Additionally, UA significantly lowered reactive oxygen species (ROS). Molecular docking studies, PCR analysis and microscale thermophoresis (MST) supported the results that UA acts through serotonin receptors to enhance stress resistance.. Considering the urgent need for new and safe medications in the treatment of depression and anxiety disorders, our results indicate that UA may be a promising new drug candidate.

Topics: Animals; Antioxidants; Caenorhabditis elegans; Depression; Disease Models, Animal; Hot Temperature; Longevity; Models, Molecular; Molecular Docking Simulation; Mutation; Naphthoquinones; Osmotic Pressure; Reactive Oxygen Species; Receptors, Serotonin; Serotonin; Stress, Physiological; Triterpenes; Ursolic Acid

Microbial reactor sensors (based on freshly harvested intact microbial cells) or microbial membrane sensors (based on immobilized microbial cells) can be used as convenient instruments for studying processes that cause the response of a biosensor, such as the properties of enzymes or the characteristics of metabolism. However, the mechanisms of the formation of biosensors responses have not yet been fully understood to study only one of these processes. In this work, the results of studies on the formation of a response to juglone for intact and immobilized bacterial cells used as receptors are presented. It was shown that the contribution of reactive oxygen species (ROS) to the formation of the biosensor response depends on the culture receptor and the form of juglone, quinone, or phenolate used. The response to the quinone form of juglone both for intact and immobilized cells of catalase-positive actinobacterium is formed regardless of the presence of ROS. The response of freshly harvested intact actinobacterial cells was caused by the rate of the enzymatic conversion of juglone. The rate of the response of immobilized actinobacterial cells was influenced by the activity of transport systems and metabolism. The response of immobilized pseudomonad cells was caused by the transport of juglone into cells, the inhibitory effect of juglone-induced ROS, and juglone metabolism.

Topics: Bacteria; Biosensing Techniques; Cells, Immobilized; Cytotoxins; Naphthoquinones; Reactive Oxygen Species

Therapies for lethal castration-resistant prostate cancer (CRPC) are an unmet medical need. One mechanism underlying CRPC and resistance to hormonal therapies is the expression of constitutively active splice variant(s) of androgen receptor (AR-Vs) that lack its C-terminus ligand-binding domain. Transcriptional activities of AR-Vs and full-length AR reside in its N-terminal domain (NTD). Ralaniten is the only drug proven to bind AR NTD, and it showed promise of efficacy in Phase 1 trials. The peptidyl-prolyl isomerase Pin1 is frequently overexpressed in prostate cancer. Here we show that Pin1 interacted with AR NTD. The inhibition of Pin1 expression or its activity selectively reduced the transcriptional activities of full-length AR and AR-V7. Combination of Pin1 inhibitor with ralaniten promoted cell cycle arrest and had improved antitumor activity against CRPC xenografts in vivo compared to individual monotherapies. These findings support the rationale for therapy that combines a Pin1 inhibitor with ralaniten for treating CRPC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Checkpoints; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Male; Mice, Inbred NOD; Mice, SCID; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; PC-3 Cells; Prostatic Neoplasms, Castration-Resistant; Protein Domains; Receptors, Androgen; Signal Transduction; Tretinoin; Tumor Burden; Xenograft Model Antitumor Assays

Juglone, mainly isolates from the green walnut husks of Juglans mandshurica, exhibits anti-cancer and anti-inflammaroty activities. But its protection on ulcerative colitis (UC) has never been explored. In this study, we first evaluated whether juglone ameliorated UC, and investigated its effects on gut microbiota and Th17/Treg balance in DSS-induced UC mice model. The model was established by administrating 2.7% DSS for seven days. Juglone was given daily by gavage for ten days, once a day. The disease activity index (DAI) decrease and pathological characteristics improvement demonstrated that the UC in mice was alleviated by juglone. Juglone treatment significantly inhibited the protein levels of IL-6, TNF-α and IL-1β, improved the protein expression of IL-10. In addition, juglone altered microbial diversity and gut microbiota composition, including the enhancement of the ratio of Firmicutes to Bacteroidota and the abundance of Actinobacteriota, and decrease of the abundance of Verrucomicrobiota. Juglone treatment also inhibited the protein expressions of IL-6, STAT3 and RORγt, meanwhile improved the protein level of FOXP3. Furthermore, juglone inhibited Th17 development and increased Treg generation, beneficial to Th17/Treg balance. Together, we herein provided the first evidence to support that juglone, especially the high dose, possibly protected mice against UC by modulating gut microbiota and restoring Th17/Treg homeostasis.

Topics: Animals; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Drug Evaluation, Preclinical; Gastrointestinal Microbiome; Humans; Intestinal Mucosa; Male; Mice; Naphthoquinones; T-Lymphocytes, Regulatory; Th17 Cells

Environmental friendly products particularly natural dyes are going to be much popular around the globe due to their non-toxic and bio-degradable nature. The current study was planned to enhance the dyeability of walnut bark having juglone as a reddish-brown natural dye under ultrasonic radiation as an environment-friendly and green tool After conducting series of experiments, it has been found that wool (RW) and extract (RE) after ultrasonic treatment for 45 min, when dyed for 45 min at 55°C using an acidic bath of 3 pH has given good color strength on the wool fabric. To develop the new shades, sustainable and eco-label chemicals (Fe, Al, and tannic acid) and four bio-mordants such as Acacia bark, Turmeric, Henna, and Pomegranate were also applied at optimum conditions. It is studied that 3% of turmeric extract as pre-bio-mordant and 5% of Acacia extract as post-bio-mordant has given excellent color characteristics as compared to their synthetic. It is concluded that ultrasonic treatment being an eco-friendly tool has a great potential to improve the dyeability of natural reddish-brown dye from walnut bark and the inclusion of sustainable biosources as a color modifier has value-added the natural dyeing process with excellent color ratings.

Topics: Animals; Coloring Agents; Juglans; Naphthoquinones; Plant Bark; Wool; Wool Fiber

We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone (3,5-dihydroxy-1,4-naphthoquinone). The anticancer activity of three synthesized compounds, named 4 (RC10), 5 (RCDFC), and 6 (RCDOH) was evaluated in vitro against two metastatic prostate cancer (PCa) cell lines, DU145 and PC3, using MTT assays. We found that 4 (RC10) and 5 (RCDFC) induced cytotoxicity against DU145 and PC3 cells. Flow cytometry analysis revealed that these two compounds promoted cell cycle arrest in G1/S and G2/M phases, increased Sub-G1 peak and induced inhibition in cell viability. We also showed that these effects are cell-type context dependent and more selective for these tested PCa cells than for HUVEC non-tumor cells. The two BNQ compounds 4 (RC10) and 5 (RCDFC) displayed promising anticancer activity against the two tested metastatic PCa cell lines, DU145 and PC3. Their effects are mainly associated with inhibition of cell viability, possibly through apoptotic cell death, besides altering the SubG1, G1/S and G2/M phases of cell cycle. 5 (RCDFC) compound was found to be more selective than 4 (RC10), when comparing their cytotoxic effects in relation to HUVEC non-tumoral cells. Future work should also test these compounds in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic PCa cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Humans; Male; Naphthoquinones; PC-3 Cells; Prostatic Neoplasms

Inflammatory bowel diseases (IBDs) are serious disorders of which the etiologies are not, as yet, fully understood. In this study, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein was shown to be dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Interestingly,

Topics: Animals; Anti-Inflammatory Agents; Colitis; Colon; Cytokines; Dextran Sulfate; Disease Models, Animal; Enzyme Inhibitors; Inflammation Mediators; Intestinal Mucosa; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase

Topics: Circular Dichroism; DNA; Humans; Molecular Docking Simulation; Naphthoquinones; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thermodynamics

Ferroptosis is a novel iron-dependent cell death pathway mainly caused by an abnormal redox state and associated with various diseases including cancer. Recently, much attention has been paid to natural compounds that are involved in its activation and inhibition. This is the first ever study to demonstrate the role of juglone isolated from Carya cathayensis green peel in inducing autophagy and inhibiting endometrial cancer (EC) cell migration. Subsequently, Fe2+ accumulation, lipid peroxidation, GSH depletion, the upregulation of HMOX1, and heme degradation to Fe2+ were reported. Juglone was involved in inducing autophagy and inhibiting cell migration and endoplasmic reticulum stress, which are the new hallmarks of cancer treatment. Collectively, our data indicate that juglone as a functional food ingredient induces the programmed cell death of EC cells by activating oxidative stress and suggest a novel therapeutic approach for the treatment and prevention of EC.

Topics: Apoptosis; Autophagy; Carya; Cell Death; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Female; Ferroptosis; Humans; Iron; Lipid Peroxidation; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Phagocytosis

Pin1, as the peptidyl-prolyl isomerase, plays a vital role in cellular processes. However, whether it has a regulatory effect on renal ischemia and reperfusion (I/R) injury still remains unknown.. The hypoxia/reoxygenation (H/R) model in human kidney (HK-2) cells and the I/R model in rats were assessed to investigate the role of Pin1 on I/R-induced acute kidney injury. Male Sprague-Dawley rats were used to establish the I/R model for 15, 30, and 45 min ischemia and then 24 h reperfusion, with or without the Pin1 inhibitor, to demonstrate the role of Pin1 in acute kidney injury. HK-2 cells were cultured and experienced the H/R model to identify the molecular mechanisms involved.. In this study, we found that Pin1 and oxidative stress were obviously increased after renal I/R. Inhibition of Pin1 with juglone decreased renal structural and functional injuries, as well as oxidative stress. Besides, Pin1 inhibition with the inhibitor, juglone, or the small interfering RNA showed significant reduction on oxidative stress markers caused by the H/R process in vitro. Furthermore, the results indicated that the expression of p38 MAPK was increased during H/R in vitro and Pin1 inhibition could reduce the increased expression of p38 MAPK.. Our results illustrated that Pin1 aggravated renal I/R injury via elevating oxidative stress through activation of the p38 MAPK pathway. These findings indicated that Pin1 might become the potential treatment for renal I/R injury.

Topics: Acute Kidney Injury; Animals; Cell Line; Enzyme Inhibitors; Humans; Kidney; Male; MAP Kinase Signaling System; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). There is currently no satisfactory treatment for this disease. Pin1 is the only known peptidyl-prolyl cis/trans isomerase (PPIase) that is involved in many cellular processes, including immune responses. Numerous studies have shown that juglone effectively inhibits Pin1 activity. However, the effect of Pin1 inhibitor juglone on autoimmune diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), remain incomplete. So the present study aimed to explore the therapeutic effects of the Pin1 inhibitor juglone on EAE. EAE was induced in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG)

Topics: Animals; Central Nervous System; Encephalomyelitis, Autoimmune, Experimental; Mice; Mice, Inbred C57BL; Naphthoquinones

Juglone (JUG), a natural product found in walnut trees and other plants, shows potent antioxidant, antimicrobial, and immunoregulatory activities. However, it remains unknown whether JUG can alleviate ulcerative colitis. This study aims to explore the effect of JUG on dextran sulfate sodium (DSS)-induced colitis in mice. The mice were randomly assigned into three groups: the vehicle group, the DSS group, and the JUG group. The experiments lasted for 17 days; during the experiment, all mice received dimethyl sulfoxide (DMSO, 0.03% v/v)-containing water, while the mice in the JUG group received DMSO-containing water supplemented with JUG (0.04 w/v). Colitis was induced by administering DSS (3% w/v) orally for 10 consecutive days. The results showed that the JUG treatment significantly ameliorated body weight loss and disease activity index and improved the survival probability, colon length, and tissue damage. JUG reversed the DSS-induced up-regulation of proinflammatory cytokines, including interleukin (IL)-6, 12, 21, and 23, and tumor necrosis factor-alpha, and anti-inflammatory cytokines, such as IL-10 and transforming growth factor-beta, in the serum of the colitis mice. Additionally, the activation of mitochondrial uncoupling protein 2 and phospho-Nuclear Factor-kappa B p65 and the inhibition of the kelch-like ECH-associated protein 1 and NF-E2-related factor 2 induced by DSS were also reversed under JUG administration. Although the JUG group possessed a similar microbial community structure as the DSS group, JUG enriched potential beneficial microbes such as

Topics: Animals; Colitis, Ulcerative; Inflammation; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Oxidative Stress

SARS-CoV-2 as a positive-sense single-stranded RNA coronavirus caused the global outbreak of COVID-19. The main protease (M

Topics: Animals; Binding Sites; Catalytic Domain; Cell Survival; Chlorocebus aethiops; COVID-19; COVID-19 Drug Treatment; Drug Design; Drug Evaluation, Preclinical; Humans; Hydrogen Bonding; Molecular Docking Simulation; Naphthoquinones; Protease Inhibitors; SARS-CoV-2; Structure-Activity Relationship; Vero Cells; Viral Matrix Proteins

Topics: Animals; Antihypertensive Agents; Blood Pressure; Endothelium, Vascular; Hypertension; Male; Naphthoquinones; Rats; Rats, Sprague-Dawley; Vasodilation

Juglone (5-hydroxy-1, 4-naphthoquinone), is a natural phenolic compound that has been shown to relax smooth muscle. Therefore the aim of this study was to determine the effect of juglone on vascular tone using porcine coronary artery (PCA). Segments of PCA, with or without endothelium, were mounted for isometric tension recording in isolated tissue baths and precontracted with the thromboxane A

Topics: Animals; Calcium; Coronary Vessels; Cyclic GMP; Indoles; Ionomycin; Naphthoquinones; Nitric Oxide; Potassium Channel Blockers; Swine; Tetradecanoylphorbol Acetate; Vasoconstriction; Vasodilation; Vasodilator Agents

Cytochrome b

Topics: Apoptosis; Cytochrome-B(5) Reductase; Cytochromes b5; Electron Transport; Erythrocytes; Humans; NAD; Naphthoquinones; Superoxides

Colorectal cancer (CRC) is the third most common fatal cancer. Indomethacin, a nonsteroidal anti-inflammatory drug, is known to reduce the occurrence of CRC. This study evaluated the potential anticolon cancer effects of juglone (5-hydroxy-1,4-naphthoquinone) in combination with indomethacin. Human colon adenocarcinoma cells (HT29) were subjected to treatment with indomethacin, juglone, and a combination of both. Morphological analysis, cell cycle regulation, and dual staining using acridine orange and ethidium bromide in control and treated cells revealed the apoptotic potential of these compounds. Bcl2 and inflammatory molecules (tumor necrosis factor-α, nuclear factor kappa B, and Cox-2) were found to be decreased with a concomitant increase in the expression of proapoptotic molecules (Bad, Bax, cytochrome c, and PUMA) as a result of the molecular regulation of Wnt, Notch, and peroxisome proliferator-activated receptor-γ signaling. Treatment with juglone was not as effective as with indomethacin; however, a combination of both was shown to be more effective, suggesting that juglone may be considered for therapeutic intervention of colon cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Checkpoints; Cell Survival; Colonic Neoplasms; Drug Synergism; HT29 Cells; Humans; Indomethacin; Inflammation Mediators; Inhibitory Concentration 50; Naphthoquinones; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; Receptors, Notch; Signal Transduction; Wnt Signaling Pathway

Due to the resistance to drugs, studies involving the combination and controlled release of different agents are gradually increasing. In this study, two different active ingredients, known to have antibacterial and antiparasitic activities, were encapsulated into single polymeric nanoparticles. After co-encapsulation their antibacterial and antileishmanial activity was enhanced approximately 5 and 250 times, respectively. Antibacterial and antileishmanial activities of caffeic acid phenethyl ester and juglone loaded, multifunctional nanoformulations (CJ4-CJ6-CJ8) were also evaluated for the first time in the literature comparatively with their combined free formulations. The antibacterial activity of the multifunctional nanoformulation (CJ8) were found to have a much higher activity (MIC values 6.25 and 12.5 μg ml

Topics: Anti-Bacterial Agents; Antiparasitic Agents; Caffeic Acids; Escherichia coli; Leishmania; Microbial Sensitivity Tests; Nanoparticles; Naphthoquinones; Particle Size; Phenylethyl Alcohol; Polylactic Acid-Polyglycolic Acid Copolymer; Staphylococcus aureus

In this study, juglone nanoparticles were prepared by single emulsion solvent evaporation method and their effect against Candida albicans biofilm was investigated in comparison with the free juglone and Fluconazole by performing XTT, crystal violet, standard plate count, confocal microscopy and membrane depolarization analyses. Juglone nanoparticles and free juglone were found to inhibit biofilm formation and pre-established biofilms (98-100%) at all doses tested, whereas Fluconazole did not cause a significant inhibition, even at the highest dose applied, especially against pre-established biofilms. Membrane depolarization analysis showed that free juglone and juglone loaded nanoparticles were effective on C. albicans membrane structure and have fluorescence quenching effect on DiSC

Topics: Antifungal Agents; Biofilms; Candida albicans; Drug Liberation; Nanoparticles; Naphthoquinones; Polylactic Acid-Polyglycolic Acid Copolymer

BRAF inhibitors are some of the most effective drugs against melanoma; however, their clinical application is largely limited by drug resistance. Juglone, isolated from walnut trees, has demonstrated anti‑tumour activity. In the present study, it was investigated whether juglone could enhance the responses to a BRAF inhibitor in melanoma cells (A375R and SK‑MEL‑5R) with an acquired resistance. These cells were treated with juglone alone, BRAF inhibitor (PLX4032) alone, or juglone combined with PLX4032. It was demonstrated that the combination of juglone and PLX4032 had synergistic effects on BRAF inhibitor‑resistant melanoma cells. Juglone potentiated PLX4032‑induced cytotoxicity and mitochondrial apoptosis in both A375R and SK‑MEL‑5R cells, which was accompanied by a decline in mitochondrial membrane potential and a decrease in Bcl‑2/Bax ratio. Moreover, juglone combined with PLX4032 markedly increased the intracellular level of reactive oxygen species (ROS) and activated p38 and p53, as compared with juglone alone or PLX4032 alone. Pre‑treatment with N‑acetyl‑L‑cysteine, a ROS scavenger, completely reversed the cytotoxicity induced by juglone combined with PLX4032. In conclusion, juglone potentiated BRAF inhibitor‑induced apoptosis in resistant melanoma cells, and these effects occurred partially through ROS and the p38‑p53 pathway, suggesting the potential of juglone as a sensitizer to BRAF inhibitors in the treatment of melanoma.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; MAP Kinase Signaling System; Melanoma; Naphthoquinones; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53; Vemurafenib

Topics: Anti-Bacterial Agents; Ascomycota; Biosynthetic Pathways; Cell Proliferation; Colonic Neoplasms; Genome, Fungal; Gram-Positive Bacteria; HCT116 Cells; Humans; Naphthoquinones; Secondary Metabolism

Juglone and thymoquinone are cytotoxic to pancreatic cancer cells. The aim of this study was to investigate, using an analysis of isobolograms, the type and degree of interactions between juglone and thymoquinone on MIA PaCa-2 pancreatic cancer cells. Cell viability was evaluated using the MTT assay. Cell death was determined by flow cytometry. The IC

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Naphthoquinones; Pancreatic Neoplasms

Topics: Animals; Cell Line; Inflammation; Interleukin-18; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; Naphthoquinones; Nitric Oxide; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species

Juglone, a naphthoquinone isolated from many species of the Juglandaceae (walnut) family, has been used in traditional Chinese medicine for centuries for its various pharmacological effects. Our previous research found its toxic effects on oocytes maturation. But we still know a little about its toxic effects on embryo development. Here, we used mouse embryo as a model to explore the effects of juglone on early mammalian embryo development. Exposure to juglone significantly decreased the development rate in early mouse embryos in vitro. Moreover, juglone exposure led to developmental arrest by disturbing mitochondrial function, producing abnormal epigenetic modifications, inducing high levels of oxidative stress and DNA damage, and increasing the rate of embryonic cell apoptosis. However, vitamin C (VC) ameliorated the toxic effects of juglone to a certain extent. Overall, juglone has a toxic effect on early embryo development through the generation of ROS and apoptosis. But VC was able to protect against these juglone-induced defects. These results not only give a new perspective on juglone's pharmacological effects on early mammalian embryo development, but also provide ideas for the better application of this agent in traditional Chinese medicine.

Topics: Animals; Apoptosis; Ascorbic Acid; DNA Damage; Embryo, Mammalian; Embryonic Development; Female; Male; Mice, Inbred ICR; Naphthoquinones; Protective Agents; Reactive Oxygen Species; Vitamins

Carnitine is required for transporting fatty acids into the mitochondria for β-oxidation. Carnitine has been used as an energy supplement but the roles in improving health and delaying aging remain unclear. Here we show in

Topics: Aging; Amyloid beta-Peptides; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carnitine; DNA-Binding Proteins; Forkhead Transcription Factors; Humans; Hydrogen Peroxide; Longevity; Naphthoquinones; Organic Cation Transport Proteins; Oxidative Stress; Paraquat; Reactive Oxygen Species; Receptor, Insulin; Receptors, Notch; Stress, Physiological; Transcription Factors

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.

Topics: Animals; Apoptosis; Blastocyst; Cattle; Cytotoxins; Embryo, Mammalian; Embryonic Development; Female; In Vitro Oocyte Maturation Techniques; Mitochondria; Naphthoquinones; Oocytes; Oxidative Stress; Pregnancy; Reactive Oxygen Species

A new oxidovanadium(IV) complex VO(L)(Jug) (HL = 5-methoxy-1,3-bis (1-methyl-1H-benzo[d]imidazol-2-yl)benzene, Jug = juglone) was synthesized and characterized. Interactions of the V(IV) complex with calf thymus DNA (CT DNA) and human serum albumin were studied using different techniques such as UV-vis and fluorescence emission spectroscopy. The experimental results were confirmed by the molecular docking study. The oxidovanadium(IV) complex can efficiently cleave pUC19 DNA in the presence of Hydrogen peroxide. Also, the in vitro cytotoxicity properties of the oxidovanadium(IV) complex was evaluated against MCF-7, HPG-2 and HT-29 cancer cell lines and HEK293 non-malignant fibroblasts were evaluated and compared with free ligands, VOSO

Topics: Binding Sites; Cell Death; Cell Line, Tumor; DNA; DNA Cleavage; Humans; Molecular Conformation; Molecular Docking Simulation; Naphthoquinones; Protein Binding; Serum Albumin, Human; Spectrometry, Fluorescence; Vanadium

Human papillomaviruses (HPVs), for instance, HPV 16 and HPV 18, are concerned associated with cervical cancer. Thus, it is essential to suppress HPVs-in HPV-positive cervical cancer for treating cervical cancer. The purpose of this study was to explore the proposed molecular mechanisms, which that underlies the antintumor potential of juglone to treat of HPV-positive on cervical cancer cells. The results showed that juglone suppressed HPV-positive cell growth in a dose- and time-dependent way. In addition, cell invasion and metastasis were also inhibited by juglone. Nevertheless, when pin 1 was knocked down in HPV-positive cells, cell proliferation, invasion and metastasis were reduced. This study was designed to acquire an understanding of the mechanism of invasion and metastasis in HPV-positive cells suppressed by juglone. It provides evidence of the advantageous use of juglone in the future.

Topics: Cell Line, Tumor; Cell Proliferation; Female; HeLa Cells; Humans; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Papillomaviridae; Papillomavirus Infections; Uterine Cervical Neoplasms

Three natural naphthoquinones were screened to find new anti-virulence agents as inhibitors against sortase A from Staphylococcus aureus (SaSrtA) by quantifying the increase in fluorescence intensity upon substrate cleavage at various concentrations. The 5-hydroxy-1,4-naphthalenedione derivatives, juglone and plumbagin, demonstrated a potent inhibitory effect, with IC

Topics: Aminoacyltransferases; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Catalytic Domain; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enterococcus faecalis; Inhibitory Concentration 50; Models, Molecular; Molecular Docking Simulation; Naphthoquinones; Protein Binding; Staphylococcus aureus; Staphylococcus epidermidis

Topics: Aged; Blister; Dermatitis, Irritant; Hand Dermatoses; Humans; Hyperpigmentation; Juglans; Male; Naphthoquinones

Juglonols A-C (

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Antineoplastic Agents; Cell Line; Humans; Juglans; Molecular Structure; Naphthoquinones; Plant Extracts; Spectrum Analysis; Tetralones

Topics: Animals; BCG Vaccine; CD8-Positive T-Lymphocytes; Cell Line; Cells, Cultured; Cytokines; Cytotoxins; Immunoglobulin G; Immunomodulation; Macrophages; Mice; Mice, Inbred C57BL; Naphthoquinones; T-Lymphocytes, Regulatory

The effective wound management strategies depends on identification and manipulation of the molecular defects in the pathophysiology of wound. Poor vascularization, protease susceptibility and microbial invasion at wound site affect the early wound closure. Hence, an efficient wound dressing material needs to promote angiogenesis, control proteolytic activity and microbial attack. The present study, describes designing and developing a novel wound dressing material by stabilization of collagen with juglone functionalized silver nanoparticle. Stabilization of collagen with juglone functionalized silver nanoparticles enhanced thermal properties, influenced the uniform alignment of collagen fibrils which enhanced collagen's ability to promote cell proliferation. FTIR and CD analyses revealed that juglone functionalized silver nanoparticles did not induced any structural changes in the collagen molecule. Juglone functionalized silver nanoparticles controlled the proteolytic activity in a spatio-temporal manner and elicited the angiogenic response by upregulation of cell adhesion molecules like β catenin and VE cadherin which had promoted the cell attachment and cell-cell contact. It had also promoted the expression of angiogenic signaling molecules like VEGF and VEGFR2. Further, the in vivo studies proved that the juglone functionalized silver nanoparticles had a potential role in rapid wound closure due to the cumulative property of juglone and silver nanoparticles.

Topics: Cell Adhesion; Cell Line; Collagen; Endothelial Cells; Gene Expression Regulation; Humans; Metal Nanoparticles; Naphthoquinones; Silver; Tissue Scaffolds; Wound Healing

The elimination of cyanobacteria blooms has become an urgent concern in aquatic environmental protection. Allelopathic control is considered a potential approach because of its exclusive and ecological safety properties. The present study evaluated the allelopathic effect of juglone, a derivative from the genus Juglans, on the toxic Microcystis aeruginosa. Juglone at 3.0-9.0 mg L

Topics: Antioxidants; Cyanobacteria; Microcystins; Microcystis; Naphthoquinones

Tyrosine kinase inhibitor (TKI)-based therapy has created promising results among much chronic myeloid leukemia (CML) patients. Imatinib as a relatively specific inhibitor of Bcr-Abl is at present one of the undisputed therapeutic agent for newlydiagnosed patients with CML. However, the occurrence of imatinib-resistance enlightens the urgent need to identify other therapeutic agents against CML. Juglone (5-hydroxy-2-methyl-1, 4-naphthoquinone) exerts cytotoxic effects against various human cancer cell lines. However, the mechanisms through which Juglone induces anticancer effects in CML especially in comparison with imatinib treatment remain unknown. Our results revealed that Juglone-inhibited K562 cells growth through inducing apoptosis. Based on our Western blot analyses, Juglone significantly reduced p-Akt levels and increased the expression level of Forkhead box O1 (FoxO1) and FoxO3a proteins. Moreover, hairy/enhancer of split-1 (Hes1) protein, overexpressed under the influence of Juglone, is apparently involved in Juglone-induced apoptosis among K562 cells. Conversely, treatment with imatinib attenuated Hes1 protein expression. Considering the different functional mechanism of Juglone compared with imatinib, it seems that Juglone treatment could be a useful alternative strategy for the treatment of patients with imatinib-resistance.

Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; Cell Survival; Forkhead Box Protein O1; Forkhead Box Protein O3; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Naphthoquinones; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Transcription Factor HES-1

Human papillomaviruses (HPVs), such as HPV 16 and HPV 18 are related to cervical cancer. Therefore, it is important to inhibit HPV-positive cervical cancer for treating cervical cancer. This study is aiming at investigating the proposed molecular mechanism, which underlies the antineoplastic potential of the aqueous extract of juglone of HPV-positive cervical cancer cells. According to the results, it is showed that, juglone prohibited HPV positive cervical cancer cells' growth through dose-dependent way. Nevertheless, when pin 1 was knocked down, the proliferation inhibition reduced. The detection of apoptosis and cell cycle also illustrated that juglone influenced HPV positive cells. Western blot expressed the influence mechanism that it affected the B-cell lymphoma 2 (Bcl-2) family and later activated the Caspase-depended apoptosis way. It is contributable for this study to understand the mechanism of inhibiting HPV positive cells by juglone and it also provides an effective strategy for the application of it in the future.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Female; Gene Expression Regulation; Human papillomavirus 16; Human papillomavirus 18; Humans; Mitochondria; Naphthoquinones; Up-Regulation; Uterine Cervical Neoplasms

Signal transducer and activator of transcription 3 (STAT3) is an oncogene constitutively activated in hepatocellular carcinoma (HCC) cells and HCC cancer stem cells (CSCs). Constitutively activated STAT3 plays a pivotal role in holding cancer stemness of HCC CSCs, which are essential for hepatoma initiation, relapse, metastasis and drug resistance. Therefore, STAT3 has been validated as a novel anti-cancer drug target and the strategies targeting HCC CSCs may bring new hopes to HCC therapy. This study aimed to isolate and identify small-molecule STAT3 signaling inhibitors targeting CSCs from the ethyl acetate (EtOAc) extract of the roots of Polygonum cuspidatum and to evaluate their in vitro anti-cancer activities.. The chemical components of the EtOAc extract and the subfractions of P. cuspidatum were isolated by using various column chromatographies on silical gel, Sephadex LH-20, and preparative HPLC. Their chemical structures were then determined on the basis of spectroscopic data including NMR, MS and IR analysis and their physicochemical properties. The inhibitory effects of the isolated compounds against STAT3 signaling were screened by a STAT3-dependent luciferase reporter gene assay. The tyrosine phosphorylation of STAT3 was examined by Western Blot analysis. In vitro anti-cancer effects of the STAT3 pathway inhibitor were further evaluated on cell growth of human HCC cells by a MTT assay, on self-renewal capacity of HCC CSCs by the tumorsphere formation assay, and on cell cycle and apoptosis by flow cytometry analysis, respectively.. The EtOAc extract of the roots of P. cuspidatum was investigated and a novel juglone analogue 2-ethoxystypandrone (1) along with seven known compounds (2-8) was isolated. Among the eight isolated compounds 1-8, 2-ethoxystypandrone was a novel and potent STAT3 signaling inhibitor (IC. A novel juglone analogue 2-ethoxystypandrone was identified from the EtOAc extract of the roots of P. cuspidatum and was demonstrated to be a potent small-molecule STAT3 signaling inhibitor, which strongly blocked STAT3 activation, inhibited proliferation, and induced cell apoptosis of HCC cells and HCC CSCs. 2-Ethoxystypandrone as a STAT3 signaling inhibitor might be a promising lead compound for further development into an anti-CSCs drug.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Fallopia japonica; Hep G2 Cells; Humans; Naphthoquinones; Neoplastic Stem Cells; Plant Extracts; Signal Transduction; STAT3 Transcription Factor

Binding interactions between the drug Juglone (JUG) and Lysozyme (LYZ) have been explored in details from spectroscopic studies aided by in silico calculations. UV-Vis, steady state and time resolved fluorescence spectroscopic studies indicate the formation of LYZ-JUG complex in the ground state. Quenching of corrected fluorescence spectra of LYZ in presence of JUG at varied concentrations in different temperature range have been estimated from Stern-Volmer (SV) plots. Time resolved fluorescence spectroscopic studies confirm the mechanism of quenching to be of static type. Binding constant associated with the LYZ-JUG complex has been estimated from Scatchard plot. The number of binding sites, thermodynamic parameters and the modes of interaction are also estimated. Synchronous fluorescence spectra monitored at two discrete wavelength windows confirm the prominent role of Tryptophan residues towards quenching of fluorescence in LYZ. The circular dichroism (CD) spectra signify alterations in the population of α-helical content within the secondary structure of LYZ in presence of JUG molecules. REES of LYZ in the presence of JUG further signify definite impact of the drug JUG molecule on the Trp residues of the protein. The experimental observations are supported by in silico molecular docking and molecular dynamics simulations.

Topics: Binding Sites; Circular Dichroism; Hydrogen Bonding; Molecular Docking Simulation; Muramidase; Naphthoquinones; Protein Binding; Protein Conformation, alpha-Helical; Spectrometry, Fluorescence; Temperature; Thermodynamics

Juglone, a naphthoquinone isolated from many species of the Juglandaceae family, has been used in traditional Chinese medicine for centuries because of its antiviral, antibacterial, and antitumor activities. However, the toxicity of juglone has also been demonstrated. Here, we used porcine oocytes as a model to explore the effects of juglone on oocyte maturation and studied the impact of vitamin C (VC) administration on juglone exposure-induced meiosis defects. Exposure to juglone significantly restricted cumulus cell expansion and decreased the first polar body extrusion. In addition, juglone exposure disturbed spindle organization, actin assembly, and the distribution of mitochondria during oocyte meiosis, while the acetylation level of α-tubulin was also reduced. These defects were all ameliorated by VC administration. Our findings indicate that juglone exposure induced meiotic failure in porcine oocytes, while VC protected against these defects during porcine oocyte maturation by ameliorating the organization of the cytoskeleton and mitochondrial distribution.

Topics: Acetylation; Animals; Ascorbic Acid; Cumulus Cells; Cytoskeleton; Female; In Vitro Oocyte Maturation Techniques; Meiosis; Mitochondria; Naphthoquinones; Oocytes; Polar Bodies; Swine; Tubulin

Metabolic reprogramming is an important feature of host-pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite

Topics: Animals; Antiprotozoal Agents; Biological Transport; Carrier Proteins; Cattle; Cell Line, Transformed; Cell Transformation, Neoplastic; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Host-Pathogen Interactions; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphocytes; Membrane Proteins; Metabolic Networks and Pathways; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Protozoan Proteins; RNA, Small Interfering; Signal Transduction; Theileria; Thyroid Hormone-Binding Proteins; Thyroid Hormones

A 70% ethanol extract from the root portion of Reynoutria japonica afforded one new and three known juglone derivatives, namely, 2-methoxy-6-acetyl-7-methyljuglone (1), 2-ethoxy-6-acetyl-7-methyljuglone (2), 2-methoxy-7-acetonyljuglone (3), and 3-acetyl-7-methoxy-2-methyljuglone (4) together with two phenolics (5 and 6), an anthraquinone (7), a stilbene (8) and a phthalide (9). Their structures were elucidated on the basis of comprehensive spectroscopic studies including IR, MS, and

Topics: Anti-Bacterial Agents; Ethanol; Helicobacter pylori; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Plant Roots; Polygonaceae

Myeloid-derived suppressor cells (MDSCs) contribute to immune activity suppression and promote the tumor progression. Elimination of MDSCs is a promising cancer therapeutic strategy, and some chemotherapeutic agents have been reported to hamper tumor progression by suppressing MDSCs. Juglone has been showed to exert a direct cytotoxic effect on tumor cells. However, the effect of juglone on MDSCs and anti-tumor immune statue has remained unexplored. In our study, we observed that juglone suppressed tumor growth and metastasis markedly, and the tumor growth suppression in immunocompetent mice was more drastic than that in immunodeficient mice. Juglone reduced the accumulation of MDSCs and increased IFN-γ production by CD8

Topics: Animals; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Fluorouracil; Interferon-gamma; Interleukin-1beta; Liver; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Myeloid-Derived Suppressor Cells; Naphthoquinones; Neoplasms

The molecular mechanism of Juglone-induced cell cycle arrest and apoptosis in human endometrial cancer cells was investigated. Juglone was purified from the green husk of Carya cathayensis Sarg and identified by HPLC, LC-MS/MS, and NMR. At an IC

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carya; cdc25 Phosphatases; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Endometrial Neoplasms; Female; Humans; Naphthoquinones; Plant Extracts

Juglone (JG) exhibits a broad-spectrum of cytotoxicity against some cancer cells. However, its molecular mechanisms have not been investigated well. Here, the present results showed that JG significantly inhibited tumor growth in vivo. CCK-8 assays, flow cytometric analysis, western blotting and immunohistochemistry revealed that JG effectively inhibited cell proliferation and induced apoptosis through extrinsic pathways. We also observed that JG treatment induced autophagy flux via activiting the AMPK-mTOR signaling pathway. In addition, we found that JG enhanced p53 activation by increasing down-regulation of ubiquitin-mediated degradation. Inhibition of p53 by siRNA attenuated JG-induced cell death and autophagy. Moreover, JG enhanced the generation of hydrogen peroxide (H

Topics: Apoptosis; Autophagy; Flow Cytometry; Gene Expression; Hep G2 Cells; Humans; Naphthoquinones; Polymerase Chain Reaction; Reactive Oxygen Species; Tumor Suppressor Protein p53

Caesalpinia mimosoides, a vegetable consumed in Thailand, has been reported to exhibit in vitro antioxidant properties. The in vivo antioxidant and anti-aging activities have not been investigated. The aim of this research was to study the antioxidant activity of C. mimosoides extracts in Caenorhabditis elegans, a widely used model organism in this context.. C. elegans were treated with C. mimosoides extracts in a various concentrations. To investigate the protective effects of the extract against oxidative stress, wild-type N2 were used to determine survival rate under oxidative stress and intracellular ROS. To study underlying mechanisms, the mutant strains with GFP reporter gene including TJ356, CF1553, EU1 and LD4 were used to study DAF-16, SOD-3, SKN-1 and GST-4 gene, respectively. Lifespan and aging pigment of the worms were also investigated.. A leaf extract of C. mimosoides improved resistance to oxidative stress and reduced intracellular ROS accumulation in nematodes. The antioxidant effects were mediated through the DAF-16/FOXO pathway and SOD-3 expression, whereas the expression of SKN-1 and GST-4 were not altered. The extract also prolonged lifespan and decreased aging pigments, while the body length and brood size of the worms were not affected by the extract, indicating low toxicity and excluding dietary restriction.. The results of this study establish the antioxidant activity of C. mimosoides extract in vivo and suggest its potential as a dietary supplement and alternative medicine to defend against oxidative stress and aging, which should be investigated in intervention studies.

Topics: Animals; Antioxidants; Body Size; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Caesalpinia; Flavonoids; Free Radical Scavengers; Longevity; Methanol; Naphthoquinones; Phenols; Plant Extracts; Plant Leaves; Reactive Oxygen Species; Reproduction

The objective of this work was to analyse the antiradical capacity of juglone (5-hydroxy-1,4,-naphthoquinone). The influence of oxidation and reduction on juglone was investigated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV), as well as spectrophotometric based methods. The role of juglone in oxidation processes, as either an antioxidant in browning reactions, was examined. These processes are characterized by a high chemical reactivity in redox. Juglone is irreversibly oxidized in at least one electrode step and reduced quasi-reversibly in at least three electrode steps. These results demonstrate that walnut genotypes have different radical scavenging powers. In addition, on the basis of thermogravimetry, it was demonstrated that 5-hydroxy-1,4-naphthalenedione has high thermal stability above 500 °C. The generation of reactive oxygen species and activity in redox processes show the properties of naphthoquinones that render these compounds interesting leads for the development of novel biomolecules for potential use in various therapeutic settings.

Topics: Antioxidants; Electrochemistry; Juglans; Naphthoquinones; Oxidation-Reduction

Naphthoquinones are known for their broad range of biological activities. Given the increasing demands of consumers in relation to food quality and growing concerns about the impact of synthetic herbicides, it is necessary to search for new agrochemicals. Natural products and allelopathy provide new alternatives for the development of pesticides with lower toxicity and greater environmental compatibility.. A structure-activity relationship to evaluate the effect of bioavailability was performed. A total of 44 O-acyl and O-alkyl derivatives of juglone and lawsone with different linear chain lengths were prepared. These compounds were tested on etiolated wheat coleoptiles, standard target species (STS) and four weeds, Echinochloa crus-galli L., Lolium rigidum Gaud., Lolium perenne L. and Avena fatua L. The results showed a strong influence of lipophilicity and, in most cases, the data fitted a logP-dependent quadratic mathematical model.. The effects produced were mostly stunting and necrosis caused by growth inhibition. The potential structure and activity behaviour is described. © 2017 Society of Chemical Industry.

Topics: Avena; Echinochloa; Herbicides; Lolium; Naphthoquinones; Plant Weeds; Structure-Activity Relationship

Transient elevations in blood glucose level may lead to changes in vascular function. Herein, we investigated the effects of high-glucose or high-fructose challenge, as well as potential influence of juglone or resveratrol on vascular reactivity, Akt/eNOS, and insulin signaling effectors in rat aorta. Aortic segments of rats were incubated with high glucose (30 mmol/L) or high fructose (2 mmol/L) in the absence and presence of juglone (5 μmol/L) or resveratrol (10 μmol/L). Acute high-glucose incubation markedly decreased acetylcholine-induced relaxation, which is further inhibited by juglone, but ameliorated by resveratrol. Incubation with high glucose caused significant reduction in pAkt/total Akt and peNOS/total eNOS ratios, as well as in the expression of some genes involved in insulin signaling. Juglone produced a further impairment, whereas resveratrol resulted in an improvement on the expression profiles of these proteins and genes. Acute exposure of aortic segments to high glucose causes a reduction in acetylcholine-induced relaxation in association with suppression of Akt/eNOS pathway, as well as several genes in insulin signaling pathway. Juglone and resveratrol have opposite actions on vascular relaxation and the above signaling targets. These findings could be relevant for the treatment of hyperglycemia-induced vascular complications.

Topics: Acetylcholine; Animals; Aorta; Endothelium, Vascular; Fructose; Gene Expression Regulation; Glucose; In Vitro Techniques; Male; Naphthoquinones; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Wistar; Resveratrol; Signal Transduction; Stilbenes; Vasodilation

Juglone (JG), a naturally-occurring naphthoquinone of Manchurian walnut (Juglans mandshurica) was shown to inhibit proliferation in various tumor types. However, the molecular mechanisms of JG on the induction of apoptosis and autophagy in HepG2 cells have not been examined. Herein, we investigated that JG could inhibit cell proliferation by induction of G2/M phase arrest. Also, occurrence of apoptosis was closely related with loss of mitochondrial membrane potential, the changes of apoptosis-related proteins after treatment with JG. In addition, we found that JG caused autophagy, as evidenced by increased expressions of LC3-II and Beclin-1. Interestingly, inhibition of JG-induced autophagy by 3-methyladenine (3-MA) and wortmannin (WT) significantly decreased apoptosis, whereas the apoptosis inhibitor z-VAD-fmk slightly enhanced autophagy. Furthermore, the induction of autophagy and apoptosis was associated with activation of MAPK family members (p38 and JNK) and production of reactive oxygen species (ROS). Both JNK inhibitor (SP600125) and ROS scavenger (N-acetylcysteine, NAC) could attenuate JG-induced autophagy and apoptosis. However, the p38-specific inhibitor SB203580 enhanced autophagic and apoptotic death. Moreover, the ROS scavenger NAC prevented phosphorylation of both p38 and JNK. Collectively, our data revealed that JG induced G2/M phase arrest, apoptosis, and autophagy through the ROS-dependent signaling pathway.

Topics: Acetylcysteine; Adenine; Amino Acid Chloromethyl Ketones; Androstadienes; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Enzyme Activation; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Wortmannin

Naphthoquinones are among the most active natural products obtained from plants and microorganisms. Naphthoquinones exert their biological activities through pleiotropic mechanisms that include reactivity against cell nucleophiles, generation of reactive oxygen species (ROS), and inhibition of proteins. Here, we report a mechanistic antiproliferative study performed in the yeast Saccharomyces cerevisiae for several derivatives of three important natural naphthoquinones: lawsone, juglone, and β-lapachone. We have found that (i) the free hydroxyl group of lawsone and juglone modulates toxicity; (ii) lawsone and juglone derivatives differ in their mechanisms of action, with ROS generation being more important for the former; and (iii) a subset of derivatives possess the capability to disrupt mitochondrial function, with β-lapachones being the most potent compounds in this respect. In addition, we have cross-compared yeast results with antibacterial and antitumor activities. We discuss the relationship between the mechanistic findings, the antiproliferative activities, and the physicochemical properties of the naphthoquinones.

Topics: Anti-Bacterial Agents; Antifungal Agents; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Humans; Mitochondria; Molecular Structure; Naphthoquinones; Oxidative Stress; Saccharomyces cerevisiae; Staphylococcus aureus; Structure-Activity Relationship

Anaplastic thyroid carcinoma (ATC) requires more innovative approaches as the current regimes for therapy are inadequate, also most anticancer drugs cause general suppression of physiological functions. However, therapy with limited nontarget tissue damage is desirable. In the present study, we show prooxidant ability of ascorbic acid, which enhances cytotoxicity induced by juglone. We decipher that juglone-ascorbate combination induces reactive oxygen species-mediated apoptosis leading to cell death in ARO cell line originated from ATC. This combination also affects enzyme activity of catalase, glutathione reductase, and superoxide dismutase destabilizing redox balance in cell and thereby making juglone effective at a lower dose. We also show that juglone-ascorbate combination suppresses cell migration, invasion, and expression of tumor-promoting, and angiogenic genes in ARO cell line, thereby disrupting epithelial-mesenchymal transition ability of the cells. Overall, we show that ascorbic acid increases cytotoxic potency of juglone through redox cycling when used in synergy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Inhibitory Concentration 50; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Proteins; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; RNA Interference; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis-trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/- mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/- mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Topics: Acrocephalosyndactylia; Animals; Core Binding Factor Alpha 1 Subunit; Cranial Sutures; Craniosynostoses; Disease Models, Animal; Female; Gain of Function Mutation; Gene Expression Regulation; Humans; Mice; Morphogenesis; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Osteoblasts; Pregnancy; Primary Cell Culture; Receptor, Fibroblast Growth Factor, Type 2; Signal Transduction

This study aimed to investigate the effects of juglone on the human bladder carcinoma cell lines TCC-SUB and RT-4 in monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. The activity of caspase-3 was detected in vitro using a caspase-3 colorimetric assay kit according to the manufacturer's instructions. The bromodeoxyuridine (BrdU) labeling index was used to determine the cells of the synthesis phase. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was used to determine the death of cells in both the monolayer and spheroid cultures. The control group had a large S-phase fraction and many of the TCC-SUB and RT-4 cells nuclei were observed to be positive for BrdU. The dead cell count was higher in the TCC-SUB and RT-4 cell lines with juglone applied than in the controls. We conclude that juglone significantly inhibits the proliferation and induces the apoptosis of TCC-SUB and RT-4 cells in vitro.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Naphthoquinones

Pitanga, a fruit of the pitangueira tree (Eugenia uniflora L.), is native to Brazil and has a high antioxidant capacity due to the elevated amount of anthocyanins. The present study aimed to investigate the chemical composition of the purple pitanga fruit and to evaluate its antioxidant effect in the nematode Caenorhabditis elegans. We observed that the ethanolic extract of purple pitanga did not cause any toxic effects but notably increased worm lifespan. The extract improved the survival, reproduction and lifespan of the worms in pre- and post-exposure to stressors H

Topics: Animals; Anthocyanins; Antioxidants; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Eugenia; Forkhead Transcription Factors; Fruit; Hydrogen Peroxide; Longevity; Naphthoquinones; Oxidative Stress; Phenols; Plant Extracts; Spectrophotometry, Ultraviolet; Tandem Mass Spectrometry

The major aim of the present study was to investigate the influence of juglone (JU; 5-hydroxy-1,4-naphthoquinone) treatments on the expression level of Cat1, Cat2 and Cat3 genes, encoding the respective catalase isozymes in maize (Zea mays L.) and wheat (Triticum aestivum L.) seeds. In parallel, germination efficiency, catalase (CAT) activity and hydrogen peroxide (H

Topics: Catalase; Enzyme Induction; Gene Expression Profiling; Gene Expression Regulation, Plant; Germination; Hydrogen Peroxide; Isoenzymes; Naphthoquinones; Plant Proteins; Seeds; Transcriptome; Triticum; Zea mays

Juglone (JL), as one of the major bioactive components present in the bark of Juglans mandshruica Maxim, exhibits versatile bioactivities, especially anti-cancer activity. To better understand the pharmacokinetic properties of juglone, the protein binding rate of juglone was determined by ultrafiltration method, and the binding affinity and mechanism between JL and human serum albumin (HSA) was investigated in vitro through multi-spectroscopic, thermodynamic, and molecular modeling methods. The binding degree of JL was measured more than 99.0% which suggested that JL had high binding ability to serum albumin. Fluorescence data showed that juglone quench the intrinsic fluorescence of HSA upon forming the JL-HSA nonfluorescent complex at 1:1 stoichiometric proportion, and the complex formation had a high affinity of 10

Topics: Binding Sites; Circular Dichroism; Humans; Models, Molecular; Molecular Docking Simulation; Naphthoquinones; Protein Binding; Serum Albumin; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Thermodynamics; Ultrafiltration

Juglone is a natural pigment, which has cytotoxic effect against various human tumor cells. However, its cytotoxicity to glioma cells, especially to tumor stem-like cells (TSCs) has not been demonstrated.. TSCs of glioma were enriched from U87 and two primary cells (SHG62, and SHG66) using serum-free medium supplemented with growth factors, including bFGF, EGF and B27. After treatment of juglone with gradient concentrations (0, 10, 20, and 40 μM), the viability and apoptosis of TSCs were evaluated by WST-8 assay and flow cytometry. Reactive oxygen species (ROS) was labeled by the cell-permeable fluorescent probe and detected with flow cytometry. ROS scavenger (NAC) and p38-MAPK inhibitor (SB203580) were applied to resist the cytotoxic effect. Caspase 9 cleavage and p38 phosphorylation (P-p38) were quantified by western blot. Juglone as well as temozolomide (TMZ) were administrated in intracranial xenografts and MR scan was performed every week to evaluate the anti-tumor effect in vivo.. Juglone could obviously inhibit the proliferation of TSCs in glioma by decreasing cell viability (P < 0.01) and inducing apoptosis (P < 0.01), which was accompanied by increased caspase 9 cleavage in a dose-dependent manner (P < 0.01). In the meantime, juglone could generate ROS significantly and increase p38 phosphorylation (P < 0.01). In addition, pretreatment with ROS scavenger or p38-MAPK inhibitor could reverse juglone-induced cytotoxicity (P < 0.01). More importantly, juglone could also suppress tumor growth in vivo and improve the survival of U87-bearing mice compared with control (P < 0.05), although TMZ seemed to have better effect.. Juglone could inhibit the growth of TSCs in gliomas through the activation of ROS-p38-MAPK pathway in vitro, and the anti-glioma effect was validated in vivo, which offers a potential therapeutic agent to gliomas.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Glioblastoma; Humans; Mice; Naphthoquinones; Neoplastic Stem Cells; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen responsible for several human diseases, including acute otitis media, pneumonia, sepsis and bacterial meningitis, and possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. With the capacity to form pores in cholesterol-rich membranes, pneumolysin (PLY) is a key virulence factor of S. pneumoniae and causes severe tissue damage during pneumococcal infection. Juglone (JG), a natural 1,4-naphthoquinone widely found in the roots, leaves, woods and fruits of Juglandaceae walnut trees, inhibits PLY-induced hemolysis via inhibition of the oligomerization of PLY and exhibits minimal anti-S. pneumoniae activity. In addition, when human alveolar epithelial (A549) cells were co-cultured with PLY and JG, PLY-mediated cell injury was significantly alleviated. These results indicate that JG directly interacts with PLY to reduce the cytotoxicity of the toxin in human alveolar epithelial cells. Hence, JG is an effective inhibitor of PLY and protects lung cells from PLY-mediated cell injury. This study also provides the basis for the development of anti-virulence drugs for the treatment of S. pneumoniae infections.

Topics: Alveolar Epithelial Cells; Bacterial Proteins; Humans; Naphthoquinones; Streptococcus pneumoniae; Streptolysins

Accumulating data show that prolylisomerase (Pin1) is overexpressed in human glioblastoma multiforme (GBM) specimens. Therefore, Pin1 inhibitors should be investigated as a new chemotherapeutic drug that may enhance the clinical management of human gliomas. Recently, juglone, a Pin1 inhibitor, was shown to exhibit potent anticancer activity in various tumor cells, but its role in human glioma cells remains unknown. In the present study, we determined if juglone exerts antitumor effects in the U251 human glioma cell line and investigated its potential underlying molecular mechanisms. Cell survival, apoptosis, migration, angiogenesis and molecular targets were identified with multiple detection techniques including the MTT cell proliferation assay, dual acridine orange/ethidium bromide staining, electron microscopy, transwell migration assay, chick chorioallantoic membrane assay, quantitative real-time polymerase chain reaction and immunoblotting. The results showed that 5-20 µM juglone markedly suppressed cell proliferation, induced apoptosis, and enhanced caspase-3 activity in U251 cells in a dose- and time-dependent manner. Moreover, juglone inhibited cell migration and the formation of new blood vessels. At the molecular level, juglone markedly suppressed Pin1 levels in a time-dependent manner. TGF-β1/Smad signaling, a critical upstream regulator of miR-21, was also suppressed by juglone. Moreover, the transient overexpression of Pin1 reversed its antitumor effects in U251 cells and inhibited juglone-mediated changes to the TGF-β1/miR-21 signaling pathway. These findings suggest that juglone inhibits cell growth by causing apoptosis, thereby inhibiting the migration of U251 glioma cells and disrupting angiogenesis; and that Pin1 is a critical target for juglone's antitumor activity. The present study provides evidence that juglone has in vitro efficacy against glioma. Therefore, additional studies are warranted to examine the clinical potential of juglone in human gliomas.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Chick Embryo; Chorioallantoic Membrane; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Naphthoquinones; Neovascularization, Pathologic; NIMA-Interacting Peptidylprolyl Isomerase; Signal Transduction

Allelopathy is a phenomenon, where one species releases compounds able to inhibit the growth of other species. Juglone, 5-hydroxy-1,4-naphtoquinone, is an allelochemical produced by walnut trees. The main mode of juglone toxicity is the formation of semiquinone radicals, able to reduce O

Topics: Allelopathy; Antioxidants; Carotenoids; Chlamydomonas reinhardtii; Chlorophyll; Electron Spin Resonance Spectroscopy; Lipid Peroxidation; Naphthoquinones; Oxidative Stress; Plastoquinone; Tocopherols

The aim of the study was to examine the effect of different process parameters which including; initial juglone amount, initial poly(d,l-lactide co-glycolide) amount, polyvinyl alcohol volume and polyvinyl alcohol concentration on encapsulation of juglone to poly(d,l-lactide co-glycolide) nanoparticles.. The synthesized nanoparticle formulations were analyzed for reaction yield, encapsulation efficiency, particle size, polydispersity, zeta potential and juglone release. In conjunction with the highest encapsulation rate, the highest amount of juglone release was obtained for F4 formulation, which has 281·8 nm particle size, 0·217 polydispersity index, and -19·55 mV zeta potential. After the detailed physicochemical characterization of this formulation, the four different kinetic models were used and it was found that juglone release mechanism controlled by Fickian diffusion method. According to antimicrobial activity results, minimal inhibitory concentration (MIC) values of both F4 and free juglone is higher for Gram negative bacteria than Gram positive bacteria. Inhibition zone diameters in the quantitative methods are found 15 and 16 mm for Staphylococcus aureus, 9 and 7 mm for Bacillus cereus, respectively, for F4 and free juglone. Moreover, the MIC values for qualitative methods were found 31·5 μg ml. It was found that the antibacterial activity of the juglone nanoparticles was higher and longer than the free juglone. Additionally, a similar antimicrobial effect with a lower juglone amount (obtained from controlled release study) indicates that nanoparticle formulation is more effective.. The use of nanoparticle formulations of juglone in biological systems and applications could be more beneficial than its free form due to its toxicity.

Topics: Anti-Bacterial Agents; Bacteria; Drug Compounding; Microbial Sensitivity Tests; Nanoparticles; Naphthoquinones; Particle Size; Polyglycolic Acid

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑based cancer therapy offers promise as TRAIL can kill cancer cells without apparent toxicity towards normal cells. However, intrinsic or acquired resistance to TRAIL inseveral types of cancer cell has become a major challenge in TRAIL‑based cancer therapy. Juglone is a natural compound isolated from walnut trees. In the present study, it was demonstrated that juglone sensitized melanoma cells to TRAIL‑induced cytotoxicity by MTT and crystal violet assays. Flow cytometry analysis indicated that juglone potentiated TRAIL‑induced cell death. Western blot assay demonstrated that the expressions of cleaved poly(ADP‑ribose) polymerase (PARP) and cleaved caspase 3 were markedly increased in the juglone combined with TRAIL group. Exposure to TRAIL alone did not induce the production of reactive oxygen species (ROS), activation of p38 orincrease of p53 in the TRAIL‑resistant melanoma cells, as determined by flow cytometry and western blot analysis. However, exposure to TRAIL in combination with juglone markedly increased the production of ROS, activated p38 and increased p53, compared with the cells treated with either juglone or TRAIL alone. Pretreatment with N‑acetyl cysteine, a ROS scavenger, significantly reduced the cytotoxicity of juglone in combination with TRAIL, which further supported that ROS was involved in the juglone‑induced sensitization of TRAIL. In conclusion, juglone potentiated TRAIL‑induced apoptosis in melanoma cells, and these effects were partially mediated through the ROS‑p38‑p53 pathway. These findings suggested that juglone may be a potential sensitizer for TRAIL therapy in the treatment of melanoma.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Humans; Melanoma; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term survival to patients. Natural bioactive compounds represent a precious source of molecules with antiproliferative activity, potentially effective also against glioma cells. Among these, Juglone is a known allelopathic compound extracted from the eastern black walnut (Juglans nigra) whose antimitotic effect has been extensively described in mammalian cells. We investigated the antiproliferative effect of a synthetic derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We compared this molecule and its effect with the natural reference compound and with newly synthesised derivatives to build a preliminar structure-activity relationship. Biological assays and NMR-based redox experiments confirmed that DiNAF is a promising lead and supported the hypothesis of a redox mechanism underlying its cytotoxic activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Glioma; Juglans; Magnetic Resonance Spectroscopy; Mitosis; Models, Molecular; Naphthoquinones; Rats; Rats, Wistar; Structure-Activity Relationship

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Naphthoquinones; Nerve Tissue Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Trastuzumab

Survival of patients with glioblastoma remains within the range of several months despite advances in therapeutic options. We have already shown that 5-hydroxy-1,4-naphthalenedione (juglone) exerts antiproliferative, anti-invasive, and cytotoxic effects on glioma C6 cells. Here, we further revealed that juglone is relatively selective to glioma cells as compared to the normal glial culture, and investigated its mechanisms of action. The incubation of glioma C6 cells with juglone generated high levels of reactive oxygen species (ROS). The produced ROS accounted for the anticancer effect of juglone as antioxidant N-acetylcysteine reduced both cytotoxic and antiproliferative activities of juglone. Furthermore, high resolution respirometry revealed that juglone decreased oxygen consumption mainly by affecting pyruvate/malate- and glutamate/malate-induced mitochondrial respiration. The inhibition of respiratory complex I by amytal decreased juglone-triggered generation of ROS and diminished its anticancer activity. Thus, our results indicate that juglone generates ROS through interaction with respiratory complex I in glioma C6 cells, and, in turn, induces the anticancer effects.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Electron Transport; Glioma; Humans; Mitochondria; Mitochondrial Membranes; Naphthoquinones; Rats, Wistar; Reactive Oxygen Species

Topics: Acute Lung Injury; Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Cells, Cultured; Drug Evaluation, Preclinical; Enzyme Inhibitors; Intestinal Mucosa; Intestines; Male; Mitochondria; Molecular Targeted Therapy; Naphthoquinones; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Src Homology 2 Domain-Containing, Transforming Protein 1; Translocation, Genetic

Since reactive oxygen species (ROS) play diverse roles in cancer, modulating the redox status of cancerous cells seems to be a promising therapeutic approach. Oxidant-targeted therapy appears logical for intervention with the acquired adaptive response to oxidative stress in cancer. In this study, we investigated the cytotoxic effects of juglone (J) and tamoxifen (T) and also the combination of each with ascorbate (A): tamoxifen/ascorbate (TA) and/or juglone/ascorbate (JA) on MCF7 cancerous cells. The results revealed that the growth inhibitory effects of juglone and tamoxifen were each associated with enhanced levels of ROS production and lipid peroxidation. These effects were markedly intensified in tamoxifen/ascorbate and juglone/ascorbate co-treatments. On the other hand, the intracellular anti-oxidant components such as reduced glutathione (GSH), catalase, superoxide dismutase (SOD), and glutathione peroxidase significantly declined in cells subjected to combination treatments compared to that in cells exposed solely to tamoxifen, juglone, and the untreated control cells. In addition, ascorbate association induced more apoptotic and necrotic or necrotic-like cell death than cells treated with each drug alone. These results were further confirmed by comparing the Bax/Bcl2 ratio in combination-treated cells. Additionally, ascorbate was able to potentiate the cytotoxic effects of combination therapy via activation of ROS-responsive factors including Foxo family members.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Catalase; Cell Proliferation; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Lipid Peroxidation; MCF-7 Cells; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Tamoxifen

Atherosclerosis is a major cause of mortality in patients with diabetes. However, novel breakthrough therapies have yet to be approved in this setting. Prolyl-isomerase-1 (Pin1) is emerging as a key molecule implicated in vascular oxidative stress and inflammation. In the present study, we investigate whether pharmacological inhibition of Pin1 may protect against diabetes-induced oxidative stress, endothelial dysfunction and vascular inflammation.. Experiments were performed in human aortic endothelial cells (HAECs) exposed to normal (5 mmol/L) or high glucose (25 mmol/L) concentrations, in the presence of Pin1 inhibitor Juglone (10 μM) or vehicle (<1% ethanol). In parallel, streptozotocin-induced diabetic mice were treated with Juglone i.p. every other day for 30 days (1mg/Kg). Organ chamber experiments were performed in aortic rings to assess endothelium-dependent relaxations to acetylcholine (Ach 10(-9) to 10(-6)mol/L). Mitochondrial oxidative stress, organelle integrity as well as NF-kB-dependent inflammatory signatures were determined both in HAECs and aortas from diabetic mice. In HAECs, ambient hyperglycemia increased mitochondrial superoxide anion generation while treatment with Juglone prevented this phenomenon. Pharmacological inhibition of Pin1 also preserved mitochondrial integrity, nitric oxide availability and endothelial expression of adhesion molecules. Interestingly enough, endothelial dysfunction, oxidative stress and NF-kB-driven inflammation were significantly attenuated in diabetic mice chronically treated with Juglone as compared to vehicle-treated animals.. Pharmacological inhibition of Pin1 by Juglone prevents hyperglycemia-induced vascular dysfunction. Taken together, our findings may set the stage for novel therapeutic approaches to prevent vascular complications in patients with diabetes.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Angiopathies; DNA; Endothelium, Vascular; Enzyme Inhibitors; Humans; Immunohistochemistry; Male; Mice; Mitochondria, Muscle; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Oxidative Stress; Peptidylprolyl Isomerase; Real-Time Polymerase Chain Reaction; Vasodilation

Juglone 1 an plumbagin 2 are plant secondary metabolites nowadays well known for their anticancer properties. In this study we synthesized analogues of 1 and 2 deriving from the functionalization of the OH group in position 5 with different side chains in form of esters and ethers. Therefore the growth inhibitory activities of these adducts were evaluated in vitro on six cancer cell lines using the MTT colorimetric assays along with the two natural parent compounds. The data revealed that these latter displayed the strongest growth inhibitory activities in vitro. Quantitative videomicroscopy analyses were then carried out on human U373 glioblastoma cells, which are characterized by various level of resistance to pro-apoptotic stimuli. We compared the naturally occurring reference compounds 1 and 2 with the derivatives exerting the best activities in terms of IC50 growth inhibitory values. These analyses showed that both juglone and plumbagin had a cytostatic effect on U373 cells and were able to overcome the intrinsic resistance of U373 cancer cells to pro-apoptotic stimuli.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Naphthoquinones

Topics: Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Fibroblasts; Humans; Keratinocytes; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Resveratrol; Signal Transduction; Sirtuin 1; Skin; Stilbenes; Ultraviolet Rays; Up-Regulation

Redox signaling plays a fundamental role in maintaining cell physiological activities. A deregulation of this balance through oxidative stress or nitrosative stress has been implicated in cancer. Here, we reported that 2-methoxy-6-acetyl-7-methyl juglone (MAM), a natural naphthoquinone isolated from Polygonum cuspidatum Sieb. et Zucc, caused hydrogen peroxide (H2O2) dependent activation of JNK and induced the expression of inducible nitric oxide synthase (iNOS), thereby leading to nitric oxide (NO) generation in multiple cancer cells. Nitrosative stress induced necroptosis in A549 lung cancer cells, but resulted in caspase-dependent intrinsic apoptosis in B16-F10 melanoma and MCF7 breast cancer cells. In addition, a decrease in GSH/GSSG levels accompanied with increased ROS production was observed. Reversal of ROS generation and cell death in GSH pretreated cells indicated the involvement of GSH depletion in MAM mediated cytotoxicity. In summary, a natural product MAM induced NO-dependent multiple forms of cell death in cancer cells mediated by H2O2-dependent JNK activation in cancer cells. GSH depletion might play an initial role in MAM-induced cytotoxicity.

Topics: Animals; Apoptosis; Cell Line, Tumor; Glutathione; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; MCF-7 Cells; Melanoma, Experimental; Mice; Naphthoquinones; Necrosis; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Reactive Oxygen Species

In this study we evaluated the impact of juglone on rat glioma C6 cell culture viability, proliferation and invasiveness in vitro. Juglone induced C6 cell death with EC50 concentrations equal to 10.4 ± 1.6 µM after 24h incubation. At relatively low concentrations juglone significantly decreased cell proliferation, reduced spheroid invasiveness and suppressed "wound" healing. In addition, generation of intracellular reactive oxygen and nitrogen species (RS) was detected in cells treated with juglone. Noteworthy, juglone was relatively stable in cell culture medium and levels of H2O2 generated from juglone due to its probable reaction with medium components were not sufficient to affect the viability of glioma cells. Moreover, addition of catalase to the cell medium did not reduce the cytotoxicity of juglone. Therefore, we propose that cell death may be induced through the action of RS other than H2O2, However the direct effect of juglone on the cellular targets could not be excluded either. In conclusion, juglone exerted cytotoxic, anti-proliferative and anti-invasive effects on C6 rat glioma cells in vitro.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glioblastoma; Humans; Hydrogen Peroxide; Naphthoquinones; Neoplasm Invasiveness; Rats; Reactive Oxygen Species

Amyloid-β, one of the hallmarks of Alzheimer's disease, is toxic to neurons and causes cell death in the brain. Oxidative stress is known to play an important role in Alzheimer's disease, and there is strong evidence linking oxidative stress to amyloid-β. The herbal plant "Tiew kon" (Cratoxylum formosum ssp. pruniflorum) is an indigenous vegetable that is grown in Southeast Asia. Many reports suggested that the twig extract from C. formosum possesses an antioxidant property. The purpose of this study was to investigate the protective effect of the twig extract from C. formosum against amyloid-β toxicity using the transgenic Caenorhabditis elegans model. This study demonstrated that the extract significantly delayed amyloid-β-induced paralysis in the C. elegans model of Alzheimer's disease. Using a genetic approach, we found that DAF-16/FOXO transcription factor, heat shock factor 1, and SKN-1 (Nrf2 in mammals) were required for the extract-mediated delayed paralysis. The extract ameliorated oxidative stress by reducing the level of H2O2, which appeared to account for the protective action of the extract. The extract possesses antioxidant activity against juglone-induced oxidative stress as it was shown to increase survival of the stressed worms. In addition, C. formosum decreased the expression of the heat shock protein-16.2 gene which was induced by thermal stress, indicating its ability to reduce cellular stress. The results from this study support the C. elegans model in the search for disease-modifying agents to treat Alzheimer's disease and indicate the potential of the extract from C. formosum ssp. pruniflorum as a source for the development of anti-Alzheimer's drugs.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Antioxidants; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Clusiaceae; Disease Models, Animal; DNA-Binding Proteins; Forkhead Transcription Factors; Naphthoquinones; Oxidative Stress; Paralysis; Plant Extracts; Protective Agents; Transcription Factors

Alternative approaches have been promoted to reduce the number of vertebrate and invertebrate animals required for the assessment of the potential of compounds to cause harm to the aquatic environment. A key philosophy in the development of alternatives is a greater understanding of the relevant adverse outcome pathway (AOP). One alternative method is the fish embryo toxicity (FET) assay. Although the trends in potency have been shown to be equivalent in embryo and adult assays, a detailed mechanistic analysis of the toxicity data has yet to be performed; such analysis is vital for a full understanding of the AOP. The research presented herein used an updated implementation of the Verhaar scheme to categorize compounds into AOP-informed categories. These were then used in mechanistic (quantitative) structure-activity relationship ((Q)SAR) analysis to show that the descriptors governing the distinct mechanisms of acute fish toxicity are capable of modeling data from the FET assay. The results show that compounds do appear to exhibit the same mechanisms of toxicity across life stages. Thus, this mechanistic analysis supports the argument that the FET assay is a suitable alternative testing strategy for the specified mechanisms and that understanding the AOPs is useful for toxicity prediction across test systems.

Topics: Animals; Aquatic Organisms; Embryo, Nonmammalian; Hydrophobic and Hydrophilic Interactions; Linear Models; Naphthoquinones; Quantitative Structure-Activity Relationship; Species Specificity; Toxicity Tests; Zebrafish

The mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a key enzyme in redox regulation, antioxidant defense, and cellular growth. TrxR1 can catalyze efficient reduction of juglone (5-hydroxy-1,4-naphthoquinone; walnut toxin) in a reaction which, in contrast to reduction of most other substrates of TrxR1, is not dependent upon an intact selenocysteine (Sec, U) residue of the enzyme. Using a number of TrxR1 mutant variants, we here found that a sole Cys residue at the C-terminal tail of TrxR1 is required for high-efficiency juglone-coupled NADPH oxidase activity of Sec-deficient enzyme, occurring with mixed one- and two-electron reactions producing superoxide. The activity also utilizes the FAD and the N-terminal redox active disulfide/dithiol motif of TrxR1. If a sole Cys residue at the C-terminal tail of TrxR1, in the absence of Sec, was moved further towards the C-terminal end of the protein compared to its natural position at residue 497, juglone reduction was, surprisingly, further increased. Ala substitutions of Trp407, Asn418 and Asn419 in a previously described "guiding bar", thought to mediate interactions of the C-terminal tail of TrxR1 with the FAD/dithiol site at the N-terminal domain of the other subunit in the dimeric enzyme, lowered turnover with juglone about 4.5-fold. Four residues of Sec-deficient TrxR1 were found to be easily arylated by juglone, including the Cys residue at position 497. Based upon our observations we suggest a model for involvement of the juglone-arylated C-terminal motif of TrxR1 to explain its high activity with juglone. This study thus provides novel insights into the catalytic mechanisms of TrxR1. One-electron juglone reduction by TrxR1 producing superoxide should furthermore contribute to the well-known prooxidant cytotoxicity of juglone.

Topics: Amino Acids; Catalysis; Cysteine; Humans; Mutation; NADPH Oxidases; Naphthoquinones; Oxidation-Reduction; Point Mutation; Selenocysteine; Substrate Specificity; Superoxides; Thioredoxin Reductase 1

A series of seven oxyprenylated phenylpropanoids and naphthoquinones were tested regarding their ability to activate transient receptor potential ankyrin subtype 1 channel (TRPA1). Three of the assayed compounds, namely, boropinal (3), juglone (5), and plumbagin (7), acted as strong modulators of TRPA1 channels with EC50 values of 9.8, 1.7, and 0.5 μM, respectively, as assessed by Ca(2+) assays. Moreover, the compounds elicited TRPA1 currents in electrophysiological whole cell recordings. We additionally provide evidence that plumbagin activated TRPA1-positive neurons isolated from mouse dorsal root ganglion neurons but did not affect sensory neurons from TRPA1-deficient mice. The high potencies of plumbagin and juglone to activate TRPA1 channels may explain the molecular basis of the mucosal irritant properties of these compounds as well as of related naphthoquinones and phytopreparations, as widely reported in the literature.

Topics: Animals; Ankyrins; Calcium; Coumarins; Female; Ganglia, Spinal; HEK293 Cells; Humans; Male; Mice; Molecular Structure; Naphthoquinones; Phenylpropionates; Sensory Receptor Cells; Transient Receptor Potential Channels; TRPA1 Cation Channel

Juglone, a natural component, is shown to have cytotoxic and apoptotic effects in several cancer cell lines. However, little is known about its effects on invasion and metastasis. In this study, we aimed to determine the antimetastatic effect of juglone in the BxPC-3 and PANC-1 pancreatic cancer cell lines. Cytotoxic effect of juglone was evaluated by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test. The cells were treated with juglone at

Topics: Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Cell Survival; Gene Expression; Humans; Metalloendopeptidases; Naphthoquinones; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,β-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

Topics: Animals; Antineoplastic Agents; Antioxidants; Catalytic Domain; Cattle; Drug Design; Drug Screening Assays, Antitumor; Fibroblasts; Glutathione Peroxidase; Glutathione Reductase; HCT116 Cells; Humans; Indoles; Inhibitory Concentration 50; MCF-7 Cells; Mice; Naphthoquinones; Oxidative Stress; Protein Domains; Rats; Reactive Oxygen Species; Recombinant Proteins; Selenocysteine; Thioredoxin-Disulfide Reductase; Thioredoxins

Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1's potential role as a therapeutic target.. The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated.. We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo.. This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients.

Topics: Animals; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; HeLa Cells; Herpesvirus 4, Human; Humans; Immunohistochemistry; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

The proportion of foodborne disease caused by pathogenic microorganisms is rising worldwide, with staphylococcal food poisoning being one of the main causes of this increase. Juglone is a plant-derived 1,4-naphthoquinone with confirmed antibacterial and antitumor activities. However, the specific mechanism underlying its antibacterial activity against Staphylococcus aureus remains unclear. To elucidate the mechanism underlying its antibacterial activity, isobaric tags for relative and absolute quantitation methods of quantitative proteomics were applied for analysis of the 53 proteins that were differentially expressed after treatment with juglone. Combined with verification experiments, such as detection of changes in DNA and RNA content and quantification of oxidative damage, our results suggested that juglone effectively increased the protein expression of oxidoreductase and created a peroxidative environment within the cell, significantly reducing cell wall formation and increasing membrane permeability. We hypothesize that juglone binds to DNA and reduces DNA transcription and replication directly. This is the first study to adopt a proteomic approach to investigate the antibacterial mechanism of juglone.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Food Preservatives; Naphthoquinones; Oxidoreductases; Proteome; Staphylococcus aureus

The purpose of the study was to obtain further in vivo data of antitumor effects and mechanisms triggered by juglone and Q7 in combination with ascorbate. The study was done using Ehrlich ascites tumor-bearing mice. Treatments were intraperitoneal every 24 h for 9 days. Control group was treated with excipient. Previous tests selected the doses of juglone and Q7 plus ascorbate (1 and 100 mg/kg, respectively). Samples of ascitic fluid were collected to evaluate carbonyl proteins, GSH and activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Hypoxia inducible factor HIF-1α, GLUT1, proteins driving cell cycle (p53, p16 and cyclin A) and apoptosis (poly-ADP-polymerase PARP, Bax and Bcl-xL) were assessed by western blot. Tumor cells were categorized by the phase of cell cycle using flow cytometry and type of cell death using acridine orange/ethidium bromide. A glucose uptake assessment was performed by liquid scintillation using Ehrlich tumor cells cultured with (14)C-deoxyglucose. Treatments caused increased protein carbonylation and activity of antioxidant enzymes and decreased levels of GSH, HIF-1α, GLUT1 and glucose uptake in tumor cells. They also caused increased number of tumor cells in G1, p53 and p16 activation and decreased cyclin A, but only when combined with ascorbate. Apoptosis was induced mostly when treatments were done with ascorbate, causing PARP and Bax cleavage, and increased Bax/Bcl-xL ratio. Juglone and Q7 in combination with ascorbate caused inhibition of tumor progress in vivo by triggering apoptosis and cell cycle arrest associated with oxidative stress, suppression of HIF-1 and uncoupling of glycolytic metabolism.

Topics: Aminophenols; Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Carcinoma, Ehrlich Tumor; Disease Progression; Male; Mice; Mice, Inbred BALB C; Naphthoquinones

Topics: Animals; beta Catenin; Blotting, Western; Carbon Tetrachloride Poisoning; Cell Differentiation; Dental Pulp; Fluorescent Antibody Technique; Hepatocytes; Humans; Hydroxyproline; In Vitro Techniques; Liver Cirrhosis; Male; Mice; Mice, Nude; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Stem Cell Transplantation; Stem Cells; Wnt Signaling Pathway

This study investigated the nature and mechanism of juglone-induced apoptosis in the human breast cancer cell line MCF-7. The inhibitory effect of juglone on MCF-7 cell growth was evaluated by the dimethylthiazol tetrazolium assay. Morphological apoptotic changes were characterized using an inverted microscope, Hoechst 33258 fluorescence staining, and Giemsa staining. The rate of cell apoptosis, intracellular levels of reactive oxygen species (ROS), and mitochondrial membrane potential were detected using flow cytometry. Intracellular Ca(2+) concentrations were detected using laser scanning confocal fluorescence microscopy. Expression of the proteins Bcl-2, Bax, and cytochrome C was assessed by western blotting. Caspase-3 activity was quantified using a caspase-3 activity kit. Juglone inhibited the growth of MCF-7 cell line with an IC50 of 11.99 μM. The rates of MCF-7 cell apoptosis at 24 h after exposure to 5, 10, and 20 μM juglone were 9.29, 20.67, and 28.39%, respectively; compared to unexposed cells, juglone-exposed cells exhibited significant elevation in intracellular ROS level, decrease in mitochondrial membrane potential, and increase in intracellular Ca(2+) concentration. Juglone upregulated the expression of Bax, and downregulated the expression of Bcl-2, promoting the release of cytochrome C, thereby upregulating the activity of caspase-3. The results suggest that the mechanism of juglone-induced apoptosis in MCF-7 cells is characterized by elevated ROS levels, reduced Bcl-2 expression, increased Bax expression, decreased mitochondrial membrane potential, increased intracellular Ca(2+) concentration, outer mitochondrial-membrane rupture, cytochrome C release, and caspase-3 activation.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Juglone is a plant-derived 1,4-naphthoquinone with confirmed antibacterial activity. However, the mechanism of action of juglone against Staphylococcus aureus remains unclear. Possible mechanisms were explored by a proteomic analysis of S. aureus proteins that are inhibited by juglone. Two-dimensional gel electrophoresis revealed that 21 protein spots were differentially expressed between juglone-treated and untreated cells of which 13 were identified. A bioinformatic analysis revealed that proteins participating in protein synthesis, the tricarboxylic acid cycle, as well as DNA and RNA synthesis were inhibited by juglone, thus leading to cell collapse. These findings provide clues regarding the mechanism of action of juglone, which can be effective for treating cases of S. aureus infection.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Humans; Naphthoquinones; Proteomics; Staphylococcal Infections; Staphylococcus aureus

This study aimed to synthesize and characterize juglone-entrapped poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles and compare the antifungal properties of free juglone with its PLGA nanoparticle formulation for the first time. The juglone-loaded nanoparticles prepared using the oil-in-water (o/w) single-emulsion solvent evaporation method were characterized by the reaction yield (RY), encapsulation efficiency (EE), polydispersity index (PDI), particle size, zeta potential (ZP), FT-IR, and in vitro release properties and evaluated for their morphological features using SEM. The nanoparticle formulation had size, RY, ZP, EE, and PDI values of 212 nm, 66.91 ± 2.4%, -16.3 ± 0.7 mV, 70.66 ± 3.1%, and 0.083 ± 0.024, respectively. In vitro release showed a triphasic pattern with initial burst followed by sustained release and dormant phase over the study period, releasing about 72.8% in total after 42 days. The antifungal studies against Aspergillus flavus, Candida albicans, and Fusarium spp. using agar dilution and top agar dilution methods indicated that the juglone-encapsulated nanoparticle was more effective than free juglone. This study showed that the top agar method, which was applied for the first time on antifungal activity, is more suitable for the nanoparticular system based on sustained release. Therefore, PLGA nanoparticle formulations may be an important tool for application in many areas for the effective and beneficial use of hydrophobic compounds such as juglone.

Topics: Antifungal Agents; Aspergillus flavus; Candida albicans; Fusarium; Lactic Acid; Nanoparticles; Naphthoquinones; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Spectroscopy, Fourier Transform Infrared

Three novel copper(II), cobalt(II), and nickel(II) complexes of juglone (Jug) containing 1,10-phenanthroline (phen) ligand, [M(Jug)

Topics: Antineoplastic Agents; Cobalt; Coordination Complexes; Copper; DNA; DNA Cleavage; Drug Stability; HeLa Cells; Hep G2 Cells; HT29 Cells; Humans; Inhibitory Concentration 50; Molecular Structure; Naphthoquinones; Nickel; Phenanthrolines; Spectrometry, Fluorescence

Therapeutic management of diabetic myocardial fibrosis remains an unsolved clinical problem. Pin1, a peptidyl-prolyl isomerase, impacts diverse cellular processes and plays a pivotal role in regulating cardiac pathophysiology. Here we investigate the potential mechanism of action of Pin1 and its role in diabetes-induced myocardial fibrosis and dysfunction in mice. Cardiac Pin1, transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA) and extracellular matrix deposits (collagen I and III) are found to be increased in diabetic mice, which are effectively prevented by Pin1 inhibition by juglone. Pin1 inhibition alleviates cardiac fibrosis and dysfunction. In vitro, high glucose increases Pin1 expression with an accompanying increase in phospho-Akt (Ser 473), p-Smad2, p-Smad3, TGF-β1, and α-SMA in cardiac fibroblasts (CFs). These increases are effectively prevented by the inhibition of Pin1 by juglone. Furthermore, Pin1 inhibition inhibits HG-induced CF proliferation and migration. Our results indicate that Pin1 inhibition attenuates cardiac extracellular matrix deposition by regulating the phosphorylation of Akt, TGF-β1/Smads, MMP activities, and α-SMA expression in diabetic mice.

Topics: Actins; Animals; Animals, Newborn; Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Collagen Type III; Diabetes Mellitus, Experimental; Enzyme Inhibitors; Fibroblasts; Fibrosis; Glucose; Male; Mice, Inbred C57BL; Microscopy, Confocal; Myocardium; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Phosphorylation; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta1

Investigating the effects of juglone loaded P188/phospholipid mixed micelles (J-MM) in breast cancer.. In vitro cytotoxicity, apoptotic effects, in vivo therapeutic efficacy and toxicity were used to assess its antitumour effect. Uptake and imaging were used to evaluate the effect on the uptake and passive targeting.. Mixed micelle carrier enhanced the targeting and uptake by MB-231 cells. The tumour inhibition rates in tumour xenograft models for paclitaxel, juglone, J-MM (10mg/kg) and J-MM (40mg/kg) were 46%, 27%, 39% and 53%, respectively. J-MM (10mg/kg) exhibited lower toxicity compared with that by free juglone or high dose J-MM.. J-MM exhibited low toxicity, improved cellular uptake, passive targeting and anti-cancer effects in breast cancer model.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Female; Humans; Micelles; Naphthoquinones; Paclitaxel; Phospholipids; Poloxamer

This study discusses the similarities and differences between the antifungal activity of extracts from walnut green husks of Lake, Koszycki, UO1, UO2 and non-grafted cultivars as well as juglone against the plant pathogenic fungi such as Alternaria alternata, Rhizoctonia solani, Botrytis cinerea, Fusarium culmorum, Phytophthora infestans as well as Ascosphaera apis causing chalkbrood disease in honey bees. The obtained data show that the antifungal activities of the extracts do not always depend on the antifungal activity of juglone, and that they can be modulated by their other components. This fact allows us to conclude that juglone is not the only component of walnut green husk extracts which is responsible for the inhibition of mycelial growth. Phenolic compounds were found to be responsible for activity of the extracts and they can modify antifungal activity of juglone.

Topics: Animals; Antifungal Agents; Bees; Fungi; Inhibitory Concentration 50; Juglans; Microbial Sensitivity Tests; Naphthoquinones; Phytochemicals; Plant Extracts

In this study the minimal inhibitory concentration (MICs) of tetracycline (Tet), erythromycin (Ery) and benzalkonium chloride (BC) in absence and in presence of a sub-MIC of juglone (Jug) were determined. In addition, the Ethidium bromide (EtBr) efflux assay was performed to assess the effect of Jug on EtBr cells accumulation. Our results showed a selective antimicrobial activity of Jug against the tested strains. A synergistic effect of Jug, drugs (Tet and Ery) and disinfectant (BC) was noticed with a reduction rate varied from 2 to 16-fold. In addition, the efflux of EtBr was inhibited depending on the Jug concentration. In the presence of Jug, a decrease in loss of EtBr from bacteria was observed. The concentration inducing 50 % of EtBr efflux inhibition after 15 min was about 182 μg ml

Topics: Anti-Bacterial Agents; Benzalkonium Compounds; Biological Transport, Active; Child; Disinfectants; Drug Synergism; Enzyme Inhibitors; Erythromycin; Ethidium; Humans; Microbial Sensitivity Tests; Mouth; Naphthoquinones; Staphylococcus aureus; Tetracycline; Tunisia

Juglone, a quinone isolated from Juglans mandshurica Maxim, has previously been shown to be effective against malignancy. The effect is at least partially due to stimulation of suicidal death or apoptosis of tumour cells. On the other hand, juglone has been shown to counteract apoptosis, for example, of neurons. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca(2+) activity [(Ca(2+) )i]. This study explored whether juglone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from FITC annexin V binding, ceramide abundance from binding of fluorescent antibodies in flow cytometry and cytosolic ATP with a luciferin-luciferase-based assay. As a result, a 24-hr exposure of human erythrocytes to juglone (5 μM) significantly decreased erythrocyte forward scatter. Juglone (1-5 μM) significantly increased the percentage of annexin V binding cells. Juglone (5 μM) significantly increased ceramide abundance at the erythrocyte surface and decreased erythrocyte ATP concentration. The effect of juglone (10 μM) on annexin V binding was slightly but significantly blunted by removal of extracellular Ca(2+) and by addition of protein kinase C (PKC) inhibitor staurosporine (1 μM). In conclusion, juglone stimulates suicidal erythrocyte death or eryptosis at least in part by upregulation of ceramide abundance, energy depletion and activation of PKC.

Topics: Adenosine Triphosphate; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Calcium; Cell Size; Ceramides; Erythrocytes; Humans; In Vitro Techniques; Naphthoquinones; Phosphatidylserines

Inflammatory responses and osteoclast differentiation play pivotal roles in the pathogenesis of osteolytic bone diseases such as periodontitis. Although overexpression or inhibition of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) offers a possible therapeutic strategy for chronic inflammatory diseases, the role of PIN1 in periodontal disease is unclear. The aim of the present study was to evaluate PIN1 expression in periodontitis patients as well as the effects of PIN1 inhibition by juglone or PIN1 small-interfering RNA (siRNA) and of PIN1 overexpression using a recombinant adenovirus encoding PIN1 (Ad-PIN1) on the inflammatory response and osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (PDLCs). PIN1 was up-regulated in chronically inflamed PDLCs from periodontitis patients and in LPS- and nicotine-exposed PDLCs. Inhibition of PIN1 by juglone or knockdown of PIN1 gene expression by siRNA markedly attenuated LPS- and nicotine-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, whereas PIN1 overexpression by Ad-PIN1 increased it. LPS- and nicotine-induced nuclear factor (NF)-κB activation was blocked by juglone and PIN1 siRNA but increased by Ad-PIN1. Conditioned medium prepared from LPS- and nicotine-treated PDLCs increased the number of tartrate-resistant acid phosphatase-stained osteoclasts and osteoclast-specific gene expression. These responses were blocked by PIN1 inhibition and silencing but stimulated by Ad-PIN1. Furthermore, juglone and PIN1 siRNA inhibited LPS- and nicotine-induced osteoclastogenic cytokine expression in PDLCs. This study is the first to demonstrate that PIN1 inhibition exhibits anti-inflammatory effects and blocks osteoclastic differentiation in LPS- and nicotine-treated PDLCs. PIN1 inhibition may be a therapeutic strategy for inflammatory osteolysis in periodontal disease.

Topics: Adolescent; Adult; Aged; Animals; Anti-Inflammatory Agents; Cell Culture Techniques; Cell Differentiation; Culture Media, Conditioned; Cyclooxygenase 2; Dinoprostone; Female; Gene Knockdown Techniques; Genetic Vectors; Humans; Lipopolysaccharides; Male; Mice, Inbred ICR; Middle Aged; Naphthoquinones; NF-kappa B; Nicotine; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoclasts; Peptidylprolyl Isomerase; Periodontal Ligament; Periodontitis; RNA, Small Interfering; Young Adult

Monodispersed and highly stable gold nanoparticles with a diameter between 8 and 9 nm were synthesized in a weakly alkaline medium by chemical reduction of AuCl4(-) using 5-hydroxyl-1,4-naphthoquinone, and stabilized by the simultaneously formed poly(hydroxyl-1,4-naphthoquinone). The electrochemical properties of the resultant poly(hydroxyl-1,4-naphthoquinone) stabilized gold nanoparticles (AuNQ NPs) and its electrocatalytic activity for glucose oxidation in alkaline media were then investigated using a range of techniques, including dc cyclic, rotating disk electrode and Fourier transformed large amplitude ac voltammetry. The results demonstrate that these AuNQ NP modified electrodes exhibit excellent catalytic activity toward glucose oxidation in the potential region where the premonolayer oxidation process occurs. The overall catalytic glucose oxidation process was found to be mass transport controlled under the experimental conditions employed, allowing measurements to be conducted with a high reproducibility. The AuNQ NP modified electrodes showed a high sensitivity of 183 μA mM(-1) cm(-2) with a wide linear dynamic range of 0.5-50 mM and a detection limit of 61 μM. However, despite its excellent tolerance toward ascorbic acid, significant interference from uric acid was found with this AuNQ NP modified electrode.

Topics: Chemistry Techniques, Synthetic; Drug Stability; Electric Conductivity; Electrochemistry; Electrodes; Glucose; Gold; Gold Compounds; Limit of Detection; Metal Nanoparticles; Naphthoquinones; Oxidation-Reduction; Particle Size; Polymers; Reducing Agents

To explore the effect of juglone on proliferation and apoptosis of human cervical squamous cancer SiHa cells.. Cultured SiHa cells in the exponential growth phase were grouped into blank control group and 10, 20, 50, 80 and 100 μmol/L juglone treatment groups. Methyl thiazolyl tetrazolium (MTT) assay was adopted to observe the inhibitory effect of juglone on the proliferation of SiHa cells, and then 50% inhibitory concentration (IC50) was calculated through formula. Annexin V-FITC/PI double staining and flow cytometry were used to detect the effect of 20 μmol/L juglone on SiHa cell apoptosis. Western blot was applied to determine the expressions of Bcl-2 and Bax.. MTT assay showed that, compared with the control group, treatment groups all showed significant inhibitory effects on SiHa cell growth, and IC50 was 20.4 μmol/L. Flow cytometry demonstrated that early apoptosis rate of SiHa cells in the control group was (2.46 ± 0.37)%, and after treatment with 20 μmol/L Juglone for 12 hours, the apoptosis rate was raised to (18.47 ± 2.26)%; Western blot analysis showed that the expression of Bcl-2 decreased while the expression of Bax increased significantly in SiHa cells treated with 20 μmol/L juglone.. Juglone could significantly inhibit the proliferation and induce the apoptosis of SiHa cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Drugs, Chinese Herbal; Female; Humans; Naphthoquinones; Uterine Cervical Neoplasms

A renewable-biomolecule-based electrode is developed through a facile synchronous reduction and self-assembly process, without any binder or additional conductive agent. The hybridized electrodes can be fabricated with arbitrary size and shape and exhibit superior capacity and cycle performance. The renewable-biomaterial-based high-performance electrodes will hold a place in future energy-storage devices.

Topics: Electric Conductivity; Electric Power Supplies; Electrodes; Graphite; Green Chemistry Technology; Models, Molecular; Molecular Conformation; Naphthoquinones; Oxides; Sodium

The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Damage; Histones; Humans; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Naphthoquinones; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species

Neutrophils play a key role in host defense against invading pathogens by releasing toxic agents, such as ROS and antimicrobial peptides. Human neutrophils express several TLRs that recognize a variety of microbial motifs. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to kill pathogens efficiently by increasing their activation, a process called priming. However, excessive activation can induce tissue injury and thereby, contribute to inflammatory disorders. Agonists that activate TLR7 and TLR8 induce priming of neutrophil ROS production; however, which receptor is involved in this process has not been elucidated. In this study, we show that the selective TLR8 agonist, CL075 (3M002), induced a dramatic increase of fMLF-stimulated NOX2 activation, whereas the selective TLR7 agonist, loxoribine, failed to induce any priming effect. Interestingly, CL075, but not loxoribine, induced the phosphorylation of the NOX2 cytosolic component p47phox on several serines and the phosphorylation of p38MAPK and ERK1/2. The inhibitor of p38MAPK completely blocked CL075-induced phosphorylation of p47phox Ser345. Moreover, CL075, but not loxoribine, induced the activation of the proline isomerase Pin1, and juglone, a Pin1 inhibitor, prevented CL075-mediated priming of fMLF-induced superoxide production. These results indicate that TLR8, but not TLR7, is involved in priming of human neutrophil ROS production by inducing the phosphorylation of p47phox and p38MAPK and that Pin1 is also involved in this process.

Topics: Chemotaxis, Leukocyte; Enzyme Activation; Gene Expression Regulation; Guanosine; Humans; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidase 2; NADPH Oxidases; Naphthoquinones; Neutrophils; NIMA-Interacting Peptidylprolyl Isomerase; p38 Mitogen-Activated Protein Kinases; Peptidylprolyl Isomerase; Phosphorylation; Primary Cell Culture; Quinolines; Reactive Oxygen Species; Signal Transduction; Thiazoles; Toll-Like Receptor 7; Toll-Like Receptor 8

Juglone as a natural production mainly extracted from green walnut husks of Juglans mandshurica has been defined as the functional composition among a series of compounds. It showed powerful protective effect in various diseases by inhibiting inflammation and tumor cells growth. However, studies on its anti-inflammatory effect based on high-fat diet-induced hepatitis and neuroinflammation are still not available. In this regard, we first investigated whether juglone suppresses high-fat diet-stimulated liver injury, hypothalamus inflammation and underlying mechanisms by which they may recover them. SD rats were orally treated with or without high-fat diet, 0.25 mg/kg or 1 mg/kg juglone for 70 days. Subsequently, blood, hypothalamus and liver tissue were collected for different analysis. Also, the primary astrocytes were isolated and used to analyze the inhibitory effect of juglone in vitro. Analysis of inflammatory cytokines declared that the inhibition of TNF-α, IL-1β and IL-6 could be carried by juglone in response to high-fat diet rats. Meanwhile, TLR4 expression and NF-kappa activity also have been confirmed to be the key link in the development of hepatitis and nerve inflammation. The activation was significantly suppressed in treatment group as compared with model. These results indicated that juglone prevents high-fat diet-induced liver injury and nerve inflammation in mice through inhibition of inflammatory cytokine secretion, NF-kappa B activation and endotoxin production.

Topics: Animals; Anti-Inflammatory Agents; Astrocytes; Cytokines; Diet, High-Fat; Endotoxemia; Hepatitis, Animal; Hypothalamus; Inflammation; Lipopolysaccharides; Male; Mice; Naphthoquinones; Nervous System Diseases; NF-kappa B; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Toll-Like Receptor 4

Naphthoquinones (NQs) occur naturally in a large variety of plants. Several NQs are highly active against protozoans, amongst them the causative pathogens of neglected tropical diseases such as human African trypanosomiasis (sleeping sickness), Chagas disease and leishmaniasis. Prominent NQ-producing plants can be found among Juglans spp. (Juglandaceae) with juglone derivatives as known constituents. In this study, 36 highly variable extracts were prepared from different plant parts of J. regia, J. cinerea and J. nigra. For all extracts, antiprotozoal activity was determined against the protozoans Trypanosoma cruzi, T. brucei rhodesiense and Leishmania donovani. In addition, an LC-MS fingerprint was recorded for each extract. With each extract's fingerprint and the data on in vitro growth inhibitory activity against T. brucei rhodesiense a Partial Least Squares (PLS) regression model was calculated in order to obtain an indication of compounds responsible for the differences in bioactivity between the 36 extracts. By means of PLS, hydrojuglone glucoside was predicted as an active compound against T. brucei and consequently isolated and tested in vitro. In fact, the pure compound showed activity against T. brucei at a significantly lower cytotoxicity towards mammalian cells than established antiprotozoal NQs such as lapachol.

Topics: Antiprotozoal Agents; Chromatography, Liquid; Inhibitory Concentration 50; Juglans; Least-Squares Analysis; Leishmania donovani; Mass Spectrometry; Naphthoquinones; Plant Extracts; Trypanosoma brucei rhodesiense; Trypanosoma cruzi

Prostate cancer initially develops in an androgen-dependent manner but, during its progression, transitions to being androgen-independent in the advanced stage. Pin1, one of the peptidyl-prolyl cis/trans isomerases, is reportedly overexpressed in prostate cancers and is considered to contribute to accelerated cell growth, which may be one of the major factors contributing to their androgen-independent growth. Thus, we investigated how Pin1 modulates the gene expressions in both androgen-dependent and androgen-independent prostate cancer cell lines using microarray analysis. In addition, the effects of Juglone, a commercially available Pin1 inhibitor were also examined.. Two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), were treated with Pin1 siRNA and its effects on gene expressions were analyzed by microarray. Individual gene regulations induced by Pin1 siRNA or the Pin1 inhibitor Juglone were examined using RT-PCR. In addition, the effects of Juglone on the growth of LNCaP and DU145 transplanted into mice were investigated.. Microarray analysis revealed that transcriptional factors regulated by Pin1 differed markedly between LNCaP and DU145 cells, the only exception being that Nrf was regulated in the same way by Pin1 siRNA in both cell lines. Despite this marked difference in gene regulations, Pin1 siRNA and Juglone exert a strong inhibitory effect on both the LNCaP and the DU145 cell line, suppressing in vitro cell proliferation as well as tumor enlargement when transplanted into mice.. Despite Pin1-regulated gene expressions differing between these two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), Pin1 inhibition suppresses proliferation of both cell-lines. These findings suggest the potential effectiveness of Pin1 inhibitors as therapeutic agents for prostate cancers, regardless of their androgen sensitivity.

Topics: Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasm Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Prostatic Neoplasms; Xenograft Model Antitumor Assays

The present study aimed to investigate the antifibrotic effects of juglone on dimethylnitrosamine (DMN)‑induced fibrosis in rats. Juglone, which is a quinone, significantly decreased DMN‑induced rat hepatic fibrosis, which was associated with increased superoxide dismutase (SOD) activity, decreased oxidative stress and reduced levels of α‑smooth muscle actin (α‑SMA) and collagen (Col) III in the liver. Serum levels of alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, laminin, type III precollagen and type IV collagen were significantly reduced by treatment with juglone. Liver fibrosis was induced in male Sprague‑Dawley rats by subcutaneous injections of DMN solution and hepatic fibrosis was assessed using Massons trichome staining. The expression levels of α‑SMA and Col III were determined using immunohistochemical techniques. The activities of SOD and malondialdehyde in liver homogenates were also determined. The results suggested that juglone augmented the antioxidative capability of the liver, possibly by stimulating the activity of SOD, which promoted the inactivation of hepatic stellate cells (HSCs) and decreased the accumulation of extracellular matrix collagen in the liver, thereby alleviating hepatic fibrosis. Silymarin was used as a positive control for liver fibrosis protection. It was hypothesized that juglone alleviates or mitigates oxidative stress‑mediated hepatic fibrosis by upregulating the expression of peroxisome proliferator‑activated receptor γ and inhibiting the activation of HSC.

Topics: Actins; Animals; Antioxidants; Collagen Type III; Dimethylnitrosamine; Hepatic Stellate Cells; Immunohistochemistry; Liver; Liver Cirrhosis, Experimental; Male; Malondialdehyde; Naphthoquinones; PPAR gamma; Protective Agents; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

This study aimed to evaluate the effects of Pin1 inhibitor Juglone on proliferation, migration and the angiogenic ability of breast cancer cell line MCF7Adr. MCF7Adr cells were cultured and separately treated with Pin1 inhibitor Juglone (treatment group) and DMEM without drug (control group). The cell cycle was examined by flow cytometry. Cell migration was measured by wound-healing assay. Cyclin E protein content was detected by Western blotting. The angiogenesis factor vascular endothelial growth factor (VEGF) in cell media was determined by enzyme linked immunosorbent assay. The results showed that the percentage of cells in G2/M phase in treatment group was significantly higher than that in control group (25.5% vs. 10.1%, P<0.05), and that in G0/G1 phase and S stage in treatment group was significantly lower than that in control group (40.5% vs. 48.2%, and 33.7% vs. 41.7%, P<0.05). Cyclin E protein content in treatment group was significantly lower than that in control group (39.2 ± 7.4 vs. 100 ± 23.1, P<0.05). (A0-A24)/A0 value in treatment group was significantly lower than that in control group (23.9 ± 3.8 vs. 100 ± 14.4, P<0.05). VEGF-A, -B, and -C contents in cell media of treatment group were significantly lower than those in control group (P<0.05). It was suggested that Pin1 inhibitor Juglone can effectively inhibit the proliferation, migration and the angiogenic ability of MCF7Adr cells, and can be used as an alternative drug therapy for breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin E; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Vascular Endothelial Growth Factor A

Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has not been defined yet. The aim of this study was to investigate the effect of juglone on Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba castellanii at 3-5 μM concentrations. Juglone increased the production of reactive oxygen species (ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-l-cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a significant role in the juglone-induced cell death of Acanthamoeba.

Topics: Acanthamoeba castellanii; Cell Line, Tumor; Cytotoxins; Dose-Response Relationship, Drug; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Reactive Oxygen Species; Time Factors

The constituents of walnut (Juglans regia L.) leaves are represented by tannins, phenolics, and naphthoquinones, the characteristic compound being juglone. The content of juglone in the methanolic extract of the leaves determined by the GC/MS method was 9.9 ± 0.2 mg/100 g; small amounts (1.3 ± 0.02 mg/100 g) were recorded in the infusion, whereas in the decoction it was not detected. As some studies indicate toxicity of juglone, only decoctions should be recommended for therapeutic use.

Topics: Gas Chromatography-Mass Spectrometry; Juglans; Naphthoquinones; Plant Extracts; Plant Leaves

Juglans regia has been found to exhibit significant anticancer activity against various human cancer cell lines. This study was undertaken to isolate the active chemical constituent (Juglone) and to investigate its cytotoxic activity along with its various analogs against different human cancer cell lines.. Isolation of juglone, a napthoquinone, from the chloroform extract of the root part of Juglans regia was executed by flash chromatography using silica gel as stationary phase. The isolated Juglone was used as starting material for the further synthesis of a novel series of triazolyl analogs using click chemistry approach to investigate their cytotoxic potential against different human cancer cell lines using 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay.. The different extracts of Juglans regia and the isolated compound (juglone) exhibited satisfactory cytotoxic activity against a panel of eight different human cancer cell lines namely, prostate colon (Colo-205 and HCT-116), breast (T47D), prostate (PC-3 and DU-145), skin (A-431) and lung (NCI-H322 and A549). Interestingly, all the synthesised analogs displayed enhanced and selective cytotoxic activity against lung cancer cell lines only. Of the synthesized derivatives, 15a and 16a displayed the best activity with IC50 of 4.72 and 4.67 μM against A549 cells. Both these derivatives exhibited superior potency to BEZ-235 against both the lung cancer cell lines. So far as the structural aspects are concerned, electron withdrawing substituents at the ortho position of R moiety of the triazolyl analogs seem to be essential for attaining better activity.. The present study demonstrates the selective and enhanced cytotoxic activity of the triazolyl analogs of juglone against NCI-H322 and A549 human lung cancer cell lines. Some derivatives exhibited superior potency to BEZ-235, a commercially available anticancer agent.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Juglans; Lung Neoplasms; Naphthoquinones; Plant Extracts

EPs7630(®) a water alcohol extract of the roots from Pelargonium sidoides contains several secondary metabolites including highly oxygenated coumarins, various phenolics and polyphenols. Using the DPPH assay to measure antioxidant activity a free radical scavenging activity of 14.7±0.85μg/ml (IC50) was determined. As an in vivo model Caenorhabditis elegans was applied to study the effect of EPs7630(®) on stress resistance. EPs7630(®) treatment reduces intracellular hsp-16.2::GFP expression (induced by the pro-oxidant juglone) indicating that the secondary metabolites of EPs7630(®) are bioavailable and exhibit antioxidant activities in vivo. Application of EPs7630(®) (50μg/ml) to the transgenic mutant TJ356 induced the migration of the transcription factor DAF-16 from cytosol to the nucleus, suggesting a prominent role of DAF-16/FOXO in the daf-2 pathway for stress resistance.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Forkhead Transcription Factors; Naphthoquinones; Oxidative Stress; Pelargonium; Plant Extracts; Transcription Factors

The Groucho/transducin-like Enhancer of split 1 (Gro/TLE1):Hes1 transcriptional repression complex acts in cerebral cortical neural progenitor cells to inhibit neuronal differentiation. The molecular mechanisms that regulate the anti-neurogenic function of the Gro/TLE1:Hes1 complex during cortical neurogenesis remain to be defined. Here we show that prolyl isomerase Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and homeodomain-interacting protein kinase 2 (HIPK2) are expressed in cortical neural progenitor cells and form a complex that interacts with the Gro/TLE1:Hes1 complex. This association depends on the enzymatic activities of both HIPK2 and Pin1, as well as on the association of Gro/TLE1 with Hes1, but is independent of the previously described Hes1-activated phosphorylation of Gro/TLE1. Interaction with the Pin1:HIPK2 complex results in Gro/TLE1 hyperphosphorylation and weakens both the transcriptional repression activity and the anti-neurogenic function of the Gro/TLE1:Hes1 complex. These results provide evidence that HIPK2 and Pin1 work together to promote cortical neurogenesis, at least in part, by suppressing Gro/TLE1:Hes1-mediated inhibition of neuronal differentiation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Cerebral Cortex; HEK293 Cells; Homeodomain Proteins; Humans; Mice; Naphthoquinones; Neural Stem Cells; Neurogenesis; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Protein Serine-Threonine Kinases; Repressor Proteins; Transcription Factor HES-1; Tretinoin

The effect of naturally occurring quinones including lawsone (LQ), ubiquinone (UQ), juglone (JQ), and 1,4-naphthoquinone (NQ) on the biotransformation of carbon tetrachloride (CT) in the presence of Geobacter sulfurreducens and ferrihydrite was investigated. AQDS was used as the model compound for comparison. The reductive dissolution of ferrihydrite by G. sulfurreducens was enhanced by AQDS, NQ, and LQ. However, addition of UQ and JQ had little enhancement effect on Fe(II) production. The bioreduction efficiency and rate of ferrihydrite was highly dependent on the natural property and concentration of quinone compounds and the addition of low concentrations of LQ and NQ significantly accelerated the biotransformation rate of CT. The pseudo-first-order rate constants for CT dechlorination (kobsCT) in AQDS-, LQ- and NQ-amended batches were 5.4-5.8, 4.6-7.4 and 2.4-5.8 times, respectively, higher than those in the absence of quinone. A good relationship between kobsCT for CT dechlorination and bioreduction ratio of ferrihydrite was observed, indicating the important role of biogenic Fe(II) in dechlorination of CT under iron-reducing conditions. Spectroscopic analysis showed that AQDS and NQ could be reduced to semiquinones and hydroquinones, while only hydroquinones were generated in LQ-amended batches.

Topics: Biodegradation, Environmental; Carbon Tetrachloride; Ferric Compounds; Ferrous Compounds; Geobacter; Hazardous Substances; Naphthoquinones; Oxidation-Reduction; Quinones; Solvents

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins enhanced mammalian pol inhibition and their cytotoxic activity. Plumbagin conjugated with chains consisting of more than C18-unsaturated fatty acids strongly inhibited the activities of calf pol α and human pol γ. Plumbagin conjugated with oleic acid (C18:1-acyl plumbagin) showed the strongest suppression of human colon carcinoma (HCT116) cell proliferation among the ten synthesized 5-O-acyl plumbagins. The inhibitory activity on pol α, a DNA replicative pol, by these compounds showed high correlation with their cancer cell proliferation suppressive activity. C18:1-Acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. This compound inhibited the proliferation of various human cancer cell lines, and was the cytotoxic inhibitor showing strongest inhibition towards HT-29 colon cancer cells (LD50 = 2.9 µM) among the nine cell lines tested. In an in vivo anti-tumor assay conducted on nude mice bearing solid tumors of HT-29 cells, C18:1-acyl plumbagin was shown to be a promising tumor suppressor. These data indicate that novel 5-O-acyl plumbagins act as anti-cancer agents based on mammalian DNA replicative pol α inhibition. Moreover, the results suggest that acylation of plumbagin is an effective chemical modification to improve the anti-cancer activity of vitamin K3 derivatives, such as plumbagin.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; DNA; DNA Replication; DNA-Directed DNA Polymerase; HCT116 Cells; HT29 Cells; Humans; Lethal Dose 50; Male; Mammals; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Nucleic Acid Synthesis Inhibitors; Temperature

Therapeutic management of liver fibrosis remains an unsolved clinical problem. Hepatic accumulation of extracellular matrix, mainly collagen, is mediated by the production of transforming growth factor-β1 (TGF-β1) in stellate cells. Pin1, a peptidyl-prolyl isomerase, plays an important pathophysiological role in several diseases, including neurodegeneration and cancer. Herein, we determined whether Pin1 regulates liver fibrogenesis and examined its mechanism of action by focusing on TGF-β1 signalling and hepatic stellate cell (HSC) activation.. Pin1 expression was assessed by immunohistochemistry, Western blot or real-time-polymerase chain reaction (RT-PCR) analyses of human and mouse fibrotic liver samples. The role of Pin1 during HSC activation was estimated using Pin1-null mouse embryonic fibroblast (MEF) cells and Pin1-overexpressing LX-2 human hepatic stellate cells.. Pin1 expression was elevated in human and mouse fibrotic liver tissues, and Pin1 inhibition improved dimethylnitrosamine (DMN)-induced liver fibrosis in mice. Pin1 inhibition reduced the mRNA or protein expression of TGF-β1 and α-smooth muscle actin (α-SMA) by DMN treatment. Pin1 knockdown suppressed TGFβ1 gene expression in both LX-2 and MEF cells. Pin1-mediated TGFβ1 gene transcription was controlled by extracellular signal-regulated kinase (ERK)- and phosphoinositide 3-kinase/Akt-mediated activator protein-1 (AP-1) activation. Moreover, TGFβ1-stimulated Smad2/3 phosphorylation and plasminogen activator inhibitor-1 expression were inhibited by Pin1 knockdown.. Pin1 induction during liver fibrosis is involved in hepatic stellate cell activation, TGFβ1 expression, and TGFβ1-mediated fibrogenesis signalling.

Topics: Animals; Cell Communication; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation, Neoplastic; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice, Inbred ICR; Mice, Knockout; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta1

The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Proliferation; DNA, Neoplasm; Electrophoresis, Agar Gel; Fluorescence; HeLa Cells; Humans; Juglans; Naphthoquinones; Necrosis; Plant Extracts; Silver Staining; Staining and Labeling; Telomerase

The hydroxyl radical ((●)OH)-induced oxidation reactions of isomeric hydroxy naphthoquinones (generally having anti-tumor activities) namely, lawsone and juglone, were carried out and the reaction mechanism was elucidated.. The degradation products from the reaction of (●)OH (produced by H(2)O(2)/UV) with lawsone and juglone were analyzed using a liquid chromatography quadrupole-time-of-flight mass spectrometer (LC-Q-TOF-MS). The transient intermediate studies were investigated using picosecond pulse radiolysis technique.. Mono hydroxylated and dihydroxylated adducts of both lawsone and juglone were identified from the product analysis. The isomeric mono-hydroxylated adducts of lawsone were confirmed using survival yield (SY) analysis. The hydroxylated adducts of lawsone also underwent dimerization reaction. The transient spectral analysis using pulse radiolysis studies revealed the formation of hydroxycyclohexadienyl type radical of both lawsone and juglone as the initially formed intermediate.. The (●)OH-induced reactions of both lawsone and juglone result in the mono and di-hydoxylated derivatives. The demonstration of the various isomeric products using mass spectrometry is a clear proof of the addition probability of (●)OH at different positions of lawsone and juglone, which is generally a difficult task using other analytical techniques.

Topics: Chromatography, Liquid; Hydroxyl Radical; Models, Chemical; Molecular Structure; Naphthoquinones; Oxidation-Reduction; Pulse Radiolysis; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Ultraviolet Rays

Nature has been the best source of medicines for a long time. Many plant extracts have been used as drugs. Juglone occurs in all parts of the Juglandaceae family and is found extensively in the black walnut plants. It possesses antifungal, antimalarial, antibacterial and antiviral properties besides exhibiting cytotoxic effects. Juglone has gained interest by the researchers for its anticancer properties. This article elucidates the antiviral activity of the Juglone by the computational method.

Topics: Antiviral Agents; Binding Sites; Computer Simulation; Drug Design; Enzyme Activation; HIV Protease; Models, Chemical; Molecular Docking Simulation; Naphthoquinones; Plant Extracts; Protease Inhibitors; Protein Binding

In this work, we report a reagentless electrochemical peptide (AVPFAQKG) sensor to directly detect the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP-BIR3). The bioreceptor was based on a conducting copolymer film electrosynthesized from juglone and a juglone-peptide conjugate (JP) newly designed. The peptide-protein interactions generated an important increase of steric hindrance at the interface and a current decrease (signal off) of the redox reaction from quinone embedded in the polymer backbone as evidenced by Square Wave Voltammetry. This allowed a specific and sensitive detection of XIAP-BIR3 with a detection limit of 1 nM (13 ng mL(-1)). The peptide-protein complex could be then dissociated by adding the free precursor peptide (AVPFAQKG) into solution, causing a shift-back on the signal, i.e. an increase in the current intensity (signal-on). This "off-on" detection sequence was used in this work as a double verification of the specificity and this approach can be employed as a general way to increase the reliability of the results. In general, the approach described in this work may be inspired to develop other direct and reagentless electrochemical protein assays with high specificity and sensitivity.

Topics: Amino Acid Sequence; Biosensing Techniques; Electrochemical Techniques; Equipment Design; Limit of Detection; Models, Molecular; Naphthoquinones; Oligopeptides; Protein Structure, Tertiary; Reproducibility of Results; X-Linked Inhibitor of Apoptosis Protein

A series of ferrocene and (arene)ruthenium(II) complexes attached to the naturally occurring anticancer naphthoquinones plumbagin and juglone was tested for efficacy against various cancer cell lines and for alterations in the mode of action. The plumbagin ferrocene and (p-cymene)Ru(II) conjugates 1c and 2a overcame the multi-drug drug resistance of KB-V1/Vbl cervix carcinoma cells and showed IC50 (72 h) values around 1 μM in growth inhibition assays using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT). They were further investigated for their influence on the cell cycle of KB-V1/Vbl and HCT-116 colon carcinoma cells, on the generation of reactive oxygen species (ROS) by the latter cell line, for their substrate character for the P-glycoprotein drug eflux pump via the calcein-AM efflux assays, and for DNA affinity by the electrophoretic mobility shift assay (EMSA). The derivatives 1c and 2a increased the number of dead cancer cells (sub-G0/G1 fraction) in a dose- and time-dependent manner. ROS levels were significantly increased upon treatment with 1c and 2a. These compounds also showed a greater affinity to linear DNA than plumbagin. While plumbagin did not affect calcein-AM transport by P-glycoprotein the derivatives 1c and 2a exhibited a 50% or 80% inhibition of the P-glycoprotein-mediated calcein-AM efflux relative to the clinically established sensitizer verapamil.

Topics: Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Coordination Complexes; Cymenes; DNA; Female; Ferrous Compounds; Humans; Metallocenes; Monoterpenes; Naphthoquinones; Ruthenium; Uterine Cervical Neoplasms

Six novel 5,6-fused hybrids such as dihydrobenzofuran-quinone (4a and 4b), benzofuran-quinone (5a and 5b) and chromene-quinone (6a and 6b) of juglone based 1,4-naphthoquinones were synthesized by employing a three step protocol with the cyclisation of o-allyl phenol as the key step. The anticancer activity of the newly synthesized compounds was evaluated in vitro against seven human cancer cell lines including cervix (ME-180 and HeLa), breast (MCF-7, MDA-MB-453 and MDA-MB-231), prostate (PC-3) and colon (HT-29) by using MTT assay. The screening results showed that majority of the synthesized compounds exhibited significant anticancer activity. In particular, compounds 6a and 6b showed potent activities than the standard drug etoposide against prostate and breast cancer cell lines respectively. Flow cytometric analysis revealed that compounds 6a and 6b induced apoptosis and arrested the cell cycle at G2/M phase in PC-3 and MDA-MB-453 cells respectively.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Chemistry Techniques, Synthetic; Drug Screening Assays, Antitumor; Humans; Naphthoquinones

Naphthoquinones represent the group of plant secondary metabolites with cytotoxic properties based on their ability to generate reactive oxygen species and interfere with the processes of cell respiration. Due to this fact, the possible cytotoxic mechanisms on cellular and subcellular levels are investigated intensively. There are many targets of cytotoxic action on the cellular level; however, DNA is a critical target of many cytotoxic compounds. Due to the cytotoxic properties of naphthoquinones, it is necessary to study the processes of naphthoquinones, DNA interactions (1,4-naphthoquinone, binapthoquinone, juglone, lawsone, plumbagin), especially by using modern analytical techniques. In our work, the Raman spectroscopy was used to determine the possible binding sites of the naphthoquinones on the DNA and to characterize the bond of naphthoquinone to DNA. Experimental data reveals the relationships between the perturbations of structure-sensitive Raman bands and the types of the naphthoquinones involved. The modification of DNA by the studied naphthoquinones leads to the nonspecific interaction, which causes the transition of B-DNA into A-DNA conformation. The change of the B-conformation of DNA for all measured DNA modified by naphthoquinones except plumbagin is obvious.

Topics: DNA; DNA, B-Form; Naphthoquinones; Plants; Reactive Oxygen Species; Secondary Metabolism; Spectrum Analysis, Raman

Pseudouridine is a noncanonical C-nucleoside commonly present in RNA, which is not metabolized in mammals, but can be recycled by the unique enzyme family of bacterial pseudouridine glycosidases such as YeiN from Escherichia coli. Here, we present rigorous bioinformatic and biochemical analyses of the protein family in order to find sequences that might code for nonpseudouridine glycosidase activities. To date, the only other function reported for the enzyme family occurs during the biosynthesis of the antibiotic alnumycin A in Streptomyces species, where AlnA functions as an unusual C-glycosynthase. Bioinformatics analysis of 755 protein sequences identified one group of sequences that were unlikely to harbour pseudouridine glycosidase activities. This observation was confirmed in vitro with one representative protein, IdgA from Streptomyces albus, which was unable to synthesize pseudouridine monophosphate, but was able to attach d-ribose-5-phosphate to juglone. Furthermore, our analyses provide evidence for horizontal gene transfer of pseudouridine glycosidases that may have occurred in Streptomyces and Doria species. Inspection of the genomic loci in the vicinity of pseudouridine glycosidases revealed that in 77% of the strains a kinase gene putatively involved in the phosphorylation of pseudouridine was found nearby, whereas the sequences encoding nonpseudouridine glycosidases coexisted with a phosphatase of the haloacid dehalogenase enzyme family. The investigation suggested that these unknown sequences might be involved in the biosynthesis of soluble blue pigments because of the presence of genes homologous to nonribosomal peptide synthetases.

Topics: Amino Acid Sequence; Burkholderia; Conserved Sequence; Escherichia coli; Escherichia coli Proteins; Evolution, Molecular; Gene Transfer, Horizontal; Genes, Bacterial; Glycoside Hydrolases; Naphthoquinones; Phylogeny; Streptomyces; Uracil

Juglone, 5-hydroxy-1,4-naphthoquinone, is the plant secondary metabolite with allelopathic properties, which was isolated especially from the plant species belonging to family Juglandaceae A. Rich. ex Kunth (walnut family). The mechanism of phytotoxic action of juglone was investigated on lettuce seedlings Lactuca sativa L. var. capitata L. cv. Merkurion by determining its effect at different levels. We have found that juglone inhibits mitosis (mitotic index 8.5 ± 0.6% for control versus 2.2 ± 0.9% for 250 μM juglone), changes mitotic phase index with accumulation of the cells in prophase (56.5 ± 2.6% for control versus 85.3 ± 5.0% for 250 μM juglone), and decreases meristematic activity in lettuce root tips (51.07 ± 3.62% for control versus 5.27 ± 2.29% for 250 μM juglone). In addition, juglone induced creation of reactive oxygen species and changed levels of reactive nitrogen species. Amount of malondialdehyde, a product of lipid peroxidation, increased from 24.0 ± 4.0 ng g(-1) FW for control to 55.5 ± 5.4 ng g(-1) FW for 250 μM juglone. We observed also changes in cellular structure, especially changes in the morphology of endoplasmic reticulum. Reactive oxygen species induced damage of plasma membrane. All these changes resulted in the disruption of the mitochondrial membrane potential, increase in free intracellular calcium ions, and DNA fragmentation and programmed cell death that was revealed by two methods, TUNEL test and DNA electrophoresis. The portion of TUNEL-positive cells increase from 0.96 ± 0.5% for control to 7.66 ± 1.5% for 250 μM juglone. Results of the study indicate complex mechanism of phytotoxic effect of juglone in lettuce root tips and may indicate mechanism of allelopathic activity of this compound.

Topics: Lactuca; Meristem; Mitosis; Naphthoquinones; Plant Roots; Reactive Oxygen Species; Seedlings

To explore the effects of Juglone on proliferation and apoptosis of human cervical cancer Caski cells, and to further study the related mechanism of cell apoptosis.. Cultured Caski cells were incubated with 20, 40, 60, 80 and 100 μmol/L juglone for 24 h. The proliferation of Caski cells was detected by methyl thiazolyl tetrazolium (MTT) assay. The cell apoptosis were detected by transmission electron microscope. The expression of Bcl-2 and Bax were detected by Western blot.. MTT results showed that in different doses of juglone groups, the Caski cell growth was greatly inhibited (P < 0.05, P < 0.01) and showed dose dependent when compared with control group except 20 μmol/L. The IC50 of juglone was 42.4 μmol/L. After treatment on Caski cells with 40 μmol/L juglone, typical apoptosis characteristics was observed by transmission electronmicro scope. The expression of Bcl-2 was decreased while the expression of Bax was increased significantly when compared with control group (P < 0.05).. Juglone significantly inhibits the proliferation and induces the apoptosis of Caski cells in vitro.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Proliferation; Female; Humans; Microscopy, Electron, Transmission; Naphthoquinones; Uterine Cervical Neoplasms

To study the anti-tumor chemical components of the pericarps of Juglans mandshurica.. The chemical constituents were isolated and purified by AB-8 macroporous adsorption resin, silica gel, Sephadex LH-20 columns and recrystallization. The structures were elucidated on the basis of physicochemical properties and NMR spectral data analysis.. From the pericarps of Juglans mandshurica, twelve compounds were separated and identified as 3-methoxy juglone(1), 3-ethoxy juglone(2), 1,8-di-hydroxy anthraquinone (3), juglone (4), 2α, 3α, 19α-trihydroxy ursolic acid (5), 1α, 3β-dihydroxy-olean-18-ene (6), methyl gallate (7), pterocarine(8), quercetin(9), kaempferol(10), daucosterol(11), and β-sitosterol(12).. Compounds 1 - 3 and 6 are isolated from the pericarps of Juglans mandshurica for the first time. Compounds 5 and 7 are isolated from Juglans genus for the first time.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Drugs, Chinese Herbal; Gallic Acid; Juglans; Kaempferols; Naphthoquinones; Phytochemicals; Seeds; Sitosterols; Triterpenes; Ursolic Acid

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Topics: Animals; Breast Neoplasms; CD24 Antigen; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Dactinomycin; Drug Resistance, Neoplasm; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mammary Glands, Human; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplastic Stem Cells; Octamer Transcription Factor-3; Paclitaxel; Proto-Oncogene Proteins c-myc; Pyrans; SOXB1 Transcription Factors; Spheroids, Cellular

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the occurrence of neurofibrillary tangles (NFTs) that are composed of abnormally hyperphosphorylated microtubule-associated protein tau. Abnormal tau hyperphosphorylation is mainly induced due to the imbalance between protein kinases and phosphatases. In the tanglerich subregions of the hippocampus and parietal cortex in the brain of AD patients, the levels of the phosphorylationdependent protein peptidyl-prolyl cis-trans isomerase (Pin1) were found to be low. Although Pin1 can regulate tau phosphorylation, it is not clear whether the inhibition of glycogen synthase kinase 3 (GSK-3), the primary mediator of tau phosphorylation in AD, could reverse tau hyperphosphorylation induced due to the down-regulation of Pin1. We found that while suppression of Pin1, either by using its inhibitor Juglone or a shRNA plasmid against Pin1, induces tau hyperphosphorylation and GSK-3β activation both in vivo and in vitro, inhibition of GSK-3β by SB216763 or LiCl reverses tau hyperphosphorylation. Our data suggest that GSK-3β activation plays an important role in tau hyperphosphorylation induced by the down-regulation of Pin1, and the inhibition of GSK-3β might be a potential therapeutic approach for AD pathology.

Topics: Animals; Down-Regulation; Enzyme Inhibitors; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Hippocampus; Humans; Indoles; Lithium Chloride; Male; Maleimides; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Protein Kinase Inhibitors; Rats; RNA, Small Interfering; tau Proteins

Although there have been many studies on bacterial removal of soluble azo dyes, much less information is available for biological treatment of water-insoluble azo dyes. The few bacterial species capable of removing Sudan dye generally require a long time to remove low concentrations of insoluble dye particles. The present work examined the efficient removal of Sudan I by Shewanella oneidensis MR-1 in the presence of redox mediator. It was found that the microbially reduced anthraquinone-2,6-disulfonate (AQDS) could abiotically reduce Sudan I, indicating the feasibility of microbially-mediated reduction. The addition of 100 μM AQDS and other different quinone compounds led to 4.3-54.7 % increase in removal efficiencies in 22 h. However, adding 5-hydroxy-1,4-naphthoquinone into the system inhibited Sudan I removal. The presence of 10, 50 and 100 μM AQDS stimulated the removal efficiency in 10 h from 26.4 to 42.8, 54.9 and 64.0 %, respectively. The presence of 300 μM AQDS resulted in an eightfold increase in initial removal rate from 0.19 to 1.52 mg h⁻¹ g⁻¹ cell biomass. A linear relationship was observed between the initial removal rates and AQDS concentrations (0-100 μM). Comparison of Michaelis-Menten kinetic constants revealed the advantage of AQDS-mediated removal over direct reduction. Different species of humic acid could also stimulate the removal of Sudan I. Scanning electronic microscopy analysis confirmed the accelerated removal performance in the presence of AQDS. These results provide a potential method for the efficient removal of insoluble Sudan dye.

Topics: Aerobiosis; Anthraquinones; Coloring Agents; Humic Substances; Microscopy, Electron, Scanning; Naphthols; Naphthoquinones; Oxidation-Reduction; Shewanella; Solubility

Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in Caenorhabditis elegans where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. gsr-1 is the sole GSR encoding gene found in C. elegans. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by gsr-1(RNAi), GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect gsr-1(RNAi) knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of C. elegans, emphasising the crucial role the GSH redox state plays in both processes.

Topics: Animals; Arsenites; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; Glutathione; Glutathione Reductase; Longevity; Naphthoquinones; Oxidants; Oxidation-Reduction; Oxidative Stress; Phenotype; RNA Interference; Stress, Physiological

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a polyphenol from red wine, has been reported to be beneficial in cases of ageing-related cardiovascular and neurodegenerative diseases owing to its property to reduce oxidative stress. Previous studies on the longevity promoting effect of resveratrol have been partly inconclusive, therefore we set out to investigate whether resveratrol at least promoted longevity in Caenorhabditis elegans under acute oxidative stress conditions.. C. elegans was cultured under standard conditions with or without resveratrol. After exposure to juglone-induced acute oxidative stress, the survival rate and hsp-16.2::GFP expression were measured. The influence of resveratrol on life span was recorded also under oxidative stress induced by high glucose concentrations in the growth medium.. No extension of the normal life span of C. elegans was observed either in liquid or solid growth media containing different concentrations of resveratrol. However, resveratrol alleviated juglone-induced lethal oxidative stress, and significantly prolonged the life span of C. elegans under conditions of acute oxidative damage and oxidative stress caused by high concentrations of glucose.. Resveratrol, as an antioxidant, ameliorated oxidative stress in vivo but did not extend the life span of C. elegans under normal conditions. However, resveratrol did extend life span under conditions of oxidative stress.

Topics: Animals; Antioxidants; Caenorhabditis elegans; Glucose; Longevity; Naphthoquinones; Oxidative Stress; Plant Extracts; Resveratrol; Stilbenes; Vitis

The structure-function relationships of the naphthoquinone phytochemicals, plumbagin, juglone, and menadione, have been studied with regard to antimutagenic and antioxidant activities. Antimutagenicity of these compounds was assessed by the Ames test and RNA polymerase B (rpoB)-based rifampicin resistance assay. Antioxidant potential was evaluated by radical scavenging assays and reducing power measurement. Protection of cells and DNA against gamma radiation-induced oxidative damage was assayed by survival analysis and gel electrophoresis profiling, respectively. On the 1,4-naphthoquinone nucleus, plumbagin possesses 5-hydroxyl and 2-methyl functional groups, whereas juglone has only the 5-hydroxyl and menadione only the 2-methyl group. Plumbagin showed strong antimutagenic (against ultraviolet and ethyl methanesulfonate) and antioxidant activities, whereas juglone displayed only strong antimutagenic, and menadione only strong antioxidant activities. Thus, these two functional groups (5-OH/2-CH3) play important roles in the differential bioactivity of naphthoquinones. Escherichia coli, microarray analysis showed upregulation of the genes rep (replication/repair), ybaK (tRNA editing), speE (spermidine synthesis), and yjfC (glutathionyl spermidine synthesis) by plumbagin or juglone, and sodC (superoxide dismutase), xthA (oxidative repair), hycB (electron carrier between hydrogenase 3 and fumarate dehydrogenase), and ligA (formation of phosphodiester bond in DNA) by plumbagin or menadione. Studies with E. coli single-gene knockouts showed that ybaK and speE, reported to prevent mistranslation, are likely to be involved in the antimutagenicity displayed by juglone, and sodC to be involved in the antioxidant activity of menadione.

Topics: Anticoagulants; Antifibrinolytic Agents; Antimutagenic Agents; Antineoplastic Agents; Antioxidants; Drug Resistance, Bacterial; Escherichia coli; Molecular Structure; Naphthoquinones; Nucleic Acid Synthesis Inhibitors; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Rifampin; Salmonella typhimurium; Structure-Activity Relationship; Vitamin K 3

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Brain; Cells, Cultured; Dopaminergic Neurons; Gene Expression; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Naphthoquinones; Neurotoxins; NIMA-Interacting Peptidylprolyl Isomerase; Parkinson Disease; Parkinson Disease, Secondary; Peptidylprolyl Isomerase; RNA Interference; RNA-Directed DNA Polymerase; Substantia Nigra; Up-Regulation

It has been shown earlier that plumbagin, a naturally occurring naphthaquinone has specific anticancer activity in BRCA1 blocked ovarian cancer cells. Plumbagin can induce estrogen dependent cell signaling and apoptosis in BRCA1 blocked ovarian cancer cells. Being a reactive oxygen species (ROS) generator and apoptosis inducing agent, plumbagin has immense potential as a promising anticancer agent. In this study we analyzed whether there would be increased anticancer activity if the positions of the functional groups on plumbagin were altered and further to analyze the detailed molecular mechanism of action of the lead molecule. Methods like MTT assay, apoptosis analysis by flow cytometry, assessment of mitochondrial membrane potential-Δψm , suppression subtractive hybridization, microarray, molecular docking and estrogen receptor-DNA binding activity by electrophoresis mobility shift assay (EMSA) were adopted for assessing the anticancer activity. Consequently we found that, plumbagin was the most potent anticancer agent when compared to structurally related compounds. The anti-cancer activities were in the order plumbagin > 1,4-naphthaquinone > juglone > lawsone > menadione. Molecular docking studies showed that plumbagin could be well docked in the receptor ligand complex of TRAIL-DR5 complexes to activate the extrinsic pathway of apoptosis. Since the antiproliferative activity of plumbagin could be reduced by inhibiting ERα, we speculated that plumbagin interferes with the binding of ERα to ERE and we confirmed this by EMSA. This study clearly indicates that plumbagin can induce multiple pathways of apoptosis and cell cycle arrest in BRCA1 blocked cells compared to unblocked cells.

Topics: Antineoplastic Agents, Phytogenic; BRCA1 Protein; Breast Neoplasms; Cell Cycle Checkpoints; Drug Screening Assays, Antitumor; Estrogen Receptor alpha; Female; Gene Knockout Techniques; Humans; Membrane Potential, Mitochondrial; Molecular Docking Simulation; Naphthoquinones; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Response Elements; Structure-Activity Relationship; Transcriptome

A high-performance liquid chromatographic method with gradient elution and diode-array detection was developed to quantify free phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic and trans-cinnamic), flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone in walnut leaves. Chromatographic separation was performed on a Hypersil Gold C18 column (5 µm particle size, 250 × 4.6 mm) and detection was conducted at three different wavelengths (254, 278 and 300 nm) according to the absorption maxima of the analyzed compounds. Validation procedures were conducted and the method was proven to be precise, accurate and sensitive. The developed method has been applied to analyze walnut leaves samples from nine different cultivars, with the same agricultural, geographical and climatic conditions. The experimental results revealed high concentrations of myricetin, catechin hydrate and rutin, and low concentrations of quercetin and epicatechin aglycones. Ellagic acid was established as the dominating phenolic acid of walnut leaves, followed by trans-cinnamic, chlorogenic and caffeic acids. Juglone content varied between 44.55 and 205.12 mg/100 g fresh weight. Significant differences were detected among cultivars for the concentration levels of phenolics.

Topics: Chromatography, High Pressure Liquid; Flavonoids; Hydroxybenzoates; Juglans; Limit of Detection; Linear Models; Naphthoquinones; Plant Leaves; Reproducibility of Results

Rooibos leaves and fine stems (Aspalathus linearis; Fabaceae) are increasingly enjoyed as herbal tea, largely in fermented (oxidised) red-brown form, but also in unfermented (unoxidised) green form. Rooibos is rich in antioxidant polyphenols, with the dihydrochalcone, aspalathin, as a major active ingredient. We used Caenorhabditis elegans as model organism to investigate the effect of rooibos extracts against oxidative stress in vivo. In a high glucose environment, C. elegans treated with rooibos extract exhibited an extended lifespan. Furthermore, green rooibos was a more potent antioxidant than red rooibos, probably due to its substantially higher aspalathin content. In addition, rooibos decreased acute oxidative damage caused by the superoxide anion radical generator, juglone, with aspalathin playing a major role in improving the survival rate of C. elegans. Quantitative real-time PCR results demonstrated that aspalathin targets stress and ageing related genes, reducing the endogenous intracellular level of ROS. These findings suggest that rooibos increases stress resistance and promotes longevity under stress, probably mediated via a regulation of the DAF-16/FOXO insulin-like signalling pathway, supporting some of the health claims put forward for rooibos tea.

Topics: Animals; Antioxidants; Aspalathus; Caenorhabditis elegans; Chalcones; Drug Evaluation, Preclinical; Glucose; Insulin; Insulin-Like Growth Factor I; Longevity; Naphthoquinones; Oxidative Stress; Phenols; Phytotherapy; Plant Extracts; Reactive Oxygen Species

Ultraviolet A (UVA) radiation (320-400 nm) is considered a major cause of human skin photoaging and skin cancer. Overexpression of cyclooxygenase-2 (COX-2) leads to prostanoid formation in skin tissue, disturbs the balance between proliferation and apoptosis, and subsequently promotes tumorigenesis. The peptidyl-prolyl isomerase Pin1 is known to be overexpressed in most cancer cell types and plays an important role in oncogenesis. Here, we studied whether exposure of JB6 Cl41 mouse epidermal cells to UVA affects COX-2 expression and the possible involvement of Pin1 activation. UVA increased COX-2 protein expression and prostaglandin E2 production in an energy-dependent manner. Pre-exposure of JB6 Cl41 cells to UVA potentiated epidermal growth factor-induced anchorage-independent growth; this effect was significantly suppressed by inhibition of COX-2. UVA-stimulated COX-2 expression was significantly decreased by inhibition of Pin1. The increased COX-2 gene transcription in response to UVA was preceded by activation of the transcription factors nuclear factor-κB (NF-κB), cAMP response element-binding protein (CREB), CCAAT/enhancer-binding proteins α and β (C/EBPα and C/EBPβ) and c-Jun/activator protein-1 (AP-1). Pin1 inhibitor treatment suppressed the activation of NF-κB, CREB, and C/EBP by UVA irradiation. Conversely, JB6 C141 cells overexpressing Pin1 showed increased basal COX-2 expression and NF-κB, CREB, C/EBP, and AP-1 activities. These results suggest that UVA-induced COX-2 expression is mediated by Pin1 activation and this is associated with malignant transformation of epidermal cells.

Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; Epidermis; Epithelial Cells; Meloxicam; Mice; Naphthoquinones; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Signal Transduction; Thiazines; Thiazoles; Transcription Factor AP-1; Ultraviolet Rays

To study the chemical constituents from the flower Juglans regia.. All compounds were isolated and purified by normal column chromatograph and polyamide chromatograph, the chemical strucures were mainly elucidated by ESI-MS and NMR spectra.. Seven compounds were identified as follows: 4,5,8-trihydroxy-alpha-tetralone 5-O-beta-D-glucopyranoside(1),4,5-dihydroxy-alpha-tetralone4-O-beta-D-glucopyranoside(2), 5-hydroxy-4-methoxytetralone (3), 5-hydroxy-1, 4-naphthoquinone (4), rutin (5), vanillin (6), tetracosanoic acid 2,3-dihydroxypropyl ester (7).. All compounds are isolated from this plant for the first time.

Topics: Benzaldehydes; Flowers; Glucosides; Juglans; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthoquinones; Rutin

Abnormal cell cycle events are increasingly becoming important attributes of neurodegenerative pathology. Pin1 is a crucial target of neurodegeneration in relation to its functions regarding these abnormal cell cycle events in neurons. Pin1 is majorly involved in many aspects of cell cycle regulation and it has also been suggested to have a neuroprotective function against neurodegenerative pathologies. Oxidative dysregulation of Pin1 affects not only normal tau regulation, eventually causing tangle formation, but also cell cycle regulation in neurons. Presence of cell cycle proteins has been shown in many neurodegenerative diseases. Importantly, many of these proteins have physical interactions with Pin1. Hence, understanding Pin1's role in abnormal cell cycle re-entry is critical in terms of finding new approaches for the future therapeutic options treating neurodegenerative pathologies. Here, we show that inhibition of Pin1 by its selective inhibitor juglone leads to up-regulation of cyclinD1, phospho-tau, and caspase 3, producing apoptosis in cultured rat hippocampal neurons. We also observed axonal retraction with a change in sub-cellular localizations of cyclins. Therefore, Pin1 dysregulation, in relation to its role in cell cycle regulation in neurons, may have profound effects in the progression of neurodegenerative pathology, making it a possible crucial target behind many neurodegenerative diseases.

Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Newborn; Apoptosis; Brain; Caspase 3; Cells, Cultured; Cyclin D; Cytotoxins; Dose-Response Relationship, Drug; Gene Expression Regulation; Indoles; Naphthoquinones; Nerve Tissue Proteins; Neurodegenerative Diseases; Neurons; Rats; Rats, Sprague-Dawley; RNA, Messenger; tau Proteins; Time Factors; Up-Regulation

The present study aimed at evaluating the anticancer and radiosensitizing potential of juglone against a chemoresistant and radioresistant tumor (B16F1 melanoma) growing on C57BL/6J mice. Volume doubling time, growth delay, and median survival were used to assess the in vivo anticancer and radiosensitizing potential of juglone. In vitro radiosensitizing potential of juglone was studied using clonogenic, comet, and reactive oxygen species induction assays. Treatment of tumor-bearing mice with sublethal doses of juglone caused a dose-dependent inhibition of tumor growth as evident from the growth delay and median survival values. Comet assay using tumor tissue and blood showed differential toxicity of juglone, where higher levels of DNA damage was seen in tumor tissue compared with blood cells. Pretreatment of tumor-bearing mice with optimum dose of juglone before radiation resulted in significant tumor growth inhibition compared with radiation alone. From the clonogenic assay, the authors observed a sensitization enhancement ratio of 1.37 for the combination treatment compared with radiation alone. Furthermore, comet assay studies revealed the potential of juglone to enhance the radiation-induced DNA damage and cause a delay in its repair. Juglone pretreatment before radiation also resulted in a significant elevation in the intracellular reactive oxygen species levels compared with radiation alone. In conclusion, the results of this study show the potential of juglone to inhibit the growth of melanoma in vivo. The study also revealed the potential of juglone to augment the radiation-induced cell death of melanoma cells, which may be attributed to oxidative stress-mediated DNA damage and its delayed repair.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Survival; DNA Damage; Female; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Naphthoquinones; Oxidative Stress; Radiation-Sensitizing Agents; Reactive Oxygen Species

Following on our discovery that pulp and paper mill effluents can interact with, and disrupt, various neurotransmitter receptors and enzymes important to fish reproduction, we tested wood and bark extracts of 14 Eastern North American hardwood trees used in pulp and paper production. Radioligand binding to neurotransmitter receptors, including the dopamine-2 receptor (D2), the gamma aminobutyric acid receptor A (GABA(A)), N-methyl-D-aspartic acid (NMDA) receptor, and muscarinic cholinergic receptor (mACh-R), were significantly changed following in vitro incubations with many but not all extracts. Activities of neurotransmitter-related enzymes monoamine oxidase (MAO), GABA-transaminase (GABA-T), acetylcholinesterase (AChE) and glutamic acid decarboxylase (GAD) were also significantly altered. Butternut wood extracts and the isolated compound juglone significantly inhibited the enzymatic activities of MAO and GAD which we suggest may be part of a mechanism that may negatively affect fish reproduction. Besides giving credence to the hypothesis that neuroactive compounds in pulp and paper effluent may originate in the trees used by mills, the results reported here also indicate important neuropharmacological activities in hardwoods which may help identify new sources of biologically active natural products.

Topics: Acetylcholinesterase; Analysis of Variance; Animals; Brain; Glutamate Decarboxylase; Goldfish; Monoamine Oxidase; Naphthoquinones; North America; Paper; Plant Bark; Plant Extracts; Radioligand Assay; Receptors, Dopamine D2; Receptors, GABA-A; Receptors, Muscarinic; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Transaminases; Trees; Wood

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold (k(cat)/K(m) increased from 105 min(-1) μM(-1) to 1331 min(-1) μM(-1) for juglone in the presence of 50 μM PQQ, mainly through a lowered apparent K(m) for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.

Topics: Animals; Enzyme Inhibitors; Escherichia coli; Glutathione Reductase; Humans; Kinetics; Mutation; Naphthoquinones; Oxidation-Reduction; Plasmids; PQQ Cofactor; Rats; Selenoproteins; Thioredoxin Reductase 1; Yeasts

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.

Topics: Alzheimer Disease; Animals; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Cytoskeleton; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Marine Toxins; Mice; Naphthoquinones; Nerve Degeneration; Neuroblastoma; NIMA-Interacting Peptidylprolyl Isomerase; Oxazoles; Peptidylprolyl Isomerase; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Purines; Rats; Receptors, GABA-A; Roscovitine; tau Proteins; Tauopathies

Juglone, a major chemical constituent of Juglans mandshruica Maxim, is a promising anticancer agent that has shown a strong activity against cancer cells in vitro. Our previous study showed that juglone inhibited the proliferation of HL-60 cells with an IC50 value ∼8 μM. To further explore the proapoptotic mechanism of juglone, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by juglone in HL-60 cells. The generation of ROS was about 2 to 8-fold as compared to control cell after treatment with juglone (2, 4 and 8 μM) for 24 h. The glutathione (GSH) depletion was consistent with ROS generation after treatment with juglone. Reversal of apoptosis in antioxidants (NAC and catalase) pretreated cells indicated the involvement of ROS in juglone-induced apoptosis. The cleavage of PARP and procaspase-3 and -9, loss of mitochondrial membrane potential (△Ψm), and release of cytochrome c (Cyt c) and Smac induced by juglone were significantly blocked by NAC. NAC also prevented the inhibition the phosphorylation of Akt and mTOR proteins by juglone. Collectively, these results indicated that ROS played a significant role in the apoptosis induced by juglone in human leukemia cell HL-60.

Topics: Acetylcysteine; Annexin A5; Antioxidants; Apoptosis; Caspases; Cell Division; Cytochromes c; Enzyme Activation; Glutathione; HL-60 Cells; Humans; Juglans; Leukemia; Membrane Potentials; Naphthoquinones; Reactive Oxygen Species

The Diels-Alder reaction of juglone with various styrenes in the presence of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was promoted by B(OAc)(3) at room temperature. The reaction proceeded smoothly and gave the products in a good yield and with excellent regioselectivity. This strategy was applied to the total syntheses of tetrangulol and anhydrolandomycinone.

Topics: Boron Compounds; Cyclization; Molecular Structure; Naphthoquinones; Oligosaccharides; Stereoisomerism; Styrenes; Temperature

Cinnamomum osmophloeum Kaneh is an indigenous tree species in Taiwan. In this study, phytochemical characteristics and antioxidant activities of the essential oils and key constituents from the leaves of two C. osmophloeum clones were investigated. The two trees possess two chemotypes, which were classified as the cinnamaldehyde type and camphor type. We demonstrated that the essential oils from C. osmophloeum leaves exerted in vivo antioxidant activities in Caenorhabditis elegans. In addition, trans-cinnamaldehyde and D-(+)-camphor, which respectively represent the major compounds in the cinnamaldehyde-type and camphor-type trees, exerted significant in vivo antioxidant activities against juglone-induced oxidative stress in C. elegans. Moreover, expressions of antioxidative-related genes, including superoxide dismutase (SOD) and glutathione S-transferase (GST), were significantly induced by trans-cinnamaldehyde and D-(+)-camphor from C. osmophloeum leaves. Our results showed that the essential oils from C. osmophloeum leaves and their major compounds might have good potential for further development as nutraceuticals or antioxidant remedies.

Topics: Acrolein; Animals; Antioxidants; Caenorhabditis elegans; Camphor; Cinnamomum; Gene Expression; Glutathione Transferase; Naphthoquinones; Oils, Volatile; Oxidation-Reduction; Oxidative Stress; Plant Leaves; Superoxide Dismutase; Taiwan

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 µM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.

Topics: Androstadienes; Animals; Cell Line; Cell Transformation, Neoplastic; Chromones; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Endothelial Growth Factors; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Morpholines; Naphthoquinones; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Ribosomal Protein S6 Kinases, 90-kDa; Skin; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Wortmannin

The effects of juglone on T24 cells were assessed in the presence and absence of ascorbate. The EC(50) value for juglone at 24 h decreased from 28.5 μM to 6.3 μM in the presence of ascorbate. In juglone-treated cells, ascorbate increased ROS formation (4-fold) and depleted GSH (65%). N-acetylcysteine or catalase restricted the juglone/ascorbate-mediated effects, highlighting the role of oxidative stress in juglone cytotoxicity. Juglone alone or associated with ascorbate did not cause caspase-3 activation or PARP cleavage, suggesting necrosis-like cell death. DNA damage and the mild ER stress caused by juglone were both enhanced by ascorbate. In cells treated with juglone (1-5 μM), a concentration-dependent decrease in cell proliferation was observed. Ascorbate did not impair cell proliferation but its association with juglone led to a clonogenic death state. The motility of ascorbate-treated cells was not affected. Juglone slightly restricted motility, but cells lost their ability to migrate most noticeably when treated with juglone plus ascorbate. We postulate that juglone kills cells by a necrosis-like mechanism inhibiting cell proliferation and the motility of T24 cells. These effects are enhanced in the presence of ascorbate.

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Urinary Bladder

A screening of plant quinones for inhibiting effects on the bacterial fire blight pathogen Erwinia amylovora was performed. The most active compound, juglone from walnuts, has a potent and specific bactericidal effect on E. amylovora and minimal inhibitory concentrations of only 2.5-10 μM, with stronger effects at lower, but still physiological, pH values. In vitro tests with juglone and inoculated flowers of apple (Malus domestica) showed an efficacy of 67% in preventing infection. In two years of field tests juglone had variable degrees of efficacy ranging from 40 to 82%, seemingly due to environmental conditions. A phytotoxic reaction to juglone, which is known for its allelopathic effect on plants, was restricted to browning of petals; later fruit russeting was not observed. Juglone is a promising candidate for the development of a new environmentally friendly plant protectant to replace the antibiotic streptomycin currently used in fire blight control.

Topics: Agrochemicals; Anti-Bacterial Agents; Drug Evaluation, Preclinical; Drug Stability; Erwinia amylovora; Flowers; Germination; Hydrogen-Ion Concentration; Malus; Microbial Sensitivity Tests; Naphthoquinones; Plant Diseases; Quinones; Toxicity Tests

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities, including anti-tumor. In the present study, HeLa cells were incubated with juglone at various concentrations. The proliferation inhibition of juglone on HeLa cells was tested by the MTT assay. Occurrence of apoptosis was detected by Hoechst 33258 staining, flow cytometry, and transmission electron microscopy. The expression of apoptotic-related proteins was examined by Western blot. The results showed that juglone inhibits the growth of HeLa cells in dose-dependent manner. Topical morphological changes of apoptotic body formation after juglone treatment were observed. The percentages of early apoptosis of Annexin V-FITC were 5.23%, 7.95%, 10.69%, and 20.92% with the concentrations of juglone (12.5, 25, 50, and 100 µmol/L), respectively. After cells were treated with juglone at the different dose for 24 h, the expression of Bcl-2 was significantly down-regulated and the expression of Bax was significantly up-regulated compared with the control. These events paralleled with activation of caspase-9, -8, -3, and PARP cleavage. The results suggest that juglone may be effective for the treatment of HeLa cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carcinoma; Caspases; Cell Proliferation; Down-Regulation; Enzyme Activation; Female; HeLa Cells; Humans; Microscopy, Electron, Transmission; Naphthoquinones; Neoplasm Proteins; Osmolar Concentration; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteolysis; Up-Regulation; Uterine Cervical Neoplasms

This study was designed to investigate the effect of juglone on the apoptosis of human gastric cancer SGC-7901 cells. The cytotoxic activity of juglone on SGC-7901 cells was tested by the sulforhodamine B (SRB) assay. The morphological changes in the cells were observed by transmission electron microscopy (TEM). The apoptotic rate, the level of reactive oxygen species (ROS), mitochondrial transmembrane potential and the expression of cytochrome c protein were detected by flow cytometry (FCM). The expression of Bcl-2 and Bax proteins were examined by Western blot. Caspase 3 activity was determined with a microplate reader. Our results were as follows: the GI(50) values for SGC-7901 cells were 36.51 ± 1.05 μmol/L (24h) and 25.37 ± 1.19 μmol/L (48 h). After 24h of exposure to juglone (5, 10, 15 and 20 μmol/L), the cells presented the typical morphological changes of apoptosis, and the rate of apoptosis was found to increase in a dose-dependent manner. After cells were treated with juglone at the same dose for 24h, the level of ROS was significantly higher, the expression of Bcl-2 was significantly down-regulated and the expression of Bax was significantly up-regulated compared to the control. The mitochondrial transmembrane potential was significantly lower, and the expression of the cytochrome c protein was significantly higher relative to the control. Caspase 3 was activated in a concentration-dependent manner. In conclusion, juglone can induce apoptosis in SGC-7901 cells through a mitochondrial pathway that seems to be mediated by the generation of ROS and a reduction in the Bcl-2/Bax ratio.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Stomach Neoplasms

Sublethal preconditioning ischemia protects neurons from lethal ischemia, and activation of ERK is associated with this protection. However, sublethal ischemia and reperfusion also results in rapid inactivation of ERK, which contributes to the dual-phase activation profile of ERK. In the present study, we observed sublethal ischemia-induced rapid inactivation of ERK was accompanied by phosphorylation of Raf-1 at Ser289/296/301 sites. Inhibition of calcium signaling by ketamine resulted in down-regulation of the Raf-1/ERK cascade and decreased phosphorylation of Raf-1 at Ser289/296/301. The MEK inhibitor U0126 suppressed ERK activity and phosphorylation of Raf-1 at Ser289/296/301 but not Raf-1 activation elicited by its dephosphorylation at S259 following ischemia. The PP2A inhibitor cantharidin but not Pin1 inhibitor juglone blocked Raf-1 dephosphorylation at Ser289/296/301 and ERK dephosphorylation and led to ERK sustained activation, which is associated with transcriptional up-regulation of genes related to differentiation. Furthermore, dual-phase activation of ERK did not alter the mRNA levels of genes related to proliferation or differentiation. These results indicate the initial robust activation of ERK phosphorylates Raf-1 at Ser289/296/301, resulting in Raf-1inhibition and then prompt inactivation of ERK following sublethal preconditioning ischemia. Dual-phase activation of ERK may exert its neuroprotection against lethal ischemia through blocking cell proliferation and differentiation.

Topics: Animals; Brain Ischemia; Calcium; Cantharidin; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Hippocampus; Ketamine; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Naphthoquinones; Protein Isoforms; Protein Phosphatase 2; Proto-Oncogene Proteins c-raf; Rats; Rats, Sprague-Dawley

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.

Topics: Animals; Antioxidants; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cisplatin; Cytosol; Dithionitrobenzoic Acid; Enzyme Inhibitors; Humans; Mice; Mitochondria; Naphthoquinones; Organogold Compounds; Oxidation-Reduction; Recombinant Proteins; Substrate Specificity; Thioctic Acid; Thioredoxin Reductase 1; Thioredoxin Reductase 2; Thioredoxins

The peptidyl prolyl isomerase (Pin1) that induces cis-trans isomerization of the peptide bond involving serine/threonine-proline has recently been shown to regulate the activity of many phosphoproteins including the ones involved in damage response pathways. We investigated Pin1 as a potential target for enhancing the efficacy of anticancer therapy by studying the effects of juglone, a Pin1 inhibitor on the cytotoxicity of etoposide (a widely used anticancer drug that targets topoisomerase IIα) in human tumor cell lines. Treatment of cells with juglone synergistically enhanced the cytotoxicity of etoposide (loss of clonogenicity) with a tenfold increase when etoposide treatment preceded juglone exposure. On the other hand, the toxicity was than additive when the treatment protocol was reversed (i.e exposure to juglone followed by etoposide treatment). This suggests that Pin1 inhibition possibly reduces the induction of initial DNA damage by etoposide, which was supported by a decrease in the levels of chromatin bound topoIIα. Increase in the etoposide induced toxicity by juglone appeared to be mainly due to enhanced mitotic cell death linked to cytogenetic damage, although a moderate increase in interphase (apoptotic) death was also evident as revealed by DNA degradation (hypodiploid population and TUNEL assay). Since the level of Pin1 is found to be higher in cancer cells, this enzyme could be a potential target for developing an adjuvant to enhance the efficacy of anticancer therapies.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; DNA Damage; Drug Synergism; Enzyme Inhibitors; Etoposide; Flow Cytometry; Humans; Lung Neoplasms; Micronucleus Tests; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Tumor Cells, Cultured; Tumor Stem Cell Assay

We further examined the usefulness of previously reported Bacillus subtilis biosensors for antibacterial mode-of-action studies. The biosensors could not detect the tRNA synthetase inhibitors mupirocin, indolmycin, and borrelidin, some inhibitors of peptidoglycan synthesis, and most membrane-damaging agents. However, the biosensors confirmed the modes of action of several RNA polymerase inhibitors and DNA intercalators and provided new insights into the possible modes of action of ciprofloxacin, anhydrotetracycline, corralopyronin, 8-hydroxyquinoline, and juglone.

Topics: Amino Acyl-tRNA Synthetases; Anti-Bacterial Agents; Bacillus subtilis; Biosensing Techniques; Ciprofloxacin; Enzyme Inhibitors; Fatty Alcohols; Indoles; Mupirocin; Naphthoquinones; Oxyquinoline; Tetracyclines

Besides apple, its primary host, the codling moth Cydia pomonella uses walnut as a secondary host. Abundance of toxic naphthoquinones, among which juglone prevails, does not restrain this economically important pest insect from infesting walnut, but processes underlying the suitability of this host were yet unknown. Larvae feeding on an artificial diet supplemented with juglone at naturally occurring concentrations survived to adulthood at a similarly high proportion as those in the juglone-devoid control. However, their development time was prolonged, their weight gain was reduced, and adult sex ratio was distorted. Results from the natural system with walnut and apple fruits were in line with data gained on artificial diet. Remarkably, a twofold increase of the maximal juglone content reported from the walnut husk was lethal to the larvae. Chemical analyses showed that larvae feeding on the artificial diet supplemented with juglone concentrations present in walnut contained 1,4,5-trihydroxynaphthalene and excreted it in their frass, whereas the hemolymph contained neither detectable amounts of juglone nor the product of its reduction. Hence, effective metabolism of juglone in the intestinal system of the larvae underlies their survival on host plants containing this defensive compound.

Topics: Animals; Feeding Behavior; Female; Fruit; Juglans; Male; Moths; Naphthoquinones; Plant Diseases; Plant Extracts

The present study was aimed to formulate and compare the pharmacokinetic, biodistribution, pharmacodynamic, and toxicity profiles of free 5-hydroxy-1,4-naphthoquinone (juglone) with sterically stabilized liposomal form. The liposomes were optimized for size, zeta potential, entrapment efficiency (EE), and in vitro release properties. The optimized formulation had a mean size, zeta potential, and EE value of 137.1 nm, -43.1 mV, and 67.2%, respectively. In vitro release studies showed biphasic pattern with initial burst followed by sustained release over the study period, releasing about 61% after 24 h. In vitro cytotoxicity studies against melanoma cells indicated that liposomal juglone was more toxic than free juglone. Free juglone had short plasma half-life of about 2 h, whereas liposomal juglone exhibited significantly improved pharmacokinetics with a 12-fold increase in plasma half-life. Further, biodistribution studies indicated rapid renal elimination of free juglone, evidenced by its significant localization in kidneys. Conversely, the accumulation of liposomal juglone in kidneys reduced significantly with enhanced tumor localization, thereby resulting in enhanced antitumor activity. The histological studies revealed lower levels of nephrotoxicity for liposomal juglone compared with that of free juglone. To conclude, sterically stabilized liposomes could be a promising approach for the intravenous delivery of hydrophobic compounds such as juglone.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Chemistry, Pharmaceutical; Female; Kaplan-Meier Estimate; Liposomes; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Naphthoquinones; Particle Size; Solubility; Surface Properties; Tissue Distribution; Xenograft Model Antitumor Assays

Pin1 is a peptidyl prolyl cis-trans isomerase that only binds to and isomerizes phosphorylated serine/threonine-proline motifs, inducing conformational changes that alter target protein function and phosphorylation. We have shown previously that deficiency of another peptidyl prolyl isomerase, FK506 binding protein 12/12.6, alters endothelial NO synthase phosphorylation and causes endothelial dysfunction and hypertension. Endothelial NO synthase contains the Pin1 binding sequence at (p)serine 116-proline 117 and phosphorylation of endothelial NO synthase serine 116 inhibits NO production; however, whether Pin1 deficiency alters vascular function and blood pressure is unknown. We hypothesized that Pin1 isomerizes p-endothelial NO synthase serine 116, which enables dephosphorylation and stimulates NO production. Immunoprecipitation of endothelial NO synthase and probing for Pin1 in rat aortic endothelial cells confirmed the interaction between the two. Pin1 knockdown via small interfering RNA or inhibition by juglone increased endothelial NO synthase serine 116 phosphorylation and prevented vascular endothelial growth factor-induced serine 116 dephosphorylation in endothelial cells. Acute treatment of isolated mouse aortas with juglone increased endothelial NO synthase serine 116 phosphorylation and decreased NO production and relaxation responses. Mice treated with juglone for 2 weeks, as well as Pin1 knockout mice, exhibited increased aortic endothelial NO synthase serine 116 phosphorylation, endothelial dysfunction, and hypertension. These data demonstrate that Pin1 binds endothelial NO synthase and enables dephosphorylation of serine 116, which increases NO production and endothelium-dependent dilation, leading to blood pressure maintenance.

Topics: Amino Acid Substitution; Animals; Aorta; Binding Sites; Blood Pressure; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Hypertension; Immunoblotting; Immunoprecipitation; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide; Nitric Oxide Synthase Type III; Peptidylprolyl Isomerase; Phosphorylation; Protein Binding; Rats; RNA Interference; Serine; Vasodilation

Many plant species produce toxic secondary metabolites that limit attacks by herbivorous insects, and may thereby constrain insect expansion to new hosts. Walnut is a host for the codling moth Cydia pomonella, which efficiently detoxifies the main walnut defensive compound juglone (5-hydroxy-1,4-naphthoquinone). The oriental fruit moth Grapholita molesta, which also belongs to the tribe Grapholitini, does not feed on walnut. We tested the performance of G. molesta, a highly invasive species, on artificial diets containing juglone at levels mimicking those found in walnut over the growing season. Juglone-fed G. molesta survived relatively well to adulthood, but larval and adult body weights were reduced, and larval developmental time was prolonged in a dose-dependent fashion. Chemical analysis of frass from larvae that had been fed a juglone-containing diet suggests that G. molesta reduces juglone to non-toxic 1,4,5-trihydroxynaphthalene in its gut. This unexpected tolerance of G. molesta to high levels of juglone may facilitate expansion of the host range beyond the current rosacean fruit trees used by this invasive pest.

Topics: Animals; Fruit; Herbivory; Host-Parasite Interactions; Juglans; Moths; Naphthoquinones; Rosaceae

We previously found that vitamin K(3) (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on juglone (5-hydroxy-1,4-naphthoquinone), which is a 1,4-naphthoquinone derivative, and chemically synthesized novel juglones conjugated with C2:0 to C22:6 fatty acid (5-O-acyl juglones). The chemically modified juglones enhanced mammalian pol inhibition and their cytotoxic and anti-inflammatory activities. The juglone conjugated with oleic acid (C18:1-acyl juglone) showed the strongest inhibition of DNA replicative pol α activity and human colon carcinoma (HCT116) cell growth in 10 synthesized 5-O-acyl juglones. C12:0-Acyl juglone was the strongest inhibitor of DNA repair-related pol λ, as well as the strongest suppression of the production of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in the compounds tested. Moreover, this compound caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. C12:0- and C18:1-Acyl juglones selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. These data indicate that the novel 5-O-acyl juglones target anti-cancer and/or anti-inflammatory agents based on mammalian pol inhibition. Moreover, the results suggest that acylation of juglone is an effective chemical modification to improve the anti-cancer and anti-inflammation of vitamin K(3) derivatives, such as juglone.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Cell Line, Tumor; DNA Polymerase beta; Enzyme Inhibitors; Humans; Inflammation; Lipopolysaccharides; Mice; Naphthoquinones; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Juglone is a phenolic compound used in popular medicine as a phytotherapic to treat inflammatory and infectious diseases. However, it also acts as an uncoupler of oxidative phosphorylation in isolated liver mitochondria and, thus, may interfere with the hepatic energy metabolism. The purpose of this work was to evaluate the effect of juglone on several metabolic parameters in the isolated perfused rat liver. Juglone, in the concentration range of 5 to 50μM, stimulated glycogenolysis, glycolysis and oxygen uptake. Gluconeogenesis from both lactate and alanine was inhibited with half-maximal effects at the concentrations of 14.9 and 15.7μM, respectively. The overall alanine transformation was increased by juglone, as indicated by the stimulated release of ammonia, urea, l-glutamate, lactate and pyruvate. A great increase (9-fold) in the tissue content of α-ketoglutarate was found, without a similar change in the l-glutamate content. The tissue contents of ATP were decreased, but those of ADP and AMP were increased. Experiments with isolated mitochondria fully confirmed previous notions about the uncoupling action of juglone. It can be concluded that juglone is active on metabolism at relatively low concentrations. In this particular it resembles more closely the classical uncoupler 2,4-dinitrophenol. Ingestion of high doses of juglone, thus, presents the same risks as the ingestion of 2,4-dinitrophenol which comprise excessive compromising of ATP production, hyperthermia and even death. Low doses, i.e., moderate consumption of natural products containing juglone, however, could be beneficial to health if one considers recent reports about the consequences of chronic mild uncoupling.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Alanine; Animals; Dose-Response Relationship, Drug; Energy Metabolism; Glycogenolysis; Glycolysis; Liver; Male; Mitochondria, Liver; Naphthoquinones; Oxygen Consumption; Rats; Rats, Wistar

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6h exposure to 10μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC(50) value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.

Topics: Adult; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Leukocytes, Mononuclear; Mitochondria; Naphthoquinones; Oligopeptides; Reactive Oxygen Species; Reference Values; Time Factors

Juglone (5-hydroxy-1,4-naphthoquinone) is known allelochemical, but its molecular mode of action is not well understood. We found that juglone induced reactive oxygen species production and calcium accumulation. To gain more insight into these cellular responses, we performed large-scale analysis of the rice transcriptome during juglone stress. Exposure to juglone triggered changes in transcript levels of genes related to cell growth, cell wall formation, chemical detoxification, abiotic stress response and epigenesis. The most predominant transcription-factor families were AP2/ERF, HSF, NAC, C2H2, WRKY, MYB and GRAS. Gene expression profiling of juglone-treated rice roots revealed upregulated signaling and biosynthesis of abscisic acid and jasmonic acid and inactivation of gibberellic acid. In addition, juglone upregulated the expression of two calcium-dependent protein kinases (CDPKs), 6 mitogen-activated protein kinase (MAPK) genes and 1 MAPK gene and markedly increased the activities of a CDPK-like kinase and MAPKs. Further characterization of these juglone-responsive genes may be helpful for better understanding the mechanisms of allelochemical tolerance in plants.

Topics: Gene Expression Regulation, Plant; Mitogen-Activated Protein Kinases; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Oryza; Plant Proteins; Plant Roots; Protein Kinases; Signal Transduction; Transcriptome

Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants.

Topics: Gene Expression Regulation, Plant; Glutathione Transferase; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; Seedlings; Zea mays

In Caenorhabditis elegans pretreatment with juglone, a generator of reactive oxygen species (ROS) provides a subsequently increased ROS-resistance. We investigated whether juglone at low or high concentrations when provided via the oral route in a liquid axenic medium affects normal lifespan of C. elegans. High juglone concentrations led to premature death, low concentrations were tolerated well and caused a prolongation of lifespan. Lifespan extension under moderate oxidative stress was associated with increased expression of small heat-shock protein HSP-16.2, enhanced glutathione levels, and nuclear translocation of DAF-16. Silencing or deletion of DAF-16 prevented the juglone-induced adaptations. RNA-interference for SIR-2.1 had the same effects as the deletion of DAF-16 but did not affect nuclear accumulation of DAF-16. Our studies demonstrate that DAF-16- and SIR-2.1-dependent alterations in gene expression after a ROS challenge lead to a lifespan extension in C. elegans as long as the stressor concentration does not exceed the saturable protective capacity.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Forkhead Transcription Factors; Gene Deletion; Heat-Shock Proteins; Kaplan-Meier Estimate; Longevity; Models, Animal; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; RNA Interference; Sirtuins; Transcription Factors

Prooxidant activity of naphthoquinone compounds was analyzed by lipid peroxidation, and the formation of base adduct in DNA. Naphthoquinones with electron-repelling hydroxyl group in the benzene moiety such as juglone and shikonin of lower concentrations stimulated the microsomal lipid peroxidation, but lawsone and lapachol with hydroxyl group in the quinone moiety did not enhance the formation of lipid peroxides. Naphthoquinone-dependent lipid peroxidation was closely related to the enhancement of Fe(2+) autooxidation. Treatment of DNA with juglone a representative of 5-hydroxylated naphthoquinone stimulated the formation of 8-hydroxy-2'-deoxyguanosine, whereas lawsone and lapachol showed negligible formation of DNA base adduct. ESR spectra showed that juglone can form semiquinone radical in the presence of ferrous ion, but lawsone cannot. Biological toxicity of juglone with the potent electron-repelling group at 5-position may be due to the reactive oxygen species formed by semiquinone radical, but naphthoquinone compounds with an electron-repelling group in the quinone moiety, lawsone shows weak toxicity with only a little ability producing reactive oxygen species by the negligible formation of semiquinone.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Survival; Deoxyguanosine; DNA Adducts; Dogs; Electron Spin Resonance Spectroscopy; Free Radicals; Hydroxylation; Iron; Lipid Peroxidation; Lipids; Microsomes, Liver; Naphthoquinones; Oxidants; Oxidation-Reduction

We developed a method to graft a tripeptide (glutathione) onto 5-hydroxy-1,4-naphthoquinone, an electropolymerizable molecule. The resulting thin conducting polymer presents a well-defined and stable electroactivity in neutral buffered solution, due to the embedded quinone group, and is able to covalently graft amino-modified DNA probe strands. It is shown that the bioelectrode presents positive current change following DNA hybridization. This makes a "signal-on" direct electrochemical DNA sensor. The results were obtained with low target concentration (50nM) and the selectivity is excellent as a single-mismatch sequence can be discriminated from the full-complementary target.

Topics: Amines; Base Sequence; Biosensing Techniques; DNA; DNA Probes; Electric Conductivity; Electrochemistry; Glutathione; Hydroxides; Naphthoquinones; Nucleic Acid Hybridization; Oxidation-Reduction; Photoelectron Spectroscopy; Polymers; Spectrometry, Fluorescence

The regioselectivities of several Diels-Alder reactions utilized en route to bisanthraquinone antibiotic BE-43472B are examined using density functional theory calculations. These reactions involve highly substituted dienes and juglone dienophiles, and there is an opposite regiochemical outcome for Diels-Alder reactions with beta-aryl substituted juglones when compared to reactions of unsubstituted juglone. In this article, the effect of an aromatic conjugating group bonded to juglone is explored.

Topics: Anthraquinones; Anti-Bacterial Agents; Biological Products; Crystallography, X-Ray; Molecular Conformation; Molecular Structure; Naphthoquinones; Stereoisomerism

Interest in the redox properties of natural products has led to the development of various assays for the detection of antioxidant activities and ROS-scavenging properties. Here, additional modifications of the 2-deoxy-d-ribose degradation assay are introduced that specifically allow the determination of interactions of the test compound with the autoxidation of ascorbic acid and the autoxidation of the test compound itself. To illustrate this, juglone and quercetin were used as examples. The modified assay systems provide insights into their specific antioxidative and pro-oxidative properties. In additional, an extensive characterization of the redox properties of their complex with iron is possible, if iron ions are added in the free form or complexed with EDTA. The juglone-iron complex proved to be pro-oxidative in a wider range of milieus than the quercetin-iron complex.

Topics: Antioxidants; Ascorbic Acid; Deoxyribose; Free Radical Scavengers; Hydrogen Peroxide; Iron; Kinetics; Metals; Naphthoquinones; Oxidation-Reduction; Quercetin; Reactive Oxygen Species

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC(50) values in the range of 0.31 (1.7microM) in HL-60 to 0.88microg/mL (4.7microM) in SF-295 and IC(50) of 0.69microg/mL (3.7microM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC(50) significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 7; Caspase 8; Cell Line, Tumor; DNA Fragmentation; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Mitochondria; Naphthoquinones; Phosphatidylserines

Electrophilic xenobiotics and endogenous products from oxidative stresses induce the glutathione S-transferases (GSTs), which form a large family within the phase II enzymes over both animal and plant kingdoms. The GSTs thus induced in turn detoxify these external as well as internal stresses. Because these stresses are often linked to ageing and damage to health, the induction of phase II enzymes without causing adverse effects would be beneficial in slowing down ageing and keeping healthy conditions.. We have tested this hypothesis by choosing allyl isothiocyanate (AITC), a functional ingredient in wasabi, as a candidate food ingredient that induces GSTs without causing adverse effects on animals' lives. To monitor the GST induction, we constructed a gst::gfp fusion gene and used it to transform Caenorhabditis elegans for use as a nematode biosensor. With the nematode biosensor, we found that AITC induced GST expression and conferred tolerance on the nematode against various oxidative stresses. We also present evidence that the transcription factor SKN-1 is involved in regulating the GST expression induced by AITC.. We show the applicability of the nematode biosensor for discovering and evaluating functional food substances and chemicals that would provide anti-ageing or healthful benefits.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance; Female; Gene Expression Regulation, Enzymologic; Glucuronosyltransferase; Glutathione Transferase; Green Fluorescent Proteins; Herbicides; Isothiocyanates; Male; Microscopy, Confocal; Naphthoquinones; Oxidative Stress; Paraquat; Reproduction; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors

The aim of this study was elucidation of the difference in inhibition influence of 5-hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) on jack bean urease activity. It was found that juglone acted as a strong, time and concentration dependent inactivator of urease. On the contrary, lawsone showed an inconsiderable inhibition influence. The reactivation of juglone modified urease showed the participation of reversible and irreversible contribution in the inactivation. In the presence of an excess of DTT, urease inactivated by juglone regained 70% of its activity. The reversible inactivation was attributed to oxidation of the essential urease thiols by reactive oxygen species (ROS) realizing during reduction of juglone to seminaphthoquinone. Presence of hydrogen peroxide in the incubation system was proved by direct determination and by application of catalase. The irreversible contribution in the inhibition was assumed as an arylation of urease thiol groups by juglone. The insignificant urease inhibition by lawsone was concluded as an effect of a low hydrogen peroxide generation and lawsone resistance for reaction with protein thiols. It was found that lawsone well reacted with l-cysteine, poorly with glutathione and hardly with urease thiols. The observed sequence was arranged according the rule the more complex thiol the less susceptible for reaction with lawsone. On the other hand, juglone displayed an excellent reactivity towards both thiols and urease. Thus, this indicated a significance of a steric hindrance which appeared when the hydroxyl group changing position from 5 in juglone (5-hydroxy-1,4-naphthoquinone) to 2 in lawsone (2-hydroxy-1,4-naphthoquinone).

Topics: Canavalia; Catalase; Dithiothreitol; Enzyme Inhibitors; Hydrogen Peroxide; Kinetics; Naphthoquinones; Urease

Epidemiological studies have repeatedly demonstrated that green tea protects against oxidative stress involved in many diseases. Health benefits of green tea are attributed to its principal active constituent, epigallocatechin gallate (EGCG). EGCG was shown to increase the stress resistance and lifespan of Caenorhabditis elegans. The mechanism of this action has been investigated in this study. The expression of hsp-16.1 and hsp-16.2 in EGCG-treated worms (N2), as quantified by real-time PCR, was significantly lower under oxidative stress induced by juglone than in controls without EGCG. In the strain TJ356 (DAF-16::GFP) EGCG treatment induced translocation of DAF-16 from the cytoplasm into the nucleus, suggesting that EGCG may affect the daf-2/insulin-like signaling pathway. EGCG decreased the formation of lipofuscin, an aging related pigment. Also, EGCG reduced beta amyloid (Abeta) deposits and inhibited Abeta oligomerization in transgenic C. elegans (CL2006). Thus, the use of green tea and EGCG is apparently rational alternatives for protecting against ROS-mediated and age-related diseases.

Topics: Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Antioxidants; Biological Transport; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Camellia sinensis; Catechin; Cell Nucleus; Cytoplasm; Heat-Shock Proteins; Insulin; Lipofuscin; Naphthoquinones; Oxidative Stress; Plant Extracts; Polymerization; Receptor, Insulin; Signal Transduction; Somatomedins; Transcription Factors

Endocrine therapies, which inhibit estrogen receptor signaling, are the most common and effective treatments for estrogen receptoralpha-positive breast cancer. However, the utility of these agents is limited by the frequent development of resistance, and the precise mechanisms underlying endocrine therapy resistance remain incompletely understood. Here, we demonstrate that peptidyl-prolyl isomerase Pin1 is an important determinant of resistance to tamoxifen and show that Pin1 increases E2F-4- and Egr-1-driven expression of LC-3 as a result of an increased interaction with and phosphorylation of MEK1/2. In human tamoxifen-resistant breast cancer, our results show a significant correlation between Pin1 overexpression and high levels of LC-3. Promoter activity as well as expression levels of Pin1 were drastically higher in tamoxifen-resistant MCF7 cells than control MCF7 cells, as were levels of LC-3 mRNA and protein, an autophagy marker. Pin1(-/-) mouse embryonic fibroblasts showed lower 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MEK1/2 phosphorylation than Pin1(+/+) mouse embryonic fibroblasts. Silencing of Pin1 expression inhibited TPA-induced MEK1/2 phosphorylation in MCF7 cells. Moreover, PD98059, a specific inhibitor of MEK1/2, and juglone, a potent Pin1 inhibitor, significantly suppressed the TPA-induced expression of E2F-4 as well as Egr-1 transcription factors, which control LC-3 gene expression. Importantly, 4-hydroxy tamoxifen, when used in combination with silencing of Pin1 or LC-3, increased cleaved poly(ADP-ribose) polymerase and DNA fragmentation to inhibit cologenic growth of MCF7 cells. We therefore link the Pin1-MEK pathway and LC-3-mediated tamoxifen resistance and show the therapeutic potential of Pin1 in the treatment of tamoxifen-resistant breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Mice; Microtubule-Associated Proteins; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Tamoxifen

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 microg/ml). Juglone could induce the mitochondrial membrane potential (DeltaPsim) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Caspase Inhibitors; Caspases; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Juglans; Membrane Potential, Mitochondrial; Mitochondrial Proteins; Naphthoquinones; Oligopeptides; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at β-secretase (BACE), as well as at β-amyloid peptide (Aβ) aggregation and disaggregating preformed Aβ fibrils. Compounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl derivatives (11-15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggregation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation and disaggregation preformed Aβ fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxyphenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead compounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Cell Line, Tumor; Humans; Naphthoquinones; Protease Inhibitors

Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

Topics: Blotting, Western; Cell Membrane; Cytosol; Humans; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Naphthoquinones; Neutrophils; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Protein Transport; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

Topics: Amino Acid Motifs; Amino Acid Substitution; Animals; Catalysis; Catalytic Domain; Crystallography, X-Ray; Electron Transport; Mutation, Missense; Naphthoquinones; Protein Structure, Tertiary; Rats; Recombinant Proteins; Selenocysteine; Selenoproteins; Structure-Activity Relationship; Thioredoxin Reductase 1; Thioredoxins

The antitubercular activity and cytotoxicity of juglone derivatives were analyzed with the topological and molecular surface features from a web-based server, MODEL (MOlecular DEscriptor Lab). Analysis of the structural features in conjunction with the biological endpoints in Combinatorial Protocol in Multiple Linear Regression led to the identification of seven descriptors for modeling the activity and six descriptors for that of the toxicity of the compounds. Analysis of these descriptors in PLS highlighted their relative significance in modulating the biological response. They suggested that structures with compact molecular arrangement, partial positive surface areas and increased autocorrelation with small lag values may lead to better antitubercular activity.

Topics: Animals; Antitubercular Agents; Chlorocebus aethiops; Least-Squares Analysis; Linear Models; Naphthoquinones; Quantitative Structure-Activity Relationship; Vero Cells

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1(-/-) mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared with Pin1(+/+) MEFs. Furthermore, Pin1 enhanced the insulin-induced extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected activator protein-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than green fluorescent protein-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Drug Synergism; Embryo, Mammalian; Fibroblasts; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Hypoglycemic Agents; Immunoblotting; Immunosuppressive Agents; Insulin; Liver Neoplasms, Experimental; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Sirolimus; Transcription Factor AP-1; Two-Hybrid System Techniques

Juglone, 5-hydroxy-1,4-naphthoquinone, is known for its wide range of biological activities. It has been suggested that juglone's excellent redox cycling properties contribute to this reputation. Many biological activities are nonlinear with low concentrations exerting stimulating effects, whereas only higher concentrations cause inhibition. Here, we corroborate studies on the nematode Caenorhabditis elegans that point out hormetic effects by showing that juglone may cause a nonlinear effect on postgerminative shoot and root growth of Sinapis alba. This effect was only significantly visible, however, when seedlings were stressed with methanol. Classic and modified versions of the deoxyribose assay were applied successfully to characterize antioxidative (purposeful generation of hydroxyl radicals) and prooxidative (no purposeful generation of hydroxyl radicals) activities. Variants of the assay with and without the addition of the iron chelator EDTA showed that the antioxidant activity is independent on chelation of iron ions by juglone; by contrast, the strength of the prooxidative activity depended on the chelation of iron ions by juglone. The hormetic effects of lower concentrations on germination of Sinapis alba, thus, may be caused by the antioxidant activities of this compound, which are especially effective when the test organism is subjected to higher oxidative challenge. The present study suggests that pronounced prooxidative activities, which are considerably accelerated by chelation of iron ions, may contribute to the toxic effects of juglone at higher concentrations.

Topics: Antioxidants; Edetic Acid; Germination; Iron Chelating Agents; Naphthoquinones; Oxidation-Reduction; Plant Roots; Plant Shoots; Reactive Oxygen Species; Seedlings; Sinapis

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We characterized 2 novel GPx genes from a lung fluke, Paragonimus westermani (designated PwGPx1 and PwGPx2). These single copy genes spanned 6559 and 12 371 bp, respectively, and contained each of 5 intervening introns. The PwGPx2 harboured a codon for Sec and a Sec insertion sequence motif. Proteins encoded by the Paragonimus genes demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic and glutathione-binding domains and absence of the subunit interaction domain. Expression of PwGPx1 increased gradually as the parasite matured, whereas that of PwGPx2 was temporally regulated. PwGPx2 was expressed at the basal level from the metacercariae to the 3-week-old juveniles; however, the expression was significantly induced in the 7-week-old immature worms and reached a plateau in the 12-week-old adults and eggs. PwGPx1 and PwGPx2 were largely localized in vitellocytes within vitelline glands and eggs. Oxidative stress-inducible paraquat, juglone and H2O2 substantially augmented the PwGPx1 and PwGPx2 expressions in viable worms by 1.5- to 11-fold. Our results strongly suggested that PwGPxs may actively participate in detoxification of oxidative hazards in P. westermani.

Topics: Animals; Enzyme Induction; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Helminth Proteins; Hydrogen Peroxide; Naphthoquinones; Oxidative Stress; Paragonimus westermani; Paraquat; Phospholipid Hydroperoxide Glutathione Peroxidase

The introduction of Registration, Evaluation and Authorisation of Chemicals (REACH), requires companies to register and risk assess all substances produced or imported in volumes of >1 tonne per year. Extrapolation methods which use existing data for estimating the effects of chemicals are attractive to industry, and comparative data are therefore increasingly in demand. Data on natural toxic chemicals could be used for extrapolation methods such as read-across. To test this hypothesis, the toxicity of natural chemicals and their synthetic analogues were compared using standardised toxicity tests. Two chemical pairs: the napthoquinones, juglone (natural) and 1,4-naphthoquinone (synthetic); and anthraquinones, emodin (natural) and quinizarin (synthetic) were chosen, and their comparative effects on the survival and reproduction of collembolans, earthworms, enchytraeids and predatory mites were assessed. Differences in sensitivity between the species were observed with the predatory mite (Hypoaspis aculeifer) showing the least sensitivity. Within the chemical pairs, toxicity to lethal and sub-lethal endpoints was very similar for the four invertebrate species. The exception was earthworm reproduction, which showed differential sensitivity to the chemicals in both naphthoquinone and anthraquinone pairs. Differences in toxicity identified in the present study may be related to degree of exposure and/or subtle differences in the mode of toxic action for the chemicals and species tested. It may be possible to predict differences by identifying functional groups which infer increased or decreased toxicity in one or other chemical. The development of such techniques would enable the use of read-across from natural to synthetic chemicals for a wider group of compounds.

Topics: Animals; Anthraquinones; Ecotoxicology; Emodin; Naphthoquinones; Oligochaeta; Organic Chemicals; Reproduction; Risk Assessment; Soil

Cells adapt to stressors by activating mechanisms that repair damage and protect them from further injury. Stress-induced damage accumulates with age and contributes to age associated diseases. Increased age attenuates the ability to mount a stress response, but little is known about the mechanisms by which this occurs. To begin addressing this problem, we studied hormesis in the nematode Caenorhabditis elegans. When exposed to a low concentration of the xenobiotic juglone, young worms mount a robust hormetic stress response and survive a subsequent exposure to a higher concentration of juglone that is normally lethal to naïve animals. Old worms are unable to mount this adaptive response. Microarray and RNAi analyses demonstrate that an altered transcriptional response to juglone is responsible in part for the reduced adaptation of old worms. Many genes differentially regulated in young versus old animals are known or postulated to be regulated by the FOXO homologue DAF-16 and the Nrf2 homologue SKN-1. Activation of these pathways is greatly reduced in juglone stressed old worms. DAF-16- and SKN-1-like transcription factors play highly conserved roles in regulating stress resistance and longevity genes. Our studies provide a foundation for developing a molecular understanding of how age affects cytoprotective transcriptional pathways.

Topics: Adaptation, Physiological; Age Factors; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cytoprotection; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Forkhead Transcription Factors; Gene Expression Profiling; Mutation; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Oxidative Stress; RNA Interference; Signal Transduction; Transcription Factors; Transcriptional Activation; Xenobiotics

The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-kappaB (NF-kappaB) is constitutively activated, but its role in these cancers remains to be determined, and evidence is still lacking to show that Pin1 contributes to cell transformation by Rel/NF-kappaB. Rel/NF-kappaB transcriptional and oncogenic activities are modulated by several posttranslational modifications and coregulatory proteins, and previous studies showed that cytokine treatment induces binding of Pin1 to the RelA subunit of NF-kappaB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-kappaB that are implicated in leukemia/lymphomagenesis and modulates their transcriptional and oncogenic activities. Pin1 markedly enhanced transformation of primary lymphocytes by the human c-Rel protein and also increased cell transformation by the potent viral Rel/NF-kappaB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-kappaB. Pin1 promoted nuclear accumulation of Rel proteins in the absence of activating stimuli. Importantly, inhibition of Pin1 function with the pharmacologic inhibitor juglone or with Pin1-specific shRNA led to cytoplasmic relocalization of endogenous c-Rel in human lymphoma-derived cell lines, markedly interfered with lymphoma cell proliferation, and suppressed endogenous Rel/NF-kappaB-dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-kappaB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-kappaB-dependent leukemia/lymphomas.

Topics: Amino Acid Sequence; Animals; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Humans; Lymphoma; Molecular Sequence Data; Multigene Family; Naphthoquinones; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Oncogene Proteins v-rel; Peptidylprolyl Isomerase; Protein Binding; Protein Transport; Sequence Homology, Amino Acid; Up-Regulation

The peptidyl-prolyl cis/trans isomerase Pin1 has been implicated in malignant transformation in multiple studies, however, little is known about its potential impact in head and neck cancer. This study evaluates the role of Pin1 in head and neck squamous cell carcinomas (HNSCCs). Pin1 expression and level of phosphorylation was evaluated by Western blot analysis and 2D-gel-electrophoresis. Pin1 was inhibited with juglone (5-hydroxy-1,4-naphthalenedione) or Pin1 specific siRNA and its influence on cell cycle checkpoint distribution was assessed by FACS analysis. Pin1 overexpression was found in HNSCC tissues and cell lines. 2D-gel-electrophoresis data pointed to Pin1 being hypophosphorylated in HNSCC cells which is consistent with overactivation of this rotamase. Inhibition of HNSCC cells with juglone or Pin1 siRNA induced the cell cycle inhibitor p21(WAF1/Cip1) with a concomitant reduction of cells in G2/M and an increased fraction of cells with fragmented DNA. Cell death did not correlate with significant levels of apoptosis in Pin1 depleted HNSCC cells. In summary, the data shows that Pin1 is overexpressed and hypophosphorylated in HNSCC, and that inhibition of Pin1 blocks cell cycle progression and triggers tumor cell death. Pin1 therefore could represent a new target for the development of improved HNSCC targeting drugs.

Topics: Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Case-Control Studies; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Naphthoquinones; Neoplasm Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; RNA, Small Interfering; Up-Regulation

We show that in hippocampal cultured neurons, dephosphorylation of peptidyl-prolyl cis-trans isomerase Pin1 on Ser16 is occurring during the early stages of exposure to Abeta (1-42) oligomers. This occurrence, resulting in Pin1 activation, is paralleled by Tau(Thr231) dephosphorylation, probably due to Pin1-mediated Tau isomerisation. Indeed, in the presence of the specific Pin1 inhibitor juglone, Abeta-induced Tau(Thr231)dephosphorylation is prevented. The involvement of protein phosphatase 2A (PP2A) in dephosphorylation of isomerised Tau is shown by the co-treatment of neurons with Abeta (1-42) and okadaic acid, a PP2A inhibitor, leading to Tau(Thr231) hyperphosphorylation. We also report the modulation, via Pin1, of Ser199, Ser396, Ser400 and Ser404 phosphorylation state in response to Abeta treatment. Taken together, these data suggest for the first time that an early Pin1 response might be transiently evoked by Abeta 1-42 oligomers, preventing Tau hyperphosphorylation. This evidence highlights the role of Pin1 as Tau phosphorylation modulator during Alzheimer onset.

Topics: Adaptor Proteins, Signal Transducing; Amyloid beta-Peptides; Animals; Cell Survival; Cells, Cultured; Cytarabine; Embryo, Mammalian; Enzyme Inhibitors; Formazans; Hippocampus; Immunosuppressive Agents; Microscopy, Atomic Force; Naphthoquinones; Neurons; Okadaic Acid; Peptide Fragments; Phosphorylation; Propanols; Rats; tau Proteins; Tetrazolium Salts; Time Factors

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2',7'-dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(+)/PI(+) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis.

Topics: Animals; Annexin A5; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Glutathione; Juglans; L-Lactate Dehydrogenase; Melanoma; Melanoma, Experimental; Mice; Naphthoquinones; Reactive Oxygen Species

An electrochemical and spectroelectrochemical strategy is presented for evaluating reactivity differences in the semiquinone anions from naturally occurring quinones juglone (5-hydroxy-1,4-naphthoquinone) and plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone). By employing cyclic voltammetry and in situ spectroelectrochemical electron spin resonance measurements, it was found that while semiquinone species generated from plumbagin are stable radical anions in DMSO solution, the species generated from juglone are more reactive. These latter species are involved in a self-protonation process involving a slow rate of protonation (1.8-2.1 mol L(-1)) due to the mild acidity of the OH group at the C-5 position. This result is important when considering observed differences in biochemical reactivity for these quinones, particularly in cases where mediated cytotoxic action is provoked by these agents, as is discussed in this work.

Topics: Anions; Dimethyl Sulfoxide; Electrochemical Techniques; Electron Spin Resonance Spectroscopy; Free Radicals; Molecular Structure; Naphthoquinones; Oxidation-Reduction

Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has important roles in cellular regulation. Because of its activity to switch the conformation of peptidyl-proline bonds in polypeptide chains, Pin1 operates as a binary switch, often in fate-determining pathways. Pin1 activity is usually controlled by substrate phosphorylation, but how Pin1 switches protein fates has been unclear. Here we show that Pin1 controls the degree of substrate ubiquitylation and thereby protein functions. We found that yeast Pin1 (Ess1) is essential for viability because it controls the NF-kappaB-related Spt23 transcription factor involved in unsaturated fatty-acid synthesis. High Pin1 activity results in low ubiquitylation of Spt23, which triggers Spt23 precursor processing and hence transcription factor activation. By contrast, decreased Pin1 activity leads to robust Spt23 polyubiquitylation and subsequent proteasomal degradation. Inhibition of Pin1 in mammalian cells changes the ubiquitylation status of the tumour suppressor protein p53 from oligoubiquitylation, which is known to trigger nuclear export, to polyubiquitylation, which causes nuclear p53 degradation. This suggests that the Pin1 activity is often translated into a fate-determining ubiquitylation switch, and that Pin1 may affect the degree of substrate ubiquitylation in other pathways as well.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Cell Nucleus; Enzyme Inhibitors; Humans; Immunoblotting; Immunoprecipitation; Leupeptins; Membrane Proteins; Microscopy, Fluorescence; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Saccharomyces cerevisiae Proteins; Substrate Specificity; Trans-Activators; Transcription Factors; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling quinone juglone upon exit from dauer diapause results in defective egg-laying (Egl phenotype) in the pcm-1 mutants only. Treatment with paraquat, a redox-cycling dipyridyl, causes a more severe developmental delay at the second larval stage in pcm-1 mutants than in wild-type nematodes. Finally, exposure to homocysteine and homocysteine thiolactone, molecules that can induce oxidative stress via distinct mechanisms, results in a more pronounced delay in development at the first larval stage in pcm-1 mutants than in wild-type animals. Homocysteine treatment also induced the Egl phenotype in mutant but not wild-type nematodes. All of the effects of these agents were reversed upon addition of vitamin C, indicating that the developmental delay and egg-laying defects result from oxidative stress. Furthermore, we have demonstrated that a mutation in the gene encoding the insulin-like receptor DAF-2 suppresses the Egl phenotype in pcm-1 mutants treated with juglone. Our results support a role of PCM-1 in the cellular responses mediated by the DAF-2 insulin-like signaling pathway in C. elegans for optimal protection against oxidative stress.

Topics: Animals; Antioxidants; Ascorbic Acid; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; Genotype; Homocysteine; Larva; Metamorphosis, Biological; Methyltransferases; Mutation; Naphthoquinones; Oxidants; Oxidative Stress; Paraquat; Phenotype; Receptor, Insulin

(+/-)-Catechin is a flavan-3-ol that occurs in the organs of many plant species, especially fruits. Health-beneficial effects have been studied extensively, and notable toxic effects have not been found. In contrast, (+/-)-catechin has been implicated as a 'chemical weapon' that is exuded by the roots of Centaurea stoebe, an invasive knapweed of northern America. Recently, this hypothesis has been rejected based on (+/-)-catechin's low phytotoxicity, instability at pH levels higher than 5, and poor recovery from soil. In the current study, (+/-)-catechin did not inhibit the development of white and black mustard to an extent that was comparable to the highly phytotoxic juglone, a naphthoquinone that is allegedly responsible for the allelopathy of the walnut tree. At high stress levels, caused by sub-lethal methanol concentrations in the medium, and a 12 h photoperiod, (+/-)-catechin even attenuated growth retardation. A similar effect was observed when (+/-)-catechin was assayed for brine shrimp mortality. Higher concentrations reduced the mortality caused by toxic concentrations of methanol. Further, when (+/-)-catechin was tested in variants of the deoxyribose degradation assay, it was an efficient scavenger of reactive oxygen species (ROS) when they were present in higher concentrations. This antioxidant effect was enhanced when iron was chelated directly by (+/-)-catechin. Conversely, if iron was chelated to EDTA, pro-oxidative effects were demonstrated at higher concentrations; in this case (+/-)-catechin reduced molecular oxygen and iron to reagents required by the Fenton reaction to produce hydroxyl radicals. A comparison of cyclic voltammograms of (+/-)-catechin with the phytotoxic naphthoquinone juglone indicated similar redox-cycling properties for both compounds although juglone required lower electrochemical potentials to enter redox reactions. In buffer solutions, (+/-)-catechin remained stable at pH 3.6 (vacuole) and decomposed at pH 7.4 (cytoplasm) after 24 h. The results support the recent rejection of the hypothesis that (+/-)-catechin may serve as a 'chemical weapon' for invasive plants. Instead, accumulation and exudation of (+/-)-catechin may help plants survive periods of stress.

Topics: Animals; Antioxidants; Artemia; Catechin; Cytotoxins; Deoxyribose; Fruit; Hydrogen-Ion Concentration; Mustard Plant; Naphthoquinones; Reactive Oxygen Species

To study the effect of juglone on the ultrastructure of human liver cancer BEL-7402 cells.. BEL-7402 cells were incubated in the presence of 12.5 micromol/L juglone for 24 h, and fixed in 2.5% glutaraldehyde for HE staining and Coomassie brilliant blue staining and scanning electron microscopy.. Incubation with juglone resulted in obvious changes in the cell morphology and cytoskeletal alterations of the cells. Scanning electron microscopy revealed reduced volume of the cell bodies, dissociation of the cells, curling and malformation of the microvilli on the cell surface with rupture of the intercellular junction and enlargement of the intercellular space. The formation of apoptotic bodies was observed. Transmission electron microscopy showed expansion of the endoplasmic reticula, mitochondrial cristea disintegration, nucleolar fragmentation and formation of the apoptotic bodies after the exposure to juglone for 24 h.. Juglone can cause ultrastructural changes of human liver cancer BEL-7402 cells and induce their apoptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Naphthoquinones

The selenoprotein thioredoxin reductase 1 (TrxR1) is currently recognized as a plausible anticancer drug target. Here we analyzed the effects of TrxR1 targeting in the human A549 lung carcinoma cell line, having a very high basal TrxR1 expression. We determined the total cellular TrxR activity to be 271.4 +/- 39.5 nmol min(-1) per milligram of total protein, which by far exceeded the total thioredoxin activity (39.2 +/- 3.5 nmol min(-1) per milligram of total protein). Knocking down TrxR1 by approx 90% using siRNA gave only a slight effect on cell growth, irrespective of concurrent glutathione depletion (> or = 98% decrease), and no increase in cell death or distorted cell cycle phase distributions. This apparent lack of phenotype could probably be explained by Trx functions being maintained by the remaining TrxR1 activity. TrxR1 knockdown nonetheless yielded drug-specific modulation of cytotoxic efficacy in response to various chemotherapeutic agents. No changes in response upon exposure to auranofin or juglone were seen after TrxR1 knockdown, whereas sensitivity to 1-chloro-2,4-dinitrobenzene or menadione became markedly increased. In contrast, a virtually complete resistance to cisplatin using concentrations up to 20 microM appeared upon TrxR1 knockdown. The results suggest that high overexpression of TrxR has an impact not necessarily linked to Trx function that nonetheless modulates drug-specific cytotoxic responses.

Topics: Adenocarcinoma; Apoptosis; Auranofin; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cisplatin; Dinitrochlorobenzene; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Naphthoquinones; RNA, Small Interfering; Thioredoxin Reductase 1; Vitamin K 3

Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3' UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP-PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1-/- mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Cytotoxins; Humans; Hyperparathyroidism, Secondary; Kidney Failure, Chronic; Male; Mice; Mice, Knockout; Naphthoquinones; Parathyroid Glands; Parathyroid Hormone; Rats; Receptors, Glucocorticoid; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Trans-Activators

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-alpha, and IL-1beta). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-kappaB, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-kappaB, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-kappaB, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.

Topics: Animals; Ankle Joint; Arthritis, Rheumatoid; Cell Line; Cells, Cultured; Chondrocytes; Collagen Type II; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Genes, Reporter; Humans; Interleukin-1beta; Mice; Naphthoquinones; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide Synthase Type II; Peptidylprolyl Isomerase; Steroid Isomerases; Transfection; Tumor Necrosis Factor-alpha

The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo.

Topics: Animals; Cattle; Cell Cycle; CHO Cells; Cricetinae; Cricetulus; Cyclin B; Cyclin B1; Cysteine; Cytoplasm; Enzyme Inhibitors; Fibroblasts; Mice; Microtubules; Mitosis; Naphthoquinones

To investigate the inhibition features of the natural product juglone (5- hydroxy-1,4-naphthoquinone) against the three key enzymes from Helicobacter pylori (cystathionine gamma-synthase [HpCGS], malonyl-CoA:acyl carrier protein transacylase [HpFabD], and beta-hydroxyacyl-ACP dehydratase [HpFabZ]).. An enzyme inhibition assay against HpCGS was carried out by using a continuous coupled spectrophotometric assay approach. The inhibition assay of HpFabD was performed based on the alpha-ketoglutarate dehydrogenase-coupled system, while the inhibition assay for HpFabZ was monitored by detecting the decrease in absorbance at 260 nm with crotonoyl-CoA conversion to beta -hydroxybutyryl-CoA. The juglone/FabZ complex crystal was obtained by soaking juglone into the HpFabZ crystal, and the X-ray crystal structure of the complex was analyzed by molecular replacement approach.. Juglone was shown to potently inhibit HpCGS, HpFabD, and HpFabZ with the half maximal inhibitory concentration IC50 values of 7.0 +/-0.7, 20 +/-1, and 30 +/-4 micromol/L, respectively. An inhibition-type study indicated that juglone was a non-competitive inhibitor of HpCGS against O-succinyl- L-homoserine (Ki=alphaKi=24 micromol/L), an uncompetitive inhibitor of HpFabD against malonyl-CoA (alphaKi=7.4 micromol/L), and a competitive inhibitor of HpFabZ against crotonoyl-CoA (Ki=6.8 micromol/L). Moreover, the crystal structure of the HpFabZ/juglone complex further revealed the essential binding pattern of juglone against HpFabZ at the atomic level.. HpCGS, HpFabD, and HpFabZ are potential targets of juglone.

Topics: Crystallization; Enzyme Inhibitors; Helicobacter pylori; Naphthoquinones; Protein Conformation; X-Ray Diffraction

Topics: Adaptor Proteins, Signal Transducing; Animals; Asthma; Drug Delivery Systems; Mice; Naphthoquinones; Rats

Topics: Adaptor Proteins, Signal Transducing; Ambrosia; Animals; Cytokines; Enzyme Inhibitors; Fibroblasts; Hypersensitivity; Interleukin-4; Mice; Mice, Knockout; Naphthoquinones; Pneumonia; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thymic Stromal Lymphopoietin; Tumor Necrosis Factor-alpha

SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function.. Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs.. We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

Topics: Animals; Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; HeLa Cells; Humans; Naphthoquinones; Oxidants; Protein Structure, Tertiary; Rats; Reactive Oxygen Species; Selenocysteine; Thioredoxin-Disulfide Reductase

Current UN International Maritime Organization legislation mandates the phased introduction of ballast water treatment technologies capable of complying with rigorous standards related to removal of waterborne organisms. Doubts concerning mechanical treatments at very high ballasting rates have renewed interest in chemical treatment for very large vessels. High removal rates for biota require broad spectrum biocides that are safe to transport and handle and pose no corrosion problems for ships' structure. The current study focuses on the naphthoquinone group of compounds and extends a previously reported set of screening bioassays with an investigation of the toxicity of four naphthoquinones to select protists and prokaryotes, representative of typical ballast water organisms. Vegetative dinoflagellate cysts exposed to 2.0 mg/L of the naphthoquinones juglone, plumbagin, menadione and naphthazarin showed varying degrees of chloroplast destruction, with menadione demonstrating the most potency. Laboratory and mesocosm exposures of various phytoplankton genera to menadione showed toxicity at 1.0 mg/L. Juglone demonstrated the most bactericidal activity as judged by a Deltatox assay (Vibrio fischeri) and by acridine orange counts of natural bacterial populations.

Topics: Aliivibrio fischeri; Animals; Bacteria; Biological Assay; Dinoflagellida; Environmental Monitoring; Microscopy, Fluorescence; Naphthoquinones; Phytoplankton; Ships; Vitamin K 3; Waste Disposal, Fluid

BACKGROUND/ABSTRACT: Immune responses initiated by T cell receptor (TCR) and costimulatory molecule mediated signaling culminate in maximal cytokine mRNA production and stability. The transcriptional responses to co-stimulatory T cell signalling involve calcineurin and NF-AT, which can be antagonized by interference with the cis-trans peptidyl-prolyl isomerases (PPIase), cyclophilin A and FKBP. Signalling molecules downstream of CD28 which are essential for the stabilization of cytokine mRNAs are largely unknown.. We now show that Pin1, a third member of the PPIase family mediates the post-transcriptional regulation of Th1 cytokines by activated T cells. Blockade of Pin1 by pharmacologic or genetic means greatly attenuated IFN-gamma, IL-2 and CXCL-10 mRNA stability, accumulation and protein expression after cell activation. In vivo, Pin1 blockade prevented both the acute and chronic rejection of MHC mismatched, orthotopic rat lung transplants by reducing the expression of IFN-gamma and CXCL-10. Combined transcriptional and post-transcriptional blockade with cyclosporine A and the Pin1 inhibitor, juglone, was synergistic.. These data suggest Pin1 inhibitors should be explored for use as immunosuppressants and employed with available calcineurin inhibitors to reduce toxicity and enhance effectiveness.

Topics: Adaptor Proteins, Signal Transducing; Animals; Chemokine CXCL10; Cyclosporine; Graft Rejection; Interferon-gamma; Interleukin-2; Lung Transplantation; Mice; Mice, Inbred C57BL; Mice, Knockout; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Rats; Rats, Inbred F344; Rats, Inbred WKY; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Th1 Cells; Transcription, Genetic; Transplantation, Homologous

Several benzo-, naphtho- and anthraquinones were tested for their efficacy as biocides in controlling aquatic nuisance species in ships' ballast water. A requirement of this application was broad spectrum aquatic toxicity, coupled with a relatively rapid rate of degradation, in order to comply with coastal discharge requirements. Compounds were screened using a suite of toxicity bioassays designed to establish their relative toxicity to an array of planktonic organisms including larval bivalves Dreissena and Crassostrea, various developmental stages of the estuarine copepod Eurytemora affinis, brine shrimp larvae (Artemia salina), the freshwater invasive water flea Bythotrephes, larval sheepshead minnows CCyprinodon variegates) and two unicellular algal genera Isochrysis and Neochloris.. The majority of the data were recorded as the lowest concentration of the test compound resulting in complete mortality or inactivation of test organisms (LC ,m). The naphthoquinones juglone, plumbagin, menadione and naphthazarin showed the highest toxicity to the broadest range of organisms, often at levels much less than 1 mg l(-1), and most of the attention was focused on this group. While plumbagin and juglone appeared overall to be the most toxic compounds, it was concluded that menadione was probably the most cost-effective candidate compound for shipboard use for controlling invasive species in ballast water, particularly in view of the large volumes of water that would require treatment.

Topics: Animals; Biological Products; Cyprinidae; Disinfectants; Invertebrates; Larva; Molecular Structure; Naphthoquinones; Pest Control; Phaeophyceae; Quinones; Ships; Toxicity Tests; Vitamin K 3

Flavonoids present in many herbal edibles possess a remarkable spectrum of biochemical and pharmacological actions and they are assumed to exert beneficial effects to human health. Although the precise biological mechanisms of their action has not been elucidated yet many of the protective properties of flavonoids are attributed to their antioxidative activity since oxidative stress is regarded as a main factor in the pathophysiology of various diseases and ageing. Oxidative stress results from excessive generation of reactive oxygen species (ROS) or diminished antioxidative defence and thus antioxidants are able to counteract such situations. We used the multicellular model organism Caenorhabditis elegans that is conserved in molecular and cellular pathways to mammals to examine the effects of the flavonoids kaempferol and fisetin with respect to their protective action in individual living worms. Both flavonoids increased the survival of C. elegans, reduced the intracellular ROS accumulation at lethal thermal stress, and diminished the extent of induced oxidative stress with kaempferol having a stronger impact. Kaempferol but not fisetin attenuated the accumulation of the ageing marker lipofuscin suggesting a life prolonging activity of this flavonoid. In addition to these effects that may be attributed to their antioxidative potential kaempferol and fisetin caused a translocation of the C. elegans FoxO transcription factor DAF-16 from the cytosol to the nucleus indicating a modulatory influence of both flavonoids on signalling cascade(s).

Topics: Adaptation, Physiological; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Nucleus; Flavonoids; Flavonols; Fluoresceins; Forkhead Transcription Factors; Glutathione Transferase; Green Fluorescent Proteins; Hot Temperature; Kaempferols; Lipofuscin; Microscopy, Fluorescence; Molecular Structure; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; Recombinant Fusion Proteins; Signal Transduction; Time Factors; Transcription Factors

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Cell Nucleus; Ganglia, Spinal; Genes, Dominant; Glutamic Acid; Humans; Models, Biological; Naphthoquinones; Neurofilament Proteins; Neurons; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Protein Binding; Protein Structure, Quaternary; Protein Transport; Rats; RNA, Small Interfering; Spinal Cord; Transfection

Infiltration, accumulation, and degranulation of eosinophils in the lung are hallmarks of active allergic asthma. The pulmonary response to inhaled allergen triggers the secretion of eosinophil chemoattractants and antiapoptotic cytokines, including GM-CSF, IL-3, IL-4, IL-5, and eotaxin, among others. We recently showed that in vitro Pin1 regulated eosinophil production of and response to GM-CSF.. We sought to determine the effect of Pin1 inhibition on pulmonary eosinophilia after allergen challenge.. The Pin1 inhibitor juglone (5-hydroxy-1,4-naphthoquinone) was administered to allergen-sensitized and allergen-challenged Brown Norway rats. Bronchoalveolar lavage fluid and lungs were assessed for inflammation, cytokine expression, and Pin1 activity.. Juglone-treated rats showed a dramatic reduction (approximately 75%) in bronchoalveolar lavage fluid and pulmonary eosinophilia but no change in lymphocyte, monocyte/macrophage, or neutrophil numbers. GM-CSF and IL-5 expression were also significantly reduced, whereas Pin1-independent cytokines, such as eotaxin or IL-4, as well as housekeeping mRNAs and proteins, including actin, were unaffected by juglone. The eosinophils present in the lung in juglone-treated rats showed significantly greater apoptosis.. These data suggest that in vivo Pin1 blockade attenuates GM-CSF and IL-5 production and can selectively reduce eosinophilic allergic inflammation.. Eosinophils can be selectively reduced by Pin1 blockade, despite allergen challenge.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Eosinophils; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-5; Mice; Mice, Mutant Strains; Naphthoquinones; Pulmonary Eosinophilia; Rats; Rats, Inbred Strains; Respiratory Hypersensitivity

This study was part of a broader investigation of low molecular weight quinones under consideration as biocides for the control of aquatic nuisance species (ANS). Preliminary investigations identified the 2-ring naphthoquinones as broad spectrum biocides controlling a wide range of aquatic organisms. All biocides were relatively short-lived in saline waters, with half-lives between 5 and 30h. Juglone (5-hydroxy 1,4-naphthoquinone) and plumbagin (5-hydroxy-2-methyl-1,4- naphthoquinone) showed the greatest toxicity against most aquatic organisms. These qualities formed the basis for a patent focusing on these two compounds as biocides for ANS control, with juglone identified as the more cost-effective of the two. Although juglone has been extensively studied as a plant toxin and reducing agent, remarkably little information exists on its use as an aquatic biocide. We describe the toxicity of juglone over the range of water quality parameters likely to be encountered in ballast water, a major vector for ANS. Tests indicated that its molecular stability was enhanced in freshwater and particularly under neutral to acid conditions. This was supported by results of bioassays on the freshwater cladoceran Daphnia magna that indicated enhanced juglone toxicity at pHs of < or =6.7. A low octanol:water partition coefficient for juglone indicated little capacity for these compounds to be adsorbed by suspended particulates and for bioaccumulation. These properties together with their relatively rapid degradation (t1/2 < or =30h), particularly in the marine environment, indicated a low the risk of residual toxicity associated with the release of juglone-treated water.

Topics: Animals; Chromatography, High Pressure Liquid; Daphnia; Half-Life; Naphthoquinones; Water; Water Pollutants, Chemical

The insecticidal active components in Juglans mandshurica Maxim leaves were extracted by conventional method. The alcohol extract and its chloroform-extraction phase showed insecticidal activities in contact toxicity and stomach toxicity against Lymantria dispar L. and Mamestra brassicae L. larvae. When the concentration of the extract was above 10 g x L(-1), the corrected mortality was higher than 50% 5 days after applying the extract. Alcohol extract had higher insecticidal activities than its chloroform-extraction phase. The GC-MS analysis on the active components in the chloroform-extraction phase showed that the main component was juglone (5-hydroxy-1,4-naphthalenedione), and the others were 2,2'-methylenebis-6-(1,1-dimethylethyl)-4-methyl-diphenol and 2-methoxy-4-vinylphenol.

Topics: Animals; Chloroform; Dose-Response Relationship, Drug; Ethanol; Gas Chromatography-Mass Spectrometry; Insecticides; Juglans; Larva; Moths; Naphthoquinones; Plant Extracts; Plant Leaves

This study investigated the transformation of carbon tetrachloride (CT) by goethite, hematite, magnetite, and kaolinite treated with bisulfide to form coatings of iron monosulfide (FeS) and other Fe(II) species. These coatings contribute to abiotic natural attenuation in anaerobic environments. Batch kinetic experiments were performed under anoxic conditions at pH 8.0. Surface-area-normalized pseudo-first-order rate constants for CT transformation did not differ significantly for the three HS- treated iron oxides, but the rate of CT transformation by bisulfide-treated kaolinite was significantly lower, most likely due to kaolinite's lower iron content. The yield of chloroform (CF) from CT transformation was typically approximately 1%. There was negligible or only slight adsorption of several natural organic matter (NOM) model compounds to the surface of HS- treated goethite, and these compounds had no influence on CT transformation rate constants or CF yields. Juglone, on the other hand, adsorbed to a greater extent, and also significantly influenced the CF yield, increasing it by a factor of approximately 20 for HS- treated hematite. We speculate that juglone or its HS- addition product adsorbed to the mineral surface and acted as a hydrogen atom donor that reacted with the trichloromethyl radical intermediate, increasing the CF yield.

Topics: Adsorption; Carbon Tetrachloride; Carboxylic Acids; Catechols; Chloroform; Ferric Compounds; Ferrosoferric Oxide; Hydroquinones; Hydroxybenzoates; Iron Compounds; Kaolin; Minerals; Naphthoquinones; Oxidation-Reduction; Sulfides; Water Pollutants, Chemical

A reagentless lactate biosensor is described, based on an electropolymerized copolymer film poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-acetic acid-1,4-naphthoquinone). The quinone group, as part of the polymer backbone, is electroactive and very stable in neutral aqueous medium. It can therefore act as an immobilized mediator for the enzyme recycling, at a working potential much lower than those commonly reported in the literature for other mediators. Experimental conditions for amperometric measurements (temperature, pH) are studied, especially the interference between quinone and molecular oxygen to investigate the enzyme/quinone recycling kinetic. Some well-known interferents are shown to have no measurable effect on the amperometric curves.

Topics: Biosensing Techniques; Electric Conductivity; Electrochemistry; Enzymes, Immobilized; Equipment Design; Equipment Failure Analysis; Lactic Acid; Mixed Function Oxygenases; Naphthoquinones; Polymers

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.

Topics: Animals; Apoptosis; Binding Sites; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Dinitrobenzenes; Dithionitrobenzoic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; Molecular Structure; Naphthoquinones; Neoplasms; Nitrogen Compounds; Oxidation-Reduction; Proto-Oncogene Proteins c-bcl-2; Sulfhydryl Reagents; Superoxides; Thioredoxin-Disulfide Reductase

Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress-induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr-231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1-deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Animals; Catalysis; Enzyme Activation; Enzyme Inhibitors; Hydrogen Peroxide; Microtubules; Naphthoquinones; Neurons; Peptides; Phosphorylation; Rats; tau Proteins

Juglone is the active ingredient of the green flesh of walnuts and is known to be a strong irritant. We report the first two paediatric cases of contact pigmentation and acute irritant contact dermatitis due to the juice of green walnut husks in two young nursery-school playmates.

Topics: Child, Preschool; Dermatitis, Contact; Humans; Juglans; Male; Naphthoquinones

Leaves of Lomatia hirsuta are used in traditional medicine in Chile under the common name of "radal". A tea of radal is traditionally used for treatment of cough, bronchial troubles, and asthma. In a preliminary screening, extracts of the leaves revealed antifungal activity, and the present phytochemical study was undertaken to explain this activity and support the traditional use.. Along with the traditional tea, extracts of the leaves were screened for antifungal and toxic activities. The profile of secondary constituents was obtained using GC-MS.. 2-Methoxyjuglone was isolated from the leaves of Lomatia hirsuta and found to be active against the pathogenic fungus Candida albicans (MIC = 8 microg/mL). Cinnamic acid and vanillic acid were identified as major constituents in the tea by GC-MS. The tea was found not to be toxic against Artemia salina.. The presence of phenolic acids with antimicrobial properties supports the traditional use of Radal, and encourages further studies.

Topics: Animals; Antifungal Agents; Artemia; Candida albicans; Cinnamates; Ethnopharmacology; Microbial Sensitivity Tests; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Leaves; Plants, Medicinal; Proteaceae; Tea; Vanillic Acid

The aim of this study was to characterize the inhibitory mechanism in teak (Tectona grandis) bark and to determine its effectiveness against Listeria monocytogenes and methicillin resistant Staphylococcus aureus (MRSA).. Methanol extracts of teak bark were inhibitory to L. monocytogenes and MRSA by means of disc diffusion. Gas chromatography-mass spectrometry, and (1)H and (13)C nuclear mass resonance analyses revealed that the inhibitory compound had a molecular weight of 174, and a structure of 5-hydroxy-1,4-naphthalenedione (Juglone).. 5-hydroxy-1,4-naphthalenedione (Juglone) inhibited L. monocytogenes and MRSA.. A compound in an extract of teak bark was inhibitory to L. monocytogenes and MRSA.

Topics: Anti-Bacterial Agents; Gas Chromatography-Mass Spectrometry; Listeria monocytogenes; Magnetic Resonance Spectroscopy; Methanol; Methicillin Resistance; Molecular Weight; Naphthoquinones; Pigments, Biological; Plant Bark; Plant Extracts; Staphylococcus aureus; Verbenaceae

Phenolic acids (chlorogenic, caffeic, p-coumaric, ferulic, sinapic, ellagic, and syringic acid) as well as syringaldehyde and juglone were identified in ripe fruits of 10 walnut cultivars: Adams, Cisco, Chandler, Franquette, Lara, Fernor, Fernette, Alsoszentivani 117 (A-117), Rasna, and Elit. Analyses were done using a high-performance liquid chromatograph equipped with a diode array detector. Significant differences in the contents of identified phenolics were observed among cultivars. Phenolics were determined separately in the kernel and in the thin skin of the walnut, termed the pellicle. Not only in the kernel but also in the pellicle did syringic acid, juglone, and ellagic acid predominate (average values of 33.83, 11.75, and 5.90 mg/100 g of kernel; and 1003.24, 317.90, and 128.98 mg/100 g of pellicle, respectively), and the contents of ferulic and sinapic acid (average values of 0.06 and 0.05 mg/100 g of kernel and 2.93 and 2.17 mg/100 g of pellicle, respectively) were the lowest in all cultivars. The highest differences in the sum of all identified phenolics were observed between Rasna and Fernette fruits; in Rasna there were >2-fold higher contents of identified phenolics in both kernel and pellicle. It was found that the walnut pellicle is the most important source of walnut phenolics. The ratio between the contents in pellicle and kernel varied by at least 14.8-fold for caffeic acid (cv. Adams) and by up to 752.0-fold for p-coumaric acid (cv. Elit).

Topics: Acids, Carbocyclic; Benzaldehydes; Chromatography, High Pressure Liquid; Fruit; Juglans; Naphthoquinones

Juglone (5-hydroxy-1,4-naphtoquinone) is a natural toxin produced by walnut trees. In this study we show that juglone differentially reduces viability of human cells in culture. Normal fibroblast were found to be especially sensitive to juglone and lost viability primarily through a rapid apoptotic and necrotic response. This response may have been triggered by DNA damage since juglone induced a rapid and strong phosphorylation of H2AX in all phases of the cell cycle. Furthermore, juglone inhibits mRNA synthesis in human fibroblasts in a dose-dependent manner. Surprisingly, juglone caused a drastic reduction of the basal level of p53 in human fibroblasts and this loss could not be fully rescued by proteasome and calpain I inhibitors. However, when cells were pretreated with UV light or ionizing radiation, juglone was not able to reduce the cellular levels of activated p53. Our results show that juglone has multiple effects on cells such as the induction of DNA damage, inhibition of transcription, reduction of p53 protein levels and the induction of cell death.

Topics: Apoptosis; Blotting, Western; Calpain; Cell Death; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytotoxins; Fibroblasts; Flow Cytometry; Histones; Humans; Naphthoquinones; Necrosis; Phosphorylation; Proteasome Endopeptidase Complex; RNA, Messenger; Tumor Suppressor Protein p53; Ultraviolet Rays

Juglone (5-hydroxy-1,4-naphthoquinone) and plumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone) are yellow pigments found in black walnut (Juglans regia). Herbal preparations derived from black walnut have been used as hair dyes and skin colorants in addition to being applied topically for the treatment of acne, inflammatory diseases, ringworm, and fungal, bacterial, or viral infections. We have studied the cytotoxicity of these quinones to HaCaT keratinocytes. Exposure to juglone or plumbagin (1-20 microM) resulted in a concentration-dependent decrease in cell viability. The cytotoxicity of these quinones is due to two different mechanisms, namely, redox cycling and reaction with glutathione (GSH). Redox cycling results in the generation of the corresponding semiquinone radicals, which were detected by electron paramagnetic resonance. Incubation of keratinocytes with the quinones generated hydrogen peroxide (H(2)O(2)) and resulted in the oxidation of GSH to GSSG. Depletion of GSH by buthionine sulfoximine enhanced semiquinone radical production, increased H(2)O(2) generation, and produced greater cytotoxicity, suggesting that GSH plays an important protective role. Both quinones decreased the intracellular levels of GSH. However, plumbagin stoichiometrically converted GSH to GSSG, indicating that redox cycling is its main metabolic pathway. In contrast, much of the GSH lost during juglone exposure, especially at the higher concentrations (10 and 20 microM), did not appear as GSSG, suggesting that the cytotoxicity of this quinone may also involve nucleophilic addition to GSH. Our findings indicate that topical preparations containing juglone and plumbagin should be used with care as their use may damage the skin. However, it is probable that the antifungal, antiviral, and antibacterial properties of these quinones are the result of redox cycling.

Topics: Benzoquinones; Cell Line; Chromatography, High Pressure Liquid; Cytotoxins; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Glutathione; Glutathione Disulfide; Humans; Hydrogen Peroxide; Juglans; Keratinocytes; Naphthoquinones; Oxidation-Reduction; Pigments, Biological

The fragment of tau containing the first and third tubulin-binding motifs, involved in self-assembly of tau, was phosphorylated by protein kinase A (PKA). In the presence of hydroxynonenal (HNE) or in the presence of quinones such as juglone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q(0) or DMM), or menadione, the polymerization of this phosphorylated tau fragment is catalyzed, whereas polymerization of the unmodified fragment takes place in a lesser extent. The quinones coenzyme Q(0) and menadione are found in every cell, including neural cells, and may interact with tau protein to facilitate its assembly into filamentous structures. These tau filaments, assembled in the presence of quinones, have a fibrillar morphology very similar to that of paired helical filaments present in the brains of patients with Alzheimer's disease.

Topics: Amino Acid Motifs; Amino Acid Sequence; Benzoquinones; Cyclic AMP-Dependent Protein Kinases; Humans; Molecular Sequence Data; Naphthoquinones; Neurofibrillary Tangles; Peptide Fragments; Phosphorylation; Polymers; Protein Binding; Protein Processing, Post-Translational; Quinones; Recombinant Proteins; tau Proteins; Tubulin; Ubiquinone; Vitamin K 1; Vitamin K 3

Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

Topics: Enzyme Inhibitors; Glycine max; Naphthoquinones; Oxygen; Peptidylprolyl Isomerase; Plant Roots; Potassium Channel Blockers; Proton-Translocating ATPases; Water-Electrolyte Balance; Zea mays

The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation. Pin1 isomerizes the peptide bond of specific phosphorylated serine or threonine residues preceding proline in several proteins involved in various cellular events including mitosis, transcription, differentiation and DNA damage response. Many Pin1 substrates are antigens of the phosphodependent monoclonal antibody MPM-2, which reacts with a subset of proteins phosphorylated at the G2/M transition.. As MPM-2 is not a general marker of mitotic phosphoproteins, and as most mitotic substrates are phosphorylated more than once, we used a different phosphodependent antibody, mAb CC-3, to identify additional mitotic phosphoproteins and eventual Pin1 substrates by combining affinity purification, MALDI-TOF mass spectrometry and immunoblotting. Most CC-3-reactive phosphoproteins appeared to be known or novel MPM-2 antigens and included the RNA-binding protein p54nrb/nmt55, the spliceosomal protein SAP155, the Ki-67 antigen, MAP-1B, DNA topoisomerases II alpha and beta, the elongation factor hSpt5 and the largest subunit of RNA polymerase II. The CC-3 mitotic antigens were also shown to be Pin1 targets. The fine CC-3- and MPM-2-epitope mapping of the RNA polymerase II carboxy-terminal domain confirmed that the epitopes were different and could be generated in vitro by distinct kinases. Finally, the post-mitotic dephosphorylation of both CC-3 and MPM-2 antigens was prevented when cellular Pin1 activity was blocked by the selective inhibitor juglone.. These observations indicate that the mitotic phosphoproteins associated with Pin1 are phosphorylated on multiple sites, suggesting combinatorial regulation of substrate recognition and isomerization.

Topics: Antibodies, Monoclonal; Antigens; Enzyme Inhibitors; Epitope Mapping; HeLa Cells; Humans; Mitosis; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphoproteins; Phosphorylation; Protein Structure, Tertiary; RNA Polymerase II

The accumulation of molecular damage from attack by reactive oxygen species is one cause of aging. Therefore, some mutant organisms showing increased resistance to reactive oxygen species should live longer. We show that selection for Caenorhabditis elegans mutants that are resistant to juglone, a reactive oxygen species-generating compound, leads to the identification of long-lived mutants. Indeed, four of six resistant mutants isolated were also long-lived. This study illustrates once more the strong relationship between oxidative stress and the aging processes.

Topics: Aging; Animals; Caenorhabditis elegans; Crosses, Genetic; DNA Transposable Elements; Drug Resistance; Enzyme Inhibitors; Free Radicals; Genetic Techniques; Mutation; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Oxygen; Reactive Oxygen Species; Stress, Physiological; Time Factors; Ultraviolet Rays

The plant hormone auxin can regulate gene expression by destabilizing members of the Aux/IAA family of transcriptional repressors. Auxin-induced Aux/IAA degradation requires the protein-ubiquitin ligase SCF(TIR1), with auxin acting to enhance the interaction between the Aux/IAAs and SCF(TIR1). SKP1, Cullin, and an F-box-containing protein (SCF)-mediated degradation is an important component of many eukaryotic signaling pathways. In all known cases to date, the interaction between the targets and their cognate SCFs is regulated by signal-induced modification of the target. The mechanism by which auxin promotes the interaction between SCF(TIR1) and Aux/IAAs is not understood, but current hypotheses propose auxin-induced phosphorylation, hydroxylation, or proline isomerization of the Aux/IAAs. We found no evidence to support these hypotheses or indeed that auxin induces any stable modification of Aux/IAAs to increase their affinity for SCF(TIR1). Instead, we present data suggesting that auxin promotes the SCF(TIR1)-Aux/IAA interaction by affecting the SCF component, TIR1, or proteins tightly associated with it.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Base Sequence; Benzamides; DNA, Plant; Indoleacetic Acids; Macromolecular Substances; Naphthols; Naphthoquinones; Plants, Genetically Modified; Protein Structure, Tertiary; Recombinant Fusion Proteins; SKP Cullin F-Box Protein Ligases

The toxic naphthoquinone juglone (5-hydroxy-1,4-naphthoquinone) is efficiently degraded by the ligninolytic fungus Pleurotus sajor-caju, as demonstrated by the total bleaching within 9 d of a conventional liquid culture medium supplemented with 0.6 mM juglone. The oxidative degradation involves the production of hydrogen peroxide arising from both enzymic and non-enzymic oxidation reactions, promoted by the fungus. Juglone is not directly attacked by the oxidative enzymes of the ligninolytic machinery of P. sajor-caju, such as laccase, manganese peroxidase and arylalcohol oxidase. On the other hand, this naphthoquinone is a good substrate for a reductase, which triggers an auto-oxidative process producing reactive oxygen species and leading to juglone degradation. The degradation process continues to completion by means of a direct, presumably non-catalysed reaction with hydrogen peroxide.

Topics: Alcohol Oxidoreductases; Biodegradation, Environmental; Lignin; Naphthoquinones; Peroxidases; Pleurotus; Reactive Oxygen Species

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.

Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; Cytochrome c Group; Electrochemistry; Electron Transport; Intracellular Membranes; Kinetics; Membrane Proteins; Molecular Sequence Data; Multienzyme Complexes; Naphthoquinones; Nitric Oxide; Oxidation-Reduction; Oxidoreductases; Protein Subunits

There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2',7'-dichlorofluorescin (DCFH) and following the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300-400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm(2) UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm(2) UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm(2) UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H(2)O(2) or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H(2)O(2)). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.

Topics: Cell Line; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Fluoresceins; Free Radicals; Humans; Hydrogen Peroxide; Models, Chemical; Naphthoquinones; Oxidation-Reduction; Photochemistry; Reactive Oxygen Species; Time Factors; Ultraviolet Rays

The concentration of 7-methyljuglone was studied in the round-leaved sundew Drosera rotundifolia L. collected from different regions in Northern Finland. Samples for analysis were collected from peat bogs and sandpit habitats. The mean concentration of 7-methyljuglone varied from 1.0 to 2.3% of dry weight. Variation between years in the amount of 7-methyljuglone was significant in plants growing on sand, and in the northernmost region studied. Overall, the variation in the production of 7-methyljuglone among different populations of round-leaved sundew in Northern Finland was rather low. The variation between years in the production of 7-methyljuglone was more significant.

Topics: Drosera; Environment; Finland; Naphthoquinones; Rain; Seasons; Statistics as Topic; Temperature

The plant hormone auxin regulates diverse aspects of plant growth and development. Despite its importance, the mechanisms of auxin action remain poorly understood. In particular, the identities of the auxin receptor and other signaling proteins are unknown. Recent studies have shown that auxin acts by promoting the degradation of a family of transcriptional regulators called the Aux/IAA proteins. These proteins interact with another large family of plant-specific transcription factors called Auxin Response Factors (ARF) and negatively regulate their activity. Auxin stimulates Aux/IAA degradation by promoting the interaction between a ubiquitin protein ligase (E3) called SCF(TIR1) and the Aux/IAA protein. In this report, we demonstrate that auxin promotes the interaction between the Aux/IAA proteins and SCF(TIR1) in a soluble extract free of membranes, indicating that this auxin response is mediated by a soluble receptor. In addition, we show that the response is not dependent on protein phosphorylation or dephosphorylation but rather is prevented by an inhibitor of peptidyl-prolyl isomerases.

Topics: Arabidopsis; Arabidopsis Proteins; Cell-Free System; Enzyme Inhibitors; Indoleacetic Acids; Naphthoquinones; Peptidylprolyl Isomerase; Phosphorylation; Plant Proteins; Proline; Protein Structure, Tertiary; Receptors, Cell Surface; SKP Cullin F-Box Protein Ligases; Solubility; Transcription Factors

EGb 761, a standardized extract of Ginkgo biloba leaves, has been shown to have antioxidative properties. We have previously demonstrated that EGb 761 increases stress resistance and mean life span in the model organism Caenorhabditis elegans. In this study, the molecular mechanism of EGb 761 on alleviating effects of oxidative stress is further investigated using transgenic C. elegans expressing a jellyfish green fluorescent protein (GFP)-tagged inducible small heat-shock protein gene (hsp-16-2). The expression of hsp-16-2 induced by the pro-oxidant juglone and by heat shock was significantly suppressed by 86% and 33%, respectively, in the transgenic nematode fed with EGb 761. These effects of EGb 761 correlate with its ability to increase mean survival rate of the nematode in response to acute oxidative and thermal stresses, as well as to attenuate the basal levels of hydrogen peroxide in the organism. Thus, we interpret the suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress resulting from exogenous treatments, therefore leading to a decreased transcriptional induction of the reporter transgene. These results support the hypothesis that EGb 761 augments the natural antistress system of C. elegans, thus increasing stress resistance and life span.

Topics: Animals; Animals, Genetically Modified; Antioxidants; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Ginkgo biloba; Heat-Shock Proteins; Hydrogen Peroxide; Models, Biological; Naphthoquinones; Oxidants; Oxidative Stress; Plant Extracts; Stress, Physiological

We investigated the release of iron from ferritin in aqueous solutions exposed to high-frequency ultrasound (US). Our data suggests that superoxide produced as a result of ultrasonic cavitation acts as a reducing agent, enabling the release of iron from ferritin. We also found that the release of ferritin iron during US exposure is enhanced by the addition of 5-hydroxy-1,4-naphthoquinone. We hypothesize that this quinone is ultrasonically transformed into a semiquinone radical capable of directly and indirectly reducing Fe(3+) in ferritin to soluble Fe(2+). Our proposed mechanism for the release of iron from ferritin adds new insight to the synergistic effect of quinone-containing cancer drugs with US.

Topics: Antineoplastic Agents; Combined Modality Therapy; Ferritins; Iron; Naphthoquinones; Neoplasms; Oxidation-Reduction; Superoxides; Ultrasonic Therapy; Ultrasonics

The phenol content and antioxidant activity of extra virgin olive oils (EVOOs) differing in their origins and degradation degrees were studied. The o-diphenolic compounds typical of olive oil, namely, the oleuropein derivatives hydroxytyrosol (3',4'-dihydroxyphenylethanol, 3',4'-DHPEA), the dialdehydic form of elenolic acid linked to 3',4'-DHPEA (3',4'-DHPEA-EDA), and an isomer of oleuropein aglycon (3',4'-DHPEA-EA), were analyzed by HPLC. The antioxidant activity was studied by (a) the xanthine oxidase (XOD)/xanthine system, which generates superoxide radical and hydrogen peroxide; (b) the diaphorase (DIA)/NADH/juglone system, which generates superoxide radical and semiquinonic radical; and (c) the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) test. Results showed that EVOOs with a low degradation level (as evaluated by acidity, peroxide number, and spectroscopic indices K(232), K(270), and deltaK according to the EU Regulation) had a higher content of 3',4'-DHPEA-EDA and a lower content of 3',4'-DHPEA than oils having intermediate and advanced degradation levels. EVOOs with a low degradation degree were 3-5 times more efficient as DPPH scavengers and 2 times more efficient as inhibitors of the XOD-catalyzed reaction than oils with intermediate and advanced degradation levels. The DIA-catalyzed reaction was inhibited by EVOOs having low or intermediate degradation levels but not by the most degraded oils.

Topics: Antioxidants; Benzoquinones; Biphenyl Compounds; Chromatography, High Pressure Liquid; Dihydrolipoamide Dehydrogenase; Hydrogen Peroxide; Naphthoquinones; Olive Oil; Phenols; Picrates; Plant Oils; Superoxides; Xanthine; Xanthine Oxidase

In Alzheimer's disease, the peptidyl prolyl cis/trans isomerase Pin1 binds to phospho-Thr231 on Tau proteins and, hence, is found within degenerating neurons, where it is associated to the large amounts of abnormally phosphorylated Tau proteins. Conversely, Pin1 may restore the tubulin polymerization function of these hyperphosphorylated Tau. In the present work, we investigated, both at the cellular and molecular levels, the role of Pin1 in Alzheimer's disease through the study of its interactions with phosphorylated Tau proteins. We also showed that in neuronal cells, Pin1 upregulates the expression of cyclin D1. This, in turn, could facilitate the transition from quiescence to the G1 phase (re-entry in cell cycle) in a neuron and, subsequently, neuronal dedifferentiation and apoptosis. The involvement of Pin1 in the G0/G1 transition in neurons points to its function as a good target for the development of new therapeutic strategies in neurodegenerative disorders.

Topics: Alzheimer Disease; Cell Line; Cyclin D1; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Naphthoquinones; Neuroblastoma; Neurons; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphopyruvate Hydratase; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Spectrometry, Fluorescence; tau Proteins

We demonstrate here that the nematode Caenorhabditis elegans displays broad hormetic abilities. Hormesis is the induction of beneficial effects by exposure to low doses of otherwise harmful chemical or physical agents. Heat as well as pretreatment with hyperbaric oxygen or juglone (a chemical that generates reactive oxygen species) significantly increased subsequent resistance to the same challenge. Cross-tolerance between juglone and oxygen was also observed. The same heat or oxygen pretreatment regimens that induced subsequent stress resistance also increased life expectancy and maximum life span of populations undergoing normal aging. Pretreatment with ultraviolet or ionizing radiation did not promote subsequent resistance or increased longevity. In dose-response studies, induced thermotolerance paralleled the induced increase in life expectancy, which is consistent with a common origin.

Topics: Adaptation, Physiological; Animals; Caenorhabditis elegans; Gamma Rays; Hot Temperature; Hyperbaric Oxygenation; Longevity; Naphthoquinones; Oxidative Stress; Stress, Physiological; Ultraviolet Rays

Evidence of the addition of hydrogen sulfide to 5-hydroxy-1,4-naphthoquinone (juglone) in aqueous solution was obtained by nuclear magnetic resonance spectrometry (NMR), electron paramagnetic resonance spectrometry (EPR), UV-visible absorbance spectroscopy, and kinetic measurements. Although numerous addition reactions of thiolated alkane and aromatic compounds to quinones have been previously reported, this study indicates that inorganic forms of S(-II) act as nucleophiles and electrophiles in addition reactions to the alpha,beta-conjugated system of the quinone. The results obtained are consistent with competing Michael and radical addition reactions, with radical addition favored with increasing pH. The simplest structure that simulated the NMR spectrum was a sulfur molecule containing sulfur bonded between two juglone molecules at C-2 or C-3, while EPR measurements of aqueous reaction solutions indicated the presence of a stable semiquinone that contained a sulfur substituent at C-2 or C-3. Quinones are present in trace amounts in natural organic matter, and the addition of S(-II) has important implications with respect to transport and transformation of a variety of compounds that react with natural organic matter.

Topics: Air Pollutants; Cytotoxins; Hydrogen Sulfide; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Naphthoquinones; Organic Chemicals; Sulfur

The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms.

Topics: Amino Acid Sequence; Arabidopsis Proteins; Base Sequence; Binding Sites; Blotting, Northern; Cloning, Molecular; Conserved Sequence; Databases, Factual; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kinetics; Mitosis; Molecular Sequence Data; Mutation; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phenotype; Phosphorylation; Plant Proteins; Plants; Protein Binding; Protein Structure, Tertiary; RNA; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; Time Factors; Transgenes

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Topics: Amino Acid Sequence; Blotting, Western; Cell Nucleus; Cloning, Molecular; Cytosol; Digitalis; DNA, Complementary; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Genetic Complementation Test; Humans; Kinetics; Molecular Sequence Data; Mutation; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phenotype; Phosphoserine; Phosphothreonine; Plant Proteins; Plants, Medicinal; Plants, Toxic; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Subcellular Fractions; Substrate Specificity; Temperature; Time Factors

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

Topics: DNA, Recombinant; Dose-Response Relationship, Drug; Enzyme Inhibitors; HeLa Cells; Humans; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Plasmids; RNA Polymerase II; Sulfhydryl Compounds; Transcription, Genetic

Topics: Aged; Dermatitis, Allergic Contact; Female; Humans; Hyperpigmentation; Naphthoquinones; Nuts; Patch Tests; Pigments, Biological; Plant Extracts

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.

Topics: Alanine; Alkylation; Animals; Cysteine; DNA Damage; DNA Topoisomerases, Type II; Drosophila; Humans; Intercalating Agents; KB Cells; Mutagenesis, Site-Directed; Naphthoquinones; Quinones; Sulfhydryl Compounds; Tumor Cells, Cultured; Vitamin K

The growth of the white-rot basidiomycete Pleurotus sajor-caju in malt-agar plates was inhibited by three naturally occurring, plant-derived naphthoquinones: juglone, lawsone, and plumbagin. The latter two compounds exerted the most potent antifungal activity, and lawsone killed the mycelium at concentrations higher than 200 ppm. Plates containing juglone and lawsone presented large decolorized areas extending from area of fungal growth, suggesting an extracellular enzymatic degradation of these quinones. Screening of culture plates for extracellular enzymatic activities revealed the presence of both laccase and veratryl alcohol oxidase in most plates, the diffusion of both enzymes matching the decolorized area. In agitated cultures, the presence of juglone was found to stimulate the production of veratryl alcohol oxidase in a significant manner. This is the first time degradation of plant derived naphthoquinones by a white-rot fungus is reported.

Topics: Biodegradation, Environmental; Culture Media; Microbial Sensitivity Tests; Naphthoquinones; Plants; Pleurotus

Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoquinone), 1,4 naphthoquinone (1,4-NQ), and 1,2 naphthoquinone (1,2-NQ) considerably quenched the fluorescence of zeta-crystallin, where as the commonly used substrate, 9,10-phenanthrenequinone (PQ) did not induce significant quenching. Activity measurements showed only PQ served as a substrate for camel lens zeta-crystallin, while juglone, 1,4-NQ, and 1,2-NQ were inhibitors. Thus quinones that interacted with zeta-crystallin directly inhibited the enzyme, whereas the substrate had very low affinity for the enzyme in the absence of NADPH. Another substrate, dichlorophenol indophenol (DCIP), conformed to the same pattern; DCIP did not quench the fluorescence of the enzyme significantly, but served as a substrate. This pattern is consistent with an ordered mechanism of catalysis with quinone being the second substrate. All three naphthoquinones were uncompetitive inhibitors with respect to NADPH and noncompetitive with respect to PQ. These kinetics are similar to those exhibited by cysteine- and/or lysine-modifying agents. Juglone, 1,4-NQ, and 1,2-NQ interacted with and quenched the fluorescence of camel lens alpha-crystallin, but to lesser extent than that of zeta-crystallin.

Topics: 2,6-Dichloroindophenol; Animals; Camelus; Catalysis; Crystallins; Cysteine; Dose-Response Relationship, Drug; Kinetics; Lens, Crystalline; Ligands; NADP; Naphthoquinones; Protein Binding; Spectrometry, Fluorescence

The catalytic properties of rabbit heart acetohexamide reductase (RHAR) for naphthoquinones were examined. RHAR efficiently reduced 1,4-naphthoquinone and juglone (5-hydroxy-1,4-naphthoquinone), whereas it had little or no ability to reduce menadione (2-methyl-1,4-naphthoquinone) or plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone). The structural requirements for these four naphthoquinones and one acetohexamide analog, and the kinetic mechanism for the inhibition of acetohexamide reduction by juglone led us to conclude that the 2-methyl group of menadione and plumbagin prevents access of the substrates to the catalytic site of RHAR. Five of six peptides derived from RHAR showed 30-42% residue identities with regions in the amino acid sequence of mouse lung carbonyl reductase (MLCR) belonging to the short-chain dehydrogenase/reductase (SDR) family. The catalytically important residues (Arg-39, Ser-136, Tyr-149 and Lys-153) of MLCR were found in the peptide sequences of RHAR, despite the low residue identities between the two enzymes. RHAR is probably best classified as a member of the SDR family similar to MLCR.

Topics: Acetohexamide; Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Catalysis; Enzyme Inhibitors; Heart; Hypoglycemic Agents; Kinetics; Mice; Molecular Sequence Data; Myocardium; NADP; Naphthoquinones; Oxidation-Reduction; Rabbits; Spectrophotometry, Ultraviolet; Substrate Specificity

Volatiles were isolated from whole green mature walnuts (Hartley variety) with husks still intact using dynamic headspace sweeping with trapping on Tenax. A total of 45 volatile compounds were identified by GC-MS. Major volatiles identified included (E)-4, 8-dimethyl-1,3,7-nonatriene, pinocarvone, pinocarveol, myrtenal, myrtenol, (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, caryophyllene epoxide, verbenol, verbenone, and terpinolene. Green walnuts that had been infested with codling moth showed appreciably higher amounts emitted for (E)-4,8-dimethyl-1,3,7-nonatriene, (E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, alpha- and beta-pinenes, sabinene, (E)-beta-ocimene, (E,E)-alpha-farnesene, and linalool. The infested nuts also emitted benzyl methyl ether, isobutyl cyanide, and 1-nitro-3-methylbutane, compounds not found with the healthy nuts. Volatiles from uninfested green walnuts at the maturity stage where the husk was just beginning to split were also analyzed and compared.

Topics: Gas Chromatography-Mass Spectrometry; Naphthoquinones; Nuts; Plant Extracts; Terpenes; Volatilization

The ability of the naturally-occurring naphthoquinone derivatives, juglone and plumbagin, to increase tissue activities of the Phase II detoxification enzymes quinone reductase (QR) and glutathione transferase (GT) has been investigated in rats. Groups of female Sprague-Dawley rats were dosed by oral intubation on 5 consecutive days with either juglone or plumbagin at 12.5, 25, 50, 75, 100 or 125 mumoles/kg/day. The animals were then killed and the activities of QR and GT determined in tissue homogenates. The naphthoquinone derivatives had no significant effect on enzyme activities in the liver, spleen, heart, lung or urinary bladder. Increases in the activities of one or both enzymes were recorded, however, in the caecum, kidney, forestomach, duodenum, colon, glandular stomach and jejunum. The possibility that induction of Phase II enzymes could contribute to the previously-reported ability of juglone and plumbagin to protect animals against chemically-induced intestinal neoplasia is discussed.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Female; Glutathione Transferase; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Organ Specificity; Rats; Rats, Sprague-Dawley

Pin1, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases) has been implicated in the G2-M transition of the mammalian cell cycle. Pin1 interacts with a series of mitotic phosphoproteins, including Polo-like kinase-1, Cdc25C, and Cdc27, and is thought to act as a phosphorylation-dependent PPIase for these target molecules. Pin1 recognizes phosphorylated serine-proline or threonine-proline peptide-bonds in test substrates up to 1300-fold better than in the respective unphosphorylated peptides. To test directly whether Pin1 regulates the G2-M transition and/or progression through mitosis by catalyzing phosphorylation-dependent prolyl isomerization of essential mitotic targets, we examined the consequences of Pin1 depletion, achieved by (a) overexpression of Pin1 antisense RNA, (b) overexpression of dominant-negative Pin1, and (c) by a known small-molecule Pin1-PPIase inhibitor, juglone. The results of all of the three lines of investigation show that the catalytic activity of Pin1 is essential for tumor cell survival and entry into mitosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Catalysis; Cell Line; Cell Nucleus; Cell Survival; Chromatin; Cloning, Molecular; Enzyme Inhibitors; Histones; Humans; Interphase; Kinetics; Microscopy, Fluorescence; Mitosis; Mutation; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Paclitaxel; Peptidylprolyl Isomerase; Phosphorylation; Proline; Protein Structure, Tertiary; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

Topics: Amino Acid Isomerases; Amino Acid Substitution; Circular Dichroism; Cysteine; Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Fungal Proteins; Glutathione; Humans; Hydrolysis; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Spectrophotometry; Sulfhydryl Compounds; Transcription Factors

The effects of two naphthoquinones, juglone and plumbagin, and an isocoumarin, hydrangenol, on intestinal carcinogenesis in rats were examined by dietary exposure during the initiation phase. Starting at 5 weeks of age, male F344 rats were fed the diets containing either of the test chemicals at a concentration of 200 ppm or the control diet without the compounds. At 6 weeks of age, all animals were treated with s.c. injections of azoxymethane (AOM) (15 mg/kg body weight, once weekly for 3 weeks) or saline alone. Animals fed experimental diets were changed to the control diet 1 week after the last carcinogen treatment. Animals given plumbagin together with the carcinogen had a lower incidence (41%) and smaller multiplicity (0.48 +/- 0.62) of tumors in the entire intestine compared with those exposed to carcinogen alone (68% and 1.04 +/- 0.62) (P < 0.05 and < 0.01, respectively). The incidence and multiplicity of tumors in the small intestine (7% and 0.07 +/- 0.25) and the multiplicity of tumors in the entire intestine (0.60 +/- 0.76) of animals treated with juglone and the carcinogen were significantly less than those of animals treated with carcinogen alone (P < 0.05 in each). Hydrangenol tended to decrease the incidence and the multiplicity of tumors in the entire intestine induced by AOM, but the effect was not statistically significant. The present data suggest that the naphthoquinones, juglone and plumbagin, could be promising chemopreventive agents for human intestinal neoplasia.

Topics: Animals; Antineoplastic Agents, Phytogenic; Azo Compounds; Benzopyrans; Colonic Neoplasms; Coumarins; Diet; Intestinal Neoplasms; Isocoumarins; Male; Naphthoquinones; Rats; Rats, Inbred F344

In plants, the naphthoquinone juglone is known to be involved in pathogenic defence mechanisms, but it may also take part in plant developmental processes. This naphthoquinone can accumulate in a glycosylated form, namely hydrojuglone beta-d-glucopyranoside. The structural configuration of this compound was shown to be 1, 5-dihydroxy-4-naphthalenyl-beta-d-glucopyranoside by means of MS, NMR and nuclear Overhauser effect spectroscopy analyses. A hydrojuglone beta-d-glucopyranoside beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from Juglans regia L. The enzyme catalysed the release of juglone from hydrojuglone beta-d-glucopyranoside with high specificity and showed Michaelis-Menten kinetics with Km=0.62 mM and Vmax=14.5 microkat/mg of protein. This enzyme also showed a higher activity towards beta-d-fucosyl than beta-d-glucosyl bonds. The purified enzyme had an apparent Mr of 64000 by SDS/PAGE and a pI 8.9 by isoelectrofocusing PAGE. The purified enzyme was inhibited by several bivalent cations, such as Cu2+, Fe2+, Hg2+, and by d-glucono-1,5-lactone, showing non-competitive inhibition of the mixed type.

Topics: beta-Glucosidase; Catalysis; Glucosides; Hydrolysis; Isoelectric Focusing; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Chemical; Naphthols; Naphthoquinones; Substrate Specificity

Actias luna and Callosamia promethea larvae were fed birch foliage supplemented with juglone (5-hydroxy-1,4-naphthoquinone) to determine whether juglone causes oxidative stress in midguts of these species. Juglone is a substituent of walnut foliage. A. luna, but not C. promethea, thrives on walnut foliage, as well as birch foliage supplemented with juglone. After 2 and 3 days on juglone-containing diets, midgut samples from these animals were compared histologically and were analyzed for GSH and GSSG content. C. promethea, but not A. luna, midguts revealed partial loss of epithelial structure. In contrast, GSH and GSSG did not change significantly in either species. In a separate experiment, live midgut explants from each species were cultured for 4 h in 0, 0.05, and 0.25% juglone. In juglone-treated explants, GSSG increased 2.1 and 5.6-fold, respectively, for A. luna, and 1.6 and 2.7-fold, respectively, for C. promethea. There was also a small dose-dependent decrease in GSH in C. promethea, but not A. luna. Although histology indicates that the midgut is a target of juglone toxicity in C. promethea, GSH analyses from either species do not support the expectation that changes in GSH/GSSG explain differences in susceptibility to juglone toxicity.

Topics: Animals; Cytotoxins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Glutathione Disulfide; Larva; Moths; Naphthoquinones; Oxidative Stress

Quinones were studied for their growth inhibitory effect on cultured malignant cells. HCT-15 cells derived from human colon carcinoma were used for these experiments. Quinones used were arbutin in the benzoquinone group, juglone and lawsone in the naphthaquinone group, alizarin, emodin, 1,8-dihydroxyanthraquinone, and anthraquinone in the anthraquinone group, and xanthone. Cultured cells were incubated with various concentrations of the quinones for four days in a 5% CO2 incubator, after which cell numbers were counted and significance of differences was analyzed by Student's t test. Anthraquinones and naphthaquinones used in these experiments were more effective than the monocyclic quinone. The 50% suppression dose was less than 12.5 micrograms/ml for them. The number of OH groups seemed to play an important role in the degree of the cell growth inhibition: anthraquinones with 2 or 3 OH groups were more effective than those with no OH group like, 9,10-dioxoanthracene and xanthone. In fact, anthraquinones with no OH group and xanthone were not significantly effective. Flow cytometric histograms revealed a specific pattern; that is, lawsone and juglone in the naphthaquinone group and alizarin and 1,8-dihydroxy-anthraquinone in the anthraquinone group blocked mainly the S phase, and emodin in the anthraquinone group blocked the G1 to S phase of the cell cycle.

Topics: Anthraquinones; Antineoplastic Agents; Arbutin; Cell Division; Colonic Neoplasms; Humans; Naphthoquinones; Quinones; Tumor Cells, Cultured; Xanthenes; Xanthones

Conformational features of naphthazarin, juglone, and naphthoquinone have been examined via ab initio (Hartree-Fock) SCF calculations at 3-21G level. The results suggest a planar structure for all the three molecules and internally hydrogen-bonded structure for naphthazarin and juglone to be their preferred conformation. The optimized structural features are essentially the same as their crystal geometries. Molecular electrostatic potential (MEP) calculations using ab initio SCF methods ranging from 3-21G to 6-31G levels have been performed to visualize their three-dimensional pharmacophoric patterns and topography. The results indicate that two factors--(i) the depth, extent, and relative location of negative potential around hydroxyl and quinonoid oxygens, and (ii) a gradual loss of negative potential over the molecular plane due to the presence and orientation of the hydroxyl groups in the phenolic part of the molecules--are crucial for recognition interaction of the compounds with their receptors. Aqueous solvation seems to have significant influence on the MEP profiles of the molecules. Although intrinsic nucleophilicity increases for all the compounds, including the different conformers, due to aqueous solvation, the intrinsic electrophilicity shows remarkable decrease for all. It appears that the acidic nature of the hydrogens in these compounds and conformers decreases sharply along with shifts of positions while going from the gas phase to the aqueous phase. These observations may help to explain the mechanism of action(s) of the anthracyclin family of cytotoxic antibiotics.

Topics: Antineoplastic Agents; Doxorubicin; Models, Chemical; Naphthoquinones; Protein Conformation; Static Electricity; Structure-Activity Relationship

Guinea pig lens zeta-crystallin showed hyperbolic saturation curves with 9,10-phenanthrenequinone (PAQ). 5-hydroxy-1,4-naphthoquinone (juglone) and NADPH. Whereas camel lens zeta-crystallin showed hyperbolic saturation curves only with PAQ and NADPH, but slightly segmoidal with juglone. For both enzymes PAQ was the preferred substrate. The catalytic center activity (Kcat) values indicated that camel zeta-crystallin catalyzed the reduction of PAQ more efficiently than the guinea pig lens zeta-crystallin, although the Km values of the two enzymes for this quinone were very similar. The guinea pig lens zeta-crystallin catalyzed the reduction of Juglone far more efficiently than that of the camel lens zeta-crystallin. Juglone did not serve as an efficient substrate for both zeta-crystallins compared to PAQ and appeared to act as a potent competitive inhibitor, with Kl values of 75 nM and 20 microM for guinea pig lens zeta-crystallin and camel lens zeta-crystallin, respectively. Thus, the camel lens zeta-crystallin was less active toward juglone as a substrate as well as less sensitive to its inhibitory action, when compared with guinea pig lens zeta-crystallin. The inhibition mechanism of guinea pig and camel lens zeta-crystallin by juglone is discussed.

Topics: Animals; Camelus; Crystallins; Enzyme Inhibitors; Guinea Pigs; Kinetics; NADP; Naphthoquinones; Phenanthrenes; Quinone Reductases; Structure-Activity Relationship; Substrate Specificity

Using flow cytometric membrane potential measurements with the oxonol dye, we determined that 5.7-57 microM juglone depolarizes human lymphocytes in a dose dependent manner. The depolarizing effect of juglone was verified by patch-clamp. Juglone decreased whole-cell n-type K+ currents in human peripheral blood lymphocytes and accelerated inactivation; however, it did not influence the kinetics of activation of the K+ conductance. The percentage increase in K+ channel inactivation rate and the degree of drug induced block was independent of membrane potential, K+ channel block by juglone fully developed within 4 minutes and was not removable by washing with drug free extracellular solution. Blocking of n-type K+ channels by juglone is in concert with its depolarizing effect on human lymphocytes.

Topics: Cytotoxins; Humans; Ion Channel Gating; Lymphocytes; Membrane Potentials; Naphthoquinones; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers

The purpose of this study was to examine the possibility of using Artemia salina as a test organism in the search for compounds having the ability to protect against superoxide-mediated toxicity. The basic procedure for the assay using Artemia salina was performed as described in previous literature, with minor modifications. We found that Artemia salina are extremely sensitive to menadione bisulfite, a compound whose toxicity is probably mediated by intracellular superoxide generation. Desferrioxamine (desferal), a compound with known protective effects, was shown to display dramatic protective activity in our system. We also observed that an inhibitor of endogenous superoxide dismutase (SOD) activity increased the toxicity of menadione toward Artemia salina. In conclusion, this simple, inexpensive, and convenient assay could be a valuable addition to a screening effort in the search for compounds that will be protective against damage by superoxide or other active oxygen species.

Topics: Animals; Artemia; Biological Assay; Deferoxamine; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Drug Interactions; Hydrogen Peroxide; Naphthoquinones; Paraquat; Potassium Cyanide; Reactive Oxygen Species; Sensitivity and Specificity; Superoxide Dismutase; Superoxides; Ubiquinone; Vitamin K; Vitamin K 3

Nitroxides stable radicals are unreactive toward most diamagnetic molecules, but readily undergo one-electron redox reactions with paramagnetic species such as free radicals and transition metals, thus serving as cell-permeable antioxidants. The cytotoxicity of juglone (5-hydroxy-1,4-naphthoquinone), like that of other naphthoquinones, requires bioreduction to yield the semiquinone which in turn reduces oxygen to O2.-. Therefore, nitroxides are expected to mitigate cytotoxicity of quinone-based xenobiotics, such as naphthoquinones. In the present study, in vitro scission of isolated DNA was induced upon juglone reduction by glutathione and Fe(II) ions, however, not by xanthine oxidase or cytochrome c reductase. The DNA scission was inhibited by nitroxides, catalase and chelating agents, though not by superoxide dismutase. Juglone was more toxic toward bacterial cells under hypoxia than under air. Nitroxides < or = 2 mM protected bacterial cells from juglone-induced toxicity under both aerobic and hypoxic conditions. The cytoprotective effect of lipophilic nitroxide was greater than that of hydrophilic ones. Catalase and metal chelating agents decreased juglone-induced cell killing, whereas H2O2 increased it. The mechanisms underlying the nitroxides protective effect involve (a) the reoxidation of reduced transition metal ions, (b) the selective radical-radical reaction with juglone semiquinone, and possibly (c) under aerobic condition catalytic removal of extra- and intracellular O2.-. The present results suggest also that the cell membrane rather than DNA is the main target of juglone toxicity.

Topics: Catalase; Cyclic N-Oxides; DNA Damage; Drug Interactions; Escherichia coli; Free Radicals; Hydrogen Peroxide; Naphthoquinones; Nitrogen Oxides; Spin Labels; Spiro Compounds; Superoxide Dismutase

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.

Topics: Animals; Catalysis; Crystallins; Cyclic N-Oxides; Cytochrome c Group; Dicumarol; Electron Spin Resonance Spectroscopy; Guinea Pigs; Hydrogen Peroxide; Kinetics; Lens, Crystalline; NADP; Naphthoquinones; Nitrofurantoin; Oxygen; Quinone Reductases; Quinones; Spin Labels; Substrate Specificity

Studies of the acceptor reductase reaction of yeast glutathione reductase (EC 1.6.4.2) revealed that the competitive inhibitors for NADPH, 2',5'-ADP and Br- decrease the rate constants for the enzyme oxidation by ferricyanide, phenanthrene quinone, and juglone. A similar effect is observed when NADH which does not bind to the reduced enzyme is used as substrate. These observations support the hypothesis that non-physiological redox agents are reduced at the NADP(H)-binding center of glutathione reductase and that NADP(H) binding stimulates the reaction by displacing tyrosine-197 which protects FAD from the solvent.

Topics: Binding Sites; Ferricyanides; Glutathione Reductase; Indicators and Reagents; NADP; Naphthoquinones; Oxidation-Reduction; Phenanthrenes; Saccharomyces cerevisiae

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone) has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO2 production from 1-14C-labelled glucose. Exposure of lenses to 15 microM 1,2-naphthoquinone or 20 microM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced stimulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP+. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.

Topics: Animals; Carmustine; Crystallins; Dose-Response Relationship, Drug; Guinea Pigs; Hydrogen Peroxide; Lens, Crystalline; NADP; Naphthoquinones; Organ Culture Techniques; Pentose Phosphate Pathway; Rats; Rats, Inbred Strains

The effect of hydroxy substitution on 1,4-naphthoquinone toxicity to cultured rat hepatocytes was studied. Toxicity of the quinones decreased in the series 5,8-dihydroxy-1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 1,4-naphthoquinone greater than 2-hydroxy-1,4-naphthoquinone, and intracellular GSSG formation decreased in the order 5,8-dihydroxy-1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone much greater than 1,4-naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. The electrophilicity of the quinones decreased in the order 1,4-naphthoquinone much greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4-naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. Treatment of the hepatocytes with BSO (buthionine sulfoximine) or BCNU (1,3-bis-2-chloroethyl-1-nitrosourea) increased 5-hydroxy-1, 4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone toxicity, whereas neither BSO nor BCNU largely affected 1,4-naphthoquinone and 2-hydroxy-1, 4-naphthoquinone toxicity. Dicumarol increased the toxicity of 1,4-naphthoquinone dramatically and somewhat the toxicity of 2-hydroxy-1,4- naphthoquinone, whereas 5-hydroxy-1,4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone toxicity increased only slightly. The toxicity of 5,8-dihydroxy-1,4-naphthoquinone decreased dramatically in reduced O2 concentration, whereas 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 2-hydroxy-1,4-naphthoquinone toxicity was not largely affected. It was concluded that 5,8-dihydroxy-1,4-naphthoquinone toxicity is due to free radical formation, whereas the toxicity of 1,4-naphthoquinone and of 5-hydroxy-1,4-naphthoquinone also has an electrophilic addition component. The toxicity of 2-hydroxy-1,4-naphthoquinone could not be fully explained by either of these phenomena.

Topics: Animals; Buthionine Sulfoximine; Carmustine; Cell Survival; Dicumarol; Glutathione; Liver; Male; Methionine Sulfoximine; Mitochondria, Liver; Molecular Structure; Naphthoquinones; Oxidation-Reduction; Oxygen Consumption; Rats; Rats, Inbred Strains; Structure-Activity Relationship

Studies with four benzoquinones, viz. juglone, embelin, maesaquinone and maesanin, on rat liver mitochondria oxidative phosphorylation have been carried out. Three of the benzoquinones are uncouplers in the order juglone greater than maesoquinone greater than embelin, while maesanin is an inhibitor of electron transport and oxidative phosphorylation.

Topics: Animals; Benzoquinones; Chick Embryo; Mitochondria, Liver; Naphthoquinones; Oxidative Phosphorylation; Oxygen Consumption; Quinones

The effect of several structurally related 1,4-benzoquinones (BQ) and 1,4-naphthoquinones (NQ) on the activity of rat hepatic glutathione S-transferases (GST) was studied. For the 1,4-benzoquinones, the extent of inhibition increased with an increasing number of halogen substituents. Neither the type of halogen nor the position of chlorine-atoms was of major importance. Similarly, 2,3-dichloro-NQ demonstrated a considerably higher inhibitory activity than 5-hydroxy-NQ. 2-Methyl derivatives of NQ did not inhibit GST activity at all. The irreversible nature of the inhibition was shown both by the time-course of the inhibition as well as by the fact that removal of the inhibitor by ultrafiltration did not restore the enzymatic activity. Incubation of quinones and enzyme in the presence of the competitive inhibitor S-hexyl-glutathione, slowed the inhibition considerably, indicating an involvement of the active site. Isoenzyme 3-3 was found to be most sensitive towards the whole series of inhibitors, whereas the activity of isoenzyme 2-2 was least affected in all cases. The inhibition by quinones is probably mainly due to covalent modification of a specific cysteine residue in or near the active site. The differential sensitivities of individual isoenzymes indicates that this residue is more accessible and/or easier modified in isoenzyme 3-3 than in any of the other isoenzymes tested. The findings suggest that quinones form a class of compounds from which a selective in vivo inhibitor of the GST might be developed.

Topics: Animals; Benzoquinones; Glutathione; Glutathione Transferase; Isoenzymes; Liver; Molecular Structure; Naphthoquinones; Quinones; Rats; Structure-Activity Relationship

Black walnut toxicosis was diagnosed in 10 horses at one stable. The time from exposure to shavings to development of clinical signs was 8 to 12 hours. Most common clinical signs were moderate to severe laminitis (Obel grade 2 or 3), pitting edema of the distal portion of the limbs, and rapid respiratory rate. Two horses had clinical signs of colic and 2 other horses had anorexia and lethargy. All 10 horses recovered without complications.

Topics: Animals; Anorexia; Colic; Edema; Horse Diseases; Horses; Lameness, Animal; Male; Naphthoquinones; Pigments, Biological; Plant Poisoning; Respiratory Insufficiency; Wood

The chitin synthetase inhibitor plumbagin and its 2-demethyl derivative juglone were found to inhibit in a dose-response fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity associated with adult female Aedes aegypti, wandering stage larvae of Drosophila melanogaster, and fat body and midgut from last instar larvae of Manduca sexta. The concentration of these naphthoquinones required to elicit a 50% inhibition of the steroid hydroxylase activity in all the insects was approximately 1 x 10(-4) M.

Topics: Aedes; Animals; Aryl Hydrocarbon Hydroxylases; Chitin Synthase; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drosophila melanogaster; Female; Glucosyltransferases; Larva; Lepidoptera; Moths; Naphthoquinones; Steroid Hydroxylases

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.

Topics: Androstenedione; Animals; Cytochrome c Group; Cytochrome P-450 Enzyme System; Humans; Microsomes; Microsomes, Liver; Naphthoquinones; Oxygenases; Placenta; Rabbits; Spectrophotometry; Vitamin K

Topics: Cosmetics; Female; Humans; Leukoplakia, Oral; Lip Neoplasms; Naphthoquinones; Pigments, Biological; Trees

The mechanisms of toxicity to isolated rat hepatocytes of two structurally related naphthoquinones have been studied. Both 5-OH-1,4-naphthoquinone (5-OH-1,4-NQ; juglone) and 2-OH-1,4-naphthoquinone (2-OH-1,4-NQ; lawsone) caused a concentration-dependent cytotoxicity to hepatocytes which was preceded by a depletion of intracellular glutathione. 5-OH-1,4-NQ caused a depletion of intracellular glutathione when incubated either at 4 degrees C or 37 degrees C whereas 2-OH-1,4-NQ caused a depletion of intracellular glutathione when the hepatocytes were incubated at 37 degrees C but not at 4 degrees C. 5-OH-1,4-NQ but not 2-OH-1,4-NQ reacted with glutathione in buffered solution. These results suggested that the depletion of intracellular glutathione by 2-OH-1,4-NQ is enzyme mediated whereas in the case of 5-OH-1,4-NQ the direct chemical reaction with gluathione may be largely responsible for the depletion. A critical role for depletion of protein thiols in menadione-induced cytotoxicity has been proposed. In agreement with earlier work, menadione caused a decrease in protein sulphydryls prior to cell death, however, at cytotoxic concentrations of both 2-OH-1,4-NQ and 5-OH-1,4-NQ this decrease only accompanied rather than preceeded cell death. The mechanism of toxicity of 5-OH-1,4-NQ is similar to that of other naphthoquinones and involves formation of its corresponding naphthosemiquinone, active oxygen species and redox cycling as it stimulated a disproportionate increase in both microsomal NADPH oxidation and oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antifungal Agents; Glutathione; In Vitro Techniques; Liver; Male; Microsomes, Liver; NADP; Naphthoquinones; Oxidation-Reduction; Oxygen Consumption; Rats; Rats, Inbred Strains; Sulfhydryl Compounds

Topics: Chromatography, Gas; Gas Chromatography-Mass Spectrometry; Naphthoquinones; Plants, Medicinal; Terpenes

Using ESR spectroscopy, we show that benzoquinone, 1,4-naphthoquinone and 5-hydroxy-1,4-naphthoquinone react readily with thiol containing compounds, such as glutathione, to form their respective semiquinone anion radicals. These quinones react similarly, but less readily, with the amino group of amino acids. The therapeutic or toxicological significance of the formation of semiquinone anion radicals from the reaction of quinones with nucleophiles, such as thiols and amines, remains to be assessed.

Topics: Amino Acids; Benzoquinones; Chemical Phenomena; Chemistry; Electron Spin Resonance Spectroscopy; Glutathione; Naphthoquinones; Quinones

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.

Topics: Drug Resistance, Microbial; Escherichia coli; Kinetics; Mutation; Naphthoquinones; Oxidation-Reduction; Paraquat; Pigments, Biological; Superoxide Dismutase

Melanin biosynthesis in the human pathogen Wangiella dermatitidis was inhibited by tricyclazole, causing pentaketide melanin metabolites to accumulate in the cultures. One of these metabolites, scytalone, was racemic and thus different than the (+)-enantiomer from Verticillium dahliae. An albino mutant of W. dermatitidis metabolized scytalone to a pigment ultrastructurally identical to wild-type melanin. Cell-free homogenates of the wild type carried out typical reductive and dehydrative reactions with known melanin intermediates and the reductive reactions were inhibited by tricyclazole. Other reductive and dehydrative reactions that utilize flaviolin and 2-hydroxyjuglone were studied anaerobically with homogenates from both the wild type and the albino mutant. The homogenates converted flaviolin to 5-hydroxyscytalone and products identical to those obtained from 2-hydroxyjuglone. The albino, in culture, carried out the same reactions with 2-hydroxyjuglone but metabolized flaviolin to a number of unknown colored products apparently through oxidative reactions. Similarities between the melanin pathway and the flaviolin and 2-hydroxyjuglone branch pathways are discussed and tricyclazole is shown to inhibit reductive reactions with naphthols in the three pathways.

Topics: Melanins; Mitosporic Fungi; Naphthols; Naphthoquinones; Optical Rotation; Thiazoles

Juglone-induced cells of Pseudomonas putida J 1 were shown to contain two isofunctional juglone hydroxylases. Both enzymes were purified about 125-fold to homogeneity in polyacrylamide gel electrophoresis. The molecular masses of the native enzymes, as determined by Sephacryl S-200 gel filtration were 59 000 Da for enzyme 1 and 56 000 Da for enzyme 2. The molecular masses of the subunits were determined by dodecyl sulfate polyacrylamide gel electrophoresis as 25 000 Da (enzyme 1) and 23 500 Da (enzyme 2). Both enzymes hydroxylated juglone, naphthazarin, 1,4-naphthoquinone and 2-chloro-1,4-naphthoquinone, but they were completely inactive against naphtholes. The activity of both hydroxylases was not affected by chelating agents, thiol reagents, however, were found to be strong inhibitors. No external cofactors such as Fe2, NADH, NADPH, FAD, FMN were required for activity. Concomitant with the hydroxylation of juglone the consumption of oxygen in a molar ratio 2: 1 (juglone: oxygen) was observed but none of the enzymes incorporated 18O2 into the substrate juglone. By activity staining enzyme 1 was found to be present in induced and non-induced cells of P. putida J 1, enzyme 2, however, only in juglone-induced cells.

Topics: Enzyme Induction; Hydrogen-Ion Concentration; Kinetics; Mixed Function Oxygenases; Molecular Weight; NAD; Naphthoquinones; Oxidoreductases; Pseudomonas; Spectrophotometry, Ultraviolet; Substrate Specificity

Electron transport from H2, NADPH, NADH and succinate to O2 or ferricytochrome c in respiratory particles isolated from Anacystis nidulans in which hydrogenase had been induced was abolished after extraction of the membranes with n-pentane; oxidation of ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine remained unaffected. Incorporation of authentic ubiquinone-10, plastoquinone-9, menaquinone-7 and phylloquinone (in order of increasing efficiency) restored the electron-transport reactions. ATP-dependent reversed electron flow from NNN'N'-tetramethyl-p-phenylenediamine to NADP+ or, via the membrane-bound hydrogenase, to H+ was likewise abolished by pentane extraction and restored by incorporation of phylloquinone. Participation of the incorporated quinones in the respiratory electron-transport reactions of reconstituted particles was confirmed by measuring the degree of steady-state reduction of the quinones. Isolation and identification of the quinones present in native Anacystis membranes yielded mainly plastoquinone-9 and phylloquinone; neither menaquinone nor alpha-tocopherolquinone could be detected. Together with the results from reconstitution experiments this suggests that phylloquinone might function as the main respiratory quinone in Anacystis nidulans.

Topics: Cell Membrane; Cyanobacteria; Dibromothymoquinone; Electron Transport; Kinetics; NADP; Naphthoquinones; Oxygen Consumption; Quinones; Tetramethylphenylenediamine; Ultraviolet Rays; Vitamin K

Juglone, a toxic compound found in all parts of plants of the walnut tree family Jugans, was evaluated as the possible toxin involved in black walnut shaving-associated laminitis in the horse. Large amounts (up to 1 g) of this chemical administered per os inconsistently caused mild signs of laminitis in ponies. Topical application of juglone to the digits of horses caused local skin irritation but did not cause laminitis. Intravenous administration of juglone caused acute pulmonary edema in some individuals previously exposed to the compound per os or IV.

Topics: Animals; Foot Diseases; Hoof and Claw; Horse Diseases; Horses; Naphthoquinones; Plant Poisoning; Pulmonary Edema

Topics: Acetylcholine; Acetyltransferases; Animals; Brain; Choline; Choline O-Acetyltransferase; In Vitro Techniques; Kinetics; Male; Mice; Naphthoquinones; Naphthylvinylpyridine; Pyridines; Rabbits

Topics: Drug Therapy; Geriatrics; Health; Humans; Naphthoquinones; Neurodermatitis

Topics: Drug Therapy; Humans; Naphthoquinones; Neurodermatitis

Crushed unripe walnut hulls (Juglans nigra), when extracted with ether, yield an extract which sedates or at least depresses the movements of Daphnia magna, leopard frogs, perch, catfish, goldfish, mice, rats, and rabbits. One purified depressant compound, 5-hydroxy-1,4-naphthoquinone (juglone), has been isolated and tested on most of these species.

Topics: Animals; Daphnia; Juglans; Mice; Naphthoquinones; Nuts; Psychopharmacology; Rats

Topics: Halogens; Naphthoquinones

Topics: Naphthoquinones